Patent application title: USE OF THE GENES IN THE HOG, Ras AND cAMP PATHWAY FOR TREATMENT OF FUNGAL INFECTION
Inventors:
Yong-Sun Bahn (Seoul, KR)
Young-Joon Ko (Seoul, KR)
Shin-Ae Maeng (Seoul, KR)
Kwang Woo Jung (Seoul, KR)
Gyu Bum Kim (Gimpo-Si, KR)
IPC8 Class: AA61K39395FI
USPC Class:
4241391
Class name: Drug, bio-affecting and body treating compositions immunoglobulin, antiserum, antibody, or antibody fragment, except conjugate or complex of the same with nonimmunoglobulin material binds antigen or epitope whose amino acid sequence is disclosed in whole or in part (e.g., binds specifically-identified amino acid sequence, etc.)
Publication date: 2012-04-19
Patent application number: 20120093817
Abstract:
Provided herein are uses of genes for HOG, Ras and cAMP signal
transduction pathways to treat fungal infection. To regulate the HOG
pathway of Cryptococcus neoformans, roles of SSK1, TCO2, SSK2, PBS2,
HOG1, ENA1 and NHA1 genes were investigated to find that a biosynthesis
level of ergosterol is increased when these genes are inhibited. When the
genes are inhibited, a large amount of ergosterol is distributed on a
fungal cell membrane. Accordingly, since there are many working points of
an ergosterol-binding antifungal agent, an efficiency of the
ergosterol-binding antifungal agent can be considerably improved. To
regulate the Ras and cAMP pathways of Cryptococcus neoformans, roles of
RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 and HSP122 genes were
investigated to find that a sensitivity to a polyene- or azole-based drug
is increased when these genes are inhibited. Therefore, an antifungal
pharmaceutical composition including an inhibitor against the gene or
protein encoded by the same can be used as an excellent combined
antifungal agent which can reduce a conventional amount of an antifungal
agent used and increase an efficiency.Claims:
1. A method of treating fungal infection, comprising: injecting an
effective amount of an inhibitor against at least one protein selected
from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1
of Cryptococcus neoformans into a subject.
2. The method of treating fungal infection of claim 1, wherein the inhibitor against at least one protein selected from the group consisting of Ssk1, Ena1 and Nha1.
3. The method of treating fungal infection of claim 1, wherein an ergosterol-binding antifungal agent or azole-based antifungal agent is sequentially or simultaneously injected with the inhibitor.
4. The method of treating fungal infection of claim 3, wherein the ergosterol-binding antifungal agent is a polyene-based antifungal agent.
5. The method of treating fungal infection of claim 4, wherein the polyene-based antifungal agent is at least one selected from the group consisting of amphotericin B, natamycin, rimocidin, filipin, nystatin and candicin.
6. The method of treating fungal infection of claim 5, wherein the polyene-based antifungal agent is amphotericin B.
7. The method of treating fungal infection of claim 3, wherein the azole-based antifungal agent is at least one selected from the group consisting of ketoconazole, fluconazole, itraconazole and voriconazole.
8. A method of treating fungal infection comprising: Injecting an effective amount of an inhibitor against at least one gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of Cryptococcus neoformans into a subject.
9. The method of treating fungal infection of claim 8, wherein the inhibitor against at least one gene selected from the group consisting of SSK1, ENA1 and NHA1.
10. The method of treating fungal infection of claim 8, wherein an ergosterol-binding antifungal agent or azole-based antifungal agent is sequentially or continuously injected with the inhibitor.
11. The method of treating fungal infection of claim 10, wherein the ergosterol-binding antifungal agent is a polyene-based antifungal agent.
12. The method of treating fungal infection of claim 11, wherein the polyene-based antifungal agent is at least one selected from the group consisting of amphotericin B, natamycin, rimocidin, filipin, nystatin, and candicin.
13. The method of treating fungal infection of claim 12, wherein the polyene-based antifungal agent is amphotericin B.
14. The method of treating fungal infection of claim 10, wherein the azole-based antifungal agent is at least one selected from the group consisting of ketoconazole, fluconazole, itraconazole and voriconazole.
15. An antifungal combined formulation, comprising: an inhibitor against at least one protein selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of Cryptococcus neoformans; and an ergosterol-binding antifungal agent or azole-based antifungal agent.
16. A method of screening an antifungal agent comprising: contacting at least one protein selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of Cryptococcus neoformans with a candidate material; and determining whether the candidate material inhibits or stimulates an activity of the protein.
17. The method of claim 16, wherein the antifungal agent is an inhibitor against an Ssk1, Ena1 or Nha1 protein.
18. A method of screening an antifungal agent comprising: contacting at least one gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of Cryptococcus neoformans with a candidate material; and determining whether the candidate material inhibits or stimulates an activity of the gene.
19. The method of claim 18, wherein the antifungal agent is an inhibitor against an SSK1, ENA1 or NHA1 gene.
20. A method of treating fungal infection, comprising: injecting an effective amount of an inhibitor against at least one protein selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of Cryptococcus neoformans into a subject.
21. The method of treating fungal infection of claim 20, wherein the inhibitor is an inhibitor against a Cac1 or Pka1 protein.
22. The method of treating fungal infection of claim 20, wherein a polyene- or azole-based antifungal agent is sequentially or simultaneously injected with the inhibitor.
23. The method of treating fungal infection of claim 22, wherein the polyene-based antifungal agent is at least one selected from the group consisting of amphotericin B, natamycin, rimocidin, filipin, nystatin, and candicin.
24. The method of treating fungal infection of claim 23, wherein the polyene-based antifungal agent is amphotericin B.
25. The method of treating fungal infection of claim 22, wherein the azole-based antifungal agent is at least one selected from the group consisting of ketoconazole, fluconazole, itraconazole and voriconazole.
26. The method of treating fungal infection of claim 20, wherein an inhibitor against at least one protein selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of Cryptococcus neoformans is sequentially or simultaneously injected with the inhibitor.
27. A method of treating fungal infection, comprising: injecting an effective amount of an inhibitor against at least one gene selected from the group consisting of RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 and HSP122 of Cryptococcus neoformans into a subject.
28. The method of treating fungal infection of claim 27, wherein the inhibitor is an against CAC1 or PKA1 gene.
29. The method of treating fungal infection of claim 27, wherein a polyene- or azole-based antifungal agent is sequentially or simultaneously injected with the inhibitor.
30. The method of treating fungal infection of claim 29, wherein the polyene-based antifungal agent is at least one selected from the group consisting of amphotericin B, natamycin, rimocidin, filipin, nystatin, and candicin.
31. The method of treating fungal infection of claim 30, wherein the polyene-based antifungal agent is amphotericin B.
32. The method of treating fungal infection of claim 29, wherein the azole-based antifungal agent is at least one selected from the group consisting of ketoconazole, fluconazole, itraconazole and voriconazole.
33. The method of treating fungal infection of claim 27, wherein an inhibitor against at least one gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of Cryptococcus neoformans is sequentially or simultaneously injected with the inhibitor.
34. An antifungal combined formulation, comprising: an inhibitor against at least one protein selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of Cryptococcus neoformans; and at least one antifungal agent selected from the group consisting of a polyene-based antifungal agent, an azole-based antifungal agent, and an inhibitor against at least one protein or gene coding for the same selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of Cryptococcus neoformans.
35. A method of screening an antifungal agent comprising: contacting at least one protein selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of Cryptococcus neoformans with a candidate material; and determining whether the candidate material inhibits or stimulates an activity of the protein.
36. The method of claim 35, wherein the antifungal agent is an inhibitor against a Cac1 or Pka1 protein.
37. A method of screening an antifungal agent comprising: contacting at least one gene selected from the group consisting of RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 and HSP122 of Cryptococcus neoformans with a candidate material; and determining whether the candidate material inhibits or stimulates the activity of the gene.
38. The composition method of claim 37, wherein the antifungal agent is an inhibitor against a CAC1 or PKA1 gene.
Description:
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of Korean Patent Applications No. 10-2009-0001947, filed on Jan. 9, 2009, and No. 10-2009-0127206, filed on Dec. 18, 2009, and all the benefits accruing therefrom under 35 U.S.C. §119 which is hereby incorporated by reference as if fully set forth herein.
BACKGROUND
[0002] 1. Field
[0003] The present invention relates to uses of genes for HOG, Ras and cAMP signal transduction pathways to treat fungal infection.
[0004] 2. Description of the Related Art
[0005] The existence and proliferation of an organism in a specific environment is usually determined by an ability to react and adapt to various environmental stresses and maintain cell homeosis. Cells regulate a key process by performing a series of combined signal networks. Among these, a p38/Hog1 mitogen-activated protein kinase (MAPK) dependent signal pathway plays an important role to regulate a wide range of stress reactions in eukaryotes, for example, from yeasts to humans.
[0006] A stress-activated p38 MAPK in a mammal induces various stress-related signals limiting change in osmosis, UV radiation, programmed apoptosis, and adaptation to an immune response by generation of cytokine and control of inflammation. Similar stress-sensitive signal transduction systems have been discovered in other species. Fungi have p38-like MAPKs regulating various stress-related responses. In the budding yeast, Saccharomyces cerevisiae (S. cerevisiae), HogI MAPK regulates a stress-related response to osmotic shock, oxidative damage and heavy metal damage. The fission yeast, Schizosaccharomyces pombe (S. pombe), also has a homolog of HogI, Sty1 (also known as Spc1 and Phh1), which is associated with adaptation to various stresses including osmotic shock, heat shock, oxidative damage and heavy metal damage, carbon deficiency and UV radiation. Interestingly, Sty1 is also associated with growth control, reproduction and differentiation. Hog1 MAPK orthologs are also found in other ascomycete pathogenic fungi including Candida albicans (Hog1) and Aspergillus fumigatus (SakA), and known to mediate reactions induced by various environmental causes including osmotic shock, UV radiation, oxidative damage and high temperature.
[0007] A common molecular mechanism of the p38/HogI MAPK signal transduction network is highly conserved in many eukaryotic cells. While the p38/Hog1 MAPK is non-phosphorylated under normal growth conditions, it is activated by double phosphorylation of Thr and Tyr residues at a TGY motif using a MAPK kinase (MAPKK) activated through phosphorylation by a MAPKK kinase (MAPKKK) in a higher signal system in response to a specific environmental stress. Subsequently, the phosphorylated p38/Hog1 MAPKs are transferred to a nucleus after a dimer is formed to trigger activation of a transcription regulatory factor and induce overproduction of stress-preventing genes resistant to external stress conditions.
[0008] In spite of the conserved regulatory mechanism of the p38/Hog1 MAPK, fungi and mammals have developed a distinctive set of a higher regulatory systems. Particularly, fungi use a two-component-like phosphorelay system, which is not present in mammals, but found only in bacteria, fungi and plants. The fungal phosphorelay system is composed of three components including a hybrid sensor kinase, histidine-containing phosphotransfer protein (HPt), and a response regulator. The three components have not been observed in mammals, and thus are considered a good target for an antifungal agent.
[0009] Basidiomycetous, Cryptococcus neoformans (C. neoformans), also uses a stress-activated Hog1 MAPK system to adapt to various environmental stresses including osmotic shock, UV radiation, heat shock, oxidative damage, toxic metabolites and antifungal agents. C. neoformans is a human pathogenic fungus found everywhere in the world, causing cryptococcal disease in the skin and lungs and cryptococcal encephalomeningitis in immunocompromised patients. While C. neoformans var. grubii (antigen-type A) is the most frequently found (>90% of environmental and clinical strains), C. neoformans var. neoformans (antigen-type D) is common only in a specific region in Europe, but not frequently found (<10%). However, it has been confirmed that C. gattii, known as C. neoformans of antigen-types B and C, are primary pathogens attacking normal people who have no immune problems.
[0010] However, it is inferred that, compared with other fungal Hog1 MAPK systems, the Hog1 MAPK pathway in C. neoformans is not characteristically developed only to correspond to various environmental stresses, but also to regulate production of two pathogenic factors such as an antiphagocytic capsule and an antioxidant melanine and sexual differentiation, and thus plays a critical role as an important signal transduction regulator in C. neoformans cross-talking to another signal transduction pathway. Recently, the inventors found that most Hog1 MAPKs in many C. neoformans strains are always phosphorylated under non-stress conditions, and rapidly dephosphorylated to activate the Hog1 MAPKs in response to the osmotic shock and treatment of an antifungal agent, fluodioxonyl, which clearly contrasts with Hog1 MAPK systems in other fungi. Double phosphorylation at the TGY motif of Hog1 needs Pbs2 MAPKK. A fungus-specific phosphorelay system which is in a higher level of a Pbs2-Hog1 pathway is also found only in C. neoformans. The C. neoformans phosphorelay system includes 7 different sensor hybrid histidine kinases (TcoI-7), a Ypd1 phosphotransfer protein, and two reaction regulators (Ssk1 and Skn7). The Pbs2-Hog1 pathway is generally regulated by Ssk1, not by Skn7. Among the 7 Tco proteins, Tco1 and Tco2 play distinctive and overlapping roles to activate the Ssk1 and the Pbs2-Hog1 MAPK pathway. However, the Tco1 and Tco2 regulate some Ssk1 and Hog1-related phenotypes, and therefore other higher receptor or sensor proteins should be discovered. More recently, a protein, Ssk2 MAPKKK, serving as a linker between the phosphorelay system and the Pbs2-Hog1 MAPK pathway was identified by comparative analysis of a meiotic map between antigen-type D f1 brother strains, B3510 and B3502, showing different phosphorylation patterns of Hog1. The most noticeable fact is that interchange of Ssk2 alleles between two C. neoformans strains showing different Hog1 phosphorylation patterns changes a phenotype controlled by constitutive Hog1 phosphorylation. Unlike S. cerevisiae and S. pombe, C. neoformans has single MAPKKK and Ssk2 regulating the Hog1 MAPK. While a downstream signal transduction network of the Hog1 MAPK pathway in C. neoformans has yet to be discovered, identification and characterization of the downstream signal transduction network of the Hog1 MAPK are needed to develop a target for a new antifungal agent.
[0011] In the past, fungal infections were mainly local infections such as athlete's foot, jock itch, or oral thrush, and rarely systemic infections. However, recently, systemic infections have become as frequent, coming in fourth in frequency among total infections occurring in hospitals.
[0012] The antifungal agents which have been developed so far may be classified into two major groups: those having an azole structure and those not having an azole structure. The azole-based antifungal agents include ketoconazole, fluconazole, itraconazole and voriconazole, while the non-azole-based antifungal agents include terbinafine, flucytosine, amphotericin B and caspofungin.
[0013] The ketoconazole, fluconazole, itraconazole and voriconazole having an azole structure have similar mechanisms to allylamine-based naftifine and terbinafine. These two different antifungal agents serve to inhibit enzymes required for the conversion of lanosterol into ergosterol, which is a main component of a fungal cell membrane. The azole-based antifungal agents inhibit a microsomal enzyme, and the acrylamine-based antifungal agents inhibit a squalene epoxidase, both having a similar effect to the above-mentioned antifungal agents. Flucytocin (5-FC) is a metabolic antagonist inhibiting the synthesis of a nucleic acid, which has an antifungal reaction by non-competitively antagonizing the cause of miscoding a fungal RNA and DNA synthesis. Amphotericin B having a polyene structure has an antifungal reaction by binding to ergosterol in the fungal cell membrane to induce depolarization of the cell membrane and generating a hole to induce loss of the cell contents. An echinocandin-based antifungal agent, caspofungin, has a reaction reversibly inhibiting the formation of a fungal cell wall, and is different from those acting on the cell membrane described above.
[0014] The azole-based drug may lead to death caused by infection when being used on a patient having hypofunction of the liver, and thus a liver function test should precede administration. It is reported that flutocytosin has a dose-dependent bone marrow inhibiting action, liver toxicity, and can cause enterocolitis. Since such side effects are increased when renal insufficiency occurs, monitoring of a renal function is very important to a patient. In addition, flutocytosin is contraindicated for pregnant woman. A major toxicity of amphotericin B is a glomerulus renal toxicity induced by renal artery vasoconstriction, which is dose dependent. Therefore, when a lifetime cumulative dose is 4 to 5 g or more, a rate of permanent loss of the renal function is increased. Furthermore, the renal toxicities such as excessive loss of potassium, magnesium and bicarbonate due to toxicity of a renal tube and low production of erithropoietins may be generated. Moreover, as acute responses, symptoms such as thrombophlebitis, chills, shivering, and hyperpnea may be shown. Since the conventionally developed antifungal agents show various side effects according to kinds of drugs, development of a new therapy which can reduce such side effects and increase an antifungal effect is demanded.
[0015] Meanwhile, in pathogenic fungi distributed in the world, including Aspergillus fumigatus, Candida albicans (C. albicans) and C. neoformans, Ras- and cAMP-signal transduction pathways are evolutionarily conserved, and significantly functional and structural differences are still being found (Pukkila-Worley & Alspaugh, 2004, Rolland et al., 2002, Wong & Heitman, 1999, Thevelein & de Winde, 1999, Alspaugh et al., 1998, Lengeler et al., 2000, and Bahn et al, 2007). In C. neoformans causing fatal fungal encephalomeningitis, the cAMP-signal transduction pathway is important in producing and differentiating pathogenic factors (Idnurm et al., 2005). Like S. cerevisiae and C. albicans, it was confirmed that two major higher signal transduction regulators of adenylyl cyclase (Cac1), adenylyl cyclase-associated protein 1 (Aca1) and Gα subunit protein (Gpa1) regulate a cAMP-signal transduction pathway of C. neoformans (Bahn et al., 2004 and Alspaugh et al., 1997). The disruption of GPA1 genes leads to multiple phenotypes of cells, which include incomplete production of core pathogenic factors, melanin and a capsule, essential for survival and proliferation of C. neoformans in a host, and a decrease in mating, which is important in distribution of infectious spores (Alspaugh et al., 1997). Aca1 physically interacts with a Cac1 adenylyl cyclase, and does not regulate a basic level of cAMP but dominates most cAMP-dependent phenotypes by regulating the induction of cAMP (Bahn et al., 2004). A deletion mutant of CAC1 produces a phenotype more defected than a deletion mutant of gpalΔ or acalΔ, and gpalΔ acalΔ double deletion mutants are equivalent to the cac1Δ deletion mutant in phenotype (Bahn et al., 2004). This indicates that Cal1 is activated by both of Aca1 and Gpa1. In a lower signal system of the Cac1 of C. neoformans, two catalytic subunits of a protein kinase A (PKA), Pka1 and Pka2, and a regulatory subunit, Pkr1, are included. While Pka1 plays a dominant role for cAMP signal transduction in a background of an antigen-type A C. neoformans H99 strain, Pka2 also plays the same role in an antigen-type D C. neoformans JEC21 strain (Hicks et al., 2004). Nevertheless, a pka1Δ-pka2Δ double deletion mutant shows a phenotype the same as the cac1Δ deletion mutant, and the cAMP signal transduction from Cac1 is split into two PKA catalytic subunits (Bahn et al., 2004). Interestingly, the deletion of PDE1, not PDE2, repairs some phenotypes including the depletion of a melanin of the gpalΔ deletion mutant, which indicates that different phosphodiesterases act in various fungi (Hicks et al., 2005).
[0016] It is revealed that two Ras proteins, Ras1 and Ras2, are found in Cryptococcus, and play common and distinctive roles (Alspaugh et al., 2000, D'Souza et al., 2001, and Waugh et al., 2002). Among these proteins, Ras1 is a major C. neoformans Ras protein supporting high-temperature growth and invasive growth essential for survival and growth in a host and stimulating sexual differentiation (Alspaugh et al., 2000). Though the ras2Δ deletion mutant does not have a recognizable phenotype, the overexpression of RAS2 somewhat inhibits most of the ras1 mutation phenotypes (Waugh et al., 2002). Like S. cerevisiae, disruption of the RAS1 and RAS2 genes affects cell viability at every temperature, which indicates that the Ras protein is essential for the growth of cells in general. Among various Ras-related phenotypes, only invasive growth and mating are cAMP-dependent, but high-temperature growth is cAMP-independent and a Ras1-specific phenotype (Alspaugh et al., 2000, Waugh et al., 2003). Interestingly, Cac1 does not bear a Leucine-rich repeat (LRR) domain, which is a binding site to a GTP-binding Ras in S. cerevisiae (Shima et al., 1997). Since an adenylyl cyclase/cyclase-related protein complex can provide a secondary Ras-binding site to activate the protein complex as shown in S. cerevisiae (Shima et al., 2000), Ras1 can still interact with an Aca1/Cac1 complex for activating the Ras1 in C. neoformans. Recently, it has been reported that a GEF protein, Cdc24, is a Ras-effecter protein, and regulates the growth of C. neoformans at high temperature in a lower system of Ras1 and a higher system of Rho-like GTPase Cdc42 (Nichols et al., 2007). Consequently, C. neoformans cAMP-signal transduction pathway is regulated by three different higher signal regulators, Ras1, Gpa1 and Aca1.
[0017] Despite the presence of the common higher signal regulators (Ras1, Aca1 and Gpa1) of Cac1, functional correlation between the components and target gene regulated by each regulator in C. neoformans remains still unclear.
SUMMARY
[0018] The present invention provides to finding a new target gene to develop an antifungal agent by investigating a signal transduction network of HOG, Ras and cAMP pathways.
[0019] In one aspect, a use of an inhibitor against at least one protein or a gene coding for the same selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans to prepare an antifungal agent, an antifungal pharmaceutical composition including the inhibitor, and a method of treating fungal infection including injecting an effective amount of the inhibitor into a subject are provided.
[0020] In another aspect, a use of at least one protein or a gene coding for the same selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans to screen an antifungal agent, a composition for screening an antifungal agent including the protein or gene, and a method of screening an antifungal agent including contacting the protein or gene with a candidate material and determining whether the candidate material inhibits or stimulates an activity of the protein or gene are provided.
[0021] In still another aspect, a use of an inhibitor against at least one protein or a gene coding for the same selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of C. neoformans to prepare an antifungal agent, an antifungal pharmaceutical composition including the inhibitor, and a method of treating fungal infection including injecting an effective amount of the inhibitor into a subject are provided.
[0022] In yet another aspect, a use of at least one protein or a gene coding for the same selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of C. neoformans to screen an antifungal agent, a composition for screening an antifungal agent including the protein or gene, and a method of screening an antifungal agent including contacting the protein or gene with a candidate material and determining whether the candidate material inhibits or stimulates an activity of the protein or gene are provided.
[0023] In the present invention, to regulate a HOG pathway of C. neoformans, roles of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 genes are investigated to reveal that a biosynthesis level of ergosterol is increased when these genes are inhibited. Since a large amount of ergosterol is distributed on a fungal cell membrane when the genes are inhibited, an efficiency of an ergosterol-binding antifungal agent can be considerably increased due to many working points of the ergosterol-binding antifungal agents. In addition, in the present invention, to regulate Ras and cAMP pathways of C. neoformans, roles of RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 and HSP122 genes were investigated to reveal that sensitivity to a polyene- or azole-based drug is increased when these genes are inhibited. Thus, an antifungal pharmaceutical composition including an inhibitor against the gene or protein encoded by the same can be used as an excellent combined antibacterial drug which can reduce an amount of a conventional antifungal agent used and increase efficiency.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The above and other aspects of the inventive concept will become more readily apparent by describing in further detail exemplary embodiments thereof which reference to the accompanying drawings, in which:
[0025] FIG. 1 shows identification of genes whose expression is regulated by Hog, Ssk1 and Skn7 of C. neoformans under normal conditions with no stress on the genome level (fold change is expressed by color);
[0026] FIG. 2 shows analysis results for induction of ergosterol biosynthesis genes by disturbance of a HOG signal transduction pathway and an ergosterol content in a cell;
[0027] FIG. 3 shows analysis results showing that the inhibition of the HOG pathway gives an elevated antifungal effect with amphotericin B in C. neoformans;
[0028] FIG. 4 shows analysis results showing that the inhibition of the HOG pathway gives an antagonistic antifungal effect with respect to some azole drugs in C. neoformans;
[0029] FIG. 5 shows analysis results showing that gene coding for an efflux pump of Na.sup.+ and K.sup.+, ENA1 and NHA1, are lower-system target genes regulated by the HOG pathway, and the inhibition of these genes gives high sensitivity to polyene-based drugs and azole-based drugs;
[0030] FIG. 6 shows analysis results for transcripts of ras1Δ, aca1Δ, gpa1Δ, cac1Δ, and pka1Δ pka2Δ deletion mutants of C. neoformans (fold change is expressed by color);
[0031] FIG. 7 shows functional categories of genes differently regulated by the ras1Δ, aca1Δ, gpa1Δ, cac1Δ, and pka1Δ pka2Δ deletion mutants of C. neoformans;
[0032] FIG. 8 shows regulation of a significant ratio of Ras- and cAMP-dependent genes by environmental stress;
[0033] FIG. 9 shows an identification result of a cAMP-signal transduction pathway dependent gene in C. neoformans;
[0034] FIG. 10 shows analysis results showing that the inhibition of the Ras- and cAMP-signal transduction pathways increases a sensitivity to polyene-based or azole-based (itraconazole) antifungal agent, independent of ergosterol biosynthesis; and
[0035] FIG. 11 shows analysis results showing that the expression of HSP12 and HSP122 is up-regulated by the cAMP- and HOG-signal transduction pathways, and increases sensitivity to polyene-based antifungal agents by hsp12Δ and hsp122Δ deletion mutants.
DETAILED DESCRIPTION
[0036] Hereinafter, the present invention will be described in detail.
[0037] An antifungal pharmaceutical composition including an inhibitor against at least one protein or gene coding for the same selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans to prepare an antifungal agent is provided.
[0038] A HOG1 pathway is a cell signal transduction system regulating responses induced by various stresses. Particularly, since fungi use a two-element-like phosphorelay system composed of three elements such as a hybrid sensor kinase, a histidine-containing phosphotransfer protein (HPt) and a response regulator, which are not present in mammals, the inventors carried on an investigation of roles of genes involved in the HOG1 pathway to develop a target for a new antifungal agent. As a result, surprisingly, it was found that, in the regulation of the HOG pathway in C. neoformans, a biosynthesis level of ergosterol is increased when SSK1, TCO2, SSK2, PBS2, and HOG1 genes are inhibited. As will be confirmed in the following embodiments, when the genes are inhibited, a large amount of ergosterol is distributed on a fungal cell membrane and working points of the ergosterol-binding antifungal agent are also increased. Therefore, an efficiency of the ergosterol-binding antifungal agent can be considerably increased. In addition, when ENA1 and NHA1 genes, the expression of which is known to be regulated by the HOG signal transduction pathway, are inhibited, it is confirmed that, regardless of the change in ergosterol level, a sensitivity to polyene-based drugs such as amphotericin B and azole-based drugs are considerably increased. Thus, the antifungal pharmaceutical composition including an inhibitor against at least one protein selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans may be used as an excellent combined antibacterial drug which can reduce an amount of the conventional ergosterol-binding antifungal agent or azole-based antifungal agent used and increase efficiency.
[0039] Accordingly, a use of an inhibitor against at least one protein or gene coding for the same selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans to prepare an antifungal agent, an antifungal pharmaceutical composition including the inhibitor, and a method of treating fungal infection including injecting an effective amount of the inhibitor to a subject are provided.
[0040] In the specification, it is understood that SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 used as a target to interrupt a HOG1 signal transduction system indicates an Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 protein, or an SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 gene. Thus, it is understood that an SSK1, TCO2, SSK2, PBS2, HOG 1, ENA1 or NHA1 inhibitor includes either an inhibitor against an Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 protein or an inhibitor against an SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 gene.
[0041] In one exemplary embodiment, the inhibitor against at least one protein selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans may bind to the protein to inhibit an activity thereof, thereby interrupting signal transduction. In another exemplary embodiment, the inhibitor against at least one gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of C. neoformans may inhibit expression of the gene to interrupt signal transduction. In the specification, the SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 gene may be a DNA coding for the gene or mRNA transcripted therefrom. Thus, the inhibitor against the gene may bind to the gene itself to disturb transcription or bind to the mRNA transcripted from the gene to disturb translation of the mRNA.
[0042] In one exemplary embodiment, the Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 protein may have an amino acid sequence of SEQ ID NOs: 1-7 respectively, and the SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 gene may have a nucleic acid sequence of SEQ ID NOs: 8-14 respectively or a cDNA sequence of SEQ ID NOs:15-21 respectively. However, this is merely an example of a sequence of C. neoformans antigen-type A H99 strain, and the sequence of the SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 is not limited thereto.
[0043] In the specification, it is understood that the Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 protein or the SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 gene includes a variant or fragment thereof having substantially the same activity as the protein or gene.
[0044] In one exemplary embodiment, the antifungal pharmaceutical composition may include at least one inhibitor against at least one protein selected from the group consisting of Ssk1, Ena1 and Nha1. SSK1 may be a good target to develop an antifungal agent because it is not only an important upstream reaction regulator of HOG1, but also a gene which is not found in mammals. Therefore, the SSK1 inhibitor may reduce a possibility of generating certain side effects and increase a biosynthesis level of ergosterol in a fungus, thereby improving the efficiency of an ergosterol-binding antifungal agent. Meanwhile, ENA1 and NHA1 are defined as lower-system target genes regulated by a HOG pathway. When these genes are inhibited, sensitivity to an azole-based drug such as fluconazole, ketoconazole and itraconazole is also considerably increased as well as that to a polyene-based drug such as amphotericin B. Therefore, the inhibitors simultaneously or independently inhibiting these genes may exhibit very high antifungal activities when used in combination with the polyene- or azole-based drug.
[0045] The inhibition of the Ssk1, Tco2, Ssk2, Pbs2 or Hog1 protein or gene improves the biosynthesis of ergosterol and increases the distribution of ergosterol on a fungal cell membrane. Thus, since binding targets of the ergosterol-binding antifungal agent disrupting the fungal cell membrane by being bound to ergosterol are increased, an effective amount of the ergosterol-binding antifungal agent may be reduced and a killing ability of the ergosterol-binding antifungal agent may be increased. In addition, the inhibitors against ENA1 and NHA1 considerably increase drug sensitivities to the azole-based antifungal agent as well as the polyene-based antifungal agent, and thus amounts of these drugs used can be reduced and a killing ability may be improved. Such an antifungal activity induced by the inhibition of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 may treat cryptococcal disease and encephalomeningitis by infection of C. neoformans.
[0046] Thus, in one exemplary embodiment, use of an Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 inhibitor to prepare a drug for treating a disease such as cryptococcal disease or encephalomeningitis, a pharmaceutical composition for treating a disease such as cryptococcal disease or encephalomeningitis including the Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 inhibitor, and a method of treating a disease such as cryptococcal disease or encephalomeningitis including injecting an effective amount of the Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 inhibitor to a subject are provided.
[0047] Other than cryptococcal disease or encephalomeningitis exemplified in the specification, diseases induced by fungal infection are well known in the art. In the specification, the inhibition of the Ssk1, Tco2, Ssk2, Pbs2, Hog1 protein or gene is revealed to improve the efficiency of the ergosterol-binding antifungal agent or azole-based antifungal agent, and thus those of ordinary skill in the art may inhibit the protein or gene to prevent or treat a disease induced by fungal infection.
[0048] In the specification, the "inhibitor of the Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 protein" used to interrupt a HOG1 signal transduction system includes all inhibitors binding to the Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 protein to interrupt signal transduction. For example, such an inhibitor may be a peptide or compound binding to the Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 protein. Such an inhibitor may be selected by a screening method to be described below in analysis of a protein structure, and may be designed using a known method in the art. In one exemplary embodiment, the inhibitor may be a polyclonal or monoclonal antibody with respect to at least one protein selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 of C. neoformans. Such a polyclonal or monoclonal antibody may be prepared using a method of preparing an antibody known in the art.
[0049] In the present invention, the "inhibitor against the SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 gene" used to interrupt a HOG1 signal transduction system includes every inhibitor inhibiting the expression of the SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 gene to interrupt signal transduction. For example, such an inhibitor may be a peptide, nucleic acid or compound binding to the gene. The inhibitor may be selected by a screening method shown below in cell-based screening, and may be designed using a known method in the art. In one exemplary embodiment, the inhibitor may be an antisense oligonucleotide, siRNA, shRNA, miRNA or vector including the same with respect to at least one gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of C. neoformans. The antisense oligonucleotide, siRNA, shRNA, miRNA or vector including the same may be prepared using a known method in the art. In the specification, the "vector" refers to a gene construct including foreign DNA inserted into a genome coding for a polypeptide. The vector used herein is a vector in which a nucleic acid sequence inhibiting the gene is inserted into a genome, and may include a DNA vector, a plasmid vector, a cosmid vector, a bacteriophage vector, a yeast vector, or a viral vector.
[0050] A pharmaceutical antifungal pharmaceutical composition including an inhibitor against at least one protein or gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of C. neoformans does not exhibit an antifungal activity alone, but increases a fungal killing ability of an antifungal agent in combination with the ergosterol-binding antifungal agent or azole-based antifungal agent. Therefore, the antifungal pharmaceutical composition may be sequentially or simultaneously injected with the ergosterol-binding antifungal agent or azole-based antifungal agent.
[0051] The ergosterol-binding antifungal agent refers to an antifungal agent binding to ergosterol on a fungal cell membrane to induce depolarization of the cell membrane and forming a hole to induce the loss of contents in a cell, thereby killing fungi. Such an ergosterol-binding antifungal agent is known in the art, and any ergosterol-binding antifungal agent considerably increases the antifungal effect when used with the antifungal pharmaceutical composition. In one exemplary embodiment, the ergosterol-binding antifungal agent may be a polyene-based antifungal agent. In one aspect, the polyene-based antifungal agent may be at least one antifungal agent selected from the group consisting of amphotericin B, natamycin, rimocidin, filipin, nystatin and candicin. In the preferable embodiment, the polyene-based antifungal agent is amphotericin B. Meanwhile, the azole-based antifungal agent may be at least one antifungal agent selected from the group consisting of ketoconazole, fluconazole, itraconazole and voriconazole.
[0052] In this aspect, an antifungal combined formulation including the antifungal pharmaceutical composition including the inhibitor of the present invention; and a known ergosterol-binding antifungal agent or azole-based antifungal agent are provided.
[0053] An antifungal pharmaceutical composition including an inhibitor against at least one protein or gene coding for the same selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of C. neoformans to prepare an antifungal agent is also provided.
[0054] The inventors performed investigation on, rather than simply the genes involved in Ras and cAMP pathways, the roles of the genes involved in Ras and cAMP pathways, to develop a target for a new antifungal agent. The result newly revealed that, surprisingly, in the Ras and cAMP pathways of C. neoformans, when a RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene is inhibited, a sensitivity to one of the polyene- or azole-based drugs, an itraconazole antifungal agent, is increased. As will be confirmed in the following exemplary embodiment, when the genes are inhibited, the sensitivity to the polyene- or itraconazole antifungal agent in a fungus may be considerably increased. Thus, the antifungal pharmaceutical composition including an inhibitor against at least one protein or gene coding for the same selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of C. neoformans may be used as an excellent combined antibacterial drug which can reduce an amount of a conventional polyene-based or itraconazole antifungal agent used and improve an efficiency.
[0055] Therefore, use of an inhibitor against at least one protein or gene coding for the same selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of C. neoformans to prepare an antifungal agent, an antifungal pharmaceutical composition including the inhibitor, and a method of treating fungal infection including injecting an effective amount of the inhibitor into a subject are provided.
[0056] In the present invention, it is construed that RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 used as a target to interrupt the Ras- and cAMP signal transduction systems is a Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein, or a RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene. Accordingly, it is construed that a RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 inhibitor includes every inhibitor against the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein or inhibitor against the RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene.
[0057] In one exemplary embodiment, an inhibitor against at least one protein selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of C. neoformans may be an inhibitor that binds to the protein to inhibit an activity, thereby interrupting signal transduction. In another exemplary embodiment, an inhibitor against at least one gene selected from the group consisting of RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 and HSP122 of C. neoformans may be an inhibitor inhibiting expression of the gene, thereby interrupting signal transduction. In the present invention, the RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene may be DNA coding for the gene or mRNA transcripted therefrom. Therefore, the inhibitor against the gene may bind to the gene to interrupt transcription or bind to mRNA transcripted from the gene to interrupt translation of the mRNA.
[0058] In one exemplary embodiment of the present invention, the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein may have an amino acid sequence of one of SEQ ID NOs: 16-24 respectively, and the RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene may have a nucleic acid or cDNA sequence corresponding to the protein. However, the sequence just shows a sequence of a C. neoformans antigen-type A H99 strain, and thus the sequence of the RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 is not limited thereto.
[0059] In the present invention, it is construed that the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein, or the RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene includes a variant or fragment thereof having substantially the same activity as the protein or gene.
[0060] In one exemplary embodiment, the inhibitor may be an inhibitor against a Cac1 or Pka1 protein or gene.
[0061] Inhibition of the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein or gene coding for the same may increase a sensitivity to a polyene- or azole-based antifungal agent, and thus an effective amount of the polyene- or azole-based antifungal agent may be reduced and a killing ability of the antifungal agent may be increased. An antifungal activity caused by the inhibition of the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 may treat cryptococcal disease and encephalomeningitis induced by infection of C. neoformans.
[0062] Thus, in one exemplary embodiment of the present invention, use of a Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 inhibitor to prepare a drug for treating diseases such as cryptococcal disease and encephalomeningitis, a pharmaceutical composition for treating diseases such as cryptococcal disease and encephalomeningitis including the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 inhibitor and a method of treating diseases such as cryptococcal disease and encephalomeningitis including injecting an effective amount of the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 inhibitor into a subject are provided.
[0063] Other than the cryptococcal disease and encephalomeningitis stated herein, diseases induced by fungal infection are well known in the art. In the present invention, it is revealed that the inhibition of the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein or gene coding for the same increases the sensitivity to a polyene- or azole-based itraconazole antifungal agent, thereby increasing the efficiency of the antifungal agent. Therefore, those of ordinary skill in the art can inhibit the protein or genes to prevent or treat a disease induced by the fungal infection.
