KR101079278B1 - Use of the genes in the HOG pathway for treatment of fungal infection - Google Patents

Abstract

The present invention provides the use of an inhibitor against at least one protein or gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2 and HOG1 of Cryptococcus neoformans for the preparation of an antifungal agent, comprising the inhibitor An antifungal pharmaceutical composition, comprising a method for treating a fungal infection comprising administering an effective amount of the inhibitor to a subject. According to the present invention, inhibition of one or more proteins or genes selected from the group consisting of SSK1, TCO2, SSK2, PBS2 and HOG1 of Cryptococcus neoformans enhances the biosynthesis of ergosterol present in fungal cell membranes. Since a large amount of ergosterol is distributed on the cell membrane of the fungi, the action point of the antifungal agent that kills the fungi by binding to ergosterol is increased. Thus, inhibitors of these proteins or genes can be used as combination therapeutics that significantly increase the fungal killing effect of erosterol binding antifungal agents.

Antifungal, Ergosterol, Cryptococcus Neoforms

Description

Use of the genes in the HOG pathway for treatment of fungal infection

The present invention relates to the use of HOG signaling pathway genes for the treatment of fungal infections.

Whether organisms can survive and multiply in a particular environment is largely determined by their ability to respond and adapt to various environmental stresses and maintain cell homeostasis. Cells regulate critical processes by performing a series of complex signaling networks. Among them, the p38 / Hog1 mitogen-activated protein kinase (MAPK) -dependent signaling pathway plays a pivotal role in regulating a wide range of stress-response in eukaryotes from yeast to humans.

Mammalian stress-activated p38 MAPKs induce a variety of stress association-signals that limit adaptation to the immune response by osmotic pressure change and ultraviolet irradiation, programmed cell death, and regulation of cytokine production and inflammation. Similar stress-sensitive signaling systems have been identified in other species. Fungi have p38-like MAPKs that regulate various stress responses. In budding yeast Saccharomyces cerevisiae, Hog1 MAPK modulates the stress response to osmotic shock, oxidative damage and heavy metal damage. Cleavage yeast Schizosaccharomyces pombe also has a Hog1 homologue, Sty1 (also known as Spc1 and Phh1), which includes osmotic shock, heat shock, oxidative damage and heavy metal damage, carbon deficiency and ultraviolet radiation. It is involved in adapting to so many different stresses. Interestingly, Sty1 is also involved in growth regulation and reproduction and differentiation. Hog1 MAPK orthologs are also found in other ascomycete pathogenic fungi, including Candida albicans (Hog1) and Aspergillus fumigatus (SakA), osmotic shock, UV irradiation, and oxidization. It has been shown to mediate responses to various environmental causes, including damage and high temperatures.

The common molecular mechanisms of the p38 / Hog1 MAPK signaling network are widely conserved in many eukaryotic cells. Under normal growth conditions, p38 / Hog1 MAPK is unphosphorylated, but in response to specific environmental stresses, the TGY motif via MAPK kinase (MAPKK) is activated through phosphorylation by MAPKK kinase (MAPKKK) in its higher signaling system. Activated by double phosphorylation of Thr and Tyr residues. Phosphorylated p38 / Hog1 MAPKs then migrate into the nucleus after forming dimers, triggering activation of transcriptional regulators and inducing an excess of stress-defense genes against external stress conditions.

Despite the conserved regulatory mechanism of p38 / Hog1 MAPK, fungi and mammals have developed a unique set of higher regulatory systems. In particular, fungi utilize a two-component-like phosphorelay system that is absent in mammals and found only in bacteria, fungi and plants. The fungal phosphate delivery system consists of three components, including hybrid sensor kinases, histidine-containing phosphotransfer protein (HPt), and response regulators. All of these are not observed in mammals and are considered good antifungal targets.

Basidiomycetous Cryptococcus neoformus also utilizes a stress-activated Hog1 MAPK system for adaptation to a variety of environmental stresses, including osmotic shock, ultraviolet radiation, heat shock, oxidative damage, toxic metabolites and antifungal agents. . Cryptococcus neoformes is a human pathogenic fungus found all over the world that causes skin and lung cryptococcosis and cryptococcus meningitis in immunocompromised patients. Cryptococcus neo-performance var. grubii (antigen A) is the most commonly found (> 90% of environmental and clinical strains), while Cryptococcus neoformus var. neoformans (antigen D) are more common in certain regions of Europe and are not frequently found (<10%). However, Cryptococcus gattii, also known as antigenic types B and C Cryptococcus neoforms, has been found to be a primary pathogen that attacks normal people without abnormalities in the immune system.