[0064] In the present invention, the "inhibitor of the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein" used to interrupt the RAS or cAMP signal transduction system includes every inhibitor binding to the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein to interrupt signal transduction. For example, such an inhibitor may be a peptide or compound binding to the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein. The inhibitor may be selected by a screening method to be described below in analysis of a protein structure, and may be designed using a method known in the art. In one exemplary embodiment, the inhibitor may be a polyclonal or monoclonal antibody with respect to at least one protein selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of C. neoformans. The polyclonal or monoclonal antibody may be constructed using a known method of constructing an antibody in the art.
[0065] In the present invention, the "inhibitor of the RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene" used to interrupt the RAS or cAMP signal transduction system includes every inhibitor inhibiting the expression of the RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene to interrupt signal transduction. For example, such an inhibitor may be a peptide, nucleic acid or compound binding to the gene. The inhibitor may be selected by a screening method to be described below in analysis of a protein structure, and may be designed using a method known in the art. In one exemplary embodiment, the inhibitor may be an antisense oligonucleotide, siRNA, shRNA, miRNA or a vector including the same with respect to at least one gene selected from the group consisting of RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 and HSP122 of C. neoformans. Such an antisense oligonucleotide, siRNA, shRNA, miRNA or a vector including the same may be constructed using a known method in the art. In the present invention, the "vector" is a gene construct including foreign DNA inserted into a genome coding for a polypeptide. The vector related to the present invention may be a vector formed by inserting a nucleic acid sequence inhibiting the gene into the genome, which may be a DNA vector, plasmid vector, cosmid vector, bacteriophage vector, yeast vector or viral vector.
[0066] The antifungal pharmaceutical composition of the present invention including the inhibitor against at least one protein or gene coding for the same selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1 , Hsp12 and Hsp122 of C. neoformans increases a fungal killing ability of the antifungal agent in combination with a polyene- or azole-based antifungal agent. Thus, the antifungal pharmaceutical composition of the present invention is sequentially or simultaneously injected with the polyene- or azole-based antifungal agent. The polyene- or azole-based antifungal agent is known in the art, and any one of the polyene- or azole-based antifungal agent significantly increases the antifungal effect when used with the antifungal pharmaceutical composition of the present invention. In one aspect, the polyene-based antifungal agent may be at least one of amphotericin B, natamycin, rimocidin, filipin, nystatin and candicin. In a preferable embodiment, the polyene-based antifungal agent may be amphotericin B. In one aspect, the azole-based antifungal agent may be at least one selected from the group consisting of ketoconazole, fluconazole, itraconazole and voriconazole.
[0067] In another exemplary embodiment, the antifungal pharmaceutical composition may be sequentially or simultaneously injected along with the inhibitor against at least one protein selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of C. neoformans. As will be confirmed from the following exemplary embodiment, when expression of HOG1 is inhibited as well as expression of CAC1 or PKA1 of C. neoformans, the sensitivity to amphotericin B is proportionally increased. This is because genes of the cAMP pathway increase the sensitivity to amphotericin B by a mechanism different from that increasing the sensitivity to amphotericin B due to the increase in biosynthesis of ergosterol when the genes of the HOG pathway described above are inhibited.
[0068] According to the aspect, the present invention also provides an antifungal combined formulation including the antifungal pharmaceutical composition including the inhibitor of the present invention; and at least one antifungal agent selected from the group consisting of a polyene-based antifungal agent, an azole-based antifungal agent and an inhibitor against at least one protein or gene coding for the same selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans. Preferably, the antifungal combined formulation may include the antifungal pharmaceutical composition including the inhibitor of the present invention, a polyene-based antifungal agent, and at least one antifungal agent selected from the group consisting of inhibitors each against at least one protein or gene coding for the same selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans.
[0069] The antifungal pharmaceutical composition or antifungal combined formulation of the present invention may be prepared using a pharmaceutically suitable and physiologically available adjuvant, wherein the adjuvant may be a solubilizer such as a diluting agent, a dispersing agent, a sweetening agent, a binding agent, a coating agent, a blowing agent, a lubricant, a gliding agent or a flavoring agent.
[0070] The antifungal pharmaceutical composition of the present invention may be formulated into a pharmaceutical composition including at least one pharmaceutically available carrier other than an active component for administration.
[0071] In the composition formulated in a liquid-phase solution, a pharmaceutically available carrier may be suitable for sterilization and living organisms, and may be saline, sterilized water, Ringer's solution, buffered saline, albumine injection, dextrose solution, maltodextrin solution, glycerol, ethanol or a mixture of at least one thereof. When necessary, another conventional additive such as an antioxidant, buffer or bacteriostatic agent may be added. In addition, a diluting agent, a dispersing agent, a surfactant, a binding agent or a lubricant may be added, and thus the composition may be formulated in the form of an injectable formulation such as an aqueous solution, a suspension or an emulsion, a pill, a capsule, a granule or a tablet. Furthermore, the composition may be formulated using a suitable method in the art, which is disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton, Pa. according to diseases or components.
[0072] Types of a pharmaceutical formulation of the pharmaceutical composition of the present invention may include a granule, an acida, a coated tablet, a tablet, a capsule, a suppository, a syrup, a juice, a suspension, an emulsion, a drop or injectable liquid and a sustained-release formulation of an active compound.
[0073] The pharmaceutical composition of the present invention may be injected by a conventional method via an intravenous, intraarterial, abdominal, sternal, percutaneous, nasal, inhaling, local, rectal, oral, intraocular or intradermal route.
[0074] An effective amount of the active component of the pharmaceutical composition of the present invention indicates an amount required for preventing or treating a disease, or achieving an effect of inducing bone growth. Accordingly, the effective amount may vary depending on various factors such as kinds of a disease, severity of a disease, kinds and contents of the active component and other components contained in the composition, kinds of dosage forms and patient's age, weight, health, sex and dietary habits, injection times and routes, release rates of the composition, duration of treatment, and co-injected drugs. For adults, when the composition is injected one or more times a day, the dosages may be, but not limited to, 0.1 ng/kg to 10 g/kg for a compound, 0.1 ng/kg to 10 g/kg for a polypeptide, protein or antibody, and 0.01 ng/kg to 10 g/kg for an antisense oligonucleotide, siRNA, shRNAi or miRNA, respectively.
[0075] In the present invention, the "subject" may be, but not limited to, a human, orangutan, chimpanzee, mouse, rat, dog, cow, chicken, pig, goat or sheep.
[0076] Furthermore, the present invention provides a use of at least one protein selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, and Nha1 of C. neoformans to screen an antifungal agent, a composition for screening an antifungal agent including the protein, and a method of screening an antifungal agent including contacting the protein with a candidate material and determining whether the candidate material inhibits or stimulates an activity of the protein.
[0077] The present invention also provides a use of at least one gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of C. neoformans to screen an antifungal agent, a composition for screening an antifungal agent including the gene, and a method of screening an antifungal agent including contacting the gene with a candidate material and determining whether the candidate material inhibits or stimulates expression of the gene.
[0078] The present invention also provides a method of screening an antifungal agent by a yeast two-hybrid system capable of monitoring physical contact between SSK1 and SSK2, SSK1 and YPD1 or YPD1 and TCO2 proteins of C. neoformans. When this method is used, a large amount of the candidate materials can be screened quickly.
[0079] As described above, when SSK1, TCO2, SSK2, PBS2 or HOG1 of C. neoformans is inhibited, the HOG1 signal transduction system is interrupted, and thus biosynthesis of ergosterol is improved. Therefore, the material screened to inhibit the protein or gene may be used as an antifungal agent improving a fungal killing ability, along with an ergosterol-binding antifungal agent. The material screened to inhibit ENA1 or NHA1 may be used as an antifungal agent improving a fungal killing ability when used with an ergosterol-binding antifungal agent or azole-based antifungal agent.
[0080] The present invention also provides a use of at least one protein selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1 , Pka1, Hsp12 and Hsp122 of C. neoformans to screen an antifungal agent, a composition for screening an antifungal agent including the protein, and a method of screening an antifungal agent including contacting the protein with a candidate material and determining whether the candidate material inhibits or stimulates an activity of the protein.
[0081] The present invention also provides a use of at least one gene selected from the group consisting of RAS1, RAS2, CDC24, GPA1, ACA1, PKA1, HSP12 and HSP122 of C. neoformans to screen an antifungal agent, a composition for screening an antifungal agent including the gene, and a method of screening an antifungal agent including contacting the gene with a candidate material and determining whether the candidate material inhibits or stimulates expression of the gene.
[0082] The present invention also provides a method of screening an antifungal agent by a yeast two-hybrid system capable of monitoring physical contact between Gpa1 and Cac1 , Cac1 and Aca1, Ras1 and Cdc24 or Ras2 and Cdc24 proteins of C. neoformans. When this method is used, a large amount of the candidate materials can be screened quickly.
[0083] As described above, when RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 of C. neoformans is inhibited, the RAS or cAMP signal transduction system is interrupted, thereby increasing a sensitivity to a polyene- or azole-based antifungal agent. Thus, the material screened to inhibit the protein or gene may be used as an antifungal agent improving a fungal killing ability when used with the polyene- or azole-based antifungal agent.
[0084] Confirmation of the reaction between the protein or gene and the candidate material may be performed by a conventional method of confirming the reaction between a protein and a protein, a protein and a compound, DNA and DNA, DNA and RNA, DNA and a protein, DNA and a compound, RNA and a protein, or RNA and a compound. For example, a hybrid test for confirming a bond between the gene and a candidate material in vitro, a method of measuring an expression level of the gene through northern blotting, quantitative PCR or quantitative real time PCR after reaction of mammalian cell and a test material, a method of connecting a reporter gene to the gene to introduce the gene into a cell, reacting the cell with a test material and measuring an expression level of a reporter protein, a method of reacting the protein with a candidate material and measuring an activity, a yeast two-hybrid, searching for a phage-displayed peptide clone binding to an Idbf protein, high throughput screening (HTS) using a natural substance and a chemical library, drug hit HTS, cell-based screening or a screening method using a DNA array may be used.
[0085] The screening composition may include distilled water or a buffer stably maintaining the structure of a nucleic acid or protein, other than the protein or gene. In addition, the screening composition may include a cell expressing the protein or gene, or a cell containing a plasmid expressing the gene in the presence of a promoter regulating a transcription rate for an in vivo test.
[0086] In the screening method of the present invention, a test material may be individually a nucleic acid, a protein, a peptide, a different extract or natural substance or a compound assumed to have possibility as a drug inhibiting signal transduction through a HOG1 signal transduction system according to a conventional screening method or randomly selected.
[0087] The matters related to a genetic engineering technique in the present invention are made more clear by the literatures disclosed by Sambrook et al. [Molecular Cloning, A Laboratory Manual, Cold Spring Harbor laboratory Press, Cold Spring Harbor, N.Y. (2001)] and Frederick M. Ausubel et al. [Current protocols in molecular biology volume 1, 2, 3, John Wiley & Sons, Inc. (1994)].
[0088] While exemplary embodiments have been disclosed herein, it should be understood that other variations may be possible. Such variations are not to be regarded as a departure from the spirit and scope of exemplary embodiments of the present application, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.
EXAMPLES
Experimental Procedures
Strains and Growth Conditions
[0089] The C. neoformans strains used in this examples are listed in Table 1[Bahn Y S, Geunes-Boyer S, Heitman J (2007) Eukaryot Cell 6: 2278-2289.; Bahn Y S, Kojima K, Cox G M, Heitman J (2005) Mol Biol Cell 16: 2285-2300.; Bahn Y S, Kojima K, Cox G M, Heitman J (2006) Mol Biol Cell 17: 3122-3135.; Perfect J R, Ketabchi N, Cox G M, Ingram C W, Beiser C L (1993) J Clin Microbiol 31: 3305-3309; Kwon-Chung K J, Edman J C, Wickes B L (1992) Genetic association of mating types and virulence in Cryptococcus neoformans. Infect Immun 60: 602-605.].
[0090] The C. neoformans strains were cultured in YPD (yeast extract-peptone-dextrose) medium unless indicated separately.
TABLE-US-00001 TABLE 1 Strain Genotype Parent Serotype A H99 MATα KN99 MATa CBN45 MATα ras1Δ::NEO H99 CBN64 MATα ras1Δ::NEO RAS1::NAT CBN45 MWC12 MATα ras2Δ::URA5 H99 CBN32 MATα cdc24Δ::NEO H99 CBN33 MATα cdc24Δ::NEO CDC24::NAT CBN32 YSB6 MATα aca1Δ::NAT-STM#43 H99 YSB51 MATα ras1Δ::NAT-STM#150 H99 YSB53 MATα ras1Δ::NAT-STM#150 H99 YSB64 MATα hog1Δ::NAT-STM#177 H99 YSB123 MATα pbs2Δ::NAT-STM#213 H99 YSB261 MATα ssk1Δ::NAT-STM#205 H99 YSB264 MATα ssk2Δ::NAT-STM#210 H99 YSB349 MATα skn7Δ::NAT-STM#201 H99 YSB278 MATα tco1Δ::NAT-STM#102 H99 YSB281 MATα tco2Δ::NAT-STM#116 H99 YSB324 MATα tco1Δ::NAT-STM#102 tco2D::NEO YSB278 YSB284 MATα tco3Δ::NAT-STM#119 H99 YSB417 MATα tco4Δ::NAT-STM#123 H99 YSB286 MATα tco5Δ::NAT-STM#125 H99 YSB348 MATα tco7Δ::NAT-STM#209 H99 YSB73 MATα ras1Δ::NEO H99 YSB42 MATα cac1Δ::NAT-STM#159 H99 YSB83 MATα gpa1Δ::NAT H99 YSB188 MATα pka1Δ::NAT H99 YSB194 MATα pka2Δ::NAT-STM#205 H99 YSB200 MATα pka1Δ::NAT pka2Δ::NEO YSB188 YSB174 MATα aca1Δ::NAT-STM#43 ras1::NEO YSB278 YSB182 MATα cac1Δ::NAT-STM#159 ras1::NEO H99 YSB156 MATα hog1Δ::NAT-STM#177 cac1::NEO H99 YSB112 MATα ura5 pka1::URA5 hog1::NATSTM#177 H99 YSB58 MATa aca1Δ::NEO KN99 YSB79 MATa cac1Δ::NEO KN99 YSB81 MATa hog1Δ::NEO KN99 YSB175 MATα aca1Δ::NEO ras1Δ::NATSTM#150 YSB58 YSB187 MATα cac1Δ::NEO ras1Δ::NATSTM#150 YSB79 YSB606 MATα gre2Δ::NAT-STM#224 H99 YSB607 MATα gre2Δ::NAT-STM#224 H99 YSB609 MATα pkp1Δ::NAT-STM#224 H99 YSB610 MATα pkp1Δ::NAT-STM#224 H99 YSB599 MATα hsp12Δ::NAT-STM#224 H99 YSB600 MATα hsp12Δ::NAT-STM#224 H99 YSB603 MATα hsp122Δ::NAT-STM#224 H99 YSB604 MATα hsp122Δ::NAT-STM#224 H99 YSB590 MATα ena1Δ::NAT nha1::NEO AI167 YSB591 MATα ena1Δ::NAT nha1::NEO AI167 YSB586 MATα nha1Δ::NEO H99 YSB587 MATα nha1Δ::NEO H99 YSB588 MATα nha1Δ::NEO H99 Serotype D JEC21 MATα B-3501 MATα YSB267 MATα pbs2Δ::NAT-STM#213 JEC21 YSB139 MATα hog1Δ::NAT-STM#177 JEC21 YSB338 MATα ssk2Δ::NAT-STM#210 JEC21 YSB340 MATα ssk2Δ::NAT-STM#210 B-3501 Each NAT-STM# indicates the Natr marker with a unique signature tag.
DNA Microarray Array Analysis
[0091] For total RNA isolation used in DNA microarray, the wild-type H99, hog1Δ (YSB64), ssk1Δ (YSB261), and skn7Δ (YSB349), ras1Δ (YSB51), aca1Δ (YSB6), gpa1Δ (YSB83), cac1Δ (YSB42) and pka1Δ pka2Δ (YSB200) mutant strains were grown in 50 ml YPD medium at 30° C. for 16 hr. Then 5 ml of the overnight culture was inoculated into a 100 ml of fresh YPD medium and further incubated for 4-5 hr at 30° C. until it approximately reaches to the 1.0 of optical density (OD) at 600 nm (OD600 nm=1.0). For zero-time samples, 50 ml out of the 100 ml culture was sampled and rapidly frozen in liquid nitrogen. To the remaining 50 ml culture, 50 ml of YPD containing 2 M NaCl, 40 μg/ml fludioxonil (PESTANAL, Sigma, 100 mg/ml stock solution in dimethylsulfoxide), or 5 mM H2O2 was added (final concentration of 1 M NaCl, 20 μg/ml fludioxonil, or 2.5 mM H2O2, respectively). During incubation in each stress-inducing medium, 50 ml of the culture was sampled at 30 and 60 min, pelleted in a tabletop centrifuge, frozen in liquid nitrogen, and lyophilized overnight. The lyophilized cells were subsequently used for total RNA isolation. As biological replicates for DNA microarray, 3 independent cultures for each strain and growth condition were prepared for total RNA isolation.
Total RNA Preparation
[0092] For total RNA isolation, the lyophilized cell pellets were added with 3 ml volume of sterile 3 mm glass bead (SIGMUND LINDER), homogenized by shaking, added with 4 ml of TRizol reagent (Tri reagent, Molecular Research Center), and allowed to incubate at room temperature for 5 min. Then 800 μl of chloroform was added, incubated for 3 min at room temperature, transferred to the 15 ml of the round-bottom tube (SPL), and centrifuged by 10,000 rpm at 4° C. for 15 min (Sorvall SS-34 rotor). Two milliliter of the supernatant was transferred to the new round-bottom tube, added with 2 ml isopropanol, inverted several times, and allowed to incubate for 10 min at room temperature. Then the mixture was re-centrifuged by 10,000 rpm at 4° C. for 10 min, and its pellet was washed with 4 ml of 75% ethanol diluted with diethylpyrocarbonate (DEPC) treated water and centrifuged by 8,000 rpm at 4° C. for 5 min. The pellet was dried at room temperature and resuspended with 500 μl DEPC-treated water. Concentration and purity of total RNA sample were calculated by measuring OD260 nm and gel electrophoresis, respectively. For control total RNA (for Cy3 labeling), all of total RNAs prepared from wild-type, hog1Δ, ssk1Δ, skn7Δ, ras1Δ, aca1Δ, gpa1Δ, cac1Δ and pka1Δ pka2Δ mutant cells grown in conditions described above were pooled (pooled reference RNAs).
cDNA Synthesis and Cy3/Cy5 Labeling
[0093] For cDNA synthesis, total RNA concentration was adjusted to 1 μg/μl with DEPC-treated water, and 15 μl of the total RNA (15 μg) was added with 1 μl of 5 μg/μl oligo dT (5'-TTTTTTTTTTTTTTTTTTTTV-3')/pdN6 (Amersham)(1:1 mixture of 10 μg/μl, respectively), incubated at 70° C. for 10 min, and place on ice for 10 min. Then 15 μl of cDNA synthesis mixture {3 μl 0.1 M DTT, 0.5 μl RNasin [Promega], 0.6 μl aa-dUTP (5-(3-aminoallyl)-2'-deoxyuridine 5'-triphosphate)/dNTPs [a mixture of 6 μl dTTP (100 mM), 4 μl aa-dUTP (100 mM), 10 μl dATP (100 mM), 10 μl dCTP (100 mM), 10 μl dGTP (100 mM)], 1.5 μl AffinityScript reverse transcriptase (Stratagene), 3 μl AffinityScript buffer, 7 μl water] was added and incubated at 42° C. for 2 hrs. Then 10 μl of 1 N NaOH and 10 μl of 0.5 M EDTA (pH 8.0) were added and incubated at 65° C. for 15 min. After incubation, 25 μl of 1 M HEPES buffer (pH 8.0) and 450 μl of DEPE-treated water were added, and the whole mixture was concentrated through Microcon30 filter (Milipore) and vacuum-dried for 1 hr.
[0094] For Cy3 and Cy5 (Amersham) labeling of the prepared cDNA, Cy3 and Cy5 were dissolved in 10 μl DMSO and 1.25 μl of each dye was aliquoted into separate tubes. The cDNAs prepared as described above were added with 9 μl of 0.05 M Na-bicarbonate (pH 8.0) and incubate at room temperature for 15 min. The cDNAs prepared from pooled reference RNAs were mixed with Cy3 as a control and the cDNAs prepared from each test RNA (each experimental condition) were mixed with Cy5. Each mixture was further incubated at room temperature for 1 hr in the dark and purified by QIAquick PCR purification kit (QIAGEN).
Microarray Hybridization and Washing
[0095] C. neoformans serotype D 70-mer microarray slide containing 7,936 spots (Duke University) was pre-hybridized at 42° C. in 60 ml of pre-hybridization buffer [42.4 ml sterile distilled water, 2 ml 30% BSA (Sigma), 600 μl 10% SDS, 15 ml 20×SSC], washed with distilled water and isopropanol, and dried by brief centrifugation (110×g, 2 min). The Cy3- and Cy5-labeled cDNA samples were combined, concentrated through Microcon30 filter, and vaccum-dried. The dried cDNA samples were resuspended with 24 μl of 1× hybridization buffer [250 μl 50% formamide, 125 μl 20×SSC, 5 μl 10% SDS, 120 μl dH2O, total 500 μl], added with 1 μl polyA tail DNA (Sigma), further incubated at 100° C. for 3 min and allowed to cool for 5 min at room temperature. The microarray slides were aligned into the hybridization chamber (DieTech), removed of any dusts, and covered by Lifterslips (Erie Scientific). The Cy3/Cy5-labeled cDNA samples were applied in between Lifterslips and slides. To prevent slides from being dried, 10 μl of 3× SSC buffer was applied onto the slides, which were subsequently incubated for 16 hr at 42° C. After incubation, the microarray slides were washed with three different washing buffers [wash buffer 1 (10 ml 20×SSC, 600 μl 10% SDS, 189.4 ml dH2O, preheated at 42° C.), wash buffer 2 (3.5 ml 20×SSC, 346.5 ml dH2O), wash buffer 3 (0.88 ml 20×SSC, 349.12 ml dH2O)] for 2, 5, and 5 min, respectively, on the orbital shaker.
[0096] For each total RNA sample, 3 independent DNA microarray with 3 independent biological replicates were performed, including one-dye swap experiment.
Microarray Slide Scanning and Data Analysis
[0097] After hybridization and washing, the microarray slides were scanned by GenePix 4000B scanner (Axon Instrument) and the signals were analyzed with GenePix Pro (Ver. 4.0) and gal file (http://genome.wustl.edu/activity/ma/cneoformans). Since we used total RNAs isolated from serotype A C. neoformans strains, 70-mer oligonucleotide sequence printed on the serotype D C. neoformans slides was queried against serotype A C. neoformans genome database by blastp search (e-value cut-off: e-4) to find the corresponding serotype A gene ID. Using the serotype A gene sequence, each S. cerevisiae gene name or ID listed in the Tables was identified by blastp search (e-value cut-off: e-4).
[0098] For further hierarchical and statistical analysis, data transported from GenePix software were analyzed with GeneSpring (Agilent) by employing LOWESS normalization, reliable gene filtering, clustering (standard correlation and average linkage) and zero-transformation, and ANOVA analysis (P<0.01).
Ergosterol Assay
[0099] Ergosterol contents were measured as previously described in "Arthington-Skaggs B A, Jradi H, Desai T, Morrison C J (1999) Quantitation of ergosterol content: novel method for determination of fluconazole susceptibility of Candida albicans. J Clin Microbial 37: 3332-3337", but with slight modification. Briefly, each C. neoformans strain was grown in 100 ml YPD medium for 24 h at 30° C. The 100 ml culture was splitted into two 50 ml cultures for duplicate measurement, pelleted in a tabletop centrifuge, and washed with sterile water. The cell pellet was frozen in liquid nitrogen and lyophilized overnight. The dried cell pellet was weighed for normalization of ergosterol contents, added with 5 ml of 25% alcoholic potassium hydroxide, and transferred to a sterile borosilicated glass screw-cap tube. Subsequently, the cells were incubated at 80° C. for 1 h and allowed to cool to room temperature. Then 1 ml of sterile water and 3 ml of heptane were added and vortexed for 3 min. Then 200 μl of the heptane layer is sampled and mixed with 800 μl of 100% ethanol, and its optical density (OD) was measured at both 281.5 nm and 230 nm. Ergosterol contents were calculated as the following: % ergosterol=[(OD281.5 nm/290)×F]/pellet weight-[(OD230 nm/518)×F]/pellet weight, where F is the ethanol dilution factor and 290 and 518 are the E values (in percentages per centimeter) determined for crystalline ergosterol and 24(28)dehydroergosterol, respectively.
Stress Sensitivity Test
[0100] Each strain was incubated overnight at 30° C. in YPD medium, washed, serially diluted (1 to 104 dilutions) in dH2O, and spotted (3 μl) onto solid YPD medium containing indicated concentrations of stress-inducing agents or antifungal drugs as previously described in "Bahn Y S, Kojima K, Cox G M, Heitman J (2005) Mol Biol Cell 16: 2285-2300." and "Bahn Y S, Kojima K, Cox G M, Heitman J (2006) Mol Biol Cell 17: 3122-3135". To examine antifungal drug sensitivity, the cells were spotted on agar-solid YPD media containing amphotericin B(Sigma), fluconazole(Sigma), itraconazole(Sigma), ketoconazole (Sigma) and fludioxonil. Then spotted cells were incubated at 30° C. for 2-4 days and photographed.
Disruption of CAMP-Signaling Dependent Genes
[0101] For gene disruption, information of genomic DNA structure (exon and intron) for each gene was obtained from serotype A C. neoformans genome database (http://www.broadinstitute.org/annotation/genome/cryptococcus_ne- oformans/MultiHome.ht ml). The GRE2 (CNAG--02182.2), HSP12 (CNAG--03143.2), HSP122 (CNAG--01446.2) and PKP1 (CNAG--00396.2) genes were deleted by overlap PCR or double joint PCR PCR) with split markers and biolistic transformation in the C. neoformans serotype A H99 strain as previously described (Bahn et al., 2005, Davidson et al., 2002). Primers for generation of the 5' and 3' flanking regions of each gene and dominant selectable nourseothricin resistant marker (NAT, nourseothricin acteryltransferase) were described in the supplemental table 1. Gold microcarriers beads (0.8˜1.2 μm [Bioworld Inc] or 0.6 -μm [BioRad]) were coated with gel-extracted deletion cassettes produced by overlap PCR and biolistically transformed into the strain H99. Stable transformants selected on YPD medium containing nourseothricin or G418 were subject to the first screening by diagnostic PCR with primers listed in Table 2. Positive mutants were further confirmed by Southern blot analysis using gene-specific probes prepared by primers listed in Table 2.
TABLE-US-00002 TABLE 2 Primer Name Sequence Description B79 TGTGGATGTCTGGCGGAGGATA Screening primer on ACT promtre B1026 GTAAAACGACGGCCAGTGAGC M13 forward (extended) B1027 CAGGAAACAGCTATGACCATG M13 reverse (extended) B1614 TGTTTAGCACCAGCGGAGTC HSP12-5' screening primer B1615 CACGATGAAAGTGCGTTGAAG HSP12-left flanking primer 1 B1616 GCTCACTGGCCGTCGTTTTACACTGTCGGTGAAAGATTGC HSP12-left flanking primer 2 B1617 CATGGTCATAGCTGTTTCCTGAGAACGACAACCAGGAGTC HSP12-right flanking primer 1 B1618 GCTCTGTGCTGACATTATCTGC HSP12-right flanking primer 2 B1707 GAAAGTGCGTTGAAGTGATG HSP12-probe primer 1 B1708 AGTAGAAGCAGCGGACTAAAG HPS12-probe primer 2 B1619 GCGTAGTGGAGATTGGTTTC GRE2-5' screening primer B1620 ATCCCCTCCACTTTACCTCC GRE2-left flanking primer 1 B1621 GCTCACTGGCCGTCGTTTTACAAGTCTCCCTTAGCGATAG GRE2-left flanking primer 2 B1622 CATGGTCATAGCTGTTTCCTGACCACACCCCTGAAGAAAC GRE2-right flanking primer 1 B1623 AACTGTTTCGTCTTGTGTGTC GRE2-right flanking primer 2 B1705 ATAGCAACTTCTTCCGTCG GRE2-probe primer 1 B1706 TGTTGCCTGTGCTCACTTG GRE2-probe primer 2 B1629 CCTCTGACAGCCACATACTG PKP1-5' screening primer B1630 AATGAAGTTCCTGCGACAG PKP1-left flanking primer 1 B1631 GCTCACTGGCCGTCGTTTTACAATGGGATGAGAACGCAC PKP1-left flanking primer 2 B1632 CATGGTCATAGCTGTTTCCTGAGCATTTTCCAGCATCAGC PKP1-right flanking primer 1 B1633 GGTGTGGAACATCTTTTGAG PKP1-right flanking primer 2 B1711 CTGGTTCATCTTGGGTGTC PKP1-probe primer 1 B1712 TCTGAGCATACCACTCCTTTAC PKP1-probe primer 2 B1666 TCTCATTCGCATCCTCTG HSP122-5' screening primer B1667 GTTGGGCAGATAATGTTTGTG HPS122-left flanking primer 1 B1668 GCTCACTGGCCGTCGTTTTACACGGCGTCAGACATTGTG HSP122-left flanking primer 2 B1669 CATGGTCATAGCTGTTTCCTGACAAGAGAAGTCCACTACTCAG HPS122-right flanking primer 1 B1670 GCAAGGTAATGATGAGCG HSP122-right flanking primer 2 B1709 GCGACTGAGATGTAGACCAAC HSP122-probe primer 1 B1710 CTCGGAACGACATAATAAGC HSP122-probe primer 2 B1673 CACACCTGGTAAGAGATAGCG NHA1-left flanking primer 1 B1674 GCTCACTGGCCGTCGTTTTACAGTGGTAGAAGTAGGGCAGC NHA1-left flanking primer 2 B1675 CATGGTCATAGCTGTTTCCTGACAGGGTCCAACAAGGATG NHA1-right flanking primer 1 B1676 TGCTACGATTGTGGTCAGCC NHA1-right flanking primer 2 B1677 GGACGAGACGAGTTATCAAAC NHA1-screening primer B1698 CTTCATCAACTTGCGTGC NHA1-probe primer
Example 1
DNA Microarray Analysis of C. neoformans hog1Δ, ssk1Δ, and skn7Δ Mutants
[0102] To investigate Hog1 signaling pathway in C. neoformans, we performed comparative transcriptome analysis of serotype A wild-type (WT, H99) strain, hog1Δ, ssk1Δ, and skn7Δ mutants under both normal growth conditions and stressed conditions, such as in the presence of osmotic shock (1 M NaCl), oxidative stress (2.5 mM H2O2), and antifungal drug fludioxonil (40 μg/ml), by using DNA microarray analysis. We isolated total RNAs from cells growing in each stress condition after zero (non-stress condition), 30, and 60 min incubation. We prepared 3 independent RNA samples for each condition as biological replicates for DNA microarray analysis. As a control RNA for common Cy3 labeling, we used reference RNAs that were pooled from all RNA samples prepared in this study. We used 70-mer serotype D C. neoformans DNA microarray chips containing total 7,936 spots, based on information from the C. neoformans genome database.
[0103] For basic validation of our array quality, we monitored expression levels of HOG1, SSK1, and SKN7 genes, and known Hog1-regulated genes, such as GPP1 (Glycerol-3-phosphatase) and GPD1 (Glycerol-3-phosphate dehydrogenase), in our array data.
[0104] FIG. 1 shows identification of genes whose expression is regulated by Hog, Ssk1 and Skn7 of C. neoformans under normal conditions with no stress on the genome level (fold change is expressed by color). FIG. 1A shows relative expression levels of HOG1, SSK1, and SKN7 genes in each corresponding mutant compared to WT strain. FIG. 1B shows condition tree analysis result in WT, hog1Δ, ssk1Δ, skn7Δ mutant. FIG. 1C shows clustering analysis result of 950 genes which are exhibited significantly different expression patterns in hog1Δ, ssk1Δ, or skn7Δ mutants compared to WT (ANOVA test, P<0.01) under normal growth condition(YDP, 30quadrature). FIG. 1D shows Venn diagram presenting HOG1, SSK1, and SKN7-dependent genes that include genes up- or down-regulated over 2 folds.
[0105] As expected, relative expression levels of HOG1, SSK1, and SKN7 genes in each corresponding mutant compared to WT strain were very low(FIG. 1A). In addition, expression of GPD1 (glycerol-3-phosphate dehydrogenase, CNAG--01745) and GPP1 (DL-glycerol-3-phosphatase, CNAG--01744) homologous genes, which are well-known Hog1-regulated stress defense genes in other fungi, was highly reduced (4.5-fold and 2.5-fold reductions, respectively) in hog1Δ and ssk1Δ mutants, further supporting the quality of our array data.
[0106] We monitored how HOG1, SSK1, and SKN7 mutations affect gene expression patterns in C. neoformans under unperturbed normal conditions. Among 7,936 spots monitored, 3,858 spots were found to be reliable based on Cross-gene error model (cutoff 10). Supporting the previous finding, the transcription profile of the hog1Δ mutant was considerably similar to that of the ssk1Δ mutant, based on the condition tree analysis (FIG. 1B). A total of 950 genes in the reliable genes exhibited significantly different expression patterns in hog1Δ, ssk1Δ, or skn7Δ mutants compared to WT (ANOVA test, P<0.01) (FIG. 1C), indicating that about 15% of the whole C. neoformans genes could be transcriptionally affected by perturbation of the two-component system and HOG signaling pathways even under unstressed, normal conditions. Among them, 559 genes exhibited more than 2-fold induction in at least one of the mutants (FIG. 1D). Several key findings were made as the following. First, a majority of the genes (555 genes, 99%) were up- or down-regulated by either Ssk1 or Hog1 under unstressed conditions while only 51 genes (9%) were regulated by Skn7. Among the Skn7-dependent genes, only 4 genes were found to be Skn7-specific (FIG. 1D). Thus it appears to be clear that HOG1 and SSK1 mutations alter genome-wide transcription profiles under normal conditions in a greater scale than the SKN7 mutation (FIG. 1D). Second, there exist significantly higher overlaps between Ssk1- and Hog1-dependent genes (422 out of 555 genes, 76%) than between Skn7- and Hog1-dependent genes (45 out of 467 genes, 10%), further corroborating that Ssk1 is the major upstream regulator of the Hog1 MAPK. Third, regardless of the significant overlap in genes regulated by Ssk1 and Hog1, there were a number of Ssk1-specific (90 genes) and Hog1-specific (40 genes) genes, strongly suggesting that Ssk1 and Hog1 are not strictly in the linear pathway and could have other target(s) or upstream regulators, respectively (FIG. 1D). This explains why the ssk1Δ mutant exhibits slightly different phenotypes (i.e. higher sensitivity to hydrogen peroxide) compared to hog1Δ mutants and Hog1 can still be phosphorylated in the absence of Ssk1 response regulator under exposure to NaCl.
[0107] Genes regulated by Hog1 and Ssk1 cover a wide variety of functional categories, including energy production and conversion, amino acid/carbohydrate/lipid transport and metabolism, translational and protein biosynthesis, post-translational modification, signal transduction, stress-defense mechanisms, and others (Supplementary table 2), indicating that active remodeling of various aspects of cellular functions could occur simply by perturbation of the HOG pathway even without external stresses. Furthermore it should be noticed that more than one third of Hog1 and Ssk1-dependent genes do not have any functional orthologs in other organisms, indicating that C. neoformans appears to develop many cryptococcus-specific Hog1/Ssk1-dependent genes.
[0108] Among Ssk1- and Hog1-regulated genes identified by our array analysis, several groups of genes provided novel insights into the potential mechanism of the HOG pathway in controlling virulence factor and sexual reproduction of C. neoformans. First, a group of genes involved in iron transport and regulation were found to be highly induced in the ssk1Δ and hog1Δ mutants compared to the wild-type strain. These genes include SIT1 (CNAG--00815 and CNAG--07138) encoding siderophore-transporters, CFO1 (CNAG--06241) and CFO2 (CNAG--02958) encoding ferroxidases, and CFT1 (CNAG--06242) encoding Fe transporter. The C. neoformans Sit1 are homologous to the S. cerevisiae Arn3/Sit1 having high affinity for the hydroxamate siderophore ferrioxamine and C. neoformans Cfo1/Cfo2 and Cft1 are homologous to high-affinity iron permease/multicopper ferroxidase complex (Ftr1-Fet3) in S. cerevisiae. Since iron transport regulation and melanin synthesis seem to be closely related in C. neoformans, increased melanin synthesis observed in both hog1Δ and ssk1Δ mutants could be correlated with increased expression of a group of genes involved in iron transport.
[0109] Second, the GPA2 gene (CNAG--00179), encoding a G-protein α-subunit in the pheromone responsive MAPK pathway, is dramatically upregulated upon ssk1Δ or hog1Δ mutation (12.1- and 13.3-fold increases, respectively). This finding suggests that increased pheromone production and sexual reproduction found in ssk1Δ and hog1Δ mutants may result from enhanced expression of Gpa2 that is induced during mating and promotes the mating process of C. neoformans.
[0110] Third, several genes involved in oxidative stress defense were differentially regulated by HOG1 and SSK1 mutation. As expected from the previous finding that the hog1Δ and ssk1Δ mutants exhibit hypersensitivity to hydrogen peroxide, two genes (CNAG--04981 and CNAG--00575), which are homologous to the CTA1 gene encoding catalase A that detoxifies H2O2 to H2O, was drastically downregulated in both mutants (Supplementary Table 1). Furthermore, basal expression levels of the SOD2 gene (mitochondrial superoxide dismutase) were decreased in both hog1Δ and ssk1Δ mutants, further corroborating the role of the HOG pathway in oxidative stress response. Interestingly, however, basal expression levels of some genes involved in oxidative stress response [TRR1 (thioredoxin reductase), TSA1 (thioredoxin peroxidase), GRX5 (glutathione-dependent oxidoreductase), CCP1 (mitochondrial cytochrome-c peroxidase)] were more than 2-fold increased (3.8, 3.1, 2.1, and 9.5 fold changes, respectively) in the hog1Δ mutant, but not in the ssk1Δ mutant (Supplementary Table 2). The SRX1 gene (sulfiredoxin) also involved in oxidative stress response was more reduced in the ssk1Δ mutant (4.2-fold reduction) than the hog1Δ mutants (1.3-fold reductions). These results may explain why the hog1Δ mutants are relatively more resistant to H2O2 than the ssk1Δ mutant.
Example 2
Ergosterol Biosynthesis Genes are Transcriptionally Upregulated by Perturbation of the HOG Signaling Pathway
[0111] Among genes upregulated by mutation of HOG1 and SSK1 genes, a gene homologous to ERG28 (CNAG--07208) was noticeable since it plays a key role in the fungal sterol biosynthesis. Previous microarray analysis performed in S. cerevisiae revealed that expression of ERG28 is tightly correlated with other ergosterol biosynthetic genes. Erg28 is an endoplasmic reticulum (ER) transmembrane scaffold, protein, which is essential for the yeast sterol biosynthesis by interacting strongly with Erg27, Erg25, Erg11, and Erg6 and weakly with Erg26 and Erg1. This finding led us to monitor expression patterns of other sterol biosynthetic genes in our array data.
[0112] FIG. 2A shows the relative expression profiles of ergosterol biosynthesis genes in hog1Δ, ssk1Δ, and skn7Δ mutants compared to WT strain. The fold change is illustrated by a color (see color bar scale) and exact value for each gene was indicated in the table placed right side of the hierarchical clustering diagram. FIG. 2B shows cellular ergosterol contents in WT (H99), skn7Δ (YSB349), ssk1Δ (YSB261), ssk2A (YSB264), and hog1Δ (YSB64) mutants. Left and right graphs demonstrate % ergosterol in each strain and relative increase of ergosterol contents compared to WT, respectively. Each bar presents the average from four independent experiments and error bar indicates the standard deviation. Asterisks (*): The ssk1Δ, ssk2Δ, pbs2Δ, and hog1Δ mutants contain significantly higher ergosterol levels compared to the WT (P<0.05, as analyzed by using the Bonferroni multiple comparison test).
[0113] Interestingly, a majority of the ergosterol biosynthetic genes were upregulated in hog1Δ and ssk1Δ mutants, but not in the skn7Δ mutant, compared to the wild-type strain (FIG. 2A). Genes, such as ERG11, ERG6, MVD1, ERG5, ERG25, ERG20, and ERG4, were upregulated in both ssk1Δ and hog1Δ mutants while genes, such as ERG27, ERG13, ERG26, ERG10, IDI1, HMG1, and ERG8, were upregulated only in the ssk1Δ mutant (FIG. 2A). In contrast, none of genes were significantly upregulated in the skn7 mutant and indeed some of genes, including ERG13, ERG1, ERG3, ERG7, and ERG2 genes, were downregulated in the skn7Δ mutant (FIG. 2A).
[0114] To verify our microarray data, we examined whether increased expression levels of some of the ergosterol biosynthesis genes indeed affect cellular ergosterol contents in the hog1Δ and ssk1Δ mutants (FIG. 2B). In accordance with our microarray data, cellular ergosterol contents were much higher in the hog1Δ and ssk1Δ mutants than WT and skn7Δ mutants (FIG. 2B), suggesting that increased expression of some of ergosterol biosynthetic genes leads to enhanced production of cellular ergosterol. The ssk2Δ (MAPKKK) and pbs2Δ (MAPKK) mutants in the HOG pathway were also found to contain significantly higher levels of cellular ergosterol than WT and skn7Δ mutants (FIG. 2B), further corroborating our array data.
[0115] This finding prompted us to investigate the susceptibility of the mutants in the two-component system and the HOG pathway to antifungal drugs that are targeted to the ergosterol biosysnthetic genes or ergosterol itself. First we have examined the susceptibility of the ssk1Δ, skn7Δ, ssk2Δ, pbs2Δ, and hog1Δ mutants made in the serotype A H99 strain background to the polyen antifungal drug, amphotericin B, which binds to ergosterol in the fungal cell membrane and ultimately causes lethality by disrupting the membrane integrity. We hypothesized that increased ergosterol contents observed in ssk1Δ, ssk2Δ, pbs2Δ, and hog1Δ mutants could render them to be hypersensitive to amphotericin B due to the increased number of drug targets.
[0116] FIG. 3 shows analysis results showing that the inhibition of the HOG pathway confers synergistic antifungal effects with amphotericin B in C. neoformans. FIG. 3A-3B show pictures photographed after incubation at 30° C. for 72 h of each C. neoformans strain spotted on YPD agar containing indicated concentrations of amphotericin B. FIG. 3C shows pictures photographed after incubation at 30° C. for 72 h of C. neoformans serotype A strains and serotype D strains spotted on YPD agar containing indicated concentrations of amphotericin B.
[0117] Confirming our hypothesis, the ssk1Δ, ssk2Δ, pbs2Δ, and hog1Δ mutants exhibited dramatic hypersensitivity to amphotericin B treatment compared to WT (FIG. 3A), which is in good agreement with the finding that ergosterol contents were significantly higher in the HOG pathway mutants than WT (FIG. 2B). In contrast, the skn7Δ mutant showed WT-levels of resistance to amphotericin B (FIG. 3A), which can be also explained by the previous data showing that cellular ergosterol contents in the skn7Δ mutants are similar to those of WT (FIG. 2B).
[0118] We also monitored amphotericin B-susceptibility of C. neoformans strains having mutation hybrid sensor kinases (Tco1, Tco2, Tco3, Tco4, Tco5, and Tco7), which act upstream of the Ssk1 response regulator. Previously we have shown that Tco1 and Tco2 play redundant and distinct roles in controlling a subset of Hog1-dependent phenotypes. Here we found that Tco1 and Tco2 play discrete roles in sensing and responding to amphotericin B. Among Tco proteins, only Tco2, which is double hybrid sensor kinases containing two response regulator domains and two histidine kinase domains in a single polypeptide, showed hypersensitivity to amphotericin B (FIG. 3B), indicating that Tco2 is involved in sensing and responding to amphotericin B for conferring the drug-resistance via the HOG pathway. However, the fact that the degree of hypersensitivity observed in the tco2Δ mutant is lesser than the ssk1Δ mutant suggests other possibilities. One possibility is that other unknown receptor/sensors may exist to respond to the amphotericin B. The other possibility is that constitutively phosphorylated Hog1 may repress ergosterol biosynthetic pathway under normal conditions hypersensitivity regardless of the presence of receptors/sensors since Ssk1, Ssk2, and Pbs2, but not Tco2 proteins, are all involved in constitutive phosphorylation levels of Hog1.
[0119] To test the hypothesis, we have also examined the amphotericin B sensitivity of other C. neoformans strains, such as JEC21 and B3501-A, showing differential Hog1 phosphorylation levels. To support our second hypothesis, the JEC21 strain where Hog1 is not constitutively phosphorylated exhibited hypersensitivity to amphotericin B even more than the ssk2Δ mutant in the H99 strain background (FIG. 3C). In the JEC21 strain background, mutation of SSK2, PBS2, and HOG1 genes did not affect sensitivity to amphotericin B (FIG. 3C). In contrast, the B3501 strain where Hog1 is constitutively phosphorylated, albeit to a lesser extent than in the H99 strain, exhibited higher resistance to amphotericin B than the JEC21 (FIG. 3C). Similar to the H99 strain, mutation of the SSK2 MAPKKK that abolishes the Hog1 phosphorylations increased the amphotericin B sensitivity (FIG. 3C). All these data strongly indicate that constitutively phosphorylated Hog1 represses ergosterol biosynthetic pathway under normal conditions.
[0120] To further support this finding, we also examined the susceptibility of the mutants to azole compounds, including triazoles (fluconazole and itraconzaole) and imidazole (ketoconazole), which inhibit the fungal cytochrome P450 enzyme 14α-demethylase and eventually prevent conversion of lanosterol to ergosterol.
[0121] FIG. 4 shows analysis results showing that the inhibition of the HOG pathway confers antagonistic antifungal effects with some azole drugs in C. neoformans. It shows pictures photographed after incubation at 30° C. for 72 h of each C. neoformans strain spotted on YPD agar containing indicated concentrations of fluconazole, ketoconazole, and itraconazole.
[0122] We had expected that the ssk1Δ and hog1Δ mutants having increased expression of many ergosterol biosynthesis genes, particularly including ERG11, should show higher resistance to azole compounds. The ssk1Δ, ssk2Δ, pbs2Δ, and hog1Δ mutants all exhibited hyper-resistance to fluconazole and ketoconazole, but not to itraconazole (FIG. 4). Interestingly, the skn7Δ mutants also showed higher resistance to fluconazole and ketoconazole than WT (FIG. 4). Among hybrid sensor kinases, only Tco1 and Tco2 display differential sensitivity to azole compounds. Although to a lesser extent than the HOG mutants, the tco2Δ mutant exhibited higher resistance to fluconazole and ketoconazole than WT (FIG. 4). In contrast, the tco1Δ mutant exhibits hypersensitivity to all azole drugs (FIG. 4), indicating that Tco1 may regulate the HOG pathway in C. neoformans in an opposite manner to Tco2. In conclusion, inactivation of the HOG pathway increases ergosterol contents by induction of ergosterol biosynthesis genes and therefore confers synergistic effects with amphotericin B treatment, but antagonistic effects with fluconazole and ketoconazole.
Example 3
Finding and Characterizing the Downstream Target Genes Controlled by the HOG Pathway
[0123] We found ENA1 (serotype A ID: CNAG--00531.2) and NHA1 (serotype A ID: CNAG_01678.2) genes as the downstream target genes controlled by the HOG pathway and performed an additional experiment. Cells excrete H+(proton) out of cell membrane using H+-ATPase pump such as Pma1, thereby playing a role in maintaining membrane potential essential to cell growth in a normal condition. On the contrary, potassium ion(K+), an ion useful to cell growth, flows into cells using K+ influx pump such as Trk1/Trk2. Na+, unlike K+, is classified as a toxic ion. When high concentration of Na+ is present in a cell, it should be excreted via efflux pump. Since K+ also has toxicity when it presents in high concentration, an efflux pump is needed. These are Ena1 and Nha1 which play a role as an efflux pump for Na+and K+.
[0124] The result showed that the two genes coding for the two efflux pumps are controlled by the HOG pathway. As shown in FIG. 5A, when the WT, skn7Δ, ssk1Δ and hog1Δ mutant strains were exposed to osmotic stress, the expression level of ENA1 and NHA1 was dependent on the deletion mutant of the HOG pathway genes.
[0125] Thus, in order to identify a characteristic of two genes, we prepared deletion mutant of each gene and double mutant (ena1Δ nha1Δ) eliminating both two genes. And then, we examined sensitivity of the mutants to the polyene-based antifungal agent such as amphotericin B (AmpB), and the azole-based antifungal agent such as fluconazole, ketoconazole and itraconazole. The ena1Δ and nha1Δ mutants did not show high sensitivity to the AmpB. However, surprisingly, the ena1Δ nha1Δ double mutant showed considerably increased sensitivity to AmpB (FIG. 5B). Although lower sensitivity than hog1Δ, high AmpB sensitivity of the ena1Δ nha1Δ suggests that these two efflux pumps play an important role in the polyene-based drug resistance. It is more noteworthy that ena1Δ and ena1Δ nha1Δ mutants also show high sensitivity to the azole-based drugs (FIG. 5C). It is a distinguished from the hog1Δ mutant which has high resistance to the azole-based antifungal agents and verify that the inhibitors simultaneously or independently targeting Ena1 and Nha1 may exhibit very high antifungal activities when used in combination with the polyene- or azole-based antifungal agents.
Example 4
Comparative Transcriptome Analysis of C. neoformans ras1Δ, aca1Δ, gpa1Δ, cac1Δ, and pka1Δ pka2Δ Mutants
[0126] To compare the downstream signaling network of Ras1-, Aca1-, and Gpa1-dependent signaling pathways, we performed comparative transcriptome analysis of the serotype A wild-type (WT, H99) strain, ras1Δ, aca1Δ, gpa1Δ, cac1Δ, and pka1Δ pka1Δ mutants by employing DNA microarray analysis as described in Materials and Methods. For basic validation of our array quality, we checked expression levels of the RAS1, ACA1, GPA1, CAC1, PKA1, and PKA2 genes in our array data. The relative expression levels of RAS1, ACA1, GPA1, CAC1, PKA1, and PKA2 in each corresponding mutant were very low compared to those in the wild type strain (0.08, 0.03, 0.09, 0.06, 0.07, and 0.12, respectively) (FIG. 6A), which supported the quality of our array.
[0127] From total 7,936 genes monitored by this DNA microarray, 565 genes exhibited differential expression patterns in the Ras- and cAMP mutants at statistically significant levels compared to the wild type strain (ANOVA test, P<0.05) (FIG. 6B). The hierarchical clustering analysis of the Ras- or cAMP-dependent genes revealed several important facts. First, the transcriptome patterns governed by the Ras1-signaling pathway were distinct from those controlled by the cAMP/PKA-signaling pathway. The statistical analysis indicated that basal expression levels of total 400 genes changed significantly in the ras1Δ mutant compared to the WT, whereas expression levels of 132 genes changed significantly in the aca1Δ, gpa1Δ, cac1Δ, and pka1Δ pka2Δ mutants (FIGS. 6C and 6D). Besides the number of genes regulated, the expression patterns of a majority of the Ras1-dependent genes were also distinguished from those of the cAMP-dependent genes, which supported that the Ras1-signaling pathway is largely independent of the cAMP-signaling pathway in C. neoformans. Second, the aca1Δ and gpa1Δ mutants showed transcriptome patterns similar to those of the cac1Δ and pka1Δ pka2Δ mutants, indicating that Aca1Δ and Gpa1 are the two major signaling modulators of the cAMP-signaling pathway (FIG. 6D). However, there were a small group of genes whose expression is differentially regulated between the aca1Δ and gpa1Δ mutants. This indicates that Aca1 and Gpa1 could have other minor signaling branches (FIG. 6D). As expected, the cac1Δ mutant exhibited transcriptome patterns almost identical to that of the pka1Δ pka2Δ mutant, further suggesting that Pka1 and Pka2 are necessary and sufficient protein kinase downstream of the adenylyl cyclase in C. neoformans (FIG. 6D).
[0128] The genes regulated by the Ras- and cAMP-signaling pathways cover a wide variety of cellular functions (FIG. 7). The cAMP-signaling dependent genes were over-represented for those involved in signal transduction mechanisms (15.2%), carbohydrate transport and metabolism (9.6%), and amino acid transport and metabolism (8.0%). These findings were rather expected results since the cAMP-pathway is one of central signal transduction cascades that regulate growth, differentiation, and virulence of C. neoformans and is known to sense glucose and amino acids (Bahn et al., 2004, Xue et al., 2006). Similarly, genes involved in signal transduction mechanisms were most over-represented in the ras1Δ mutant (12.1%) (FIG. 7). In contrast to the cAMP-pathway, however, genes involved in cell wall/membrane/envelope biogenesis were over-represented (2.9%), which implies that Ras1 may be implicated in maintenance of cell wall integrity.
[0129] Among the Ras- and cAMP-dependent genes, a significant proportion of them were found to be environmental stress-regulated (FIG. 8). Our prior transcriptome analysis discovered a number of ESR (Environmental Stress Regulated) genes in C. neoformans (Ko et al., 2009). A total of 1,959 genes were found to be more than 2-fold up or downregulated in response to either of osmotic stress, oxidative stress, or antifungal drug (fludioxonil) treatment (Ko et al., 2009). Interestingly, our current array analysis revealed that a subset of the ESR genes (a total of 225 ESR genes) exhibited significant changes in expression levels in either the ras1Δ or cAMP mutants compared to the wild-type strain (ANOVA test, P<0.05) (FIG. 8). Among these, eighty-six ESR genes showed more than 2-fold induction or reduction in the mutants (FIG. 8). Furthermore, a total of 55 CSR (Common Stress Response) genes were found to be differentially regulated (ANOVA test, P<0.05) and 31 genes of them exhibited more than 2-fold induction or reduction in the mutants (FIG. 2B). The major proportion of the Ras- or cAMP-pathway-dependent ESR and CSR genes did not have any other homologs with significant homology (Table S6). Nevertheless, these results implied that the Ras-and cAMP-signaling pathways be implicated in diverse stress response of C. neoformans.
Example 5
Identification of the Ras- or cAMP-Dependent Genes in C. neoformans
[0130] Next we further investigated individual Ras1- and cAMP-dependent genes identified by our transcriptome analysis.
[0131] Among the selected 161 Ras-dependent genes (2-fold cutoff, FIG. 6C), a majority of them (101 genes, 63%) do not have any orthologs in other fungi (Table S4), which indicated that C. neoformans contains a unique set of Ras-dependent genes. Among the evolutionary conserved Ras-dependent genes, three genes, PXL1, RDI1, and BEM3, whose orthologs are known to be involved in regulation of Rho-GTPase Cdc42 in S. cerevisiae, were notable since the Ras1-Cdc24 signaling pathway has been reported to be controlled by one of three Cdc42 homologues in C. neoformans (Nichols et al., 2007). RDI1 and BEM3 encode Rho-GDP dissociation inhibitor and Rho-GTPase activating protein, respectively (Price et al., 2008, Zheng et al., 1994). Notably, in a good agreement with the role of Ras1 in genotoxic stress response of C. neoformans (FIG. 4), a number of genes involved in regulation of DNA damage repair were identified as Ras-dependent genes. These include RNR2/RNR3 (Ribonucleotide-diphosphate reductase), RAD3 (DNA helicase, a subunit of nucleotide excision repair factor 3), RAD14 (a subunit of nucleotide excision repair factor 1), MSH6 (a protein required for mismatch repair), MND1 (a protein required for recombination and repair of DNA double strand breaks), and DNA2 (ATP-dependent nuclease). Finally, several genes, CHS1 (Chitin synthase 1), CDA2 (Chitin deacetylase), BGL2 (glucan 1,3-β-glucosidase), and GSC2 (Glucan synthase), involved in governing cell wall integrity were also identified as Ras-dependent genes 6, which further supported the role of Ras1 in maintaining cell wall integrity of C. neoformans.
[0132] The statistical comparison of transcriptome data obtained from the cAMP mutants (aca1Δ, gpa1Δ, cac1Δ, and pka1Δ pka2Δ) with that from the WT strain (ANOVA, P<0.05) identified 163 genes (FIG. 6C). Among these, 38 genes exhibited more than 2-fold induction or reduction in the cAMP mutants, except CAC1, ACA1, PKA1, and GPA1 (FIG. 9). A majority of the cAMP-dependent genes (31 genes, 81%) do not have any known function in C. neoformans or orthologs in S. cerevisiae, which indicated that C. neoformans contains a unique set of cAMP-dependent genes similarly to the Ras-dependent genes. This observation further corroborates that C. neoformans cAMP mutants have unique phenotypic characteristics that have not been observed in other fungi. Five cAMP-dependent genes (GRE2, ENA1, HSP12, CAT1, and PKP1) in C. neoformans appear to be evolutionarily conserved in other fungi. Interestingly, the GRE2, ENA1, and HSP12 genes are known to be transcriptionally regulated by environmental stress in S. cerevisiae. In C. neoformans, it has been recently reported that Ena1 not only controls osmotic stress under carbon starvation condition (Ko et al., 2009), but also is required for survival in alkaline pH and in vivo virulence (Idnurm et al., 2009). The GRE2 (genes de respuesta a estres, stress-responsive gene), a homolog of mammalian 3-β-hydroxysteroid dehydrogenase, is strongly induced in response to a variety of stresses, including osmotic and oxidative stress, upon binding of HOG-dependent Sko1 transcription factor to CRE (cAMP response element) in the promoter region in S. cerevisiae (Garay-Arroyo & Covarrubias, 1999, Rep et al., 2001). The heat shock protein HSP12 (03143) is a small hydrophilic protein whose expression is also induced by diverse stresses and regulated by both HOG and cAMP signaling pathways (Varela et al., 1995). Here we named this gene as HSC1 (HSP12-like C. neoformans gene 1, 03143).
EXAMPLE 6
Inhibition of the Ras and cAMP-Signaling Pathway Increased Polyene Sensitivity
[0133] Gre2 is involved in regulation of some of ergosterol biosynthesis genes, including ERG6, ERG10, and ERG19/MVD1 (Warringer & Blomberg, 2006). Furthermore, GRE2 is reported to be one of six genes whose expression increased with resistance to amphotericin B (AmpB) in S. cerevisiae (Anderson et al., 2009). Therefore, we examined whether the C. neoformans Ras- and cAMP-mutants are more susceptible to AmpB treatment than WT.
[0134] As shown in FIG. 10, the ras1Δ mutant showed higher susceptibility to AmpB than WT whereas the aca1Δ mutant exhibited slightly higher AmpB susceptibility (FIG. 10A). The ras1Δ aca1Δ double mutant exhibited higher AmpB-sensitivity than each single mutant (FIG. 10B), indicating that Ras1 and Aca1 redundantly or independently control AmpB sensitivity. Cdc24 appears to work downstream of Ras1 for regulation of the polyene drug resistance (FIG. 10C). Interestingly, the ras2Δ mutant was also slightly more sensitive to AmpB than WT, indicating that both Ras proteins control resistance to polyene drugs in C. neoformans.
[0135] Notably, the gpa1Δ and cac1Δ mutants showed much higher AmpB-sensitivity than WT and even than the ras1Δ or aca1Δ mutant (FIG. 10A). Downstream of the Cac1 adenylyl cyclase, the pka1Δ mutant, but not the pka2Δ mutant, showed increased susceptibility to AmpB (FIG. 10A), strongly indicating that the Gpa1-Cac1-Pka1 signaling cascade is one of signaling circuits to control the polyene drug sensitivity. The ras1Δ cac1Δ double mutant exhibited even higher AmpB susceptibility than each single mutant (FIG. 10B), indicating that the Ras- and Gpa1-Cac1-Pka1 pathways are independently involved in AmpB susceptibility. The ras1Δ and ras1Δ cac1Δ mutants generated in MATa background (KN99 strain) exhibited the same phenotypes (data not shown).
[0136] To address whether the involvement of the Ras- and cAMP-pathways in the polyene sensitivity is related to the levels of ergosterol biosynthesis, we checked expression levels of ergosterol biosynthesis genes in the mutants from our array data. Interestingly, none of ergosterol biosynthesis genes, except ERG3 and ERG25 (<less than 2-fold), exhibited significant expression changes in the ras1Δ, aca1Δ, gpa1Δ, cac1Δ, or pka1Δ pka2Δ mutants compared to WT (Table S2 and S3). Northern blot analysis showed that expression levels of the ERG3 and ERG25 genes in the mutants were not significantly different from those of WT (FIG. 10D). We also checked cellular ergosterol contents in the Ras- and cAMP-mutants and found that cellular ergosterol contents were not significantly increased in the Ras- and cAMP-mutants compared to WT whereas the hog1Δ mutant has increased ergosterol contents as previously reported (data not shown) (Ko et al., 2009). Furthermore, expression levels of ERG11 in the ras1Δ and cAMP mutants were not significantly different from those of WT (FIG. 10D). Supporting this finding, the gpa1Δ, cac1Δ, pka1Δ, pka2Δ, and pka1Δ pka2Δ mutants were nearly as resistant to fluconazole, which target to the fungal cytochrome P450 enzyme 14α-demethylase and inhibit conversion of lanosterol to ergosterol, as the WT strain (data not shown). All these data strongly implied that the Ras and cAMP-signaling pathway independently influence the polyene sensitivity without affecting ergosterol biosynthesis.
[0137] We have found in Examples 1 to 3 that the HOG pathway controls ergosterol biosynthesis of C. neoformans under unstressed conditions and the HOG pathways mutants are hyper-sensitive to AmpB, but hyper-resistance to fluconazole because of the increased cellular ergosterol contents in the mutants (Ko et al., 2009). Therefore, it is easily conceivable that the HOG and cAMP pathways influence the polyene sensitivity in different manners. Supporting this, we found that the hog1Δ cac1Δ and hog1Δ pka1Δ double mutants were even more sensitive to AmpB than the hog1Δ, cac1Δ, or pka1Δ single mutant (FIG. 10E). Unexpectedly, the hog1Δ cac1Δ double mutants also exhibited hypersensitivity to various azole drugs, such as fluconazole, ketoconazole, and itraconazole (FIG. 10F). Interestingly, the ras1Δ, aca1Δ, gpa1Δ, cac1Δ, and pka1Δ mutants all showed increased sensitivity to itraconazole (FIG. 10G). Particularly, both Ras1 and Ras2 appear to be involved in itraconazole susceptibility in a manner dependent of Cdc24 (FIG. 10H). Taken together, these date indicate that the HOG pathway and cAMP-signaling pathways independently control polyene and azole drug susceptibility.
[0138] One of key findings made by this study was that the Ras- and cAMP-signaling pathways controlled the polyene- and azole-based drug susceptibility in C. neoformans. Both Ras1 and Ras2 appeared to be involved in polyene susceptibility by using Cdc24 as a downstream effector. Interestingly, the ras1Δ aca1Δ mutant was also hypersensitive to amphotericin B, indicating that the Ras1 and Aca1 may play a minor role in susceptibility to the polyene drugs. It could be possible that perturbed action cytoskeleton regulation and cell wall integrity by ras1 and aca1 mutation makes cell more susceptible to the polyene drugs.
[0139] The cAMP-signaling pathway was even more significantly involved in polyene sensitivity than the Ras-signaling pathway. Mutation of the GPA1, CAC1, and PKA1, rendered C. neoformans cells to be hypersensitive to the polyene drugs, such as amphotericin B (AmpB). We recently reported that perturbation of the HOG pathway also renders C. neoformans cells to be hypersensitive to AmpB (Ko et al., 2009). However, the cAMP and HOG pathways appear to work differently for modulation of the polyene drug susceptibility. Inhibition of the HOG pathway, but not the cAMP pathway, increases ergosterol biosynthesis, which enhances the polyene drug susceptibility and azole drug resistance (Ko et al., 2009). Furthermore, the hog1Δ mutant exhibited higher sensitivity to AmpB than the cAMP mutants.
Example 7
Characterization of the cAMP-Dependent Genes in C. neoformans
[0140] We also addressed the role of the cAMP-dependent genes and in diverse stress response and antifungal drug susceptibility of C. neoformans due to the involvement of the cAMP-pathway in the process that we discovered in this study.
[0141] Hypersensitivity of the cAMP mutants to the polyene drug appeared to be partly contributed by decreased expression of the two heat shock proteins Hsp12 (H99 gene ID: CNAG--03143.2), C. neoformans homologs of HSP12, and Hsp122(H99 gene ID: CNAG--01446.2) (FIG. 11).
[0142] Interestingly, however, the hsp12Δ or hsp122Δ mutant exhibited slightly higher susceptibility to AmpB than WT, although the cac1Δ mutant was more sensitive to AmpB than the hsp12Δ or hsp122Δ mutant (FIG. 11A). Therefore, it was conceivable that decreased expression of HSP12 or HSP122 contributes to hypersensitivity of the cAMP mutants to AmpB.
[0143] To further characterize the regulatory mechanism of HSP12 and HSP122, we performed Northern blot analysis to confirm that the cAMP-signaling pathway modulated expression of the HSP12 and HSP122 genes. In S. cerevisiae, HSP12 is not expressed under unstressed, glucose-rich condition, but is induced in response to environmental stresses (Praekelt & Meacock, 1990, Siderius et al., 1997). Unexpectedly, however, the HSP12 and HSP122 genes were found to be highly expressed genes in the WT strain under unstressed, glucose-rich condition (FIG. 11A). In a good agreement with the microarray data, HSP12 and HSP122 expression was significantly downregulated in the cAMP mutants, including gpa1Δ, cac1Δ, and pka1Δ pka2Δ mutants (FIG. 11A). In the aca1Δ and ras1Δ mutants, expression levels of the HSP12 and HSP122 genes were only slightly affected (FIG. 11A). These data not only confirmed our microarray data, but also indicated that HSP12 and HSP122 were positively regulated by the cAMP-signaling pathway.
[0144] Interestingly our previous array analysis showed that HSP12 and HSP122 may also be under control of the HOG pathway. HSP12 and HSP122 expression levels were considerably low in the hog1Δ and ssk1Δ, but not in the skn7Δ mutant (FIG. 11B). To confirm this, we performed Northern blot analysis and found that expression levels of HSP12 and HSP122 were very high in the WT and skn7Δ mutants, but was undetectable in the hog1Δ and ssk1Δ (FIG. 11B). All these data strongly indicated that the HSP12 and HSP122 gene was co-regulated by the cAMP and HOG signaling pathways.
[0145] As discussed in the above, hypersensitivity of the cAMP mutants to the polyene drug appeared to be partly contributed by decreased expression of the heat shock protein Hsp12 and Hsp122. In S. cerevisiae, Hsp12 plays a role in stabilizing the plasma membrane as a cell wall plasticizer and water replacement molecules (Sales et al., 2000, Shamrock & Lindsey, 2008) and therefore is involved in maintaining cell wall integrity under the stressful conditions in S. cerevisiae (Shamrock et al., 2009). Therefore, the hsp2Δ mutant is unable to grow in the presence of a cell wall destabilizer, Congo red (Motshwene et al., 2004). Therefore, perturbation of the cAMP-signaling pathway reduces basal expression levels of Hsp12, which subsequently weakened cell wall integrity and membrane plasticity of C. neoformans. Similarly, hypersensitivity of the HOG pathway mutants to the polyene drug in part results from decreased expression of HSP12. However, since the cac1Δ mutant is much more sensitive to AmpB than the hsp12Δ mutant, other factors, except ergosterol biosynthesis, may affect resistance to the polyene drug. Supporting this, the hog1Δ cac1Δ or hog1Δ pka1Δ double mutant exhibited even higher polyene drug sensitivity than each single mutant, which indicated that the two pathways play an independent role in the polyene drug susceptibility. Notably, the double mutation of the HOG1 and CAC1 genes renders C. neoformans cells to be hypersensitive to most of azole drugs, including fluconazole, ketoconazole, and itraconazole, with unknown reasons.
[0146] In any case, modulation of each Ras-, cAMP/PKA-, and HOG-signaling pathway (or combination of them) may provide a novel antifungal therapeutic approach in combination with polyene and azole drugs. Simultaneous inhibition of the cAMP and HOG pathways when treated with polyene drugs such as amphotericin B could be one of the most powerful combination therapy for treatment of cryptococcosis.