However, compared with other fungal Hog1 MAPK systems, the Cryptococcus neoform Hog1 MAPK pathway not only responds to various environmental stresses, but also supports the production and sexual differentiation of two virulence factors, such as anti-naphtha capsules and antioxidant melanin. Uniquely developed to regulate, suggesting that Hog1 MAPK plays a pivotal role as an important signaling regulator in Cryptococcus neoforms that cross-talks with other signaling pathways. Recently, the inventors have found that Hog1 MAPKs in many Cryptococcus neoformus strains are always phosphorylated under non-stressed conditions and rapidly dephosphorylated for their activation in response to osmotic shock and antifungal fludioxosonyl treatment. This is in stark contrast to other fungal Hog1 MAPK systems. Dual phosphorylation of the TGY motif at Hog1 requires Pbs2 MAPKK. Fungal specific phosphate delivery systems, which are located above the Pbs2-Hog1 pathway, are also found in Cryptococcus neoformes. The Cryptococcus neoform phosphate delivery system includes seven different sensor hybrid histidine kinase (Tco1-7), Ypd1 phosphotransfer protein, and two reaction modulators (Ssk1 and Skn7). The Pbs2-Hog1 pathway is usually regulated by Ssk1 but not by Skn7. Of the seven Tco proteins, Tco1 and Tco2 play a differential and simultaneous overlapping role in activating the Ssk1 and Pbs2-Hog1 MAPK pathways. However, since Tco1 and Tco2 regulate some phenotypes associated with Ssk1- and Hog1, other higher receptor or sensor proteins remain to be identified. More recently, we compared the meiotic maps between B-3501 and B-3502 antigenic D f1 sibling strains showing different phosphorylation patterns of Hog1, thus linking the linkage between the phosphate delivery system and the Pbs2-Hog1 MAPK pathway. Ssk2 MAPKKK was identified. Most notable is that the interchange of Ssk2 alleles between two Cryptococcus neoformes strains showing different Hog1 phosphorylation patterns alters the phenotype controlled by persistent Hog1 phosphorylation. Unlike S. servige and S. pombe, Cryptococcus neoformus has a single MAPKKK, Ssk2, which regulates Hog1 MAPK. The downstream signaling network of the Hog1 MAPK pathway in Cryptococcus neoforms has not yet been identified, but the development and targeting of new antifungal agents requires identification and characterization of the downstream signaling network of Hog1 MAPK.

In the past, fungal infections have caused many local infections such as leukoplakia, erythematosus, thrush, and thrush. Systemic fungal infections have rarely occurred, but recently, they are the most common cause of infection in the entire hospital.

Antifungal agents developed to date can be classified into antifungal agents having chemically azole structures and antifungal agents having no azole structure. The azole antifungal agents include ketoconazole, fluconazole, itraconazole and voriconazole, and the non-azole antifungal agents are terbinafine and flucytosine. , Amphotericin B, caspofungin, and the like.

Ketoconazole, fluconazole, itraconazole, voriconazole, and allylamine-based naphthypine and terbinapine having an azole structure have similar mechanisms of action. Both classes of antifungal agents inhibit the enzymes necessary for the conversion of lanosterol to ergosterol, a major component of fungal cell membranes. The azole antifungal agent inhibits microsomal enzymes, and the allylamine antifungal agent inhibits squalene epoxidase, thereby exhibiting the above effects. Flucytosine (5-FC) is a metabolic antagonist that inhibits nucleic acid synthesis, and has antifungal action by uncompromising antagonism of fungal RNA and DNA synthesis, and shows antifungal action, and has a polyene structure. Binds to ergosterol inside the fungal cell membrane to induce depolarization of the cell membrane, forms pores, and causes loss of intracellular contents, thereby exhibiting antifungal action. Caspofungin, an antifungal agent of the Echinocandins family, reversibly inhibits the formation of fungal cell walls and differs from the antifungal agents acting on the cell membranes in that they act on the cell walls.

Since azole drugs may cause hepatitis death when used in patients with reduced liver function, hepatic function tests must be performed prior to administration. Flucytosine has been reported to be dose-dependently inhibiting myelosuppression, hepatotoxicity, and small intestinal colitis, and these side effects increase when renal function is lowered. Therefore, monitoring renal function of patients is very important. It is also contraindicated in pregnant women. Representative toxicity of amphotericin B is glomerular neotoxicity following renal artery contraction, which is dose dependent, resulting in an increased incidence of permanent renal insufficiency at lifetime cumulative doses of 4-5 g or more. In addition, nephrotoxicity such as excessive loss of potassium, magnesium, and bicarbonate due to tubular toxicity and a decrease in hematopoietic hormone production may occur. In addition, acute reactions may cause symptoms such as thrombophlebitis, chills, tremors and hyperventilation. As such, the antifungal agents developed in the past have various side effects according to the types of drugs, and thus, there is a demand for the development of new treatments that can reduce the side effects and enhance the antifungal effect.

In the present invention, by examining the signaling network of the HOG pathway, it aims to discover a new target gene for the development of antifungal agents.