Sequence CWU
1
6811309PRTCryptococcus neoformans serotype A H99
strainPEPTIDE(1)..(1309)amino acid sequence of SSK1 1Met Trp Gly Ser Asn
Ala Ser Ile Ala Ala Ser Glu Ser Thr Asp Ser1 5
10 15Leu Ser Pro Ala Pro Ser Gln Ser Ala Ala Val
Glu Phe Pro Leu Pro 20 25
30Val Ser Ser Arg Pro Ser Leu Thr Ser Ala Ala His Pro Ser Gln Met
35 40 45Ser Ala Ser Ser Ser Ser Thr Ser
Ser Gln Pro Leu Phe Asp Trp Arg 50 55
60Ile Pro Lys Pro Thr Ser Pro Arg Thr Arg Met Asp Pro Phe Asp Thr65
70 75 80 Phe Asp Pro Val
Ser Ser Ser Ser Glu Asp Asp Pro Val Pro Gln Glu 85
90 95Ser Arg Arg Ala Gly His Gln Arg Ser Val
Thr Asp Pro Leu Leu Arg 100 105
110Asp Gly Gln Pro Leu Asp Met Glu Phe Thr Thr Ala Gly Pro Pro Ile
115 120 125Gln Ser Tyr Asp Phe Glu Gln
Pro Pro Thr Phe Ser Arg Thr Leu Ser 130 135
140Ser Pro Leu Pro Ala Lys Val Gly Ser Leu Arg His Pro Met Pro
Phe145 150 155 160 Thr
Ile Asp Asp Leu Ser Ser Arg Asn Val Asn Ser Thr His Arg Pro
165 170 175Gln Pro Thr Thr Pro Leu His
Ser Ile Ser Val Glu Leu Ala Asp Ser 180 185
190Leu Gln Ser Ala Ile Gln Thr Leu Leu His Leu Ser Pro Pro
His Leu 195 200 205Leu Asp Asn Ala
Lys Glu Gln Tyr Ser Gly Cys Thr Val Gln Ile Pro 210
215 220Ala Thr Ser Leu Ser Ala Leu Leu Thr Ser Met Arg
Gly Leu Asn Phe225 230 235
240 Leu Ser Ala His Ala Glu Glu Leu Val Asp Met Ser Ala Arg Gly Asp
245 250 255Pro Pro Val Leu His
Gln Glu Asp Phe Asp Val Gly Glu Leu Leu Gln 260
265 270Asn Val Ala Asp Met Leu Ser Gly Glu Ala Ala Glu
Lys Arg Ile Asp 275 280 285Phe Val
Leu Phe His Gly Asp Val Ala Met Arg His Val Ser Val Tyr 290
295 300Gly Asp Ser Asp Gly Ile Ser Tyr Thr Leu Ser
His Val Ile Arg Gln305 310 315
320 Ile Leu Ala Val Ala Asn Tyr Asp Asp Thr Ile Glu Leu Gly Leu Gln
325 330 335Val Ile Pro Gln
Ser Pro Ser Leu Ala Ser Ala Val Gly Leu Pro Leu 340
345 350Thr Ser Ala Asp Val Ser Gly Gly Gly Gly Val
Lys Ser Ala Ser Thr 355 360 365Ser
Arg Ser Gly Ser Pro Asn Asn Ser Leu Ser Arg Ser Asn Ser Val 370
375 380His Asp Gly Pro Leu Leu Cys Val Phe Glu
Ile Val His Asn Ile Tyr385 390 395
400 Gln Pro Pro Pro Ser Ser Ala Ser Ala Thr Pro Lys Ala Glu Leu
Asn 405 410 415Pro Phe Thr
His Leu Ala Glu Glu Thr Glu Ala Leu Lys Pro Arg Leu 420
425 430Asp Thr Ala Phe Cys Lys Asn Leu Leu His
Arg Gln Asn Ala Val Leu 435 440
445Lys Val Asp Val Gln Pro Ser Ser Pro Leu Gly Ser Gly Met Pro Arg 450
455 460Arg Ala Tyr Ala Leu Ser Val Leu
Leu Pro Arg Gly Lys Pro Ile Thr465 470
475 480 Glu Pro Ala Ile Leu Ser Lys Glu Glu Gln Glu Val
Arg Gln Pro Phe 485 490
495Ser Ser His Val Leu Ala Arg Glu Pro Thr Leu Asn Glu Leu Ser Glu
500 505 510Phe Ala Glu Ser Leu Arg
Gly Arg Lys Val Phe Ile His Ala Asn Leu 515 520
525Ser Ser Val Phe Ala Arg His Leu Thr Ser Tyr Leu Ala Ala
Trp Gly 530 535 540Met Asp Ile Ser His
Leu Pro Thr Asp Gly Asp Glu Ala Asp Lys Leu545 550
555 560 Lys Asp Val Ala Ala Lys His Asp Ser Ala
Tyr Thr Gly Ser Met Gly 565 570
575Val Ser Gly Gly Thr Thr Ser Ser Ala Glu Thr Pro Tyr Ser Ile Lys
580 585 590Pro Thr Gly Val Thr
Ala Val Gln Pro Gly His Phe Val Ile Ile Asp 595
600 605Asp Asp Val Ala Val Leu Arg Arg Glu Leu Val Arg
Ile Arg Ser Glu 610 615 620Leu Leu Pro
Ile Leu Phe Lys Pro Arg Leu Ser Lys Arg Pro Thr Met625
630 635 640 Thr Ser Arg Thr Arg Ser Thr
Pro Ser Leu Arg Gln Val Pro Pro Arg 645
650 655Ser Ser Ser Gly Ser Val Leu Ile His Phe Thr Ser
Leu Ala Asn Tyr 660 665 670Asn
Arg Val Arg Asp Ala Ile Ala Ser Phe Val Gly Ala Pro Gly Leu 675
680 685Thr Asn Pro Glu Thr Tyr Val Gln Pro
Glu Val Ile Val Ile Pro Lys 690 695
700Pro Val Gly Pro Arg Arg Phe Leu Thr Ala Leu His Thr Ala Val Lys705
710 715 720 Gln Pro Met Val
Asp Pro Phe Phe Ser Pro Ile Ala Thr Ser Pro Arg 725
730 735Ser Pro Gly Gly Gly Tyr Phe Gly Gly Leu
Arg Thr Pro Thr Glu Arg 740 745
750Glu Ser Gly Phe Phe Asp Ser Val Ala Glu Glu Pro His Glu Glu Ala
755 760 765Asp Ser Arg Pro Asp Tyr Ala
Thr Val Gln Lys Ala Arg Ser Pro Leu 770 775
780Gly Glu Phe Pro Pro Ser Ala Ala Gln Ile Val Arg Thr Asn Gln
Gly785 790 795 800 Leu
His Leu Ser Leu Pro Thr Pro Asn Glu Ile Met Thr Thr Pro Ala
805 810 815Pro Glu Tyr Phe Ser Gly Ser
Ser Lys Ser Pro Ser Ser Gly Ala Ser 820 825
830Gly Val Val Met Gln Ser Pro Asp Gly Arg Pro Phe Gly Met
Phe Phe 835 840 845Glu Pro Pro Ile
Lys Asn Glu Arg Arg Gly Ser Thr His Arg Thr Pro 850
855 860Ser Asp Ser Ile Arg Arg Lys Gln Ala Asn Arg Arg
Ala Ser Thr Ser865 870 875
880 Asp Glu Pro Phe Ser Ser Pro Ser Thr Ala Leu Pro Pro Arg Arg Ser
885 890 895Ser Thr Ile Ser Thr
Thr Gly Asn Glu Glu His Arg Ser Ser Pro Ile 900
905 910Ala Asn Val Thr Asp Arg Pro Thr His Ser Arg Val
Asn Ser Arg Arg 915 920 925Lys Asn
Asn Leu Pro Ala Ala Glu Gln Pro Ile Leu Ala Val Gly Arg 930
935 940Ala Lys Gly Arg Glu Arg Ser Glu Thr Val Thr
Lys Gly Gly Asp Leu945 950 955
960 Gly Ser Arg Lys Gly Thr Pro Ala Ala Ser Pro Arg Ile Glu Glu Lys
965 970 975Lys Glu Leu Glu
Arg Gly Glu Lys Thr Lys Ser Leu Ala Pro Ser Thr 980
985 990Ala Pro Thr Lys Lys Asn Ala Lys Val Asp Val
Val Val Pro Pro Ile 995 1000
1005Asn Val Leu Ile Val Glu Asp Asn Pro Ile Asn Gln Asn Ile Leu
1010 1015 1020Ser Met Phe Leu Arg Lys
Lys Lys Ile Lys Asn Ser Ser Ala Lys 1025 1030
1035Asp Gly Ala Glu Ala Val Glu Lys Trp Arg Thr Gly Gly Phe
His 1040 1045 1050Leu Ile Leu Met Asp
Ile Gln Leu Pro Val Met Asp Gly Ile Ala 1055 1060
1065Ala Thr Lys Glu Ile Arg Arg Leu Glu Arg His Asn Asn
Ile Gly 1070 1075 1080Val Phe Pro Ser
Thr Pro Ala Ala Glu Leu Pro Arg Gly Gln Asn 1085
1090 1095Val Ala Asp Ser Pro Pro Pro Ser Ser Pro Phe
Arg Ser Ser Val 1100 1105 1110Ile Ile
Val Ala Leu Thr Ala Ser Ser Leu Gln Ser Asp Arg Val 1115
1120 1125Ala Ala Leu Ala Ala Gly Cys Asn Asp Phe
Leu Thr Lys Pro Val 1130 1135 1140Ser
Leu Lys Trp Leu Asp Lys Lys Ile Val Glu Trp Gly Cys Met 1145
1150 1155Gln Ala Leu Ile Asp Phe Asp Gly Trp
Arg Arg Trp Lys Ser Ser 1160 1165
1170Asp Thr Lys Asn Pro Ser Glu Thr Lys Gln Gly Phe Ser Val Gly
1175 1180 1185Pro Gln Gln Ala Ala Arg
Ser Leu Ala Ser Arg Leu Arg Ile Glu 1190 1195
1200Arg Lys Gly Ser Arg Ser Pro Ala Ala Pro Val Ser Thr Pro
Arg 1205 1210 1215Leu Asn Leu Gln Ser
Ala Thr Pro Asp Arg Pro Glu Thr Pro Pro 1220 1225
1230Asp Ser Thr Ser Gln Met Pro Lys Ala Pro Pro Val Ala
Ala Ser 1235 1240 1245Asp Pro Pro Leu
Ser Pro Lys Ser Leu Asn Lys Thr Val Asn Asp 1250
1255 1260Val Phe Glu Gln Ala Asp Ala Arg Leu Glu Asn
Ala Arg Glu Glu 1265 1270 1275Gln Gly
Val Ser Ser Gln Lys Glu Asn Thr Ser Leu Thr Asp Ser 1280
1285 1290Thr Asn Thr Thr Ile Thr Pro Ser Lys Thr
Tyr Pro Ala Pro Pro 1295 1300
1305Pro21691PRTCryptococcus neoformans serotype A H99
strainPEPTIDE(1)..(1691)amino acid sequence of TCO2 2Met Ile Leu Gly Thr
Asp Ile Asp Leu Ser Ser Ile Pro Thr Ala Phe1 5
10 15Leu Glu Ala Tyr Pro Phe Pro Ala Val Val Phe
Val Ile Asp Ser Pro 20 25
30Pro Ser Pro Arg Pro Arg Leu His Ser Arg Asn Thr Asp Thr Thr Ile
35 40 45Arg Arg Thr Asp Gly Gln Ile Ser
Pro Leu Thr Gly Pro Pro Val Gln 50 55
60Gln Phe Ala Ser Ala Pro Val Val Trp Gly Asn Gln Arg Trp His Glu65
70 75 80Leu Ala Gln Gly Lys
Thr Ile Ala Glu Cys Val Asp Val Ala Ser Gln 85
90 95Asn Lys Leu Gln Thr Trp Val Glu Asn Asp Thr
Gly Asp Lys Ser Glu 100 105
110Ser Leu Ala Leu Asp Leu Lys Val Pro Gln Gly Val Thr Leu His Leu
115 120 125Ala Lys Thr Ile Leu Pro Leu
Ser Pro Pro Ser Ser Ser Gln Ser Leu 130 135
140Cys Ile Leu Ile Ser Gln Tyr Ile Asp Lys Pro Glu Ser Phe Ala
Pro145 150 155 160Pro Ile
Ser Ser Gly Asp Ile Leu Phe Ser Ser Leu Ser Arg Leu Ser
165 170 175Gln Thr Phe Ser Arg Ser Ser
Ser Phe Ser Ser Asn Pro Arg Lys Ser 180 185
190Ile Asp Val Pro Ala Ser Leu Ser Glu His Arg Gly Ser Ala
Thr Ser 195 200 205Thr Ser Ser Asn
Leu Arg Ser Ser Ile Asp Leu Thr Ser Pro Asn Ser 210
215 220Gln Pro Ser Pro Leu Asn Arg Glu Gln Ser Thr Tyr
Phe Thr His Gly225 230 235
240Ser Ala Thr Arg Glu Glu Arg Pro Ser Val Arg Arg Arg Arg Ser Pro
245 250 255Pro Ile Ser Met Thr
Arg Pro Lys Pro Leu Glu Ser His Ala Gln Glu 260
265 270Cys Trp Asp Leu Val Glu Asn Phe Asp Trp Ser Lys
Thr Ala Leu Gly 275 280 285Pro Arg
Glu Gln Trp Met Asp Ala Leu Asp Pro Val Leu Ala Ile Thr 290
295 300Phe Glu Ser Arg Thr Ala Asp Cys Ala Trp Leu
Gly Pro Asp Leu Glu305 310 315
320Leu Val Tyr Asn Lys Ala Tyr Gln Glu Leu Val Asp His Pro Asn Ala
325 330 335Phe Gly Lys Pro
Ala Arg Gln Val Trp Ala Thr Asn Trp Asp Tyr Leu 340
345 350Glu Pro Leu Val Lys Arg Cys Leu Ser Gly Thr
Pro Val Tyr Lys Asp 355 360 365Asn
Asp Pro Leu Phe Trp Arg Arg Tyr Gly Asn Gly Arg Leu Leu Glu 370
375 380His Tyr His Thr Trp Arg Tyr Val Pro Ile
Thr Gly Lys Asp Gly Ser385 390 395
400Val Leu Gly Ile Phe Asn Gln Ser Ile Glu Val Thr Asp Ser Val
Leu 405 410 415Leu Glu Arg
Arg Met Gly Thr Thr Arg Glu Leu Ser Glu His Met Ser 420
425 430Phe Ile Arg Thr Thr Glu Asp Phe Phe Ser
Ser Val Ala Asp Val Phe 435 440
445Ser Gln Asn Pro Thr Asp Ile Pro Phe Ala Leu Cys Tyr Arg Val Arg 450
455 460Gln Val Asp Thr Asp Gly Thr Phe
Val His Leu Asp Val Ser Leu Gln465 470
475 480Ser Ser Val Gly Val Pro Glu Gly His Pro Ser Ala
Pro Asp Gln Ile 485 490
495Pro Val Ser Phe Leu Asn Gly Asn Pro Tyr Pro Ser Asn Val Glu Arg
500 505 510Ser Phe Ser Pro Ala Phe
Ser Ile Val Ser Ile His Ser Ser Ser Ser 515 520
525His Arg Val Cys His Val Ser Glu Asp Thr Thr Gln Trp Pro
Ile Ala 530 535 540Lys Ala Leu Gln Arg
Arg Gln Cys Val Ile Ile Glu Glu Cys Ser Gln545 550
555 560Leu Ile Glu Gly Tyr Pro Ile Arg Arg Trp
Asp Gly Leu Pro Phe Ser 565 570
575Ala Ile Val Val Pro Ile Cys Ser Glu Gly Ser Pro Glu Ile Pro Asp
580 585 590Ala Val Val Ile Leu
Gly Leu Asn Val Arg Arg Cys Phe Asp His Glu 595
600 605Tyr Asp Ser Trp Ile His Ser Ile Arg Ser Gln Leu
Ser Ser Ala Leu 610 615 620Val Met Val
Lys Ala Arg Glu Ala Glu Gln Lys Met Val Glu Glu Ser625
630 635 640Ala Arg Met Glu Lys Ala Lys
Val Ala Trp Phe Arg Gly Ala Ala His 645
650 655Asp Leu Arg Ser Pro Leu Thr Leu Val Ala Gly Pro
Leu Ala Asp Val 660 665 670Leu
Asp Ser Asp Leu Asn Ser Ser Gln Arg Thr Ala Leu Thr Val Ala 675
680 685Gln Arg Asn Leu Asp Arg Leu Val Arg
Leu Val Asn Ala Leu Met Asp 690 695
700Phe Ser Arg Val Glu Ala Gly Arg Met Glu Gly Arg Phe Val Pro Thr705
710 715 720Asn Leu Ser Gln
Phe Val Thr Gln Leu Ala Ala Leu Phe Lys Pro Ala 725
730 735Ile Glu Arg Leu Gly Leu Glu Tyr Val Leu
Asp Val Gln Pro Ser Glu 740 745
750Glu Leu Val Phe Ile Asp Pro Val Leu Phe Glu Thr Val Val Ser Asn
755 760 765Leu Ile Gly Asn Ala Leu Lys
Tyr Thr Glu Thr Gly Ser Ile Thr Val 770 775
780Arg Val Gln Tyr Thr Asp Tyr Ala Glu Val Ser Val Ile Asp Thr
Gly785 790 795 800Val Gly
Ile Pro Lys Asn Glu Leu Ala Leu Val Thr Glu Trp Phe His
805 810 815Arg Ala Ser Thr Ala Ile His
Ser Gly Thr Gln Gly Thr Gly Leu Gly 820 825
830Leu Ala Leu Ala Lys Glu Leu Leu Lys Leu His Lys Gly Glu
Leu Leu 835 840 845Val Glu Ser Gln
Thr Ala Asn Glu Ser Gly Gly Pro His Gly Ser Ile 850
855 860Phe Thr Ala Lys Ile Pro Leu Asp Phe Lys Pro Ser
Pro Ser Ala His865 870 875
880Ile Ile Pro Ser Val Glu Ser His Lys Thr Phe Gly Lys Tyr Ser Lys
885 890 895Ala Val Ala Asp Glu
Ala Met Arg Trp Val Gly Asp Ser Asp Ala Ala 900
905 910Ser Glu Ala Tyr Asp Met Ser Ser Gly Thr Gly Val
Ser Ser Ala Gly 915 920 925Ser Gly
Ser Gly Asn Thr Thr Thr Phe Gly Pro Lys Phe Ala Asp Ala 930
935 940Phe Leu Phe Asp Lys Asn Asp Ile Val Leu Ile
Val Glu Asp Asn Val945 950 955
960Asp Met Arg Glu Tyr Ile Arg Gln Leu Phe Ala Pro Tyr Cys Thr Val
965 970 975Leu Glu Ala Ser
Asn Gly Glu Gln Ala Tyr Asn Met Ala Thr Gln Asn 980
985 990Pro Pro Asn Leu Ile Leu Ser Asp Val Leu Met
Pro Lys Leu Ser Gly 995 1000
1005Met Glu Leu Leu Gln Arg Ile Arg Ser His Pro Asp Thr Arg Ile
1010 1015 1020Val Pro Met Val Leu Ile
Ser Ala Ile Ala Gly Asp Glu Ser Arg 1025 1030
1035Val Glu Ala Leu Leu Asn Gly Ala Asp Asp Tyr Leu Ala Lys
Pro 1040 1045 1050Phe Lys Pro Lys Glu
Leu Ile Ala Arg Val His Leu His Met Gln 1055 1060
1065Val Gly Lys Lys Arg Ala Lys Leu Glu Ala Leu Tyr Ala
Gln Arg 1070 1075 1080Glu Thr Glu Leu
Thr Ala Leu Ser Asp Tyr Cys Pro Ile Gly Ile 1085
1090 1095Phe Arg Gly Asp Lys Tyr Gly His Ile Val Tyr
Ala Asn Ala Ala 1100 1105 1110Trp Arg
Ala Gln Ser Gly Leu Leu Val Gly Asp Pro Asn Asp Trp 1115
1120 1125Ala Ser Tyr Val His Pro Asp Ser Lys Ala
Gln Leu Leu Glu Gln 1130 1135 1140Trp
Asn Gln Trp Leu Arg Gly Asp Leu Lys Glu Phe Arg Ala Ala 1145
1150 1155Trp Arg Trp Ser Asn Gly Ile Pro Val
Arg Ser Ile Leu Val Arg 1160 1165
1170Leu Asp Asp Val Lys Glu Gly Phe Ser Gly Leu Ile Gly Cys Val
1175 1180 1185Val Asp Val Ser His Glu
Glu Arg Arg Leu Ile Glu Ala Glu Glu 1190 1195
1200Arg Arg Lys Glu Ala Glu Glu Ser Lys His Gln Gln Glu Leu
Leu 1205 1210 1215Ile Asp Leu Thr Ser
His Glu Ile Arg Thr Pro Val Ser Ala Ile 1220 1225
1230Leu Gln Cys Ser Asp Leu Val Lys Glu Asn Leu Val Ala
Leu Lys 1235 1240 1245Asp Gln Leu Arg
Gly Ala Gly Pro Lys Gly Phe Val Pro Ser Gln 1250
1255 1260Glu Leu Leu Ala Asp Leu Glu Gln Asp Val Glu
Ala Leu Glu Ser 1265 1270 1275Ile Tyr
Gln Cys Gly Leu Val Gln Glu Arg Ile Ala Gly Asp Val 1280
1285 1290Leu Ser Leu Ala Arg Ile Gln Leu Asp Met
Leu Ser Leu His Asp 1295 1300 1305Ile
Asp Val Asn Leu Arg Arg Glu Gly Arg Lys Val Ser Ser Ile 1310
1315 1320Phe Ala Ser Glu Ala Lys Met Lys Asp
Ile Asp Leu Gln Leu Glu 1325 1330
1335Phe Gly Pro Thr Ile Glu Gln Ser Lys Val Leu Ala Ile Lys Thr
1340 1345 1350Asp Pro Val Arg Leu Gly
Gln Val Val Thr Asn Leu Ile Ser Asn 1355 1360
1365Ala Ile Arg Phe Thr Ser Ser Ser Asp Val Arg Lys Ile Thr
Ile 1370 1375 1380Gln Tyr Asp Val Ser
Phe Val Pro Pro Ala Asp Asp Ser Cys Ala 1385 1390
1395Leu Pro Ser Ser Val Gly Leu Pro Asp Ile Leu Pro Val
Lys Glu 1400 1405 1410Asn Thr Pro Leu
Trp Leu Phe Val Ser Val Thr Asp Ser Gly Pro 1415
1420 1425Gly Met Thr Glu Gln Glu Leu Ser Val Leu Phe
Gln Arg Phe Ala 1430 1435 1440Gln Gly
Asn Lys Met Ile His Thr Lys Tyr Gly Gly Ser Gly Leu 1445
1450 1455Gly Leu Phe Ile Cys Arg Lys Ile Thr Glu
Leu Leu Gly Gly Arg 1460 1465 1470Ile
Glu Val Leu Ser Gln Val Gly His Gly Ser Val Phe Arg Phe 1475
1480 1485Phe Ile Lys Thr Arg Ala Val Ala Pro
Pro Ser Ala Ile Ala Ala 1490 1495
1500Leu Val Glu Ser Ser Pro Leu Lys Pro Val Ser Ala Thr Ser Pro
1505 1510 1515Ser Ser Ser Leu Ala Met
Ser Arg Ser Ser Ser Arg Ser Thr Asn 1520 1525
1530Val Thr Thr Pro Ile Glu Gly Gly Gly Thr Glu His Val Leu
Ile 1535 1540 1545Val Glu Asp Asn Leu
Ile Asn Gln Thr Val Leu Lys Arg Gln Leu 1550 1555
1560Val Lys Ala Gly Leu Ser Cys Asn Val Ala Ser Asn Gly
Leu Glu 1565 1570 1575Ala Leu Asn Val
Ile Arg Glu Val His Arg Gln His Arg Arg Gly 1580
1585 1590Gly Pro Asn Arg Lys Arg Leu Phe Asp Val Val
Leu Met Asp Leu 1595 1600 1605Glu Met
Pro Val Met Asp Gly Ile Thr Ala Val Arg Glu Ile Arg 1610
1615 1620Gln Ser Glu Ala Ala Gly Thr Leu Gly Arg
Asn Met Val Ile Ala 1625 1630 1635Leu
Thr Gly Asn Ala Arg Gln Gly Gln Ile Asp His Ala Leu Ala 1640
1645 1650Ser Gly Phe Asp Asp Val Val Ile Lys
Pro Tyr Ile Leu Val Asp 1655 1660
1665Leu Leu Asn Lys Ile Lys Ser Met Lys Val Arg Lys Leu Glu Leu
1670 1675 1680Glu Thr Ala Lys Ala Gln
Glu Glu 1685 169031417PRTCryptococcus neoformans
serotype A H99 strainPEPTIDE(1)..(1417)amino acid sequence of SSK2 3Met
Ser Asn Pro Thr Ser Pro Ser Asn Pro Ser Asp Thr Gly Pro Ser1
5 10 15Ser Ala Ser Asn Val Thr Ser
Ser Ser Ser Lys Thr Gly Arg Arg Ser 20 25
30Val Arg Leu Phe Ala Pro Asp Glu Glu Asp Ser Ser Asp Glu
Asp Gly 35 40 45Gly Leu Ile Gly
Val Pro Ala Glu Thr Thr Phe Lys Asp Asp Glu Ile 50 55
60Pro Pro Ser Asn Pro Arg Ser Ala Ser Tyr Pro Gly Pro
Pro Ala His65 70 75
80Thr Ser Pro Thr Ser Lys Ile Ser Thr Ile Val Ser Ser Ala Ser Ala
85 90 95Ala Gln Pro Lys Leu Ala
Arg Ser Ile Thr Tyr Val Ala Pro Asn Ala 100
105 110Val Ser Ser Arg Pro Ala Tyr Pro Leu Asn Pro Ala
Gly Ser Glu Thr 115 120 125Leu His
Ala Ser Gly Arg Ser Tyr Thr Asp Pro Asp Ile Gly Tyr Phe 130
135 140Ser His Asp Ala Gly Asp Asp Gly Trp Gly Ser
Asp Asp Asp Asp Glu145 150 155
160Leu Arg Ser Pro Gly Trp Gly Ile Ser His His Asn Met Asp Ser Gly
165 170 175Gly Lys Thr Asn
Gly Ser Pro Gln Leu Pro Ile Lys Pro Ala Asp Val 180
185 190Thr Glu Asp Glu Gly Gln Glu Arg Leu Asp Trp
Gln Gly Met Leu Glu 195 200 205Ser
Val Leu Asn Ser Asp Val Leu Lys Val Glu Glu Gln Arg Ile Tyr 210
215 220Asn Ser Met Pro Thr Asp Ser Phe Arg Glu
Glu Ile Gly Lys Thr Leu225 230 235
240Trp Trp Gln Ile Arg Ala Lys Leu Arg Gly Arg Thr Glu Ala Glu
Glu 245 250 255Lys Lys Arg
Val Gln Glu Arg Arg Ala Arg Val Val Asp Pro Val Leu 260
265 270Glu Glu Ile Asn Glu Phe Lys Tyr Asp Pro
Lys Asn Asn Pro Glu Gly 275 280
285Glu Glu Asp Ser Asp Gly Asp Pro Gln Asp Ala Thr Ser Thr Ala Ala 290
295 300Pro Gln Ser Lys Ala Leu Asn Gln
Val Asn Thr Val Leu Ala Lys Leu305 310
315 320His Ala Ile Lys Gly Leu Tyr Pro Asn Leu Ala Ala
Met Arg Ala Asp 325 330
335Lys Val Leu Tyr Thr Asp Glu Asn Phe Arg Lys Arg Ala Asp Ala Leu
340 345 350Thr Ser Trp Ser Ile Ile
Val Ser Ser Leu Gln Thr Gln Leu Lys Leu 355 360
365Leu Gln Lys Trp Thr Gly Ser Asp Glu Leu Asp Ile Thr Lys
Pro Asn 370 375 380Thr Thr His Glu Lys
Ala Leu Val Gly Lys Tyr Lys Tyr His Ser Ile385 390
395 400Asp Ser Lys Gly Thr Pro Gly Arg Asp Ala
Ala Asp Asp Ser Ser Phe 405 410
415Leu Asp Arg Val Ile Lys Glu Asp Asn Leu Gln Arg Thr Phe Glu Arg
420 425 430Arg Ala Phe Val Asp
Met Ile Asn Leu Val Arg Asn Ala Lys Glu Thr 435
440 445Val Ile Ser Tyr Leu Pro Gln Phe Gln Glu Gln Asn
Leu Pro Asp Phe 450 455 460Gln Tyr Glu
Ile Val Arg Leu Ile Gly Phe Pro Gly Arg Leu Ile Ile465
470 475 480Glu Ala Val Lys Val Arg Leu
Asp Ala Ala Ser Arg Leu Leu Asp Pro 485
490 495Asn Pro Met Val Val Glu Asp Phe Ile Glu Asn Leu
Arg Leu Ser Ile 500 505 510Ser
Leu Ala Val Leu Ile Arg Lys Gln Tyr Asp Glu Ile Met Ala Pro 515
520 525Asp Ala Glu Gly Arg Trp Lys Ile Pro
His Cys Leu Pro Thr Glu Tyr 530 535
540Asn Asp Val Leu Leu Asp Ala Leu Arg Thr Phe Phe Lys Leu Leu His545
550 555 560Trp Arg Leu Arg
Gly Val Gly Lys Ala Ser Tyr Tyr Lys Glu Thr Glu 565
570 575Val Leu Glu Glu Glu Ala Pro Phe Leu Tyr
Glu Ala Ala Glu Ala Ile 580 585
590Val Gly Gly Asp Met Val Val Ala Glu Gln Tyr Cys Ala Leu Ser Asn
595 600 605Lys Leu Leu Ile Arg Ser Ala
Asn Tyr Leu Asp Gln Gln Leu Arg Val 610 615
620Pro Ile His Ser Pro Ser Arg Asp Lys Glu Arg Gly Asp Lys Glu
Arg625 630 635 640Asp Gly
Ser Ser Ser Ser Gln Arg Asn Arg Asp Gly Arg Asp Ser Ser
645 650 655Leu Pro Gly Pro Pro Lys His
Met Lys Val Glu Glu Leu Phe Ser Trp 660 665
670Tyr Ser Lys Leu Leu Asp Ser Ala Arg Met Arg His Arg Lys
Thr Gln 675 680 685Arg Phe Cys Arg
Lys Leu Thr Gln Arg Phe Asp Asn Ser Ala Glu Tyr 690
695 700Ser Ile Glu Glu Thr Glu Met Asp Met Leu Val Glu
Thr Leu Gln Asp705 710 715
720Thr Gly His Phe Leu Val Tyr Thr Gly Lys Phe Glu Ala Asn Gly Thr
725 730 735Tyr Ile Val Ala Asp
Gly Ser Leu Trp Gly Gln Pro Asp Asp Val Arg 740
745 750His Leu Leu Lys Arg Val Phe Ser Val Thr Ile Pro
Gly Ser Arg Val 755 760 765Arg Pro
Arg Gln Thr Thr Ser Gln Val Ser Val Gly Gly Ala Ser Pro 770
775 780Ser Asn Gly Gln Val Ala Ala Gln His Asp Pro
Ala Asp Pro Tyr Pro785 790 795
800Glu Ala Asp Asp Phe Asp Asp Glu Ala Leu Ala Ala Tyr Ile Leu Leu
805 810 815Ile Ser Pro Arg
Gln Ser Phe Val Trp Ser Gly Ala Val Met Thr Leu 820
825 830Asp Val Asp Tyr Ile Asp Tyr Glu Leu Pro Asp
Asn Arg Val Arg Leu 835 840 845Ile
Ala Asp Gly Pro Thr Lys Arg Leu Ala Leu Cys Lys Leu Tyr Phe 850
855 860Lys Gln Ala Leu Ile His Pro Asp Thr Gly
Glu Thr Ile Asp Leu Pro865 870 875
880Cys Val Val Glu Ala Gln Ala His Leu Pro Thr Ile Gln Lys Gln
Leu 885 890 895Val Lys Ile
Ala Lys Ser Ser Tyr Arg Leu Ser Glu Cys Ile Val Gln 900
905 910Ser Ala Pro Leu Val Arg Asn Ala Phe Arg
Gly Lys Pro Gly Ser Gln 915 920
925Glu Leu Val Glu Asn Trp Tyr Ser Phe Ala Thr Glu His Gly Thr Arg 930
935 940Val Leu Ile His Ile Glu Pro Ser
Val Trp Glu Arg Phe Asn Arg Leu945 950
955 960Leu Met Arg Leu Ala Ile Ser Trp Ile Ser Phe Ile
Ser Gln Glu Cys 965 970
975Asn Pro Thr Asp Arg Lys Thr Phe Arg Trp Thr Val Ala Ala Leu Thr
980 985 990Tyr Ala Phe Asn Met Thr
Arg Gly Ser Asn Ile Leu Ala Leu Asp Arg 995 1000
1005Ser Glu Phe Ser Leu Leu Arg Arg Ser Ser Met Glu
Ala Lys Lys 1010 1015 1020Glu Ala Asp
Arg Ile Glu Ala Met Arg Arg Leu Gln Arg Leu Gln 1025
1030 1035Glu Asn Leu Asp Asp Glu Phe Leu Pro Arg Thr
Pro Thr Glu Ser 1040 1045 1050Gly Asp
Gln Pro Arg Ile Asp Arg Ser Ile Arg Leu Thr Val Glu 1055
1060 1065Glu Arg Leu Arg Leu Ile Ala Glu Leu Glu
Ala Arg Arg Asp Glu 1070 1075 1080Leu
Ala Pro Ala Pro Val Gly Gln Val Leu Asp Glu Glu Val Ser 1085
1090 1095Glu Asp Arg Ala Leu Val Phe Leu Ala
Ala Ser Lys Ser Asn Ile 1100 1105
1110Ser Met Arg Trp Gln Gln Gly Ala Tyr Ile Gly Gly Gly Ala Ser
1115 1120 1125Gly Ser Val Tyr Leu Gly
Tyr Ser Leu Gln Asp Asn Thr Val Phe 1130 1135
1140Ala Val Lys Ile Leu Pro Thr Val Asp Leu Gln Ser Ser Pro
Ala 1145 1150 1155Leu Tyr Glu Ser Ile
Lys Arg Glu Ser Asp Val Met Ser Leu Leu 1160 1165
1170Ser His Pro Asn Ile Val Gly Phe Leu Gly Leu Glu Val
His Arg 1175 1180 1185Asn Arg Val Cys
Leu Phe Gln Glu Tyr Cys Glu Gly Gly Ser Leu 1190
1195 1200Ala Gly Met Leu Glu Tyr Gly Lys Ile Asp Asp
Glu Glu Val Val 1205 1210 1215Gly Ala
Phe Thr Ile Gln Leu Leu Arg Gly Leu Glu Tyr Leu His 1220
1225 1230Thr Asn Arg Ile Glu His Arg Asp Leu Lys
Pro Glu Asn Ile Leu 1235 1240 1245Ile
Gly Ala Asn Ser Val Leu Lys Leu Ala Asp Phe Gly Thr Ala 1250
1255 1260Lys Ile Ile Lys Ser Asn Lys Thr Leu
Ala Arg Thr Arg Gly Gly 1265 1270
1275Ala His Ala Lys Met Glu Gly Leu Glu Gly Thr Pro Met Tyr Met
1280 1285 1290Ala Pro Glu Met Ile Lys
Asn Gln Arg Thr Gly Lys Leu Gly Ala 1295 1300
1305Cys Asp Ile Trp Gly Leu Gly Cys Ile Val Leu Gln Met Ile
Thr 1310 1315 1320Gly Arg Lys Pro Trp
Ser Phe Leu Asp Phe Asp Asn Glu Trp Ala 1325 1330
1335Ile Met Phe His Leu Gly Ala Thr Lys Glu Pro Pro Pro
Leu Pro 1340 1345 1350Asp Pro Asn Glu
Met Ser Asp Gln Gly Ile Glu Phe Ile Asp Gln 1355
1360 1365Cys Leu Ser Leu Asp Pro Glu Ala Arg Pro Val
Ala Ser Glu Leu 1370 1375 1380Leu Gln
Asp Glu Trp Leu Val Pro Met Leu Glu Gln Met Val Ser 1385
1390 1395Cys Leu Ser Cys Arg Ala Gly Ala Arg Ile
Pro Arg Tyr Ile Gly 1400 1405 1410Asp
Gly Pro Lys 14154609PRTCryptococcus neoformans serotype A H99
strainPEPTIDE(1)..(609)amino acid sequence of PBS2 4Met Thr Asp Pro Thr
Pro Pro Ala Leu Asp Ser Leu Ser Leu Ala Asp1 5
10 15Lys Ala Pro Thr Pro Glu Glu Ser Pro Glu Asp
Ala Ala Glu Gln Pro 20 25
30Lys Pro Ala Ala Ser Pro Ser Ala Gly Thr Pro Gly His Asp Ala Gln
35 40 45Ser Ser Ser Thr Ser Pro Pro Gln
Arg Pro Gln Ser Met Gln Thr Asn 50 55
60Asp Lys Ala Pro Asp Thr Ser Ala Pro Ala Ser Arg Pro Gln Pro Gln65
70 75 80His Val Pro Ala Ser
Ala Pro Ala Leu Pro Ser Thr Asn Pro Val Arg 85
90 95Pro Gln Pro Gly Ala Arg Pro Gly Ala Ala Arg
Gly Met Pro Ala Pro 100 105
110Met Gly Met Arg Ala Gln Ala Gly Arg Gly Ala Gly Gly Pro Gln Met
115 120 125Gln Thr Lys Met Leu Pro Ser
Leu Gln Ala Lys Met Asp Lys Ile Ala 130 135
140Ala Ser Arg Gln Gly Pro Pro Pro Ser Ser Gly Met His Asp Pro
Asn145 150 155 160Ala Thr
Ser Met Gly Ala Leu Leu Arg Ser Gln Ala Leu Arg Ala Pro
165 170 175Gly Ala Ser Gln Ala Pro Pro
Gly Pro Gly Pro Ala Ser Gly Pro Phe 180 185
190Gly Leu Ala Ala Arg Arg Ala Ala Ala Gly Gly Pro Pro Arg
Pro Asn 195 200 205Leu Gly Met Met
Gly Met Gly Ala Ser Ala Pro Gly Ala Val Gly Arg 210
215 220Gly Ser Gly Leu Ala Gly Arg Arg Gly Pro Pro Gly
Gly Leu Thr Leu225 230 235
240Ser Gly Met Lys Gly Ala Ile Lys Asp Glu Gly Asn Lys Phe Ser Asp
245 250 255Phe Gln Gly Val Met
Asp Pro Ser Gly Ser Leu Arg Phe Ser Lys Lys 260
265 270Ala Val Leu His Ala Lys Gly Val Asp Phe Glu Asp
Gly Gln Ser Phe 275 280 285Lys Ile
Asn Met Asp Glu Ile Glu Val Leu Gly Glu Leu Gly Lys Gly 290
295 300Asn Tyr Gly Ser Val His Lys Val Phe His Arg
Pro Thr Gly Val Thr305 310 315
320Met Ala Met Lys Glu Ile Arg Leu Glu Leu Asp Asp Ser Lys Leu Asn
325 330 335Gly Ile Ile Met
Glu Leu Asp Ile Leu His Arg Ala Val Ala Pro Glu 340
345 350Ile Val Glu Phe Tyr Gly Ala Phe Thr Ile Glu
Ser Cys Val Tyr Tyr 355 360 365Cys
Met Glu Tyr Met Asp Ala Gly Ser Leu Asp Ser Leu Thr Gly Gly 370
375 380Gly Val Ala Ala Lys Asp Gln Thr Lys Asp
Glu Glu Asn Asp Ala Thr385 390 395
400Lys Arg Val Pro Glu Asp Val Leu Arg Arg Ile Thr Ala Arg Ile
Val 405 410 415Lys Gly Leu
Arg Phe Leu Lys Asp Glu Leu Gln Ile Ile His Arg Asp 420
425 430Val Lys Pro Thr Asn Val Leu Ile Asn Gly
Lys Gly Glu Val Lys Met 435 440
445Cys Asp Phe Gly Val Ser Gly Gln Leu Glu Lys Ser Leu Ala Lys Thr 450
455 460Asn Ile Gly Cys Gln Ser Tyr Met
Ala Pro Glu Arg Ile Lys Ser Glu465 470
475 480Thr Ala Asn Gln Asn Pro Thr Tyr Thr Val Ser Ser
Asp Val Trp Ser 485 490
495Val Gly Leu Ser Ile Val Glu Leu Ala Lys Gly Cys Tyr Pro Tyr Pro
500 505 510Pro Glu Thr Tyr Ala Asn
Val Phe Ala Gln Leu Gln Ala Ile Val His 515 520
525Gly Thr Pro Pro Thr Leu Pro Pro Gly Tyr Ser Asp Asn Ala
Asn Asp 530 535 540Phe Val Ala Lys Cys
Leu Glu Lys Asp Pro Asn Arg Arg Pro Thr Tyr545 550
555 560Ala Gln Leu Leu Glu His Pro Phe Leu Val
Ala Asp Lys Gly Ala Glu 565 570
575Val Asp Met Val Gly Trp Val Glu Gly Ala Leu Lys Arg Lys Ala Glu
580 585 590Arg Gly Ile Ala Ser
Leu Asn Pro Ile Gln Pro Pro Val Pro Leu Glu 595
600 605Pro 5365PRTCryptococcus neoformans serotype A H99
strainPEPTIDE(1)..(365)amino acid sequence of HOG1 5Met Ala Asp Phe Val
Lys Leu Ser Ile Phe Gly Thr Val Phe Glu Val1 5
10 15Thr Thr Arg Tyr Val Asp Leu Gln Pro Val Gly
Met Gly Ala Phe Gly 20 25
30Leu Val Cys Ser Ala Lys Asp Gln Leu Ser Gly Thr Ser Val Ala Ile
35 40 45Lys Lys Ile Met Lys Pro Phe Ser
Thr Pro Val Leu Ser Lys Arg Thr 50 55
60Tyr Arg Glu Leu Lys Leu Leu Lys His Leu Arg His Glu Asn Ile Ile65
70 75 80Ser Leu Ser Asp Ile
Phe Ile Ser Pro Leu Glu Asp Ile Tyr Phe Val 85
90 95Thr Glu Leu Leu Gly Thr Asp Leu His Arg Leu
Leu Thr Ser Arg Pro 100 105
110Leu Glu Lys Gln Phe Ile Gln Tyr Phe Leu Tyr Gln Ile Leu Arg Gly
115 120 125Leu Lys Tyr Val His Ser Ala
Gly Val Val His Arg Asp Leu Lys Pro 130 135
140Ser Asn Ile Leu Val Asn Glu Asn Cys Asp Leu Lys Ile Cys Asp
Phe145 150 155 160Gly Leu
Ala Arg Ile Gln Asp Pro Gln Met Thr Gly Tyr Val Ser Thr
165 170 175Arg Tyr Tyr Arg Ala Pro Glu
Ile Met Leu Thr Trp Gln Lys Tyr Asp 180 185
190Val Ala Val Asp Ile Trp Ser Thr Gly Cys Ile Phe Ala Glu
Met Leu 195 200 205Glu Gly Lys Pro
Leu Phe Pro Gly Lys Asp His Val Asn Gln Phe Ser 210
215 220Ile Ile Thr Glu Leu Leu Gly Thr Pro Pro Asp Asp
Val Ile Gln Thr225 230 235
240Ile Ala Ser Glu Asn Thr Leu Arg Phe Val Gln Ser Leu Pro Lys Arg
245 250 255Glu Lys Val Pro Phe
Ser Thr Lys Phe Pro Asn Ala Asp Pro Val Ser 260
265 270Leu Asp Leu Leu Glu Lys Met Leu Val Phe Asp Pro
Arg Thr Arg Ile 275 280 285Ser Ala
Ala Glu Gly Leu Ala His Glu Tyr Leu Ala Pro Tyr His Asp 290
295 300Pro Thr Asp Glu Pro Val Ala Ala Glu Val Phe
Asp Trp Ser Phe Asn305 310 315
320Asp Ala Asp Leu Pro Val Asp Thr Trp Lys Val Met Met Tyr Ser Glu
325 330 335Ile Leu Asp Phe
His Asn Leu Gly Asp Ile Ser Gln Asn Glu Ala Glu 340
345 350Gly Pro Val Thr Gly Glu Val Pro Ala Ala Pro
Ala Ser 355 360
36561090PRTCryptococcus neoformans serotype A H99
strainPEPTIDE(1)..(1090)Amino acid sequence of ENA1(CNAG_00531.2) 6Met
Ser Ser Glu Lys Gly Gln Ser Asn Thr Asn Glu Lys Gln Leu Ile1
5 10 15Asn Arg Ala Asp Thr Gly Lys
Thr Ala Val Ser Asp Ser Pro Leu Pro 20 25
30Phe Lys Pro His Thr Ala Leu Ser Gly Lys Ile Leu Glu Ala
Leu Gly 35 40 45Ser Asn Val Thr
Ser Gly Leu Ser Asp Asp Glu Ala Ser Arg Arg Leu 50 55
60Gln Gln Tyr Gly Pro Asn Arg Leu Lys Pro Pro Glu Arg
Pro Ser Ile65 70 75
80Leu Lys Ile Ile Ala Arg Gln Val Gly Asn Ala Met Thr Leu Val Leu
85 90 95Ile Ala Ala Met Ala Thr
Ser Leu Gly Thr Met Asp Trp Ile Ser Gly 100
105 110Gly Val Ile Ala Ala Leu Val Ile Leu Asn Val Ser
Val Gly Ala Tyr 115 120 125Thr Glu
Trp Gln Ala Glu Lys Thr Val Ala Ser Leu Glu Ser Val Gly 130
135 140Ala Pro Gln Ala Thr Val Val Arg Thr Arg Asn
Gly Ser Arg Glu Ala145 150 155
160Thr Val Lys Ile Ile Pro Val Glu Glu Val Val Pro Gly Asp Ile Ile
165 170 175Gln Leu Lys Asn
Gly Asp Ile Val Pro Ala Asp Gly Arg Ile Leu Asp 180
185 190Gly His Leu Ser Asn Leu Glu Ala Asp Glu Ala
Phe Leu Thr Gly Glu 195 200 205Ser
Leu Pro Val Ala Lys Gln Thr Glu Pro Ile Asp Glu Glu Asp Cys 210
215 220Pro Val Gly Asp Arg Val Cys Met Val Phe
Ser Gly Ser Gln Ile Thr225 230 235
240Lys Gly Arg Ala Arg Ala Val Ile Thr Ser Thr Gly Met Gly Thr
Glu 245 250 255Ile Gly Lys
Ile Ala Gln Ala Leu Glu Ser Lys Ala Lys Asn Lys Asn 260
265 270Arg Gly Phe Ala Ala Phe Trp Trp Lys Val
Lys Val Ile Leu Gly Val 275 280
285Glu Glu Thr Thr Pro Leu Gln Ile Lys Leu Asn Lys Leu Ala Tyr Phe 290
295 300Leu Leu Ala Cys Ala Leu Val Ile
Ala Val Ile Val Val Ala Ser Thr305 310
315 320Gly Phe Asn Asp Val Pro Leu Ser Ile Ala Thr Tyr
Ala Val Ala Ala 325 330
335Ala Val Ser Ile Leu Pro Ala Ser Leu Ile Ala Val Val Ser Leu Thr
340 345 350Leu Ala Arg Ala Ser Thr
Asp Leu Ala Ser Arg His Ala Leu Val Arg 355 360
365Arg Met Asp Ala Ile Glu Ala Leu Ala Gly Val Glu Asn Val
Cys Ser 370 375 380Asp Lys Thr Gly Thr
Leu Thr Val Gly Arg Met Val Val Arg Lys Val385 390
395 400Trp Val Pro Ala Leu Asp Trp Arg Pro Asn
Glu Phe Ala Pro Leu Asp 405 410
415Thr Ser Gly Gly Gln Ala Tyr Ser Phe Glu Thr Gly Ser Asp Pro Phe
420 425 430Tyr Pro Arg Gly Glu
Val Leu Ala Asp Ser Gln Lys Ile Thr Gly Thr 435
440 445Ala Glu Thr Leu Asp Leu Lys Gln Pro Arg Asp Gln
Ser Asp Ser Ser 450 455 460Ser Ser Asp
Ser Asp Pro Asp Glu Arg Asp Val Glu Glu Gln Glu Arg465
470 475 480Val Ile His Val Glu Asp Met
Glu Asn Asn Leu Arg Asp Leu Ala Leu 485
490 495Cys Ile Ser Leu Cys Asn Gln Ala Thr Leu Thr Arg
Pro Val Asn Gln 500 505 510Asp
Gly Gln Trp Glu Ala Asn Gly Asp Pro Thr Glu Thr Ala Leu Gln 515
520 525Val Ala Ala His Lys Leu Gly His Gly
Lys Pro Phe Leu Thr His Ala 530 535
540Ala Lys Pro Ser His Arg Ala Asp Ser Ile Arg Ser Gly His Ser Ser545
550 555 560Arg Pro Leu Val
Ala Gly Ile Arg Gly His Phe Val Pro Ile Ile Glu 565
570 575His Pro Phe Asp Ser Thr Val Lys Arg Met
Ser Ile Ala Tyr Lys Phe 580 585
590Val Ser Glu Asp Pro Gln Asp Ser His Ile Leu Cys Leu Leu Lys Gly
595 600 605Ala Ile Glu Arg Val Phe Glu
Arg Cys Thr Lys Ile Gln Gly Gln Pro 610 615
620Ile Thr Glu Glu His Lys Lys Asn Ile Met Val Lys Val Asp Ala
Leu625 630 635 640Ala Ala
Gln Gly Leu Arg Val Leu Ala Leu Cys Gly Lys Arg Leu Pro
645 650 655Val Ser Met Val Asp Glu Val
Lys Ser Thr Pro Arg Asp Ala Phe Glu 660 665
670Ala Asp Phe His Phe Leu Gly Leu Ala Gly Ile Phe Asp Pro
Pro Arg 675 680 685Lys Glu Ser Ala
Gly Ala Val Ala Asp Cys Phe Arg Ala Gly Ile Thr 690
695 700Pro Arg Met Leu Thr Gly Asp His Pro Ala Thr Ala
Thr Ala Ile Ala705 710 715
720Leu Asn Ile Gly Ile Leu Asp Lys Thr Tyr Ser Lys Asp Ser Val Met
725 730 735Thr Gly Gln Gln Phe
Asp Ser Leu Ser Glu Asp Glu Ile Asp Gln Leu 740
745 750Pro Glu Leu Pro Leu Val Val Ala Arg Cys Ala Pro
Glu Thr Lys Val 755 760 765Arg Met
Val Asp Ala Ile His Arg Arg Gly Gln Ser Thr Val Met Thr 770
775 780Gly Asp Gly Val Asn Asp Ser Pro Ala Leu Lys
Arg Ala Asp Val Gly785 790 795
800Val Gly Met Gly Thr Gly Ser Asp Val Ala Lys Gln Ser Ala Arg Ile
805 810 815Val Leu Ser Asp
Asp Asn Phe Ser Thr Ile Ile Arg Ala Ile Arg Lys 820
825 830Gly Arg Ser Val Phe Lys Asn Leu Ser Lys Phe
Leu Leu Tyr Leu Leu 835 840 845Ser
Gly Asn Leu Ala Glu Ile Ile Val Leu Met Ile Gly Leu Ala Phe 850
855 860Lys Asp Asp Asn Gly Gln Ala Val Phe Pro
Leu Ser Pro Val Ala Ala865 870 875
880Leu Trp Ile Asn Thr Leu Ala Ala Gly Pro Pro Ala Leu Ala Leu
Gly 885 890 895Leu Glu Pro
Thr Ala Ile Asp Ala Met Glu Gln Gly Pro Glu Val Tyr 900
905 910His Arg Ile Phe Thr Leu Glu Phe Tyr Val
Asp Leu Ile Phe Tyr Gly 915 920
925Phe Leu Met Gly Ser Ile Ser Leu Val Asn Phe Val Ile Val Leu Trp 930
935 940Gly Tyr Tyr Pro Gly Asp Leu Gly
Arg Leu Cys Asn Glu Asp Asp Pro945 950
955 960Ser Ile Cys Asp Pro Val Tyr Gln Ala Arg Ala Ala
Cys Phe Ala Thr 965 970
975Leu Val Ile Val Leu Met Ile His Ala Leu Glu Cys Lys His Leu Ser
980 985 990Lys Gly Leu Ala Gln Ile
Asn Leu Arg Asp Asn Lys Val Leu Leu Trp 995 1000
1005Cys Val Val Ala Leu Ser Leu Ser Thr Phe Pro Val
Val Tyr Ile 1010 1015 1020Pro Val Ile
Asn Asn Lys Val Phe Leu Leu Asn Gly Pro Arg Trp 1025
1030 1035Glu Trp Gly Ile Ile Phe Gly Met Ile Leu Val
Tyr Leu Ser Ala 1040 1045 1050Thr Glu
Leu Tyr Lys Trp Ile Lys Arg Ile Trp Ile Arg Arg His 1055
1060 1065Ala Pro Pro Ser Lys Gly Pro Ser Asp Lys
Thr Leu Arg Met Glu 1070 1075 1080Ser
Thr Ile Ala Pro Pro Val 1085 10907916PRTCryptococcus
neoformans serotype A H99 strainPEPTIDE(1)..(916)Amino acid sequence of
NHA1 (CNAG_01678.2) 7Met Thr Ala Phe His Pro Phe Glu Val Asn Ala Pro His
Leu Ala Tyr1 5 10 15Thr
Phe Leu Gly Gly Phe Val Val Ile Phe Gly Met Ile Ser Leu Phe 20
25 30Ile Lys Glu Lys Leu Tyr Val Gly
Glu Ala Pro Ile Ala Thr Val Val 35 40
45Gly Ile Ile Ile Gly Pro His Cys Leu Asn Phe Phe Asn Pro Ala Gly
50 55 60Trp Gly Gly Gly Glu Glu Glu Val
Ala Ser Asp Val Thr Leu Glu Phe65 70 75
80Thr Arg Val Val Ile Ala Ile Ser Val Phe Ala Val Gly
Val Glu Leu 85 90 95Pro
Lys Ala Tyr Met Lys Arg His Trp Arg Ser Leu Phe Phe Leu Leu
100 105 110Gly Pro Cys Met Val Trp Gly
Trp Met Ile Ser Ala Leu Leu Ile Trp 115 120
125Gly Leu Ile Pro Asp Leu Thr Phe Leu Ala Ser Leu Val Val Ala
Ala 130 135 140Gly Val Thr Pro Thr Asp
Pro Ile Leu Ala Gln Ala Val Ile Gly Gly145 150
155 160Lys Phe Ala Asp Lys His Val Pro Ala His Ile
Arg His Leu Leu Ser 165 170
175Ala Glu Ser Gly Ser Asn Asp Gly Ala Ala Phe Pro Phe Leu Tyr Ile
180 185 190Ala Leu Tyr Leu Leu Leu
Asp Ala Ser Pro Gly His Ala Val Gly Glu 195 200
205Trp Phe Tyr Met Thr Trp Val Tyr Glu Ile Ile Leu Gly Val
Ile Ile 210 215 220Gly Ala Ile Leu Gly
Phe Cys Ala Arg Lys Leu Met Lys Leu Ala Glu225 230
235 240Arg Lys Arg Leu Ile Asp Arg Gln Ser Tyr
Val Ala Gln Tyr Val Ser 245 250
255Leu Ala Val Leu Ser Ile Gly Val Thr Ser Leu Leu Gly Ser Asp Asp
260 265 270Leu Leu Ser Ala Phe
Ala Cys Gly Cys Ala Phe Ala Trp Asp Gly Phe 275
280 285Phe Asn Lys Ala Thr Glu Asp Ala Val Phe Ser Asn
Val Ile Asp Leu 290 295 300Leu Phe Asn
Cys Ala Ala Phe Ile Tyr Ile Gly Ala Ile Ile Pro Phe305
310 315 320Asn His Phe Asn Asp Leu Pro
Asp Leu Arg Val Trp Arg Leu Val Val 325
330 335Leu Ala Ile Leu Ile Leu Leu Val Arg Arg Leu Pro
Ser Ile Ile Ala 340 345 350Cys
Tyr Lys Phe Val Pro Asp Ile Lys Thr Phe Arg Glu Ala Leu Phe 355
360 365Thr Gly Trp Phe Gly Pro Met Gly Val
Gly Ala Val Phe Ile Ser Thr 370 375
380Leu Ala Arg Ser Ser Leu Pro Glu Gly Glu Pro Glu Gln Asn Thr Glu385
390 395 400Ala Val Asp Arg
Leu Lys Asp Val Ile Met Pro Val Thr Leu Phe Leu 405
410 415Val Leu Ser Ser Ile Val Thr His Gly Met
Ser Ile Pro Phe Phe Ser 420 425
430Leu Gly Arg Arg Val His Ser Ile Thr Tyr Thr Arg Ser Arg Asn Leu
435 440 445Ser Met Asp Thr Arg Gly Asp
Glu Pro Ala Trp Thr Thr His Ala Arg 450 455
460Arg Ile Ile Pro Gly Gln Glu Ile Ile Val Asn Arg Asp Asp Asp
Asp465 470 475 480Glu Glu
Gly Asp Leu Gly Val Arg Arg Met Asp Thr Leu Thr Ser Asp
485 490 495Ser Asn Gly Arg Ile Arg Glu
Lys Ile Glu Glu Glu Asp Ser Gly Glu 500 505
510Ser Ser Ser Ser Arg Thr Arg Gln Gly Glu Met Ile Glu Met
Thr Glu 515 520 525Lys Arg Gly Pro
Ala Arg His Gly Ser Gln Ala Ser Gln Gly Glu Ala 530
535 540Ala Glu Glu Gly Glu Arg Trp Arg Ser Ser Gly Glu
Glu Ser Ser Asp545 550 555
560Leu Ala Asn Asp Pro Glu Thr Gln Arg Glu Val Glu Glu Gly Met Glu
565 570 575Glu Val Glu Asp Lys
Glu Gly Gly Gly Arg Arg Thr Pro Pro Leu Ala 580
585 590Lys Tyr Arg Glu Gly Asn His Leu Ile Val Glu Arg
Lys Val Lys Asp 595 600 605Ser Asp
Glu Val Glu Val Glu Val Ile Arg Asn His Phe Ser Asp Asn 610
615 620Lys Lys Thr Glu Ser Asp Arg Phe Thr His Pro
His Arg Leu Lys Ser625 630 635
640Arg Glu Leu Asp Asp Leu Leu His His Leu Pro Lys Ser Leu Glu His
645 650 655Ala Thr Ser Arg
Val Gln Asn Gly Gly Lys Asp Ala Val Asp Arg Leu 660
665 670Gly Leu Gly Leu Met Ala Ile Asn Thr Pro Glu
Pro Ser Pro Ser Ile 675 680 685Glu
Ser His Gly Gly Pro Arg His Asp Tyr Val Asp Gly Leu Glu Arg 690
695 700Thr Gln Ser Pro Glu Gly Leu Ala Asp Glu
Asp Arg Asp Ser Glu Gly705 710 715
720Arg Gly Asp Val Ser His Gly Gly Asp Tyr Glu Glu Asn Glu Ala
Asp 725 730 735Tyr Glu Asp
Val Pro Asn Glu Thr Arg Arg Gln Arg Arg Lys Lys Met 740
745 750Lys Pro Pro Ala Ile Val Val Ser Arg Gln
Asn Ser Ala Gly Leu Pro 755 760
765Arg Arg Ser Ile Arg Ser Arg Leu Phe Gly Arg Arg Gln His Ser Ser 770
775 780Asn Ser Pro Ser Arg Ala Glu Glu
Gly Leu Ala Pro Pro Asn Pro Ser785 790
795 800Leu Leu Val Pro Ser Ser Ser Pro Ser Arg Pro Gln
Asn Ile Ala Ala 805 810
815Glu Pro Glu Ser Ile Leu Ala Glu Asp Ser Arg Gly Ser Ser Ser Pro
820 825 830Ser Gln Ser Gln Asn Leu
Ala Ile Pro Leu Thr Arg Thr Leu Ser Ala 835 840
845Ser Arg Ser Ser Pro Ala Val Arg Phe Ala Asp Asp Ala Ser
Pro Ser 850 855 860Ser Asp Thr Ala Pro
Gly Gln Ser Asn Tyr Gly Thr Asn Ala Pro Gly865 870
875 880Phe Lys Lys Asn Pro Ala Leu Ala Met Tyr
Arg Ser Ala Ser Val Gln 885 890
895Ser Thr Gly Ser Asn Lys Asp Gly Pro Ser Val Ser Phe Lys Glu Pro
900 905 910Glu Ile Lys Arg
91584121DNACryptococcus neoformans serotype A H99
straingene(1)..(4121)genomic DNA sequence of SSK1 8atgtggggct caaatgcttc
catcgccgcc tcggagtcga ccgactccct ttcccccgcc 60ccctcccagt ctgccgccgt
agagttcccc ctgcccgtaa gctcccgccc gtctctcact 120tccgccgctc acccctccca
gatgtccgct tcctcctcct ccacctcctc ccagcctctc 180tttgattggc gcattcccaa
gcccacctca ccccgcacac gcatggaccc attcgacact 240tttgatcctg tatcctcaag
ctcagaggat gatccggtcc ctcaagagag ccgccgtgct 300ggtcatcagc gctccgtaac
agatcctctt ttacgagatg gccaacccct cgatatggag 360ttcactactg ccgggccgcc
tatacagagc tacgactttg aacaaccgcc cacgtttagc 420agaacgttat cttcccctct
tccagctaaa gtcggctcgc ttagacaccc tatgccattc 480accattgacg atttaagctc
tcgcaatgtg aattcaaccc atcgtccgca gccgactaca 540cctctgcatt ccatatccgt
tgagttagcc gattccttgc aatcggccat tcaaacatta 600ctgcatctgt ccccacctca
cctactcgat aacgcaaaag aacagtactc tggatgcacg 660gtacagatac ctgctacctc
gctttcggcc cttttgacct ctatgagagg cctcaacttc 720ttgtcggcgc atgctgaaga
actggtcgac atgagtgcac gtggagatcc acctgtactc 780catcaagaag acttcgatgt
aggagaactt ttgcaaaacg ttgcggatat gttgagtgga 840gaagcagcag aaaaacggat
tgatttcgtt ttgttccatg gcgacgtagc gatgaggcat 900gtcagtgtgt atggagattc
tgatggaatt agctatacct tgagtcatgt aagtcctaca 960tgcacctgct gcatactgac
aaacaggtta ttcgacaaat actggcagta gccaattacg 1020atgataccat agaactcggc
cttcaagtca ttcctcaaag tccatcttta gcttccgccg 1080tcggacttcc tctaacctct
gccgatgtta gtggaggagg tggtgtcaag tcagcgtcca 1140catctcggtc aggctccccc
aataacagtc tctctcgatc taattctgtc catgacgggc 1200cccttctctg tgtgttcgaa
atagtacata acatctatca gccaccacca agctcggcat 1260ccgccactcc taaagccgag
ctgaaccctt tcactcatct tgctgaagaa accgaagcct 1320tgaaaccaag attggataca
gcattttgca aaaacctgct tcatcggcaa aatgctgtcc 1380tcaaagttga tgtgcagcct
tcatctcctt taggatccgg gatgccccgt agagcttacg 1440cgttatcagt gctcctacca
agaggtaaac ccatcactga gcctgcaata ctttctaaag 1500aggaacaaga agttcgtcaa
ccattttcat cccacgtact tgcacgagaa cccaccctca 1560atgagctctc ggaatttgct
gaatcattac gaggaagaaa ggtgtttatc catgccaatt 1620tgagtagtgt tttcgcgaga
cacctcacga gctacctagc tgcatgggga atggatatat 1680cgcatctacc gacagatggc
gatgaggctg ataaattgaa ggatgtcgcg gccaaacatg 1740actcggctta tactggatct
atgggtgtgt caggcggcac tacttccagc gcagaaacgc 1800cctattcaat taaaccgacc
ggcgtgactg ctgttcaacc tggacacttt gtcattatcg 1860acgatgatgt tgcggtcttg
cgccgtgaac tcgtgcgcat ccgttcagaa ttacttccca 1920ttctctttaa acctagactg
tcaaagcgtc ccactatgac ttctcgaacc cgttccaccc 1980cttcattgcg acaggtcccc
ccaaggtcat catcgggttc tgtacttata cactttacct 2040ctcttgccaa ttataaccga
gttcgagacg cgattgcgag ctttgtgggg gcgccagggt 2100taaccaatcc ggaaacttat
gttcagccgg aggtgatagt gatacccaag cctgttggac 2160cacgaagatt tttgactgct
ctgcataccg ctgtgaaaca gcctatggtt gacccatttt 2220tctcccctat cgccacatct
cctagatcac caggcggagg ttactttggt ggtttgcgga 2280ctccgacgga gagagaatca
ggattctttg attctgttgc agaagaacca catgaagagg 2340cggattcgcg accagattat
gccacggtgc agaaagccag atctccttta ggagaatttc 2400cgccttctgc ggcacagatc
gttcgtacca accaaggctt gcatctttcg cttcccactc 2460caaatgaaat tatgacaacg
cctgctccag aatatttttc tgggtcttcc aagtctccta 2520gctctggtgc gtccggagtc
gtgatgcaga gccctgatgg tcgtcctttc ggaatgtttt 2580tcgaaccgcc cataaaaaat
gagcgccgcg gatctactca caggacgcct tccgattcca 2640tcaggaggaa acaagcgaac
cgccgtgcgt ctacaagtga tgaacccttt tcttcacctt 2700ctaccgccct acctccccgt
cgctcgtcca caatttctac gactggcaat gaggaacacc 2760gcagttcacc tatcgctaac
gtcacagacc gtcctaccca ttcaagggta aattcaagaa 2820ggaagaacaa tcttccggcg
gcggagcaac ctattttggc tgtgggcagg gcaaaaggca 2880gggaaagatc ggagactgtc
acgaagggag gggacctcgg gtcgagaaaa ggtacaccag 2940cggcaagccc acgtatagag
gagaagaagg aattggaaag aggcgagaag actaaaagcc 3000tggctccttc aactgctcct
acgaagaaga atgctaaagt cgatgttgtg gtgccgccca 3060tcaacgtgct gattgttgaa
ggtaaatctt ccattcaaat gatttgttca aacaccgact 3120gacagataac tagacaaccc
catcaatcaa aacattttga gtatgttcct gagaaaaaag 3180aagataaaga attcctcggc
caaggatggc gcagaagctg ttgaaaagtg gaggactgga 3240ggcttccatc tgattctggt
aggctatgat ctctttcttt gattcgtgca gtacttattg 3300gacccttcgc agatggatat
ccaattgccc gtcatggatg gcatagctgc taccaaagag 3360attcgtcgac ttgaacgtca
caataacatt ggcgtttttc catcgactcc agcggccgaa 3420cttcctcggg gtcaaaatgt
tgcggattct ccaccaccat cttctccatt tcgctcgtca 3480gttatcattg ttgccctgac
agcctcgtcc ttgcaaagcg acagagtagc tgctttggct 3540gctggctgta atgacttctt
gaccaagcct gtgtctttga aatggttgga caaaaagatt 3600gtggaatggg gttgtatgca
ggttagtgat ctcttctttt tttttgatta tagctgatct 3660aaatataggc attgattgat
tttgacggct ggcgacgatg gaagagctcc gataccaaga 3720atcctagcga aactaagcag
ggcttctcag tgggccctca acaggctgct aggtcgcttg 3780ctagcagact acgtattgaa
cgcaaaggat ctcgatctcc ggcagctcca gtatcaaccc 3840cgcgactcaa tttgcagtcg
gcaaccccag ataggccaga aaccccccca gattccacgt 3900cacaaatgcc aaaggccccg
cccgttgcag cctctgaccc cccgttatct cccaagtcgc 3960tgaacaagac agttaatgat
gtcttcgagc aagcagacgc tagactcgaa aatgcgcggg 4020aggaacaagg agtatcaagt
caaaaggaaa acacaagctt aacagattca acaaacacca 4080ccattacgcc ctcaaagacc
tatccggctc ctcccccatg a 412195666DNACryptococcus
neoformans serotype A H99 straingene(1)..(5666)genomic DNA sequence of
TCO2 9atgatcttag gaaccgacat cgacctgtcg tctataccaa cggcgtttct cgaggtgtgt
60atcgctgtcc ctttcgagtt gcaccatgaa gctgaccatg agaacgcatt cgccgccaac
120ttccaaatct gttctgggct tgtcttttgg acaaccatct aggcttatcc cttcccagcg
180gttgtgttcg tgatcgattc tccccctagc ccaagaccgc ggctccattc cagaaataca
240gacacgacca ttcggcgaac ggatggccaa atatcgcctc ttacaggtcc tccagtgcaa
300cagttcgcgt cagcgcccgt ggtatggggc aatcaacgat ggcacgagct ggctcagggg
360aaaacaattg cagagtgcgt ggatgtggcg tcacagaaca agctgcaaac ttgggtggaa
420aatgacaccg gtgacaagtc ggagagtttg gccctggacc tgaaggtgcc gcaaggcgtg
480actcttcatc tggcaaagac catattgcca ttaagtccac cgtcctcctc tcagtcactt
540tgcatcctta tatcgcaata tatcgataag ccggaaagtt tcgcgccacc aatctcatct
600ggagatattc ttttctcttc tctatcgcga ctctcccaga ctttttctcg gtcatcctct
660ttttcatcca accctagaaa atcgattgat gtccctgcgt cactatccga acaccggggt
720tctgctacat cgacaagtag caatctgcgc tcttcgatcg atttgacttc ccctaattct
780caaccctctc cactaaaccg tgaacaaagc acgtacttca cccatggctc cgcgaccaga
840gaagagcgac cctcagtaag gcgtagacgg tcaccgccaa tctcaatgac gaggcccaag
900cctcttgaga gccatgctca agaatgctgg gacttggtag agaatttcga ctggtcaaaa
960acagcattag ggccgagaga acagtggatg gatgcgttag atcctgttct ggcaatcaca
1020tttgaatcca gaacggcaga ttgtgcctgg ttagggcctg atctagagct agtttagtga
1080gatactatcc gtcttgcaaa aataatggcg actaactttt atgatcagca ataaggcgta
1140tcaagagctg gttgaccatc ccaatgcttt tggaaaacct gcaagacaag tttgggctac
1200caattgggac tacttggaac ccctggtcaa gcgatgtctc agtgggaccc cggtctacaa
1260ggacaacgac ccgcttttct ggcgtcgata cggcaatggt cgacttctgg aacattacca
1320cacttggcga tatgtcccga taacgggcaa agatggctca gtgcttggca tcttcaacca
1380gtcaattgag gtcaccgact cagtactgct agagaggcga atgggcacga ccagggaact
1440ttcggaacac atgtcgttta ttcgtacaac tgaggacttt tttagctcgg ttgccgacgt
1500ctttagtcag aaccctactg acataccgtt cgcactttgt taccgggtcc gacaagttga
1560caccgatggg acatttgtcc atttggacgt ctcgcttcag tcgtccgtcg gtgtacccga
1620aggccatccg tctgctccag atcaaattcc cgtcagcttc ttaaatggta acccttaccc
1680tagcaatgtc gagcgatcat tttctcctgc tttctcaatc gtttcaatcc actcttcgag
1740cagtcatcga gtctgtcacg tctctgaaga cactacacaa tggcccatcg ccaaagccct
1800acaaaggcgg caatgtgtca tcatcgaaga atgttcgcaa ttaatagaag gatatcctat
1860ccgtcgctgg gatgggcttc cattctcagc cattgtcgtg cccatatgct ctgaagggtc
1920tcccgaaatc cctgacgccg ttgttattct tggtctcaat gtgcgacgtt gttttgacca
1980tgaatacgat tcctggattc actctattcg gtcacaacta tcttcggccc tcgtgatggt
2040caaggcgcgt gaagctgaac aaaagatggt tgaggaaagc gcacgtatgg agaaagcaaa
2100agtcgcttgg ttcagaggag ccgcgcacga ccttcgtagt ccattaaccc tcgtcgctgg
2160accgcttgcc gatgtgcttg attcggattt gaactcgagt cagcgcacgg ctttgaccgt
2220tgcgcaacgc aatcttgatc gtttagtgcg cttggtcaac gccctcatgg atttctcgag
2280ggtggaagct ggacgaatgg aaggacgatt tgttccgacg aacttgagtc aattcgtgac
2340acagttggca gctcttttca agcctgcaat agaaagattg gggttagaat acgtactaga
2400tgtccagcca agcgaggagc ttgttttcat cgatcctgtt ctgtttgaga ccgtggtatc
2460aaaccttatt ggcaatgcgc tcaaatacac tgaaacgggt tctatcactg ttcgggtgca
2520atacacggat tacgcagagg tctcggtcat cgataccggt gtgggtatac cgaaaaatga
2580gctggcactg gtgaccgaat ggttccacag ggcaagtact gccattcact cgggaaccca
2640gggaacagga ttgggactgg ctttggccaa ggaattgctc aagttgcata aaggagaatt
2700gcttgtcgag tctcaaaccg ccaatgagtc aggaggtcct catgggtcca tttttacagc
2760gaaaattcct cttgatttca agccctctcc atcggctcat atcattccgt ccgtcgaatc
2820tcacaagacg tttggcaaat acagtaaagc cgtcgcagac gaagccatgc gctgggttgg
2880ggactcagat gccgctagtg aggcgtacga catgtcgagc ggtaccggag tctcaagcgc
2940tggtagtggc tctggaaaca cgaccacctt cggacccaag tttgcagatg cctttttgtt
3000tgataagaac gacattgtgc ttattgtgga agacaatgtc gacatgcgtg aatacatacg
3060acagcttttc gccccttatt gtaccgtact cgaagcttcc aatggtgaac aggcttacaa
3120tatggctacc caaaaccctc ccaacctcat tttgtcggac gtgctcatgc ccaaattatc
3180tggtatggag ctactacaaa ggatcagatc ccatcctgac actcgcattg tgcctatggt
3240ccttatttcg gctattgctg gtgatgagtc tagggttgag gctctgctaa acggcgctga
3300tgactatctt gccaagcctt tcaaacccaa ggaactcatc gcgcgtgttc acctgcacat
3360gcaagttggc aagaaacgtg ccaagctcga agcgctatac gcccaacgcg aaacagaatt
3420gacagctcta tctgactatt gtccgatcgg tatcttccga ggagacaaat atggccatat
3480tgtttatgcg aacgcagctt ggcgtgcgca gagcggcctt ttggtgggtg accctaacga
3540ttgggcatct tatgtgcacc cggattcgaa agcgcagctc ttggaacaat ggaatcagtg
3600gttgaggggg gatttgaagg agttccgagc ggcttggaga tggtctaatg gcatccctgt
3660caggagcatc ttggtccggt tagatgacgt caaggaaggg ttttctgggt taattgggtg
3720cgtagtggat gtgtctcatg aagagagacg attaatcgaa gctgaggaaa gaagaaaaga
3780ggcggaagag agtaaacatc agcaagaact ccttattgac ttgacaagtc atgaaattag
3840gaccccggtg tcagcaatcc tgcagtgctc agatcttgtt aaagagaatc ttgtagctct
3900gaaggaccag ttgagaggag cggggccaaa gggctttgtg ccgagtcaag aattactggc
3960tgatcttgag caggatgtgg aagctttgga aagtaattca ccatcccctt tcatgctaat
4020ttcgaaacta acagtgattt tgtaaggtat ttatcagtgc ggtcttgtgc aggaacgcat
4080tgccggagat gttctttcgc tggctcgtat ccaactcgat atgctgagtt tgcacgacat
4140tgacgtcaac ttgcgccgag aaggcaggaa agtttcgtcc atctttgcat cggaagccaa
4200gatgaaggat atcgacctcc aattggaatt tggacctact atcgaacagt ccaaagtgct
4260ggccatcaag acagatcccg tgagattagg ccaggtggta acaaatctca tttccaacgc
4320cattcggttt acatcttcga gtggtgagtt tcatccaacc cataatgtgc tatagtgata
4380ctgaattgta attcatttag atgtccgaaa gattactatc caatacgacg tatcgtttgt
4440ccctcctgcc gatgactctt gcgccctccc ttcatctgtt ggcttgcccg acatacttcc
4500tgtgaaagag aatactccac tatggctgtt tgtcagtgtt accgattctg gacctggtat
4560gacagagcaa gagttatctg ttttgttcca aaggtttgcc cgtaagtcct aagcccattc
4620tctttgtcga ggttacgttg acgaatcttg atatctagag ggcaataaga tgattcatac
4680taagtatggc ggaagcggtt tgggactgtt catctgtcga agtgagtgat tggaggaggt
4740tttttttttt ttttggtggg gggatacatg atgctgaaac catctacaga gattacagag
4800cttcttggcg gtcgtatcga agtgctcagc caagtcgggc acggtagtgg tgagtgcccg
4860tgtgctctgt tatccattgc atgctgctga atcgagtgcc tttcccaagt tttccgattc
4920ttcattaaaa cgcgcgctgt cgctcctccg tccgccatcg ctgctctcgt agaatcctct
4980cctctcaaac cggtatccgc cacttcacct tcttcctcgt tagccatgag ccgatcgtct
5040tctcggagca caaacgtcac tacacctata gagggtggtg ggaccgagca cgtgttgatc
5100gtggaagata acctaatcaa tcagactgtc ctgaagcgac agctcgtcaa ggcgggttta
5160tcgtgcaacg gtgagttctc catcccatcc tctgtgattc ccacatttat cttgattgct
5220gttgtatagt cgcgagtaat ggccttgaag ctcttaatgt catccgtgaa gtccatcggc
5280aacaccgacg cggtgggccg aaccgtaaaa ggctatttga cgtggtattg atggatctcg
5340agatgccagt gatggatggt atcaccgccg tacgagagat acgacagtcc gaggccgcgg
5400ggacattggg caggaatatg gtgattgccc tgacggggaa cgcgagacaa ggacagattg
5460atcatgcctt ggcttctgga tttgacgatg gtgagttggg aagacccttt tttgggggct
5520cagaggacga tgtggattat gaacttttct gacacttgat tgtagtcgtc atcaaaccgt
5580atatcctggt agatttgctg aacaagatca aatctatgaa agttagaaaa ttggagttgg
5640aaactgcgaa agctcaagaa gagtga
5666104744DNACryptococcus neoformans serotype A H99
straingene(1)..(4744)genomic DNA sequence of SSK2 10atgtccaacc caacctcccc
ctcaaacccc tcagacaccg gcccgtcctc ggcgtccaac 60gtcacatcct cgtcctcaaa
gaccggacgc agatcagtgc gcctctttgc cccagacgag 120gaggacagct cagacgagga
cggcggcctc atcggcgtgc ccgcagagac cacattcaag 180gacgacgaga gtgagtagat
acgcaggaac caaatgcagc gcatactcac actccacagt 240ccctccttcc aacccacgtt
ccgcctccta ccccgggcca ccggcacaca cctcccccac 300ctctaaaatc tcaaccatcg
tttcgtccgc ttctgcagcc cagccaaaac ttgcacgttc 360aataacatac gtcgcaccca
atgccgtctc ctcccggccg gcatatcccc tcaatcccgc 420agggtcagaa accttacacg
cctcgtacga gacatggcga aaaacgcgat ataccctcga 480gtcattcggc agaggatcca
aaccagacgg gaagaatacg gcacaaagag gaaggtcgta 540tactgacccc gatataggat
actttagcca cgatgcagga gatgatggtt ggggctcaga 600tgatgacgac gaattgagat
cccctggctg gggcatatcc catcataaca tggactctgg 660aggcaagacg aacgggtcac
cacagttgcc tataaagccc gccgatgtca ccgaggatga 720aggacaggaa cgtttagatt
ggcaaggcat gctggaaagt gtcctcaact cggatgttct 780caaggtggag gaacaacgta
tctacaattc catgccgaca gattcattca gagaagagat 840tggaaagacc ctttggtggc
aaatccgtgc caaactgcgt gggaggacag aggcggagga 900gaagaaacgg gtgcaagagc
gacgagcgag agtggtggac ccggtgctgg aagagataaa 960cgagttcaag tacgacccaa
aaaataaccc agaaggcgaa gaagacagtg atggcgatcc 1020gcaagacgcg acttcgactg
ctgcacccca atccaaagct ctcaatcaag tcaacaccgt 1080tctcgccaaa cttcatgcaa
tcaaaggtct ttatcccaac ctcgcagcca tgcgagccga 1140caaggttctc tataccgatg
aaaatttccg caaacgcgcc gacgcattga cctcttggtc 1200catcatcgtt tcatccctcc
aaacccagct caaactcttg caaaaatgga caggttccga 1260tgagcttgac atcaccaagc
ccaacacgac ccacgagaaa gcattggtcg gcaagtacaa 1320gtatcactct atcgacagca
agggtacgcc cggcagggat gcagccgatg actcgagttt 1380cctcgatcgt gtgataaaag
aagataacct tcaacggaca ttcgagcgtc gagcgtttgt 1440agacatgatc aacctcgtgc
gcaacgccaa ggagacggtc atcagctatc tcccccagtt 1500ccaagaacaa aatcttcccg
atttccagta cgaaatcgtt cgtcttattg gtttccccgg 1560tcgacttatc attgaagctg
tcaaggttcg tttggatgct gcatcccgac tacttgaccc 1620gaaccctatg gtcgtcgaag
actttatcga aaaccttcgt ctatccattt cgctcgccgt 1680gctaatccgg aaacaatacg
acgaaatcat ggcacccgat gccgagggga gatggaaaat 1740cccgcattgc ttgccgacag
agtacaatga tgttctgctc gatgcgctga ggacattttt 1800caaattgttg cattggagat
tacgaggagt ggggaaagcg agttattaca aggaaacaga 1860agtgttggaa gaagaggcgc
cgttcttgta tgaagcggcg gaggctattg taggcggtga 1920tatggttgtt gcagagcagt
attggtgagt ttgaaatcgt atcatcctgg caaggagctt 1980agtgctaagt atcgatgtaa
atagcgcgtt atccaacaag ctccttatac gttcagcaaa 2040ttatcttgac cagcaacttc
gggtaccaat acattccccg tctcgcgaca aggaacgtgg 2100tgacaaggag cgcgatggct
cttcgtcttc tcaacgtaac cgtgacggcc gtgatagctc 2160gctgcccggc ccaccgaaac
acatgaaagt cgaagaactc ttctcatggt actccaaact 2220ccttgattcc gctcgtatgc
gacaccgtaa aacccaacgt ttctgtcgta aactcaccca 2280acgattcgat aattccgccg
aatattcaat cgaggagacg gagatggaca tgctggtgga 2340gacattgcaa gatactggtc
atttcttggt atataccggg aaatttgagg cgaatgggac 2400gtatatcgtt gcggatggga
gtctctgggg tcagccggac gatgtgagac atctgttgaa 2460gagggtgttt tcagtgacga
ttcctggatc tcgagtccgt ccaaggcaga caacctcgca 2520agtatctgtc ggaggtgcga
gcccgtccaa tggtcaagtc gcggcgcaac atgatcctgc 2580agatccgtac cccgaggcag
acgattttga cgacgaagcg ctcgcggctt acatcctcct 2640catctcccca cgccaaagtt
ttgtatggtc cggagcggtc atgacgctgg atgtggatta 2700catcgactat gaactacctg
ataaccgagt cagattgatc gctgacggtc ccaccaagcg 2760gttagcgctg tgcaaacttt
atttcaagca agcgctcatt caccctgata cgggcgaaac 2820aatcgacttg ccatgtgtgg
ttgaggctca agcgcattta ccgaccattc agaaacaact 2880tgtcaagatt gctaaatcga
gttatcgtct ttcagagtgc attgtccagt ctgcaccact 2940cgtccgcaat gcgttcaggg
gcaaaccggg atcacaagag ttggtggaga attggtacag 3000ttttgcgaca gagcatggga
cgagagtgtt gatccatatt gagcctagtg tatgggagcg 3060attcaatcgg ttgttgatgc
gtctggcgat cagttggatt agctttatca gtcaagagtg 3120taaccccaca gaccgcaaga
cgttccgatg gactgtggca gctttgacct atgcgttcaa 3180catgacgaga gggagtaaca
ttctcgcgct tgatcgatca gaattttcgc ttttgaggag 3240gtatgttggt gtttgtgtgt
cactgttggt tagccacttt gatatcctcg gcgcaaggtc 3300gagtatggag gccaaaaagg
aggcagacag gattgaggcg atgaggaggt tacaacggct 3360tcaagaaaac ctggacgacg
aattcctgcc ccggactccg acagagtctg gcgatcaacc 3420acgtatcgac cgctctataa
ggctcacagt cgaagaacgt ctccgtctca ttgccgagct 3480cgaagctcgt cgtgacgagt
tggcacccgc acccgtcggt caagtccttg atgaagaagt 3540ctctgaagac cgtgcgttgg
tgttccttgc agcttccaaa tccaacattt ctatgcgatg 3600gcagcaaggc gcgtacatcg
gtggaggtgc atcgggaagc gtgtacttgg gatactcgtt 3660gcaggataac actgtgtttg
ctgtcaagat cttgccaacg gtggatctgc agagtagtcc 3720ggcgttgtac gaaagtatca
agcgagaatc ggatgtgatg agcttgttga gtcatccgaa 3780tatcgttggt ttccttgggt
tggaagtgca taggaacaga gtttgtcttt tccaagaggt 3840aagtgcttgt tgttgtttcc
atttgtgttg ggagggtgtg gtgccaaagc tgatgttcgt 3900gattttagta ctgtgaagga
gggtcgctgg caggtatgct cgaatatggc aaaattgacg 3960atgaggaagt cgttggagcg
tttacgatcc agctgttacg cggccttgag tatctgcaca 4020ccaaccgcat cgaacaccga
gatctcaaac cagaaagtaa gctgacaccc atcttttgat 4080cctttccaac acacacacac
taactcgtgt tctccacaga tattctcatc ggcgccaatt 4140ctgtcctcaa gctggccgac
tttggtaccg ccaaaatcat caaatccaac aagacgctcg 4200cccgtacacg tggtggcgcg
cacgccaaga tggagggtct tgagggtaca ccgatgtaca 4260tggcgccaga gatgatcaag
aaccagagga ctggcaagct gggtgcttgt gatatctggg 4320gtttaggatg tatcgttttg
cagatgatca ctggtaggaa gccatggagc ttcttggact 4380ttgataatga atggtacgtc
ttttcttgca atgatgtttt ccgcgtaggg agttatgagc 4440tgataatatg attagggcaa
tcatgttcca tcttggtgcg acaaaggagc cacctcctct 4500acccgatccc aacgagatgt
ccgaccaagg tatcgaattc attgatcaat gtctttcttt 4560ggatccggaa gcgaggccgg
tggccagcga gttattgcaa gatgaatggc tggttccaat 4620gttggagcag atggtgagtt
gtctttcatg tatgtgtaaa aaatggtcag aagcttatct 4680gctttgcaaa acaggccgag
ctggagcaag aataccccga tatattggcg atgggccaaa 4740gtga
4744112202DNACryptococcus
neoformans serotype A H99 straingene(1)..(2202)genomic DNA of PBS2
11atgacagacc ctacgccccc cgccctggac agtctctccc tggcagacaa ggcgcctact
60cccgaagaaa gtcccgaaga cgccgctgaa cagcccaagc ccgcggcctc accgtccgca
120ggcacacccg gccatgacgc ccaaagctca tccacctcgc ccccgcaacg ccctcagtcc
180atgcagacaa atgacaaggc gccagataca tctgctccgg cttccaggcc ccaaccgcaa
240catgtccctg catcggcacc tgcgcttccc tctaccaacc ccgtccgtcc acagccgggc
300gcccgtcctg gagcggcgag gggtatgccc gcgcccatgg gtatgcgggc gcaagcaggc
360cgaggcgctg gcggccccca gatgcagacc aagatgctgc ccagtttgca ggctaaaatg
420gacaaggtgt gtatcgctcc atcatttatc ccgctgcata ctcatccaga ggctgtgctg
480acaaaccaca ctatgctatc attagatcgc ggcgtctcgg caagggccac ctccctcctc
540tggcatgcat gatccgaatg ccacatccat gggcgccctc ttacgctccc aagccctccg
600cgcccccggc gcatcgcaag ctcctcccgg ccccggaccg gcttcaggcc ctttcggtct
660cgccgctcgg cgcgcagctg ctgggggccc tccgagaccg aatttgggta tgatgggtat
720gggtgcaagt gcgccgggtg cggttggacg gggatcaggt ctggcgggta gacgggggcc
780ccctggagga ctgacactga gtgggatgaa gggtgcgatc aaggatgagg gaaacaagtt
840ttcagacttt cagggtgtca tgtgggttca gcagactcct tttccatgac tgtgggctga
900tctcaagtac agggacccgt ctggatcgct gagattctca aagaaggctg tcctgcatgc
960aaagggcgtg gactttgagg atgggcaaag tttcaagatc aatatggatg agatcgaggt
1020gcttggagaa ttaggaaagg gcaattacgg ttctgtgcac aaagtcttcc accgtccgac
1080aggcgtcacc atggccatga aggtgatctt attctttctt gcgtcgcttc tggtccagta
1140actaacaaac acgacaggaa atccggttag aacttgacga ttccaagctc aacggcatca
1200ttatggaact cgacatccta caccgggccg ttgctcccga aatagtcgaa ttctacggtg
1260cattcaccat tgaatcatgc gtctactact gtatggagta catggatgcc ggttcactcg
1320actctctcac cggtggcggt gtggcggcca aagatcaaac aaaggatgaa gaaaacgatg
1380cgacaaaacg agtgccggag gatgtattga ggaggattac agcgagaatc gtgaaagggt
1440tgaggttctt gaaggatgaa ttgcagatca tccatcgagg tgagttttcc atgtgcaatg
1500aaaacgggag gaaatgtgct gatatgatgt agacgtcaaa cccacaaatg tgttaatcaa
1560tggcaaggga gaggtcaaga tgtgtgactt tggcgtttca ggtcagctcg aaaagagttt
1620ggccaagacc aatatcggtt gtcaatccta catggctgta cgtctttccc tctcctccat
1680ctcaaagagc ctcccagcta acccgattcc ctctctttct ttagcccgaa cgtatcaagt
1740ctgaaactgc caaccagaat cctacatata ctgtctcttc agacgtctgg tctgtcggtc
1800tgtccattgt cgagcttgcc aaggggtgtt acccctaccc accggagacg tatgcgaatg
1860tgtttgcgca gttgcaggcg attgtgcatg gcactccgcc aacgttgcca cctgggtaca
1920gcgataatgc gaatgatttc gttgccaagt ggtacgtctc tcaccccttt ctcttcgtgt
1980ttgaatttga caatgctgat aatgagcgca atctttagtc ttgagaaaga tcccaaccga
2040cgaccgactt atgctcagct cttagaacat cctttcttgg tagcggacaa gggcgcagaa
2100gttgacatgg ttggatgggt ggaaggggcg ttgaagcgca aggcagagag ggggattgcg
2160agcctgaatc ctatccaacc acctgtccct ttggaaccat aa
2202121620DNACryptococcus neoformans serotype A H99
straingene(1)..(1620)genomic DNA sequence of HOG1 12atggccgatt ttgtcaagct
ctccatcttt ggaaccgtat gtttctttta ttgctctttc 60tcttttccca ccaccgtcat
gatctgctct tccaaccaac caacctacga acacgcggcg 120tttgtttttt ccgttggcca
ctggatcata tcgtgttgat tctgtccata cgccggatgg 180aggagatctg taaaggcaag
gccgcggacg ctgatggatg ggctttctcc atggataggt 240ttttgaggtt accacgcgtt
atgtcgacct ccaacctgtc ggtatgggcg ctttcggtct 300cgtctggtga gtcttgtttt
tctcaagcaa ctatcctttc atctggtttt tcaacccagc 360gtcgaaacag gtcgtccgac
ctttgcatgt cgatgtagag atgtgaactg acaaaaccat 420cttgtttgat gcagttccgc
caaggatcag ctgtctggaa cttctgtggc tatcaagaag 480attatgaagc ccttttcaac
ccctgttctt tccaagagga cttaccgaga gctcaagctt 540cttaagcact tgagacatga
gaacattatc tctcttagtg acattttcat ctctcctctc 600gaagatatgt gagttttgct
caatagttgc atatcaaaga aggggggagg gggcctgctg 660acatttatcc aatagctact
ttgtcaccga gctgctcggt actgaccttc atcgactcct 720tacctctcgc cctcttgaga
agcaattcat ccaatacttc ctttatcaaa tcctccgtgg 780tctcaagtat gtccactctg
ccggtgtagt ccatcgagac ttgaagcctt caaacattct 840cgtcaacgag aactgtgact
tgaagatttg cgatttcggc cttgcgagga tccaagaccc 900tcagatgact ggttatgttt
ctacgaggta ctaccgagca cccgagatca tgttgacatg 960gcaaaagtat gatgtcgcgg
gtgagtttca agttttacgt ttgggggtgg tcttttaatt 1020ggcgatccat gctgaccacg
caaaaaatca gttgacattt ggagtaccgg ctgtatcttt 1080gcggagatgc tggagggcaa
gccattattc cccggaaagg accacgtgaa ccaattctca 1140atcatcaccg aattgctcgg
tactccgccg gacgatgtca ttcaaactat cgcctctgaa 1200aacactctcc gtttcgtcca
gagtctgccc aagcgcgaaa aggtcccatt ctccaccaag 1260ttccccaacg ccgaccctgt
gtctcttgat ttgttagaga agatgctcgt gtttgaccct 1320cgtacccgta tatccgccgc
tgaaggtctc gcgcacgagt atcttgcgcc ttaccatgat 1380cctaccgatg agcctgttgc
cgccgaggtg tttgattgga gttttaacga tgcggatttg 1440ccggtggata cttggaaggt
gatgatgtat agtgaaattc ttggtaagtc tctgtgcctt 1500gccttttttt gggtattata
ctaacgtcgg actttagact tccacaacct cggagatatt 1560tcacagaacg aagcagaggg
acccgttact ggcgaagtcc ccgctgctcc tgccagctaa 1620133792DNACryptococcus
neoformans serotype A H99 straingene(1)..(3792)Genomic DNA sequence of
ENA1(CNAG_00531.2) 13atgtcttctg agaaaggaca atcaaataca aacgagaaac
aacttattaa ccgcgccgac 60actggcaaga ctgcagtgtc agactctcct ctccctttca
aacctcatac cgctctctct 120ggcaagatcc tcgaggcttt agggagtaat gttacttctg
gtctatcaga tgacgaagca 180tcaaggagac tccaacaata tggtcccaat aggctgaagc
cccctgagag acctagtatt 240ctcaagatca tcgctaggca agtgggcaat gctatgactc
ttgtcctcag taggtgttct 300ctccctttaa cgtttcttaa gctgatcaat ttcgtagtcg
ctgccatggc aacttcattg 360ggtaccatgg actggatcag cggtggcgtt attgcggctc
tggttatcct caatgtatca 420gtgggagcct acacagaatg gcaagccgaa aaggtacgtg
ttttaagatc tgcggagatc 480ccataaaccc cgaggaattc tgacgtcata aatagaccgt
ggccagtctc gagtctgttg 540gagctccgca agctactgta gtccgaactc gcaatggctc
tcgcgaggct accgtcaaaa 600ttatccccgt agaggaagtc gtacccggtg acattattca
actcaaaaat ggtgatattg 660ttcctgcgga cggaagaatc cttgacgggc acctgagtaa
cttggaagct gacgaggctt 720tcctgactgg cgaaagtctg ccggttgcaa aacagactga
gcctatcgat gaagaggact 780gtcctgttgg gtaagtagaa aagatttccg cacattcagc
cacagcctaa ttatgaggac 840agcgaccgtg tttgtatggt cttttctggt tcccagatca
ccaaaggtcg agctcgtgcc 900gtcattacca gcactggtat ggggacagag attggaaaaa
ttgctcaagc tcttgaatct 960aaagctaaaa ataagaaccg tggatttgct gctttctggt
ggaaagtcaa agttattttg 1020ggtgtcgagg agactactcc tttgcaaatc aagtatgtta
tcttgctata gtagtgtaat 1080ggttggatgg tactgacgcc gatgctagac ttaataagct
cgcatacttc cttttggcgt 1140gtgccctcgt catagccgtc attgttgtcg cctccaccgg
ttttaatgat gtccccctct 1200ctattgccac ctacgctgtc gctgccgccg tctccattct
ccccgcctct ttgattgcag 1260ttgttagttt gactttggcg cgtgcgtcaa ctgatttagc
atctcgacat gctttggtcc 1320gacgaatgga tgctattgag gctttagctg gtgttgagaa
tgtgtgctcg gacaaggtaa 1380gttaccattc aatttggctc gaaacctgtt gatacagttt
tagccggtac ccttactgtt 1440ggccgcatgg tagttcgcaa agtctgggtt cctgctcttg
actggcgccc caatgaattt 1500gctcccctcg acactagtgg tggtcaagca tatagttttg
agaccggatc tgatcctttc 1560tatcctcgtg gtgaagtcct ggccgattcc cagaagatca
ctgggactgc ggagaccctc 1620gatctcaagc aacctcgtga ccaatctgac tcttcctctt
ccgactctga ccccgatgaa 1680cgagacgtag aggaacaaga acgggtcatc cacgttgaag
acatggaaaa caaccttcga 1740gaccttgctc tctgtatttc gctttgtaat caagcgactc
tcactcgtcc tgtcaaccaa 1800gacggccaat gggaagcaaa cggtgatcct accgaaacgg
cccttcaagt tgctgcacac 1860aaacttggtc atggcaagcc ctttcttact catgctgcca
agccaagcca ccgtgcggat 1920tctatccgat ctggtcacag ttctcgtccc cttgttgctg
gtattcgtgg gcactttgtt 1980ccgataattg agcatccttt cgattccacc gtcaagcgaa
tgtcaatcgc ttataaattt 2040gtgagcgagg atcctcagga ttctcacatc ctctgtctcc
ttaagggtgc catcgagcgt 2100gtctttgaac gatgcaccaa gatccaagga cagcccatca
ccgaagagca taagaagaat 2160atcatggtca aagttgatgc tctcgccgct caaggtcttc
gggtcctcgc tctttgtgga 2220aagcgacttc ctgtcagcat ggtagacgaa gtcaaatcca
cccctcgaga cgcattcgaa 2280gccgatttcc atttcctcgg tcttgctggt atcttcgatc
cgcccagaaa ggaatctgca 2340ggcgccgttg ctgattgttt cagggctggt atcacccctc
gaatgttgac aggcgatcat 2400cctgctaccg ctacagctat cgccctcaac attggtattc
tcgataagac gtactcaaag 2460gattcagtca tgacgggtca gcagtttgac tctttgagcg
aagacgaaat tgatcaactg 2520cccgagttgc ctcttgtcgt tgctcgctgc gcccccgaaa
ccaaagtgag ctgttttaac 2580atatctaatg atgtacttgt gcctgacggt tcccagttcg
aatggtcgat gccattcatc 2640gacgaggaca aagcactgta atgactggtg atggtgtcaa
cgactctccc gccctcaagc 2700gtgctgatgt gggcgttggc atgggtactg gttccgatgt
tgccaagcag tcagcgcgta 2760tcgtcctcag tgatgacaac ttcagcacca tcattcgggc
tattaggaaa ggtcgttctg 2820tcttcaagaa cttgtctaaa ttcttgctcg tgagtaattc
aatgcatgtg atggaaacga 2880agctgatctg gcttccttag tacttgcttt ccggtaactt
ggctgaaatc atcgtcctca 2940tgattggtct cgctttcaag gatgacaatg gtcaggctgt
tttccccctg tcacctgttg 3000ccgctctttg gatgtacgtg taactacctg attctttgca
aggactttga ctgactccca 3060tttagcaaca ctctcgctgc cggacctcct gcccttgccc
taggtcttga acctacagct 3120atcgacgcca tggagcaggg acccgaggta taccatcgaa
tcttcactct tgaattttac 3180gtcgatctga tcttctacgg tttcctgatg ggctccatca
gtttggtcaa cttcgtcatt 3240gtactatggg gatactatcc tgtaagttca gtttgcatcc
ccaagaagca tccctaattt 3300atgaataggg agacttaggt cgtctttgta acgaagatga
tcccagcatc tgtgatcccg 3360tctatcaggc tcgagctgcc tgttttgcca ccctcgttat
tgtcctcatg attcatgctt 3420tggagtgtaa gcacttgagc aaagggttgg cccaaatcaa
tttgcgtgac aacaaggtgt 3480tgctgtggtg tgtcgttgcc ctcagtcttt ccactgtaag
ccctttacac catctatctg 3540gcctacgata tcagctaatg atgaacatag ttccctgtcg
tgtacattcc tgtgatcaat 3600aacaaggtgt ttttgctcaa cggtcccagg tgggaatggg
gtatcatctt cggcatgatc 3660ttggtgtatc tcagtgctac tgagctctac aagtggatca
aaagaatttg gatccgacga 3720catgcccccc cttccaaagg accttccgac aagaccctta
ggatggagag taccattgct 3780cctcctgttt ga
3792143173DNACryptococcus neoformans serotype A H99
straingene(1)..(3173)Genomic DNA sequence of NHA1 (CNAG_01678.2)
14atgactgctt tccacccctt tgaagtcaat gcccctcatc tcgcatacac gttcctcggc
60ggctttgtgg tcatctttgg catgatcagt ttgtttatca aagagaagct ctatgtcggc
120gaagcaccta tagcaactgt agtcggcatc atcattggtc cccattgcct caattttttc
180aatcctgcag gatggggtgg cggggaggaa gaggtcgcga gtgacgttac attggaattc
240actcgcgttg tcattgctat atccgtattc gccgtcggcg ttgaattgcc caaggtaggt
300ggtaacttgt gattcattgg agtagagaaa gcgctgatct tggtgcttgg gaaaggcata
360catgaagcgg cactggcgat cgctcttctt ccttcttggc ccgtgcatgg tgtggggatg
420gatgatctcc gccctgctga tctggggcct gatacctgac ctaacatttc tcgcctcgct
480cgtagttgcg gcgggcgtca cccccacaga tcctatcttg gcccaggcag ttatcggagg
540caagttcgcc gataaacatg ttcccgccca catccgccac ctcctctccg cagaaagtgg
600aagtaacgat ggggccgcct ttcccttcct ctacatcgcc ctctacctcc tactcgatgc
660gagcccaggc catgccgtcg gagaatggtt ctacatgact tggtgcgtag gcaaacctag
720agcatcctca gttacctttg ttcttgtcgc tcacatgtct ttttagggtc tacgaaatta
780ttcttggtgt tatcatcggg gccatcctgg gattctgcgc acgcaagttg atgaagttag
840cggagcgcaa acgtctcatt gataggcagt cttacgtcgc ccagtatgtc agtctggcag
900tgctgtcgat tggtaagcgt gttgaccggc cttgcgatta ttcaatgagc tgaccacgat
960gtaggcgtta caagtttgct cggcagtgac gatttgcttt ctgctttcgc ttgcggttgt
1020gcttttgcat gggagtacgt agtgcttgtg atttatctct tgactgcgct gacgacatta
1080cagcggtttc ttcaacaaag ctacggagga tgcggtgttc tcgaacgtta ttgatctact
1140tttcaattgc gccgccttca tctatatcgg cgctatcatt cctttcaatc attttaacga
1200tttgcccgat gtacgttcgc atgggctacc acatacacga actaactgga tctcagctcc
1260gagtatggcg attggttgtg ttggctatcc tcattcttct agttcgtcgt ctgccttcta
1320taatagcgtg ttacaaattc gttcccgata tcaagacgtt cagagaagct ctttttacgg
1380gatggttcgg tgagttcctt attgtgagta tcggttgggt catgtctgat gagaggttag
1440ggcctatggg cgtcggtgct gtattcatct ccactcttgc tcggtcgtct ttgccagaag
1500gggagcctga acagaataca gaagcggtgg accgcctaaa agacgtcatc atgcctgtca
1560ccttatttct tgtattgtct tcaatcgtaa ctcgtaagtc tatcccgtcg cactcactac
1620tgactcagtg tagacggcat gtcaattcca tttttctctc ttggtcgccg ggtccattcc
1680attacttata ctcgatcacg aaatctttcc atggacacgc gaggcgatga gcctgcctgg
1740acaactcatg ctcggcgtat tattccaggc caggagatca ttgtcaaccg tgatgacgac
1800gacgaagaag gcgacttggg tgttagacgg atggacacac tcacgagcga ttcaaatggt
1860cgtatcaggg aaaagattga ggaagaagat agcggagaaa gtagctcatc ccgaacaagg
1920cagggagaaa tgattgaaat gacagaaaaa cgtggcccgg ctcgccatgg tagccaggcc
1980agccagggcg aagcggcgga ggaaggagag aggtggagaa gttcgggaga agaaagctct
2040gatcttgcga atgaccctga gacacagaga gaggtggaag agggaatgga agaggtcgaa
2100gataaggaag gaggtggtag aagaacgccc cccctggcca agtacagaga aggaaaccac
2160ctcattgtgg agagaaaagt caaggacagt gacgaggtat gctatgcgga tgtcccaatt
2220gcttatcacg acttgctcat gcgtatggct tttaggttga agtcgaggtc atccgaaacc
2280atttttccga caacaagaaa acggaaagtg accgcttcac tcatccccat cgcctcaagt
2340cacgagagct tgacgatttg cttcatcacc ttcccaaaag cctcgagcat gctacttcac
2400gggttcaaaa tggcggcaaa gatgcagttg atcgtctcgg tcttgggctg atggctatta
2460acactccgga accgtcacca tcgatcgaat cgcacggcgg tccaaggcat gattatgtcg
2520atggcttgga gagaacgcag agcccagagg gtcttgcaga cgaggatagg gatagcgagg
2580gccggggcga tgtgtcccat gggggtgatt atgaagaaaa cgaggccgac tatgaggatg
2640ttccgaacga gactcgtcgg caaaggagga agaaaatgaa accaccagca attgtcgtct
2700ctcggcagaa cagcgccggg ctcccgagac gatccatccg ctccaggctg ttcggccgac
2760gacaacattc ttccaactct ccctcccgtg ccgaagaagg cttagcccct cccaatccat
2820ctcttcttgt tccatcctca tccccttcgc gtcctcaaaa cattgctgca gaacccgagt
2880ccatactggc agaagattcg cgcggatcat cctcaccttc ccaatctcaa aatcttgcga
2940tccctctcac aagaaccctt tcagctagcc gatcgtcgcc tgcggtgcgc ttcgccgacg
3000atgctagtcc ttcatcggac acagcgcctg ggcagtcaaa ttatggtact aacgctccag
3060gtttcaagaa gaatccggct ttagcaatgt atcgatcggc cagtgtacaa agtacagggt
3120ccaacaagga tgggcctagc gtatctttca aagaacctga aatcaagcgt tga
3173153930DNACryptococcus neoformans serotype A H99
strainC_region(1)..(3930)coding region sequence of SSK1 15atgtggggct
caaatgcttc catcgccgcc tcggagtcga ccgactccct ttcccccgcc 60ccctcccagt
ctgccgccgt agagttcccc ctgcccgtaa gctcccgccc gtctctcact 120tccgccgctc
acccctccca gatgtccgct tcctcctcct ccacctcctc ccagcctctc 180tttgattggc
gcattcccaa gcccacctca ccccgcacac gcatggaccc attcgacact 240tttgatcctg
tatcctcaag ctcagaggat gatccggtcc ctcaagagag ccgccgtgct 300ggtcatcagc
gctccgtaac agatcctctt ttacgagatg gccaacccct cgatatggag 360ttcactactg
ccgggccgcc tatacagagc tacgactttg aacaaccgcc cacgtttagc 420agaacgttat
cttcccctct tccagctaaa gtcggctcgc ttagacaccc tatgccattc 480accattgacg
atttaagctc tcgcaatgtg aattcaaccc atcgtccgca gccgactaca 540cctctgcatt
ccatatccgt tgagttagcc gattccttgc aatcggccat tcaaacatta 600ctgcatctgt
ccccacctca cctactcgat aacgcaaaag aacagtactc tggatgcacg 660gtacagatac
ctgctacctc gctttcggcc cttttgacct ctatgagagg cctcaacttc 720ttgtcggcgc
atgctgaaga actggtcgac atgagtgcac gtggagatcc acctgtactc 780catcaagaag
acttcgatgt aggagaactt ttgcaaaacg ttgcggatat gttgagtgga 840gaagcagcag
aaaaacggat tgatttcgtt ttgttccatg gcgacgtagc gatgaggcat 900gtcagtgtgt
atggagattc tgatggaatt agctatacct tgagtcatgt tattcgacaa 960atactggcag
tagccaatta cgatgatacc atagaactcg gccttcaagt cattcctcaa 1020agtccatctt
tagcttccgc cgtcggactt cctctaacct ctgccgatgt tagtggagga 1080ggtggtgtca
agtcagcgtc cacatctcgg tcaggctccc ccaataacag tctctctcga 1140tctaattctg
tccatgacgg gccccttctc tgtgtgttcg aaatagtaca taacatctat 1200cagccaccac
caagctcggc atccgccact cctaaagccg agctgaaccc tttcactcat 1260cttgctgaag
aaaccgaagc cttgaaacca agattggata cagcattttg caaaaacctg 1320cttcatcggc
aaaatgctgt cctcaaagtt gatgtgcagc cttcatctcc tttaggatcc 1380gggatgcccc
gtagagctta cgcgttatca gtgctcctac caagaggtaa acccatcact 1440gagcctgcaa
tactttctaa agaggaacaa gaagttcgtc aaccattttc atcccacgta 1500cttgcacgag
aacccaccct caatgagctc tcggaatttg ctgaatcatt acgaggaaga 1560aaggtgttta
tccatgccaa tttgagtagt gttttcgcga gacacctcac gagctaccta 1620gctgcatggg
gaatggatat atcgcatcta ccgacagatg gcgatgaggc tgataaattg 1680aaggatgtcg
cggccaaaca tgactcggct tatactggat ctatgggtgt gtcaggcggc 1740actacttcca
gcgcagaaac gccctattca attaaaccga ccggcgtgac tgctgttcaa 1800cctggacact
ttgtcattat cgacgatgat gttgcggtct tgcgccgtga actcgtgcgc 1860atccgttcag
aattacttcc cattctcttt aaacctagac tgtcaaagcg tcccactatg 1920acttctcgaa
cccgttccac cccttcattg cgacaggtcc ccccaaggtc atcatcgggt 1980tctgtactta
tacactttac ctctcttgcc aattataacc gagttcgaga cgcgattgcg 2040agctttgtgg
gggcgccagg gttaaccaat ccggaaactt atgttcagcc ggaggtgata 2100gtgataccca
agcctgttgg accacgaaga tttttgactg ctctgcatac cgctgtgaaa 2160cagcctatgg
ttgacccatt tttctcccct atcgccacat ctcctagatc accaggcgga 2220ggttactttg
gtggtttgcg gactccgacg gagagagaat caggattctt tgattctgtt 2280gcagaagaac
cacatgaaga ggcggattcg cgaccagatt atgccacggt gcagaaagcc 2340agatctcctt
taggagaatt tccgccttct gcggcacaga tcgttcgtac caaccaaggc 2400ttgcatcttt
cgcttcccac tccaaatgaa attatgacaa cgcctgctcc agaatatttt 2460tctgggtctt
ccaagtctcc tagctctggt gcgtccggag tcgtgatgca gagccctgat 2520ggtcgtcctt
tcggaatgtt tttcgaaccg cccataaaaa atgagcgccg cggatctact 2580cacaggacgc
cttccgattc catcaggagg aaacaagcga accgccgtgc gtctacaagt 2640gatgaaccct
tttcttcacc ttctaccgcc ctacctcccc gtcgctcgtc cacaatttct 2700acgactggca
atgaggaaca ccgcagttca cctatcgcta acgtcacaga ccgtcctacc 2760cattcaaggg
taaattcaag aaggaagaac aatcttccgg cggcggagca acctattttg 2820gctgtgggca
gggcaaaagg cagggaaaga tcggagactg tcacgaaggg aggggacctc 2880gggtcgagaa
aaggtacacc agcggcaagc ccacgtatag aggagaagaa ggaattggaa 2940agaggcgaga
agactaaaag cctggctcct tcaactgctc ctacgaagaa gaatgctaaa 3000gtcgatgttg
tggtgccgcc catcaacgtg ctgattgttg aagacaaccc catcaatcaa 3060aacattttga
gtatgttcct gagaaaaaag aagataaaga attcctcggc caaggatggc 3120gcagaagctg
ttgaaaagtg gaggactgga ggcttccatc tgattctgat ggatatccaa 3180ttgcccgtca
tggatggcat agctgctacc aaagagattc gtcgacttga acgtcacaat 3240aacattggcg
tttttccatc gactccagcg gccgaacttc ctcggggtca aaatgttgcg 3300gattctccac
caccatcttc tccatttcgc tcgtcagtta tcattgttgc cctgacagcc 3360tcgtccttgc
aaagcgacag agtagctgct ttggctgctg gctgtaatga cttcttgacc 3420aagcctgtgt
ctttgaaatg gttggacaaa aagattgtgg aatggggttg tatgcaggca 3480ttgattgatt
ttgacggctg gcgacgatgg aagagctccg ataccaagaa tcctagcgaa 3540actaagcagg
gcttctcagt gggccctcaa caggctgcta ggtcgcttgc tagcagacta 3600cgtattgaac
gcaaaggatc tcgatctccg gcagctccag tatcaacccc gcgactcaat 3660ttgcagtcgg
caaccccaga taggccagaa acccccccag attccacgtc acaaatgcca 3720aaggccccgc
ccgttgcagc ctctgacccc ccgttatctc ccaagtcgct gaacaagaca 3780gttaatgatg
tcttcgagca agcagacgct agactcgaaa atgcgcggga ggaacaagga 3840gtatcaagtc
aaaaggaaaa cacaagctta acagattcaa caaacaccac cattacgccc 3900tcaaagacct
atccggctcc tcccccatga
3930165076DNACryptococcus neoformans serotype A H99
strainC_region(1)..(5076)coding region sequence of TCO2 16atgatcttag
gaaccgacat cgacctgtcg tctataccaa cggcgtttct cgaggcttat 60cccttcccag
cggttgtgtt cgtgatcgat tctcccccta gcccaagacc gcggctccat 120tccagaaata
cagacacgac cattcggcga acggatggcc aaatatcgcc tcttacaggt 180cctccagtgc
aacagttcgc gtcagcgccc gtggtatggg gcaatcaacg atggcacgag 240ctggctcagg
ggaaaacaat tgcagagtgc gtggatgtgg cgtcacagaa caagctgcaa 300acttgggtgg
aaaatgacac cggtgacaag tcggagagtt tggccctgga cctgaaggtg 360ccgcaaggcg
tgactcttca tctggcaaag accatattgc cattaagtcc accgtcctcc 420tctcagtcac
tttgcatcct tatatcgcaa tatatcgata agccggaaag tttcgcgcca 480ccaatctcat
ctggagatat tcttttctct tctctatcgc gactctccca gactttttct 540cggtcatcct
ctttttcatc caaccctaga aaatcgattg atgtccctgc gtcactatcc 600gaacaccggg
gttctgctac atcgacaagt agcaatctgc gctcttcgat cgatttgact 660tcccctaatt
ctcaaccctc tccactaaac cgtgaacaaa gcacgtactt cacccatggc 720tccgcgacca
gagaagagcg accctcagta aggcgtagac ggtcaccgcc aatctcaatg 780acgaggccca
agcctcttga gagccatgct caagaatgct gggacttggt agagaatttc 840gactggtcaa
aaacagcatt agggccgaga gaacagtgga tggatgcgtt agatcctgtt 900ctggcaatca
catttgaatc cagaacggca gattgtgcct ggttagggcc tgatctagag 960ctagtttaca
ataaggcgta tcaagagctg gttgaccatc ccaatgcttt tggaaaacct 1020gcaagacaag
tttgggctac caattgggac tacttggaac ccctggtcaa gcgatgtctc 1080agtgggaccc
cggtctacaa ggacaacgac ccgcttttct ggcgtcgata cggcaatggt 1140cgacttctgg
aacattacca cacttggcga tatgtcccga taacgggcaa agatggctca 1200gtgcttggca
tcttcaacca gtcaattgag gtcaccgact cagtactgct agagaggcga 1260atgggcacga
ccagggaact ttcggaacac atgtcgttta ttcgtacaac tgaggacttt 1320tttagctcgg
ttgccgacgt ctttagtcag aaccctactg acataccgtt cgcactttgt 1380taccgggtcc
gacaagttga caccgatggg acatttgtcc atttggacgt ctcgcttcag 1440tcgtccgtcg
gtgtacccga aggccatccg tctgctccag atcaaattcc cgtcagcttc 1500ttaaatggta
acccttaccc tagcaatgtc gagcgatcat tttctcctgc tttctcaatc 1560gtttcaatcc
actcttcgag cagtcatcga gtctgtcacg tctctgaaga cactacacaa 1620tggcccatcg
ccaaagccct acaaaggcgg caatgtgtca tcatcgaaga atgttcgcaa 1680ttaatagaag
gatatcctat ccgtcgctgg gatgggcttc cattctcagc cattgtcgtg 1740cccatatgct
ctgaagggtc tcccgaaatc cctgacgccg ttgttattct tggtctcaat 1800gtgcgacgtt
gttttgacca tgaatacgat tcctggattc actctattcg gtcacaacta 1860tcttcggccc
tcgtgatggt caaggcgcgt gaagctgaac aaaagatggt tgaggaaagc 1920gcacgtatgg
agaaagcaaa agtcgcttgg ttcagaggag ccgcgcacga ccttcgtagt 1980ccattaaccc
tcgtcgctgg accgcttgcc gatgtgcttg attcggattt gaactcgagt 2040cagcgcacgg
ctttgaccgt tgcgcaacgc aatcttgatc gtttagtgcg cttggtcaac 2100gccctcatgg
atttctcgag ggtggaagct ggacgaatgg aaggacgatt tgttccgacg 2160aacttgagtc
aattcgtgac acagttggca gctcttttca agcctgcaat agaaagattg 2220gggttagaat
acgtactaga tgtccagcca agcgaggagc ttgttttcat cgatcctgtt 2280ctgtttgaga
ccgtggtatc aaaccttatt ggcaatgcgc tcaaatacac tgaaacgggt 2340tctatcactg
ttcgggtgca atacacggat tacgcagagg tctcggtcat cgataccggt 2400gtgggtatac
cgaaaaatga gctggcactg gtgaccgaat ggttccacag ggcaagtact 2460gccattcact
cgggaaccca gggaacagga ttgggactgg ctttggccaa ggaattgctc 2520aagttgcata
aaggagaatt gcttgtcgag tctcaaaccg ccaatgagtc aggaggtcct 2580catgggtcca
tttttacagc gaaaattcct cttgatttca agccctctcc atcggctcat 2640atcattccgt
ccgtcgaatc tcacaagacg tttggcaaat acagtaaagc cgtcgcagac 2700gaagccatgc
gctgggttgg ggactcagat gccgctagtg aggcgtacga catgtcgagc 2760ggtaccggag
tctcaagcgc tggtagtggc tctggaaaca cgaccacctt cggacccaag 2820tttgcagatg
cctttttgtt tgataagaac gacattgtgc ttattgtgga agacaatgtc 2880gacatgcgtg
aatacatacg acagcttttc gccccttatt gtaccgtact cgaagcttcc 2940aatggtgaac
aggcttacaa tatggctacc caaaaccctc ccaacctcat tttgtcggac 3000gtgctcatgc
ccaaattatc tggtatggag ctactacaaa ggatcagatc ccatcctgac 3060actcgcattg
tgcctatggt ccttatttcg gctattgctg gtgatgagtc tagggttgag 3120gctctgctaa
acggcgctga tgactatctt gccaagcctt tcaaacccaa ggaactcatc 3180gcgcgtgttc
acctgcacat gcaagttggc aagaaacgtg ccaagctcga agcgctatac 3240gcccaacgcg
aaacagaatt gacagctcta tctgactatt gtccgatcgg tatcttccga 3300ggagacaaat
atggccatat tgtttatgcg aacgcagctt ggcgtgcgca gagcggcctt 3360ttggtgggtg
accctaacga ttgggcatct tatgtgcacc cggattcgaa agcgcagctc 3420ttggaacaat
ggaatcagtg gttgaggggg gatttgaagg agttccgagc ggcttggaga 3480tggtctaatg
gcatccctgt caggagcatc ttggtccggt tagatgacgt caaggaaggg 3540ttttctgggt
taattgggtg cgtagtggat gtgtctcatg aagagagacg attaatcgaa 3600gctgaggaaa
gaagaaaaga ggcggaagag agtaaacatc agcaagaact ccttattgac 3660ttgacaagtc
atgaaattag gaccccggtg tcagcaatcc tgcagtgctc agatcttgtt 3720aaagagaatc
ttgtagctct gaaggaccag ttgagaggag cggggccaaa gggctttgtg 3780ccgagtcaag
aattactggc tgatcttgag caggatgtgg aagctttgga aagtatttat 3840cagtgcggtc
ttgtgcagga acgcattgcc ggagatgttc tttcgctggc tcgtatccaa 3900ctcgatatgc
tgagtttgca cgacattgac gtcaacttgc gccgagaagg caggaaagtt 3960tcgtccatct
ttgcatcgga agccaagatg aaggatatcg acctccaatt ggaatttgga 4020cctactatcg
aacagtccaa agtgctggcc atcaagacag atcccgtgag attaggccag 4080gtggtaacaa
atctcatttc caacgccatt cggtttacat cttcgagtga tgtccgaaag 4140attactatcc
aatacgacgt atcgtttgtc cctcctgccg atgactcttg cgccctccct 4200tcatctgttg
gcttgcccga catacttcct gtgaaagaga atactccact atggctgttt 4260gtcagtgtta
ccgattctgg acctggtatg acagagcaag agttatctgt tttgttccaa 4320aggtttgccc
agggcaataa gatgattcat actaagtatg gcggaagcgg tttgggactg 4380ttcatctgtc
gaaagattac agagcttctt ggcggtcgta tcgaagtgct cagccaagtc 4440gggcacggta
gtgttttccg attcttcatt aaaacgcgcg ctgtcgctcc tccgtccgcc 4500atcgctgctc
tcgtagaatc ctctcctctc aaaccggtat ccgccacttc accttcttcc 4560tcgttagcca
tgagccgatc gtcttctcgg agcacaaacg tcactacacc tatagagggt 4620ggtgggaccg
agcacgtgtt gatcgtggaa gataacctaa tcaatcagac tgtcctgaag 4680cgacagctcg
tcaaggcggg tttatcgtgc aacgtcgcga gtaatggcct tgaagctctt 4740aatgtcatcc
gtgaagtcca tcggcaacac cgacgcggtg ggccgaaccg taaaaggcta 4800tttgacgtgg
tattgatgga tctcgagatg ccagtgatgg atggtatcac cgccgtacga 4860gagatacgac
agtccgaggc cgcggggaca ttgggcagga atatggtgat tgccctgacg 4920gggaacgcga
gacaaggaca gattgatcat gccttggctt ctggatttga cgatgtcgtc 4980atcaaaccgt
atatcctggt agatttgctg aacaagatca aatctatgaa agttagaaaa 5040ttggagttgg
aaactgcgaa agctcaagaa gagtga
5076174254DNACryptococcus neoformans serotype A H99
strainC_region(1)..(4254)coding region sequence of SSK2 17atgtccaacc
caacctcccc ctcaaacccc tcagacaccg gcccgtcctc ggcgtccaac 60gtcacatcct
cgtcctcaaa gaccggacgc agatcagtgc gcctctttgc cccagacgag 120gaggacagct
cagacgagga cggcggcctc atcggcgtgc ccgcagagac cacattcaag 180gacgacgaga
tccctccttc caacccacgt tccgcctcct accccgggcc accggcacac 240acctccccca
cctctaaaat ctcaaccatc gtttcgtccg cttctgcagc ccagccaaaa 300cttgcacgtt
caataacata cgtcgcaccc aatgccgtct cctcccggcc ggcatatccc 360ctcaatcccg
cagggtcaga aaccttacac gcctcaggaa ggtcgtatac tgaccccgat 420ataggatact
ttagccacga tgcaggagat gatggttggg gctcagatga tgacgacgaa 480ttgagatccc
ctggctgggg catatcccat cataacatgg actctggagg caagacgaac 540gggtcaccac
agttgcctat aaagcccgcc gatgtcaccg aggatgaagg acaggaacgt 600ttagattggc
aaggcatgct ggaaagtgtc ctcaactcgg atgttctcaa ggtggaggaa 660caacgtatct
acaattccat gccgacagat tcattcagag aagagattgg aaagaccctt 720tggtggcaaa
tccgtgccaa actgcgtggg aggacagagg cggaggagaa gaaacgggtg 780caagagcgac
gagcgagagt ggtggacccg gtgctggaag agataaacga gttcaagtac 840gacccaaaaa
ataacccaga aggcgaagaa gacagtgatg gcgatccgca agacgcgact 900tcgactgctg
caccccaatc caaagctctc aatcaagtca acaccgttct cgccaaactt 960catgcaatca
aaggtcttta tcccaacctc gcagccatgc gagccgacaa ggttctctat 1020accgatgaaa
atttccgcaa acgcgccgac gcattgacct cttggtccat catcgtttca 1080tccctccaaa
cccagctcaa actcttgcaa aaatggacag gttccgatga gcttgacatc 1140accaagccca
acacgaccca cgagaaagca ttggtcggca agtacaagta tcactctatc 1200gacagcaagg
gtacgcccgg cagggatgca gccgatgact cgagtttcct cgatcgtgtg 1260ataaaagaag
ataaccttca acggacattc gagcgtcgag cgtttgtaga catgatcaac 1320ctcgtgcgca
acgccaagga gacggtcatc agctatctcc cccagttcca agaacaaaat 1380cttcccgatt
tccagtacga aatcgttcgt cttattggtt tccccggtcg acttatcatt 1440gaagctgtca
aggttcgttt ggatgctgca tcccgactac ttgacccgaa ccctatggtc 1500gtcgaagact
ttatcgaaaa ccttcgtcta tccatttcgc tcgccgtgct aatccggaaa 1560caatacgacg
aaatcatggc acccgatgcc gaggggagat ggaaaatccc gcattgcttg 1620ccgacagagt
acaatgatgt tctgctcgat gcgctgagga catttttcaa attgttgcat 1680tggagattac
gaggagtggg gaaagcgagt tattacaagg aaacagaagt gttggaagaa 1740gaggcgccgt
tcttgtatga agcggcggag gctattgtag gcggtgatat ggttgttgca 1800gagcagtatt
gcgcgttatc caacaagctc cttatacgtt cagcaaatta tcttgaccag 1860caacttcggg
taccaataca ttccccgtct cgcgacaagg aacgtggtga caaggagcgc 1920gatggctctt
cgtcttctca acgtaaccgt gacggccgtg atagctcgct gcccggccca 1980ccgaaacaca
tgaaagtcga agaactcttc tcatggtact ccaaactcct tgattccgct 2040cgtatgcgac
accgtaaaac ccaacgtttc tgtcgtaaac tcacccaacg attcgataat 2100tccgccgaat
attcaatcga ggagacggag atggacatgc tggtggagac attgcaagat 2160actggtcatt
tcttggtata taccgggaaa tttgaggcga atgggacgta tatcgttgcg 2220gatgggagtc
tctggggtca gccggacgat gtgagacatc tgttgaagag ggtgttttca 2280gtgacgattc
ctggatctcg agtccgtcca aggcagacaa cctcgcaagt atctgtcgga 2340ggtgcgagcc
cgtccaatgg tcaagtcgcg gcgcaacatg atcctgcaga tccgtacccc 2400gaggcagacg
attttgacga cgaagcgctc gcggcttaca tcctcctcat ctccccacgc 2460caaagttttg
tatggtccgg agcggtcatg acgctggatg tggattacat cgactatgaa 2520ctacctgata
accgagtcag attgatcgct gacggtccca ccaagcggtt agcgctgtgc 2580aaactttatt
tcaagcaagc gctcattcac cctgatacgg gcgaaacaat cgacttgcca 2640tgtgtggttg
aggctcaagc gcatttaccg accattcaga aacaacttgt caagattgct 2700aaatcgagtt
atcgtctttc agagtgcatt gtccagtctg caccactcgt ccgcaatgcg 2760ttcaggggca
aaccgggatc acaagagttg gtggagaatt ggtacagttt tgcgacagag 2820catgggacga
gagtgttgat ccatattgag cctagtgtat gggagcgatt caatcggttg 2880ttgatgcgtc
tggcgatcag ttggattagc tttatcagtc aagagtgtaa ccccacagac 2940cgcaagacgt
tccgatggac tgtggcagct ttgacctatg cgttcaacat gacgagaggg 3000agtaacattc
tcgcgcttga tcgatcagaa ttttcgcttt tgaggaggtc gagtatggag 3060gccaaaaagg
aggcagacag gattgaggcg atgaggaggt tacaacggct tcaagaaaac 3120ctggacgacg
aattcctgcc ccggactccg acagagtctg gcgatcaacc acgtatcgac 3180cgctctataa
ggctcacagt cgaagaacgt ctccgtctca ttgccgagct cgaagctcgt 3240cgtgacgagt
tggcacccgc acccgtcggt caagtccttg atgaagaagt ctctgaagac 3300cgtgcgttgg
tgttccttgc agcttccaaa tccaacattt ctatgcgatg gcagcaaggc 3360gcgtacatcg
gtggaggtgc atcgggaagc gtgtacttgg gatactcgtt gcaggataac 3420actgtgtttg
ctgtcaagat cttgccaacg gtggatctgc agagtagtcc ggcgttgtac 3480gaaagtatca
agcgagaatc ggatgtgatg agcttgttga gtcatccgaa tatcgttggt 3540ttccttgggt
tggaagtgca taggaacaga gtttgtcttt tccaagagta ctgtgaagga 3600gggtcgctgg
caggtatgct cgaatatggc aaaattgacg atgaggaagt cgttggagcg 3660tttacgatcc
agctgttacg cggccttgag tatctgcaca ccaaccgcat cgaacaccga 3720gatctcaaac
cagaaaatat tctcatcggc gccaattctg tcctcaagct ggccgacttt 3780ggtaccgcca
aaatcatcaa atccaacaag acgctcgccc gtacacgtgg tggcgcgcac 3840gccaagatgg
agggtcttga gggtacaccg atgtacatgg cgccagagat gatcaagaac 3900cagaggactg
gcaagctggg tgcttgtgat atctggggtt taggatgtat cgttttgcag 3960atgatcactg
gtaggaagcc atggagcttc ttggactttg ataatgaatg ggcaatcatg 4020ttccatcttg
gtgcgacaaa ggagccacct cctctacccg atcccaacga gatgtccgac 4080caaggtatcg
aattcattga tcaatgtctt tctttggatc cggaagcgag gccggtggcc 4140agcgagttat
tgcaagatga atggctggtt ccaatgttgg agcagatggt gagttgtctt 4200tcatgccgag
ctggagcaag aataccccga tatattggcg atgggccaaa gtga
4254181830DNACryptococcus neoformans serotype A H99
strainC_region(1)..(1830)coding region sequence of PBS2 18atgacagacc
ctacgccccc cgccctggac agtctctccc tggcagacaa ggcgcctact 60cccgaagaaa
gtcccgaaga cgccgctgaa cagcccaagc ccgcggcctc accgtccgca 120ggcacacccg
gccatgacgc ccaaagctca tccacctcgc ccccgcaacg ccctcagtcc 180atgcagacaa
atgacaaggc gccagataca tctgctccgg cttccaggcc ccaaccgcaa 240catgtccctg
catcggcacc tgcgcttccc tctaccaacc ccgtccgtcc acagccgggc 300gcccgtcctg
gagcggcgag gggtatgccc gcgcccatgg gtatgcgggc gcaagcaggc 360cgaggcgctg
gcggccccca gatgcagacc aagatgctgc ccagtttgca ggctaaaatg 420gacaagatcg
cggcgtctcg gcaagggcca cctccctcct ctggcatgca tgatccgaat 480gccacatcca
tgggcgccct cttacgctcc caagccctcc gcgcccccgg cgcatcgcaa 540gctcctcccg
gccccggacc ggcttcaggc cctttcggtc tcgccgctcg gcgcgcagct 600gctgggggcc
ctccgagacc gaatttgggt atgatgggta tgggtgcaag tgcgccgggt 660gcggttggac
ggggatcagg tctggcgggt agacgggggc cccctggagg actgacactg 720agtgggatga
agggtgcgat caaggatgag ggaaacaagt tttcagactt tcagggtgtc 780atggacccgt
ctggatcgct gagattctca aagaaggctg tcctgcatgc aaagggcgtg 840gactttgagg
atgggcaaag tttcaagatc aatatggatg agatcgaggt gcttggagaa 900ttaggaaagg
gcaattacgg ttctgtgcac aaagtcttcc accgtccgac aggcgtcacc 960atggccatga
aggaaatccg gttagaactt gacgattcca agctcaacgg catcattatg 1020gaactcgaca
tcctacaccg ggccgttgct cccgaaatag tcgaattcta cggtgcattc 1080accattgaat
catgcgtcta ctactgtatg gagtacatgg atgccggttc actcgactct 1140ctcaccggtg
gcggtgtggc ggccaaagat caaacaaagg atgaagaaaa cgatgcgaca 1200aaacgagtgc
cggaggatgt attgaggagg attacagcga gaatcgtgaa agggttgagg 1260ttcttgaagg
atgaattgca gatcatccat cgagacgtca aacccacaaa tgtgttaatc 1320aatggcaagg
gagaggtcaa gatgtgtgac tttggcgttt caggtcagct cgaaaagagt 1380ttggccaaga
ccaatatcgg ttgtcaatcc tacatggctc ccgaacgtat caagtctgaa 1440actgccaacc
agaatcctac atatactgtc tcttcagacg tctggtctgt cggtctgtcc 1500attgtcgagc
ttgccaaggg gtgttacccc tacccaccgg agacgtatgc gaatgtgttt 1560gcgcagttgc
aggcgattgt gcatggcact ccgccaacgt tgccacctgg gtacagcgat 1620aatgcgaatg
atttcgttgc caagtgtctt gagaaagatc ccaaccgacg accgacttat 1680gctcagctct
tagaacatcc tttcttggta gcggacaagg gcgcagaagt tgacatggtt 1740ggatgggtgg
aaggggcgtt gaagcgcaag gcagagaggg ggattgcgag cctgaatcct 1800atccaaccac
ctgtcccttt ggaaccataa
1830191098DNACryptococcus neoformans serotype A H99
strainC_region(1)..(1098)coding region sequence of HOG1 19atggccgatt
ttgtcaagct ctccatcttt ggaaccgttt ttgaggttac cacgcgttat 60gtcgacctcc
aacctgtcgg tatgggcgct ttcggtctcg tctgttccgc caaggatcag 120ctgtctggaa
cttctgtggc tatcaagaag attatgaagc ccttttcaac ccctgttctt 180tccaagagga
cttaccgaga gctcaagctt cttaagcact tgagacatga gaacattatc 240tctcttagtg
acattttcat ctctcctctc gaagatatct actttgtcac cgagctgctc 300ggtactgacc
ttcatcgact ccttacctct cgccctcttg agaagcaatt catccaatac 360ttcctttatc
aaatcctccg tggtctcaag tatgtccact ctgccggtgt agtccatcga 420gacttgaagc
cttcaaacat tctcgtcaac gagaactgtg acttgaagat ttgcgatttc 480ggccttgcga
ggatccaaga ccctcagatg actggttatg tttctacgag gtactaccga 540gcacccgaga
tcatgttgac atggcaaaag tatgatgtcg cggttgacat ttggagtacc 600ggctgtatct
ttgcggagat gctggagggc aagccattat tccccggaaa ggaccacgtg 660aaccaattct
caatcatcac cgaattgctc ggtactccgc cggacgatgt cattcaaact 720atcgcctctg
aaaacactct ccgtttcgtc cagagtctgc ccaagcgcga aaaggtccca 780ttctccacca
agttccccaa cgccgaccct gtgtctcttg atttgttaga gaagatgctc 840gtgtttgacc
ctcgtacccg tatatccgcc gctgaaggtc tcgcgcacga gtatcttgcg 900ccttaccatg
atcctaccga tgagcctgtt gccgccgagg tgtttgattg gagttttaac 960gatgcggatt
tgccggtgga tacttggaag gtgatgatgt atagtgaaat tcttgacttc 1020cacaacctcg
gagatatttc acagaacgaa gcagagggac ccgttactgg cgaagtcccc 1080gctgctcctg
ccagctaa
1098203273DNACryptococcus neoformans serotype A H99
strainC_region(1)..(3273)coding region sequence of ENA1(CNAG_00531.2)
20atgtcttctg agaaaggaca atcaaataca aacgagaaac aacttattaa ccgcgccgac
60actggcaaga ctgcagtgtc agactctcct ctccctttca aacctcatac cgctctctct
120ggcaagatcc tcgaggcttt agggagtaat gttacttctg gtctatcaga tgacgaagca
180tcaaggagac tccaacaata tggtcccaat aggctgaagc cccctgagag acctagtatt
240ctcaagatca tcgctaggca agtgggcaat gctatgactc ttgtcctcat cgctgccatg
300gcaacttcat tgggtaccat ggactggatc agcggtggcg ttattgcggc tctggttatc
360ctcaatgtat cagtgggagc ctacacagaa tggcaagccg aaaagaccgt ggccagtctc
420gagtctgttg gagctccgca agctactgta gtccgaactc gcaatggctc tcgcgaggct
480accgtcaaaa ttatccccgt agaggaagtc gtacccggtg acattattca actcaaaaat
540ggtgatattg ttcctgcgga cggaagaatc cttgacgggc acctgagtaa cttggaagct
600gacgaggctt tcctgactgg cgaaagtctg ccggttgcaa aacagactga gcctatcgat
660gaagaggact gtcctgttgg cgaccgtgtt tgtatggtct tttctggttc ccagatcacc
720aaaggtcgag ctcgtgccgt cattaccagc actggtatgg ggacagagat tggaaaaatt
780gctcaagctc ttgaatctaa agctaaaaat aagaaccgtg gatttgctgc tttctggtgg
840aaagtcaaag ttattttggg tgtcgaggag actactcctt tgcaaatcaa acttaataag
900ctcgcatact tccttttggc gtgtgccctc gtcatagccg tcattgttgt cgcctccacc
960ggttttaatg atgtccccct ctctattgcc acctacgctg tcgctgccgc cgtctccatt
1020ctccccgcct ctttgattgc agttgttagt ttgactttgg cgcgtgcgtc aactgattta
1080gcatctcgac atgctttggt ccgacgaatg gatgctattg aggctttagc tggtgttgag
1140aatgtgtgct cggacaagac cggtaccctt actgttggcc gcatggtagt tcgcaaagtc
1200tgggttcctg ctcttgactg gcgccccaat gaatttgctc ccctcgacac tagtggtggt
1260caagcatata gttttgagac cggatctgat cctttctatc ctcgtggtga agtcctggcc
1320gattcccaga agatcactgg gactgcggag accctcgatc tcaagcaacc tcgtgaccaa
1380tctgactctt cctcttccga ctctgacccc gatgaacgag acgtagagga acaagaacgg
1440gtcatccacg ttgaagacat ggaaaacaac cttcgagacc ttgctctctg tatttcgctt
1500tgtaatcaag cgactctcac tcgtcctgtc aaccaagacg gccaatggga agcaaacggt
1560gatcctaccg aaacggccct tcaagttgct gcacacaaac ttggtcatgg caagcccttt
1620cttactcatg ctgccaagcc aagccaccgt gcggattcta tccgatctgg tcacagttct
1680cgtccccttg ttgctggtat tcgtgggcac tttgttccga taattgagca tcctttcgat
1740tccaccgtca agcgaatgtc aatcgcttat aaatttgtga gcgaggatcc tcaggattct
1800cacatcctct gtctccttaa gggtgccatc gagcgtgtct ttgaacgatg caccaagatc
1860caaggacagc ccatcaccga agagcataag aagaatatca tggtcaaagt tgatgctctc
1920gccgctcaag gtcttcgggt cctcgctctt tgtggaaagc gacttcctgt cagcatggta
1980gacgaagtca aatccacccc tcgagacgca ttcgaagccg atttccattt cctcggtctt
2040gctggtatct tcgatccgcc cagaaaggaa tctgcaggcg ccgttgctga ttgtttcagg
2100gctggtatca cccctcgaat gttgacaggc gatcatcctg ctaccgctac agctatcgcc
2160ctcaacattg gtattctcga taagacgtac tcaaaggatt cagtcatgac gggtcagcag
2220tttgactctt tgagcgaaga cgaaattgat caactgcccg agttgcctct tgtcgttgct
2280cgctgcgccc ccgaaaccaa agttcgaatg gtcgatgcca ttcatcgacg aggacaaagc
2340actgtaatga ctggtgatgg tgtcaacgac tctcccgccc tcaagcgtgc tgatgtgggc
2400gttggcatgg gtactggttc cgatgttgcc aagcagtcag cgcgtatcgt cctcagtgat
2460gacaacttca gcaccatcat tcgggctatt aggaaaggtc gttctgtctt caagaacttg
2520tctaaattct tgctctactt gctttccggt aacttggctg aaatcatcgt cctcatgatt
2580ggtctcgctt tcaaggatga caatggtcag gctgttttcc ccctgtcacc tgttgccgct
2640ctttggatca acactctcgc tgccggacct cctgcccttg ccctaggtct tgaacctaca
2700gctatcgacg ccatggagca gggacccgag gtataccatc gaatcttcac tcttgaattt
2760tacgtcgatc tgatcttcta cggtttcctg atgggctcca tcagtttggt caacttcgtc
2820attgtactat ggggatacta tcctggagac ttaggtcgtc tttgtaacga agatgatccc
2880agcatctgtg atcccgtcta tcaggctcga gctgcctgtt ttgccaccct cgttattgtc
2940ctcatgattc atgctttgga gtgtaagcac ttgagcaaag ggttggccca aatcaatttg
3000cgtgacaaca aggtgttgct gtggtgtgtc gttgccctca gtctttccac tttccctgtc
3060gtgtacattc ctgtgatcaa taacaaggtg tttttgctca acggtcccag gtgggaatgg
3120ggtatcatct tcggcatgat cttggtgtat ctcagtgcta ctgagctcta caagtggatc
3180aaaagaattt ggatccgacg acatgccccc ccttccaaag gaccttccga caagaccctt
3240aggatggaga gtaccattgc tcctcctgtt tga
3273212751DNACryptococcus neoformans serotype A H99
strainC_region(1)..(2751)coding region sequence of NHA1 (CNAG_01678.2)
21atgactgctt tccacccctt tgaagtcaat gcccctcatc tcgcatacac gttcctcggc
60ggctttgtgg tcatctttgg catgatcagt ttgtttatca aagagaagct ctatgtcggc
120gaagcaccta tagcaactgt agtcggcatc atcattggtc cccattgcct caattttttc
180aatcctgcag gatggggtgg cggggaggaa gaggtcgcga gtgacgttac attggaattc
240actcgcgttg tcattgctat atccgtattc gccgtcggcg ttgaattgcc caaggcatac
300atgaagcggc actggcgatc gctcttcttc cttcttggcc cgtgcatggt gtggggatgg
360atgatctccg ccctgctgat ctggggcctg atacctgacc taacatttct cgcctcgctc
420gtagttgcgg cgggcgtcac ccccacagat cctatcttgg cccaggcagt tatcggaggc
480aagttcgccg ataaacatgt tcccgcccac atccgccacc tcctctccgc agaaagtgga
540agtaacgatg gggccgcctt tcccttcctc tacatcgccc tctacctcct actcgatgcg
600agcccaggcc atgccgtcgg agaatggttc tacatgactt gggtctacga aattattctt
660ggtgttatca tcggggccat cctgggattc tgcgcacgca agttgatgaa gttagcggag
720cgcaaacgtc tcattgatag gcagtcttac gtcgcccagt atgtcagtct ggcagtgctg
780tcgattggcg ttacaagttt gctcggcagt gacgatttgc tttctgcttt cgcttgcggt
840tgtgcttttg catgggacgg tttcttcaac aaagctacgg aggatgcggt gttctcgaac
900gttattgatc tacttttcaa ttgcgccgcc ttcatctata tcggcgctat cattcctttc
960aatcatttta acgatttgcc cgatctccga gtatggcgat tggttgtgtt ggctatcctc
1020attcttctag ttcgtcgtct gccttctata atagcgtgtt acaaattcgt tcccgatatc
1080aagacgttca gagaagctct ttttacggga tggttcgggc ctatgggcgt cggtgctgta
1140ttcatctcca ctcttgctcg gtcgtctttg ccagaagggg agcctgaaca gaatacagaa
1200gcggtggacc gcctaaaaga cgtcatcatg cctgtcacct tatttcttgt attgtcttca
1260atcgtaactc acggcatgtc aattccattt ttctctcttg gtcgccgggt ccattccatt
1320acttatactc gatcacgaaa tctttccatg gacacgcgag gcgatgagcc tgcctggaca
1380actcatgctc ggcgtattat tccaggccag gagatcattg tcaaccgtga tgacgacgac
1440gaagaaggcg acttgggtgt tagacggatg gacacactca cgagcgattc aaatggtcgt
1500atcagggaaa agattgagga agaagatagc ggagaaagta gctcatcccg aacaaggcag
1560ggagaaatga ttgaaatgac agaaaaacgt ggcccggctc gccatggtag ccaggccagc
1620cagggcgaag cggcggagga aggagagagg tggagaagtt cgggagaaga aagctctgat
1680cttgcgaatg accctgagac acagagagag gtggaagagg gaatggaaga ggtcgaagat
1740aaggaaggag gtggtagaag aacgcccccc ctggccaagt acagagaagg aaaccacctc
1800attgtggaga gaaaagtcaa ggacagtgac gaggttgaag tcgaggtcat ccgaaaccat
1860ttttccgaca acaagaaaac ggaaagtgac cgcttcactc atccccatcg cctcaagtca
1920cgagagcttg acgatttgct tcatcacctt cccaaaagcc tcgagcatgc tacttcacgg
1980gttcaaaatg gcggcaaaga tgcagttgat cgtctcggtc ttgggctgat ggctattaac
2040actccggaac cgtcaccatc gatcgaatcg cacggcggtc caaggcatga ttatgtcgat
2100ggcttggaga gaacgcagag cccagagggt cttgcagacg aggataggga tagcgagggc
2160cggggcgatg tgtcccatgg gggtgattat gaagaaaacg aggccgacta tgaggatgtt
2220ccgaacgaga ctcgtcggca aaggaggaag aaaatgaaac caccagcaat tgtcgtctct
2280cggcagaaca gcgccgggct cccgagacga tccatccgct ccaggctgtt cggccgacga
2340caacattctt ccaactctcc ctcccgtgcc gaagaaggct tagcccctcc caatccatct
2400cttcttgttc catcctcatc cccttcgcgt cctcaaaaca ttgctgcaga acccgagtcc
2460atactggcag aagattcgcg cggatcatcc tcaccttccc aatctcaaaa tcttgcgatc
2520cctctcacaa gaaccctttc agctagccga tcgtcgcctg cggtgcgctt cgccgacgat
2580gctagtcctt catcggacac agcgcctggg cagtcaaatt atggtactaa cgctccaggt
2640ttcaagaaga atccggcttt agcaatgtat cgatcggcca gtgtacaaag tacagggtcc
2700aacaaggatg ggcctagcgt atctttcaaa gaacctgaaa tcaagcgttg a
275122170PRTCryptococcus neoformans serotype A H99
strainPEPTIDE(1)..(170)an amino acid sequence of Ras1(CNAG_01672.2) 22Met
Ser Gly Asn Gly His Tyr Arg Arg Asp Gln Arg Leu Val Val Val1
5 10 15Gly Cys Gly Ala Phe Arg Glu
Tyr Asn Pro Thr Ile Glu Asp Ser Tyr 20 25
30Arg Lys Gln Val Val Val Asp Asn Glu Ala Thr Thr Leu Glu
Ile Leu 35 40 45Asp Thr Ala Gly
Gln Glu Glu Tyr Ala Ala Met Ala Asp Gln Trp Tyr 50 55
60Thr Phe Gly Ser Gly Phe Leu Leu Val Tyr Ser Leu Thr
Asp Arg Ser65 70 75
80Ser Phe Glu Glu Ile Gln Asn Phe His Arg Glu Ile Leu Arg Val Lys
85 90 95Asp Arg Asp Tyr Val Pro
Cys Val Ile Ile Cys Asn Lys Cys Asp Leu 100
105 110Gln Lys Tyr Arg Ser Val Gly Gln Leu Glu Gly Arg
Glu Leu Ala Arg 115 120 125Ser Val
His Ala Pro Phe Ile Glu Cys Ser Ala Ala Glu Arg Val Asn 130
135 140Val Asp Val Ala Phe Asn Glu Leu Val Lys Leu
Val Arg Lys Asp Glu145 150 155
160Arg Val Arg Ile Asn Tyr Asp Ile Ala Phe 165
17023227PRTCryptococcus neoformans serotype A H99
strainPEPTIDE(1)..(227)an amino acid sequence of Ras2(CNAG_04762.2) 23Met
Leu Phe Lys Ile Thr Val Leu Gly Asp Gly Gly Val Gly Lys Thr1
5 10 15Ala Ile Thr Val Gln Thr Cys
Lys Thr Tyr Asp Pro Thr Ile Glu Asp 20 25
30Cys Tyr Arg Lys Gln Trp Val Val Asp Glu Gln Pro Cys Leu
Leu Glu 35 40 45Val Leu Asp Thr
Ala Gly Gln Glu Glu Tyr Thr Ala Leu Arg Asp Gln 50 55
60Trp Ile Arg Glu Gly Glu Gly Phe Leu Ile Val Tyr Ser
Ile Thr Ser65 70 75
80Arg Pro Thr Phe Glu Arg Val Glu Arg Ile Val Glu Arg Val Leu Arg
85 90 95Val Lys Asp Glu Ser Gly
Leu Pro Leu Pro Pro Leu Ser Ser Ser Leu 100
105 110Ser Asn Asp Pro Tyr Gly Leu Ala Thr Ser Arg Ser
Thr Pro Thr Ser 115 120 125Ala Gly
Gly Gly Gly Gly Ser Gly Gly Gly Gly Met Trp Ala Ala Arg 130
135 140Val Pro Ile Val Ile Val Gly Asn Lys Lys Asp
Met Phe His Ser Arg145 150 155
160Glu Val Ser Thr Asp Glu Gly Ala Ser Leu Ala Arg Arg Leu Gly Cys
165 170 175 Glu Phe Tyr Glu
Ala Ser Ala Lys Thr Asn Ser Asn Val Glu Ala Ala 180
185 190Phe Lys Cys Leu Val Lys Lys Ile Lys Leu Ala
Lys Gln Gly Gly Val 195 200 205Ala
Val Gln Ala Glu Arg Val Gly Gly Arg Lys Lys Lys Gln Lys Cys 210
215 220Val Val Leu225241112PRTCryptococcus
neoformans serotype A H99 strainPEPTIDE(1)..(1112)an amino acid sequence
of Cdc24(CNAG_04243.2) 24Met Ser Val Ser Gly Pro Ile Ser Arg Arg Arg Ile
Gly Ser Val Ser1 5 10
15Gln Arg Gly Asn Glu Ser Leu Pro Gln Leu Asp Ile Gln Ser Ile Gln
20 25 30Met Pro Ser Asn Pro Gln Asn
Ala Leu Ala Leu Lys Thr Ala Ala Leu 35 40
45Ser Thr Ser Thr Arg Ser Leu His Gln Ile Cys Ser Ile Leu Lys
Lys 50 55 60Arg Leu Leu Cys Val Asp
Gly Phe Lys Ala Phe Leu Glu Gln Pro Pro65 70
75 80Asn Ala Glu Pro Leu Asp Val Val Ser His Met
Cys His Leu Phe Arg 85 90
95Leu Gly Ser Pro Leu Cys His Leu Tyr Asn Leu Leu Ile Pro Ser Phe
100 105 110Val Asp Cys Leu Ser Pro
Leu Tyr Ala Asp Leu Pro Ala Pro Ala Lys 115 120
125Ile Glu Tyr Asp Phe Pro Gln Phe Tyr Asp Ser Pro Asn Gly
Val Arg 130 135 140Asn Trp Ala Lys Arg
Pro Glu Asn Ala Lys Pro Cys Gln Arg Tyr Ile145 150
155 160Ala Ala Phe Cys Met Ala Met Lys Lys Arg
Ile Glu Glu Gly Arg Trp 165 170
175Thr Ser Asp Met Trp Ala Leu His Glu Leu Trp Gly Lys Ser Thr Gly
180 185 190Glu Asp Ile Glu Ala
Tyr Asp Ser Thr Gly Leu Met Lys Val Leu Ser 195
200 205Thr Val Glu Glu Met Leu Asp Asn Leu Pro Glu Ser
Ala Met Ser Pro 210 215 220Ile Ser Pro
Gln Thr Pro Phe Thr Ala Ser Gly Ser Ile Ala Gln Arg225
230 235 240Ala Gln Ser Arg Gln Ser Tyr
Asp Leu Pro Phe Ser Met Gly Gly Ile 245
250 255Gly Ser Gly Ala Ser Ala Val Ala Asn Met Ala Ala
Thr Met Asn Gly 260 265 270Gly
Val His Val Glu Thr Gly Pro Ser Glu Asn Ser Pro Thr Ala Ala 275
280 285Glu Met Gln Arg Gly Leu Ser Thr Ser
Leu Ala Glu Ala Asn Ala Phe 290 295
300Lys Ser Val Glu Glu Leu Val Ala Ser Glu Lys Ser Tyr Val Gln Glu305
310 315 320Leu Glu Ile Leu
Val Arg Cys Ser Gln Glu Met Leu Glu Ala Gln Leu 325
330 335Val Ser Thr Glu Thr Asn His Gln Ile Phe
Ser Asn Leu Ser Lys Ile 340 345
350Leu Asp Phe His Arg Lys Phe Leu Ile Lys Leu Glu Thr Glu Tyr Glu
355 360 365Pro Ile Gln Glu Arg Gly Pro
Gly Ala Trp Ala Glu Gly Val Trp Gly 370 375
380Arg Pro Phe Ile Leu Ser Glu Ala Glu Phe Asp Cys Tyr Gly Pro
Tyr385 390 395 400Cys Ala
Asn Tyr Leu Asp Ala Ile Thr Val Val Asn Glu Gln Met Pro
405 410 415Ile Leu Met Arg Gly Gln Glu
Leu Ser Pro Gly Glu Arg Pro Cys Leu 420 425
430Asp Pro Gln Arg Glu Leu Gln Ala Phe Met Ile Lys Pro Ile
Gln Arg 435 440 445Ile Thr Lys Tyr
Gly Leu Leu Leu Asp Ala Ile Leu His Ala Thr Ala 450
455 460Lys His Glu Tyr Pro Phe Arg Pro Glu Leu Glu Glu
Ala Ser Ala Ala465 470 475
480Val Lys Arg Ile Ala Ala Gly Ile Asn Glu Val Thr Asp Phe Lys Ala
485 490 495Lys Gln Ala Thr Val
Arg Glu Leu Ile Glu Arg Val Asp Asp Trp Lys 500
505 510Gly His Asp Val Asp Lys Phe Gly Pro Leu His Ile
Asp Asp His Phe 515 520 525Thr Val
Thr Lys Ala Asp Gln Pro Arg Glu Tyr His Val Phe Leu Phe 530
535 540Glu Lys Met Met Leu Cys Cys Lys Glu Ile Thr
Pro Glu Lys Lys Lys545 550 555
560Gln Asn Lys Asn Ser Ser Met Leu Arg Lys Asp Arg Gly Thr Ser Lys
565 570 575Ser Gly Pro Leu
Asp Lys Lys Lys Leu Ala Leu Lys Gly Arg Ile Phe 580
585 590Val Ser Asn Ile Lys Glu Ala Thr Ile Leu Pro
Thr Glu Pro Gly Asp 595 600 605Ala
Tyr Gly Val Ala Arg Leu Leu Ile Gly Trp Thr Ile Pro Leu Arg 610
615 620Asn Gln Asp Gly Tyr His Asp Asp Gln Glu
Asp Ser Phe Val Met Ile625 630 635
640Gly Lys Ser Glu Glu Gln Met Arg Lys Trp Ser Glu Lys Val Met
Glu 645 650 655Leu Ala Asn
Asn Glu Arg Lys Ile Gln Glu Asp Met Arg Ala Ala Arg 660
665 670Met Lys Ala Gly Arg Phe Ser Gly Ser Glu
Arg Gln Tyr Tyr Gln His 675 680
685Ser Phe Phe Gly Pro Pro Thr Pro Ala Thr Glu His Pro Pro Met Thr 690
695 700Pro Phe Asn Met Pro Pro Leu Pro
Asn Gly Ser Ala Thr Pro Tyr Tyr705 710
715 720Ser Glu Asp Glu Asp Pro Glu Gly Leu Arg Ser Gly
Arg Thr Thr Pro 725 730
735Ser Ile Leu Gly His His Pro Tyr Ala Tyr Ser Gly Gln Pro Ser Ala
740 745 750Ser Arg Arg Val Gln Ser
Gln Gln Ser Met Thr Ser Val Met Pro Thr 755 760
765Glu Leu Arg Ala Arg Ala Met Thr Glu Asp Gln Tyr Gly Pro
Ser Met 770 775 780Thr Gln Trp Arg Thr
Gln Gln Pro Met Ala Pro Pro Leu Pro Arg Leu785 790
795 800Thr Ser Ala Met Ser Gly Met Ser Val Ala
Ser Glu Leu Ser Phe Gly 805 810
815Ser Gly Pro Asn Asn Ile Gly Ile Arg Thr Gly Met Val Arg Gln Met
820 825 830Ser Ser Thr Arg Leu
Pro Arg Ala Thr Glu Val Asp Glu Ala Glu Glu 835
840 845Asn Pro Val Asp Thr Arg Asp Ser Tyr Gly Arg Tyr
Gly Ser Leu Arg 850 855 860Gly Ile Met
Arg Ala Pro Ser His Ala Met Pro Ser Val Pro His Pro865
870 875 880Pro Pro Leu Arg Asn Arg Ser
Ala Ser Ser Pro Asn Val Tyr Gln Gln 885
890 895Pro Thr Val Thr Gly Ala Ala Ser Leu Pro Tyr Thr
Ala Gly Pro Asn 900 905 910Gly
Thr Trp Thr Thr Ser Pro Leu Ala Ser Thr Leu Gln Met Ser Thr 915
920 925His Pro Tyr Val Gln Ser Thr Pro Val
Pro Gly Phe Gly Pro Ser Ser 930 935
940Ser Thr Thr Leu Val Gly Gly Thr Ala Tyr Phe Asn Lys Arg Met Ser945
950 955 960Asn Glu Lys Arg
Ser Ser Gly Glu Ser His His Ser Thr Thr Thr Thr 965
970 975Asp Thr Ser Asp Gln Thr Ser Pro Ala Thr
Pro Tyr Gly Ser Gly Asn 980 985
990Gly Asp Ile Arg Gly Pro Ser Arg Gln Asn Ser Gly Asp Asn Val Ser
995 1000 1005Gly Ser Val Leu Val Lys
Leu Arg Phe Gly Asn Asp Gln Phe Ile 1010 1015
1020Leu Gly Val Ser Gln Gly Ile Asp Phe Ile Thr Leu Tyr Gln
Lys 1025 1030 1035Ile His Lys Lys Ile
Arg Leu Cys Ser Ser Ser Asn Arg Pro Thr 1040 1045
1050Asn Glu His Asp Lys Leu Gln Ile Arg Tyr Val Asp Asn
Asp Gly 1055 1060 1065Asp Glu Ile Gln
Val Lys Phe Asp Ser Asp Val Glu Leu Met Phe 1070
1075 1080Glu Asp Ala Arg Asp Gln Ala Gly His Ile Asn
Leu Ile Ala Arg 1085 1090 1095Trp Ala
Glu Asp Arg Arg Gly Thr Pro Gln Gly Glu Ile Tyr 1100
1105 111025432PRTCryptococcus neoformans serotype A H99
strainPEPTIDE(1)..(432)an amino acid sequence of Gpa1(CNAG_04505.2) 25Met
Gly Gly Cys Met Ser Thr Pro Glu Ala Pro Arg Lys Ala Ala Glu1
5 10 15Thr Lys Gln Val Pro Ser Thr
Ser Thr Thr Ser Arg Pro Pro Gln Ala 20 25
30Ser Thr Ser Ala Thr Ala Thr Ala Ala Gly Ala Ser Thr Ser
Pro Pro 35 40 45Asn Gly Thr Ala
Asn Gly Ile Lys Gly Asp Thr Thr Ala Ala Asn Arg 50 55
60Thr Gly Ala Ser Ala Gly Gln Gly Ile Val Ala Ala Leu
Ala Ser Thr65 70 75
80Glu Pro Pro Gly Ala Gln Asp Ser Lys Gly Asn Lys Asp Arg Ser Asn
85 90 95Gln Ile Asp Arg Gln Leu
Glu Asp Asp Gln Lys Lys Phe Arg Lys Glu 100
105 110Cys Lys Ile Leu Leu Leu Gly Ser Gly Glu Ser Gly
Lys Ser Thr Ile 115 120 125Val Lys
Gln Met Lys Ile Ile His Gln Asn Gly Tyr Ser Lys Asp Glu 130
135 140Leu Leu Ser Phe Arg Gly Val Ile Tyr Lys Asn
Val Leu Asp Ser Ala145 150 155
160Gln Ala Leu Ile Met Ala Met Arg Lys Ile Gly Val Asp Pro Glu Asp
165 170 175Ala Asn Asn Arg
Ser Tyr Ala Asp Arg Ile Leu Glu Tyr Arg Met Asp 180
185 190Ala Asp Leu Asn Ala Val Ile Pro Ser Glu Ile
Leu Tyr Asn Ile Glu 195 200 205Ser
Leu Trp His Asp Pro Val Ile Pro Ser Val Met Asp Arg Ser Ser 210
215 220Glu Phe Tyr Ile Met Asp Ser Ala Thr Tyr
Phe Phe Ala Asn Ile Arg225 230 235
240Lys Ile Ala Gly Pro Asp Tyr Val Pro Asp Glu Ala Asp Val Leu
Arg 245 250 255Ala Arg Thr
Lys Thr Thr Gly Ile Ser Glu Thr Arg Phe Asn Met Gly 260
265 270Gln Leu Ser Ile His Met Phe Asp Val Gly
Gly Gln Arg Ser Glu Arg 275 280
285Lys Lys Trp Ile His Cys Phe Glu Ala Val Thr Ser Ile Ile Phe Cys 290
295 300Val Ala Leu Ser Glu Tyr Asp Gln
Val Leu Leu Glu Glu Ser Gly Gln305 310
315 320Asn Arg Met Gln Glu Ser Leu Val Leu Phe Glu Ser
Val Ile Asn Ser 325 330
335Arg Trp Phe Leu Arg Thr Ser Val Ile Leu Phe Leu Asn Lys Ile Asp
340 345 350Leu Phe Lys Gln Lys Leu
Pro Lys Val Pro Leu Val Gln Tyr Phe Pro 355 360
365Glu Tyr Thr Gly Gly Ala Asp Ile Asn Lys Ala Ala Lys Tyr
Ile Leu 370 375 380Trp Arg Phe Thr Gln
Thr Asn Arg Ala Arg Leu Ser Val Tyr Pro His385 390
395 400Leu Thr Gln Ala Thr Asp Thr Ser Asn Ile
Arg Leu Val Phe Ala Ala 405 410
415Val Lys Glu Thr Ile Leu Gln Asn Ala Leu Arg Asp Ser Gly Ile Leu
420 425 430262250PRTCryptococcus
neoformans serotype A H99 strainPEPTIDE(1)..(2250)an amino acid sequence
of Cac1(CNAG_03202.2) 26Met Pro Met Phe Arg Arg Ser Ala Ser Ser His Ser
Thr Ser Asp Ala1 5 10
15Ala Ala Pro Pro Thr Ile Thr Asn Val Thr Glu Gly Ser Pro Val Ser
20 25 30Ser Gly Ser Ile Thr Arg Gln
Lys Arg Ser Arg Ser His Gly Gly Gly 35 40
45Gly Ser Pro Ala Ser Thr Phe Ser Ser Arg Phe Gly Ile Ser Arg
His 50 55 60Leu Ser His Gln Phe Gln
Gln Pro His Glu Gln Glu Gln Gly Gln Val65 70
75 80Pro Pro Pro Glu Lys Pro Ala Thr Arg Glu Val
Pro Val Ser Leu Glu 85 90
95Pro Glu Ile Tyr Gly Ser Glu Ala Gly Glu Asp Ser Asn Phe Gly Gln
100 105 110Glu Pro Ala Gly Glu Gln
Leu Ile Pro Glu Thr Arg Ser Ser Ser Arg 115 120
125Gln Ser Arg Arg Ser Ser Arg Glu Leu Ser Val Gln Thr Ile
Glu Pro 130 135 140Ser Ser Asp Asp Glu
Ile Arg Ser Pro Glu Lys Arg Arg Val Ser Pro145 150
155 160Leu Met Lys Arg Leu Gly Glu Glu Pro Pro
Phe Met Leu Pro His Pro 165 170
175Ser Leu Ser Asp Ser Thr Tyr Gln Ala Tyr Pro Gly Val Val Ile Gly
180 185 190Ser Phe Ala Gly Gly
Gly Pro Asp Thr Leu Phe Gly Asn Gly Met Gln 195
200 205Leu Glu Gly Thr Val Asp Asp Ile Leu Asp Pro Asn
Ala Ala Arg Gln 210 215 220Glu Asp Arg
Gly Asn Gln Val Pro Ala Gly Ala Asn Ile Ala Pro Trp225
230 235 240Leu Met Asp Asp Gly Pro Pro
Ser Arg Ser Glu Asn Pro Ser Pro Ala 245
250 255Leu Ser Glu Thr Gln Glu Arg Pro Val Lys Gly Pro
Ala Ala Ala Leu 260 265 270Arg
Glu Lys Asp Pro Arg Lys Thr Ser Thr Val Leu Asn His Phe Ser 275
280 285Ser Val Pro Ser Leu Pro Lys Ile Arg
Arg His Gly Arg Ala Ala Thr 290 295
300Thr Thr Pro Asp Gln Thr Pro Arg Gly Ser Thr His Ser Gln Ser Asn305
310 315 320Leu Ala Ser Ser
Ser Ser Ser Leu Asn Asn Glu Ser Arg Glu Ser Arg 325
330 335Ala Gly Ser Asp Asp Ser Ile Gln Thr Thr
Leu Thr Gln Lys Gly Arg 340 345
350Arg Gln Ser Pro Gly Glu Trp Gly Gln Ala Ser Ala Val Pro Pro Pro
355 360 365Ser Lys Gly Thr Arg Pro Gly
Arg Phe Gly Ser Thr Ala Ser Ile Ile 370 375
380Ser Gly Thr Gly Ser Val Gly Glu Lys Lys Lys Ser Leu Phe Gly
Gly385 390 395 400Leu Leu
Lys Arg Lys Thr Asn Pro Asn Leu Ser Leu Asn Pro Ile Ser
405 410 415His Asp Phe Thr Thr Ser Glu
His Arg Gly Ser Ala Gly Ser Ile Pro 420 425
430Leu Ser Ala Ser Ser Ser Lys Leu Ser Ser Cys Ser Leu Ser
Ser Leu 435 440 445Pro Ser Lys Ser
Pro Pro Phe Thr Ser Pro Pro Glu Ala Phe Ser Arg 450
455 460Gln Phe Leu Pro Ala Asn Tyr Val His Glu Gly Ala
Val Ser Pro Leu465 470 475
480Gln Glu Ile Ser Glu Ser Pro Phe His Leu Asp Met Asn Leu Asp Asp
485 490 495Met Glu Gly Ile Ile
Asp Pro Ala Lys Ala Gly Leu Pro Ser Thr Val 500
505 510Ala Tyr Arg Pro Ser Ala Ser Ser Glu Val Thr Thr
Asp Ser Ser Ala 515 520 525Ser Glu
Ser Met Arg Leu Glu Glu Ala Leu Asn Gln Thr Ser Ser Phe 530
535 540Gly Thr Ser Val Ser Gly Ala Ser Arg Gly Ser
Asp Gly Thr Lys Met545 550 555
560Pro Gly Arg Ile Val Leu Gly Glu Ala Glu Arg Leu Pro Thr Pro Phe
565 570 575Thr Gly Thr Asp
Pro Phe Gln Gln His Arg Glu Ser Ala Ser Thr Thr 580
585 590Gly Ser Asp Gly Lys Pro Phe Ser Pro Pro Ser
Pro His Thr Leu Ser 595 600 605Pro
Lys His His Leu Pro Ser Ser Ser Ala Asn Gln Pro Arg Arg Pro 610
615 620Ser Ala Leu Arg Asn Val Glu Thr Gly Gln
Val Asp Glu Thr Pro Gln625 630 635
640Leu Ser Ala Ser Glu Gly Ser Ile Gln Pro Ile Ser Pro Ser Trp
Ala 645 650 655Gly Gly Ser
Gly Ile Thr Val Phe Asn Asp Pro Phe Ser Thr Ser Arg 660
665 670Gln Arg Gln Glu Gln Pro Pro Asn Ser Ala
Gly Leu Ser Pro Ser Thr 675 680
685Thr Ala Tyr Pro Ser Ala Val Thr Gln Pro Gly Pro Ser Thr Ala Arg 690
695 700Phe Leu Ile Thr Ala Gly Ser Thr
Thr Pro Ser Ala Ala Trp Ala Ala705 710
715 720Pro Glu Ser Trp Gly Val Glu Ala Asp Glu Ala Pro
Ala Glu Glu Ile 725 730
735Thr Ser Ser Asp Glu Asp Asp Trp Ala Gly Leu Gly Val Glu Glu Val
740 745 750Ala Ser Ala Ser Pro Thr
Ser Asp Thr Leu Pro Ser Pro Pro Thr Ser 755 760
765Pro Arg Ala Ser Ser Leu Pro Ser Pro Lys Arg Ala Pro Pro
Phe Gly 770 775 780Phe Lys Ser Gln Gln
Arg Ala Lys Pro Gly Thr Ser Gly Thr Thr Asp785 790
795 800Ser Thr Thr Ser Ala Ala Gly Arg Arg Lys
Gly Lys Arg Val Gly Ser 805 810
815Ser Gly Arg Pro Ala Thr Gly Arg Pro Gly Thr Ser Gly Ser Ala Tyr
820 825 830Asn Pro Ser Ser Leu
His Trp Ile Arg Ile Tyr Arg Ala Asp Lys Ser 835
840 845Tyr Met Leu Tyr Asn Leu Pro Leu Asn Thr Ser Thr
Gly Glu Leu Leu 850 855 860Ala Leu Leu
Ala Ala Gln Ala Glu Gln Gly Met Val Arg Gly Lys Asn865
870 875 880Val Ala Ile Asn Met Lys Leu
Tyr Ile Cys Glu Arg Gly Gln Asn Arg 885
890 895Met Leu Leu Pro Ser Glu Lys Pro Leu Thr Ile Gln
His Arg Arg Leu 900 905 910Leu
Gln Leu Gly His Thr Glu Ala Asp His Leu Asp Glu Leu Gly Lys 915
920 925Asn Asp Met Ala Val Leu Cys Arg Phe
Ile Tyr Gln Ala Pro Ile Leu 930 935
940Pro Ile Met Asp Pro Glu Glu Glu Ser Ser Tyr Asp Ser Phe Glu Phe945
950 955 960Ile Asp Ile Ala
Ser Arg Asp Leu Gln Thr Ile Pro Ile Phe Leu His 965
970 975Leu His Ala His Asp Ile Ile Ile Leu Asn
Ile Ser Lys Asn Pro Met 980 985
990Thr Asp Ile Pro Leu Asp Phe Ile Gln Ala Cys Thr Ser Leu Lys Glu
995 1000 1005Leu Arg Met Ser Asn Met
Ala Leu Lys Arg Val Pro Ile Ser Ile 1010 1015
1020Arg Ala Ser Thr Thr Leu Ala Arg Leu Asp Val Ser Cys Asn
Arg 1025 1030 1035Ile Ala Asp Leu Glu
Ser Val Ala Leu His Glu Val Glu Thr Leu 1040 1045
1050Val Ser Leu Lys Val Gln Asn Asn Lys Leu Thr Ser Met
Pro Ser 1055 1060 1065Tyr Phe Ala Gln
Met Lys Ser Leu Lys Tyr Leu Asn Ile Ser Asn 1070
1075 1080Asn Lys Phe Glu Thr Phe Pro Ser Val Val Cys
Glu Met Ser Asn 1085 1090 1095Leu Val
Asp Leu Asp Val Ser Phe Asn Asn Ile Ala Glu Leu Pro 1100
1105 1110Ala Lys Met Ser Asp Leu Lys Ser Leu Glu
Lys Leu Gly Leu Tyr 1115 1120 1125Ser
Asn Asp Ile Ser Lys Phe Pro Glu Ser Phe Cys Thr Leu Ala 1130
1135 1140Asn Leu Arg Ile Leu Asp Val Arg Arg
Asn Lys Ile Thr Asp Leu 1145 1150
1155Ser Ala Val Tyr Ala Leu Pro Asn Leu Ala Thr Leu Gln Ala Asp
1160 1165 1170Asn Asn Asn Ile Val Thr
Leu Asp Ala Gln Leu Gly Ala Asn Val 1175 1180
1185Arg Gln Phe Ser Val Pro His Asn Ser Val Thr Arg Phe Thr
Leu 1190 1195 1200Ala Pro Pro Pro Asn
Met Ala Val Val Thr Tyr Met Leu Thr Asn 1205 1210
1215Leu Asp Leu Ser His Gly Lys Ile Ser Thr Leu Ala Asp
Glu Ala 1220 1225 1230Phe Ser Gly Leu
Thr Asn Leu Val Thr Leu Asn Leu Asn Phe Asn 1235
1240 1245Gln Phe Thr Lys Leu Pro Ala Thr Leu Gly Arg
Leu Thr Ser Leu 1250 1255 1260Glu Val
Phe Ser Cys Thr Asp Asn Met Leu Asn Leu Val Pro Ala 1265
1270 1275Gly Phe Gly Lys Leu Gln Arg Leu Arg Met
Ile Asn Leu His Asn 1280 1285 1290Asn
Asn Leu Lys Ser Leu Pro Glu Asp Leu Trp Ala Cys Gly Ala 1295
1300 1305Leu Glu Val Phe Asn Ala Ser Ser Asn
Leu Leu Asp Ser Phe Ile 1310 1315
1320Pro Pro Pro Ala Asp Ile Glu Ser Val Val Gly Arg Val Gly Ser
1325 1330 1335Gly Thr Ser Gln Thr Ser
Asn Gly Arg Lys Lys Tyr Ser Val Pro 1340 1345
1350Pro Ile Gly Leu Ser Ile Arg Lys Leu Phe Leu Ala Asp Asn
Arg 1355 1360 1365Leu Asn Asp Asp Val
Phe His Trp Ile Ser Leu Met Pro Ser Leu 1370 1375
1380Arg Ile Ile Asn Leu Ser Phe Asn Asp Ile Tyr Glu Leu
Thr Asn 1385 1390 1395Leu Pro Ser Glu
Asp Leu Glu Lys Leu Gln Ser Leu Lys Val Leu 1400
1405 1410His Leu Asn Gly Asn Lys Leu Gln Thr Leu Pro
Ser Glu Leu Gly 1415 1420 1425Ala Ile
Lys Thr Leu Gln His Leu Asp Val Gly Ser Asn Val Leu 1430
1435 1440Lys Tyr Asn Ile Ala Asn Trp Pro Tyr Asp
Trp Asn Trp Asn Trp 1445 1450 1455Asn
Thr Ser Leu Arg Tyr Leu Asn Leu Ser Gly Asn Lys Arg Leu 1460
1465 1470Glu Ile Lys Pro Thr Ser Ala His Glu
Met Ser His Ala Ser Ser 1475 1480
1485Phe Arg Lys Glu Leu Ser Asp Phe Thr Ala Leu Thr Gln Leu Arg
1490 1495 1500Val Leu Gly Leu Met Asp
Val Thr Leu Arg Ile Pro Ser Leu Pro 1505 1510
1515Asp Glu Ser Glu Glu Lys Arg Val Arg Thr Ser Phe Ser Asp
Ile 1520 1525 1530Asn Asn Met Ala Tyr
Gly Ile Ser Asp Met Leu Gly Ser Ile Asp 1535 1540
1545Asn Leu Ala Met Phe Asp Leu Val Val Pro His Phe Arg
Gly Lys 1550 1555 1560Glu Asn Glu Cys
Leu Phe Gly Met Phe Gly Arg Val Thr Thr Thr 1565
1570 1575Leu Gln Gly Gly Lys Ile Ala Lys Tyr Val Gln
Glu Ile Phe Ala 1580 1585 1590Glu Thr
Leu Thr Ala His Leu His Gln Leu Glu Pro Gly Glu Glu 1595
1600 1605Pro Ser Glu Ala Leu Arg Arg Thr Phe Leu
Leu Gly Asp Arg Lys 1610 1615 1620Ala
Phe Glu Phe Phe Ser Asp Lys Leu Gln Leu Glu Lys Glu Arg 1625
1630 1635Lys Pro Ser Trp Thr Ser Phe Ala Ser
Phe Asp Ser Met Phe Arg 1640 1645
1650Gly Trp Thr Pro Gly Val Asn Ser Val Leu Arg Thr Gly Ala Ser
1655 1660 1665Gly Ala Val Val Tyr Leu
Val Asp Lys Val Leu His Val Gly Ser 1670 1675
1680Ile Gly Asp Thr Leu Val Val Leu Ser Arg Lys Gly Asp Ala
Glu 1685 1690 1695Leu Leu Ser Lys Arg
His Asp Pro Thr Asp Arg Glu Glu Ser Ala 1700 1705
1710Arg Ile Arg Lys Ala Glu Ala Trp Val Ser Thr Lys Gly
Phe Val 1715 1720 1725Asn Asp Asp Lys
Asp Leu Asp Ile Ser Arg Ala Phe Gly Tyr Trp 1730
1735 1740His Glu Cys Pro Ala Val Asn Ala Ala Pro Glu
Ile Arg Thr Arg 1745 1750 1755Arg Leu
Gln Glu Ser Asp Glu Phe Val Ile Ile Gly Asn His Ala 1760
1765 1770Leu Trp Gln Phe Cys Ser Tyr Gln Thr Ala
Val Asp Ile Ala Arg 1775 1780 1785Thr
Glu Arg Asp Asp Pro Met Met Ala Ala Gln Lys Leu Arg Asp 1790
1795 1800Phe Ala Ile Ser Tyr Gly Ala Glu Gly
Asn Val Met Val Met Val 1805 1810
1815Val Asn Val Ser Asp Leu Phe Leu Ala Lys Gly Gly Arg Ala Arg
1820 1825 1830Gly Pro Ser Lys Gln Thr
Ala Thr Asp Ala Asn Ala Asp Val Glu 1835 1840
1845Gly Tyr Ala Val Ala Lys Arg Gln Val Arg Arg Arg Tyr Asp
Glu 1850 1855 1860Val Gly Asp Arg Thr
Leu Asn Arg Leu Gln Gln Glu Ile Glu Pro 1865 1870
1875Pro Val Gly Gln Val Ala Ile Val Phe Thr Asp Ile Val
Asn Ser 1880 1885 1890Thr His Leu Trp
Glu Thr Asn Pro Ala Met Pro Thr Ala Ile Lys 1895
1900 1905Met His His Asn Leu Met Arg Arg Gln Leu Arg
Leu Asp Gly Gly 1910 1915 1920Tyr Glu
Val Lys Thr Glu Gly Asp Ser Phe Met Val Ser Phe Gln 1925
1930 1935Ser Val Ala Ser Ala Leu Leu Trp Ser Phe
Asn Cys Gln Ile Gly 1940 1945 1950Leu
Leu Gln Gln Glu Trp Pro Arg Glu Leu Leu Glu Ala His Asp 1955
1960 1965Gly Lys Val Val Tyr Asp Ser Asn Gly
Thr Ile Val Gln Arg Gly 1970 1975
1980Leu Arg Val Arg Met Gly Val His Trp Gly Ala Pro Glu Cys Glu
1985 1990 1995Lys Asp Pro Ile Thr Arg
Arg Met Asp Tyr Tyr Gly Pro Met Val 2000 2005
2010Asn Arg Ala Ala Arg Ile Asn Ala Ser Ala Asp Gly Gly Gln
Leu 2015 2020 2025Met Ala Ser Gln Asp
Val Leu Asn Glu Ile Ala Pro Leu Met Glu 2030 2035
2040Tyr Leu Asn Ser Ser Asp Glu Gln Val Leu Asn Asp Leu
Gln Gly 2045 2050 2055Asp Leu Lys Arg
Glu Val Met Glu Leu Arg Arg Ile Gly Leu Glu 2060
2065 2070Val Arg Asp Met Gly Asp Arg Lys Leu Lys Gly
Leu Glu Val Pro 2075 2080 2085Glu Arg
Leu His Leu Leu Tyr Pro Lys Thr Leu Ala Gly Arg Leu 2090
2095 2100Glu Ile Ser Asn Glu Ile Arg Ala Glu Val
Glu Val Asn Asp Ala 2105 2110 2115Arg
Lys Ser Ala Glu Arg Gln Arg Ser Val Asp Ile Asp Gln Val 2120
2125 2130Tyr Gln Leu Ser Asp Ile Ala Leu Arg
Leu Glu Ala Val Cys Cys 2135 2140
2145Tyr Asn Pro Thr Pro Ser Ser Pro Gly Asp Thr Pro Thr Ala Gly
2150 2155 2160Val Met Arg Leu His Pro
Pro Ala Ser Tyr Leu Gly Pro Ser Ile 2165 2170
2175Arg Glu Asp Met Asn Asp Glu Glu Leu Trp Thr Ile Ile Glu
Ser 2180 2185 2190Leu Val Gly Arg Ile
Glu Asn Val Met Ser Thr Leu Tyr Leu Lys 2195 2200
2205Asn Phe Gly Glu Phe Ser Ala Val Leu Ala Ala Leu Glu
Ser Ala 2210 2215 2220Thr Lys Ile Asp
Gln Lys Leu Ile Val His Ala Leu Ala Leu Met 2225
2230 2235Asn Glu Ala Met Gly Lys Asp Glu Glu Asn Ala
Ile 2240 2245 225027499PRTCryptococcus
neoformans serotype A H99 strainPEPTIDE(1)..(499)an amino acid sequence
of Aca1(CNAG_05218.2) 27Met Ala Thr Ser Gln Gly Ile His Ser Ile Ser Thr
Ile Leu Arg Leu1 5 10
15Glu Asp Ile Ala Val Thr Gln Ala Pro His Gly Ser Ser Val Lys Ser
20 25 30Pro Ala Pro Ala Ser Asp Thr
Pro Thr Gly Val Ala Pro Pro Ala Pro 35 40
45Pro Pro Pro Pro Ala Pro Glu Ala Pro Lys Ala Ala Glu Met Thr
Gln 50 55 60Pro Ala Gln Ser Pro Ala
Ser Lys Val Tyr Gln Asp Glu Ile Ile Asn65 70
75 80Gly Ala Leu Asn Asp Phe Leu Ser Lys Ser Lys
Glu Val Gly Gly Leu 85 90
95Val Ala Glu His Ser Ala Leu Ile Gly Pro Leu Cys Glu Ala Gln Leu
100 105 110Ser Phe Leu Gln Phe Ala
Ser Asn His Ala Lys Pro Ala Thr Pro Asn 115 120
125Ala Leu Ala Pro Leu Leu Glu Pro Gln Gly Lys Ala Ile Glu
Ala Ile 130 135 140Met Glu Thr Lys Asp
Lys Leu Ser Arg Ser Lys Glu Gly Arg Glu Trp145 150
155 160Gly Val Cys Phe Asn Val Leu Gly Glu Gly
Val Pro Ala Trp Gly Trp 165 170
175Val Gln Val Glu Pro Thr Pro Ala Pro Tyr Val Gly Glu Met Lys Asn
180 185 190Ala Ala Gln Phe Trp
Ser Asp Arg Val Ile Lys Gln Tyr Lys Glu Thr 195
200 205Asn Ala Ser Ala Val Ala Trp Ala Lys Ser Phe Ile
Ala Leu Ile Ala 210 215 220Ala Leu Glu
Ser Tyr Val Lys Gln Trp His Thr Thr Gly Val Val Trp225
230 235 240Asn Pro Lys Gly Ser Pro Ala
Pro Pro Ser Met Pro Lys Ala Ser Ala 245
250 255Ser Ala Pro Ser Pro Pro Pro Pro Pro Pro Ser Gly
Ser Ala Pro Ala 260 265 270Ala
Pro Thr Ser Gly Ser Gly Ala Ala Ala Leu Leu Ala Asp Leu Asn 275
280 285Arg Gly Gly Ala Val Thr Ser Gly Leu
Arg Lys Val Asp Ser Ser Gln 290 295
300Met Thr His Lys Asn Pro Ser Leu Arg Ser Ala Gly Thr Val Ser Asp305
310 315 320Asn Ala Lys Lys
Gly Pro Pro Leu Lys Pro Lys Pro Gly Ala Lys Pro 325
330 335Ala Lys Lys Pro Ala Lys Ile Glu Leu Glu
Asp Gly Asn Lys Trp Ile 340 345
350Ile Glu Asn Gln Glu Asp Asn Lys Ser Ile Lys Ile Asp Asn Thr Glu
355 360 365Leu His His Thr Val His Ile
Phe Gly Cys Val Asn Ser Val Val Gln 370 375
380Ile Ser Gly Lys Ile Asn Ala Val Thr Met Ala Gly Cys Lys Lys
Thr385 390 395 400Ser Val
Val Leu Asp Thr Ala Val Ser Ser Phe Ser Ile Thr Ser Ser
405 410 415Pro Ser Phe Glu Val Gln Ile
Ile Gly Ser Ile Pro Thr Ile Gln Ile 420 425
430Asp Thr Thr Asp Ser Gly Gln Val Tyr Leu Ser Lys Asp Cys
Met Glu 435 440 445Val Val Glu Ile
Val Thr Ser Lys Ser Ser Ser Ile Asn Ile Ser Val 450
455 460Pro Thr Gly Glu Asp Gly Asp Phe Val Glu Arg Pro
Val Pro Glu Gln465 470 475
480Met Lys Ser Arg Ile Ile Asp Gly Lys Leu Val Thr Glu Ile Val Glu
485 490 495His Ser Gly
28515PRTCryptococcus neoformans serotype A H99 strainPEPTIDE(1)..(515)an
amino acid sequence of Pka1(CNAG_00396.2) 28Met Phe Gln Lys Val Ser Asp
Lys Phe His Arg Lys Gln Gln Ser Ser1 5 10
15Thr Ser Pro Gly Lys Thr Gln Gln Val Pro Asn Ser Pro
Ser Ser Val 20 25 30Leu Ala
Lys Ala Asn Ser Gln Ala Gln Gln Ala Tyr Ser Ser Gln Asp 35
40 45His Ser Pro Met Glu Gly Ile Gln Ser Asp
Ser Thr His Ile Gln Gln 50 55 60Pro
Met Ala Thr Gln Lys Ala Pro Ile Val Gly Pro Ser Thr Ser Thr65
70 75 80Ser Leu Ser Thr Val Pro
Val Gln Asp Gly Thr Leu Pro Leu Thr Pro 85
90 95Gly Ala Gln Gly Met Leu Ala Gly Thr Thr Asp Gly
His Arg Gln Val 100 105 110Gln
Ser Pro Val Ser Arg Ser Ser Ser Ala Gly Glu Asp Lys Met Arg 115
120 125Glu Lys Ala Arg Asp Ala Gln Glu Gln
Ala Ala Gln Ala Gln Ala Asn 130 135
140Leu His Arg Val Thr Gln Gln Ala Arg Val Ala Ala Ile Asn Ala Ala145
150 155 160Ala Thr Gln Ala
Ala Leu Glu Thr Ala Thr Gln Leu Pro Ala Thr Ala 165
170 175Arg Val Pro Thr Ser Gly Thr Gly Ala Glu
Pro Gly Gln Ala Arg Arg 180 185
190Lys Thr Ala Gly Arg Tyr Ala Leu Ser Asp Phe Leu Ile Glu Arg Thr
195 200 205Leu Gly Thr Gly Ser Phe Gly
Arg Val His Leu Val Arg Ser Arg His 210 215
220Asn Gly Arg Phe Tyr Ala Val Lys Val Leu Asn Lys Glu Lys Val
Ile225 230 235 240Lys Met
Lys Gln Val Glu His Thr Asn Ser Glu Arg Glu Met Leu Val
245 250 255Arg Val Arg His Pro Phe Leu
Val Asn Leu Trp Gly Thr Phe Gln Asp 260 265
270Val Asn Asn Leu Tyr Met Val Met Asp Phe Val Ala Gly Gly
Glu Leu 275 280 285Phe Ser Leu Leu
Arg Lys Ser Gln Arg Phe Pro Asn Ser Val Ala Lys 290
295 300Phe Tyr Ala Ala Glu Val Ala Leu Ala Leu Asp Tyr
Leu His Ser Leu305 310 315
320Asp Ile Ile Tyr Arg Asp Leu Lys Pro Glu Asn Leu Leu Leu Gly Ala
325 330 335Asp Gly His Val Lys
Val Thr Asp Phe Gly Phe Ala Lys Tyr Val Pro 340
345 350Asp Ile Thr Trp Thr Leu Cys Gly Thr Pro Asp Tyr
Leu Ala Pro Glu 355 360 365Val Val
Gln Ser Lys Gly Tyr Asn Lys Ser Val Asp Trp Tyr Ala Leu 370
375 380Gly Val Leu Ile Phe Glu Met Leu Ala Gly Tyr
Pro Pro Phe Phe Thr385 390 395
400Glu Asp Gly Asn Pro Met Lys Leu Tyr Glu Lys Ile Ile Ala Gly Lys
405 410 415Val Arg Tyr Pro
Thr Tyr Phe Asp Val Leu Ala Lys Glu Leu Leu Lys 420
425 430Asn Leu Leu Ile Gly Asp Leu Thr Lys Arg Tyr
Gly Asn Leu Arg Ala 435 440 445Gly
Ser Ser Asp Ile Phe Ala His Gly Trp Phe Ala Glu Val Asp Trp 450
455 460Asp Lys Leu Tyr Arg Arg Glu Ile Pro Ala
Pro Tyr Val Pro Lys Ile465 470 475
480Asp Gly Glu Gly Asp Ala Ser Gln Phe Asp Arg Tyr Gln Glu Ala
Asp 485 490 495Val Ser Ala
Tyr Gly Lys Val Gly Asn Gly Pro Tyr Asp His Phe Phe 500
505 510Val Glu Phe 5152983PRTCryptococcus
neoformans serotype A H99 strainPEPTIDE(1)..(83)an amino acid sequence of
Hsp12(CNAG_03143.2) 29Met Ser Asp Ala Gly Arg Gln Ser Phe Thr Asp Lys Ala
Gly Ala Ala1 5 10 15Met
Lys Pro Asp Ser Glu Lys Ser Tyr Leu Glu Gln Ala Lys Asp Thr 20
25 30Ile Gly Gly Lys Ala Asp Ser Ala
Ala Ser Thr Gly Gln Pro Gln Ser 35 40
45Gln Lys Ser Tyr Thr Gln Glu Ile Gly Asp Ala Phe Ser Gly Asn Lys
50 55 60Asn Asp Asn Gln Glu Ser Leu Thr
Asp Lys Ala Lys Asn Ala Phe Gly65 70 75
80Ala Asn Gln3070PRTCryptococcus neoformans serotype A
H99 strainPEPTIDE(1)..(70)an amino acid sequence of Hsp122(CNAG_01446.2)
30Met Ser Asp Ala Gly Arg Gln Ser Leu Ala Asp Lys Ala Ser Ser Ser1
5 10 15Met Lys Pro Asp Ser Glu
Lys Ser Tyr Val Glu Gln Ala Ser Asp Phe 20 25
30Ile Ser Gly Lys Leu Asp Ser Ala Ala Ser Ala Val Gln
Pro Gln Gln 35 40 45Glu Lys Ser
Thr Thr Gln Lys Ile Gly Asp Ala Val Ser Gly Asp Asn 50
55 60Arg Asn Arg Asp Val Ala65
703120DNAArtificial SequenceSynthetic oligonucleotide 31tttttttttt
tttttttttt
203221DNAArtificial SequenceSynthetic oligonucleotide 32tgtggatgct
ggcggaggat a
213321DNAArtificial SequenceSynthetic oligonucleotide 33gtaaaacgac
ggccagtgag c
213421DNAArtificial SequenceSynthetic oligonucleotide 34caggaaacag
ctatgaccat g
213520DNAArtificial SequenceSynthetic oligonucleotide 35tgtttagcac
cagcggagtc
203621DNAArtificial SequenceSynthetic oligonucleotide 36cacgatgaaa
gtgcgttgaa g
213740DNAArtificial SequenceSynthetic oligonucleotide 37gctcactggc
cgtcgtttta cactgtcggt gaaagattgc
403840DNAArtificial SequenceSynthetic oligonucleotide 38catggtcata
gctgtttcct gagaacgaca accaggagtc
403922DNAArtificial SequenceSynthetic oligonucleotide 39gctctgtgct
gacattatct gc
224020DNAArtificial SequenceSynthetic oligonucleotide 40gaaagtgcgt
tgaagtgatg
204121DNAArtificial SequenceSynthetic oligonucleotide 41agtagaagca
gcggactaaa g
214220DNAArtificial SequenceSynthetic oligonucleotide 42gcgtagtgga
gattggtttc
204320DNAArtificial SequenceSynthetic oligonucleotide 43atcccctcca
ctttacctcc
204440DNAArtificial SequenceSynthetic oligonucleotide 44gctcactggc
cgtcgtttta caagtctccc ttagcgatag
404540DNAArtificial SequenceSynthetic oligonucleotide 45catggtcata
gctgtttcct gaccacaccc ctgaagaaac
404620DNAArtificial SequenceSynthetic oligonucleotide 46aactgtttcg
tcttgtgtgc
204719DNAArtificial SequenceSynthetic oligonucleotide 47atagcaactt
cttccgtcg
194819DNAArtificial SequenceSynthetic oligonucleotide 48tgttgcctgt
gctcacttg
194920DNAArtificial SequenceSynthetic oligonucleotide 49cctctgacag
ccacatactg
205019DNAArtificial SequenceSynthetic oligonucleotide 50aatgaagttc
ctgcgacag
195139DNAArtificial SequenceSynthetic oligonucleotide 51gctcactggc
cgtcgtttta caatgggatg agaacgcac
395240DNAArtificial SequenceSynthetic oligonucleotide 52catggtcata
gctgtttcct gagcattttc cagcatcagc
405320DNAArtificial SequenceSynthetic oligonucleotide 53ggtgtggaac
atcttttgag
205419DNAArtificial SequenceSynthetic oligonucleotide 54ctggttcatc
ttgggtgtc
195522DNAArtificial SequenceSynthetic oligonucleotide 55tctgagcata
ccactccttt ac
225618DNAArtificial SequenceSynthetic oligonucleotide 56tctcattcgc
atcctctg
185721DNAArtificial SequenceSynthetic oligonucleotide 57gttgggcaga
taatgtttgt g
215839DNAArtificial SequenceSynthetic oligonucleotide 58gctcactggc
cgtcgtttta cacggcgtca gacattgtg
395943DNAArtificial SequenceSynthetic oligonucleotide 59catggtcata
gctgtttcct gacaagagaa gtccactact cag
436018DNAArtificial SequenceSynthetic oligonucleotide 60gcaaggtaat
gatgagcg
186121DNAArtificial SequenceSynthetic oligonucleotide 61gcgactgaga
tgtagaccaa c
216220DNAArtificial SequenceSynthetic oligonucleotide 62ctcggaacga
cataataagc
206321DNAArtificial SequenceSynthetic oligonucleotide 63cacacctggt
aagagatagc g
216441DNAArtificial SequenceSynthetic oligonucleotide 64gctcactggc
cgtcgtttta cagtggtaga agtagggcag c
416540DNAArtificial SequenceSynthetic oligonucleotide 65catggtcata
gctgtttcct gacagggtcc aacaaggatg
406620DNAArtificial SequenceSynthetic oligonucleotide 66tgctacgatt
gtggtcagcc
206721DNAArtificial SequenceSynthetic oligonucleotide 67ggacgagacg
agttatcaaa c
216818DNAArtificial SequenceSynthetic oligonucleotide 68cttcatcaac
ttgcgtgc 18
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