The present invention provides the use of an inhibitor against at least one protein or gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2 and HOG1 of Cryptococcus neoformans for the preparation of an antifungal agent, comprising the inhibitor An antifungal pharmaceutical composition, comprising a method for treating a fungal infection comprising administering an effective amount of the inhibitor to a subject.

The present invention also provides the use of one or more proteins or genes selected from the group consisting of SSK1, TCO2, SSK2, PBS2 and HOG1 of Cryptococcus neoformans for the screening of antifungal agents, comprising the protein or gene An antifungal agent screening composition, and a method of screening an antifungal agent comprising contacting a candidate with the protein or gene and determining whether the candidate inhibits or promotes the activity of the protein or gene.

In the present invention, the role of SSK1, TCO2, SSK2, PBS2 and HOG1 genes in the HOG pathway regulation of Cryptococcus neoforms was investigated, and newly discovered that the biosynthesis of ergosterol is enhanced when these genes are inhibited. Inhibiting the genes causes a large amount of ergosterol to be distributed on the fungal cell membranes, thereby increasing the effect of the ergosterol-binding antifungal agent and significantly increasing the efficacy of the ergosterol-binding antifungal agent. Therefore, an antifungal pharmaceutical composition comprising an inhibitor against at least one protein selected from the group consisting of Cryptococcus neoformus SSK1, TCO2, SSK2, PBS2 and HOG1 can be used while reducing the dosage of existing ergosterol-binding antifungal agents. It can be used as an excellent complex antimicrobial agent that can increase the efficacy.

Hereinafter, the present invention will be described in more detail.

The present invention provides an antifungal pharmaceutical composition comprising an inhibitor against at least one protein or gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2 and HOG1 of Cryptococcus neoformans for the preparation of antifungal agents. to provide.

The HOG1 pathway is a cell signaling pathway that regulates responses to various stresses. In particular, fungi include hybrid sensor kinases, histidine-containing phosphotransfer proteins (HPt), and response regulators that are not present in mammals in the HOG1 pathway. Because we use a two-component-like phosphorelay system consisting of three elements, the inventors investigate the role of genes involved in the HOG1 pathway for the development of new antifungal targets. It was. As a result, it was surprisingly found that the inhibition of the SSK1, TCO2, SSK2, PBS2 and HOG1 genes in the regulation of HOG pathway of Cryptococcus neoformus enhanced the biosynthesis of ergosterol. As can be seen in the following examples, since inhibiting the genes are a large distribution of ergosterol on the cell membrane of the fungi, there is a lot of action point of the ergosterol-binding antifungal agent to significantly increase the efficacy of the ergosterol-binding antifungal agent It becomes possible. Therefore, an antifungal pharmaceutical composition comprising an inhibitor against at least one protein selected from the group consisting of Cryptococcus neoformus SSK1, TCO2, SSK2, PBS2 and HOG1 can be used while reducing the dosage of existing ergosterol-binding antifungal agents. It can be used as an excellent combination antimicrobial agent that can increase the efficacy.

Therefore, the present invention provides the use of an inhibitor against at least one protein or gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2 and HOG1 of Cryptococcus neoformus for the preparation of an antifungal agent, an antifungal medicament comprising the inhibitor A method of treating a fungal infection comprising administering to a subject a composition, an effective amount of said inhibitor.

In the present invention, SSK1, TCO2, SSK2, PBS2 or HOG1 used as a target for blocking the HOG1 signaling system means SSK1, TCO2, SSK2, PBS2 or HOG1 protein or SSK1, TCO2, SSK2, PBS2 or HOG1 gene It is interpreted as meaning. Thus, an SSK1, TCO2, SSK2, PBS2, or HOG1 inhibitor is interpreted to include both inhibitors against SSK1, TCO2, SSK2, PBS2, or HOG1 proteins or inhibitors against SSK1, TCO2, SSK2, PBS2, or HOG1 genes.

 In one embodiment, the inhibitor for at least one protein selected from the group consisting of SSK1, TCO2, SSK2, PBS2 and HOG1 of Cryptococcus neoformes is an inhibitor that blocks signaling by inhibiting activity by binding to the protein. Can be. In another embodiment, the inhibitor for one or more genes selected from the group consisting of SSK1, TCO2, SSK2, PBS2 and HOG1 of Cryptococcus neoforms may be inhibitors that inhibit signaling by inhibiting the expression of the gene. . In the present invention, the SSK1, TCO2, SSK2, PBS2 or HOG1 gene may be DNA encoding them or mRNA transcribed therefrom. Thus, the inhibitor for the gene may be an inhibitor that binds to the gene itself to interfere with transcription or binds to mRNA transcribed from the gene and interferes with the translation of the mRNA.

In one embodiment of the invention, the SSK1, TCO2, SSK2, PBS2 or HOG1 protein may have an amino acid sequence of SEQ ID NO: 1 to 5, respectively, the SSK1, TCO2, SSK2, PBS2 or HOG1 gene is SEQ ID NO: 6 It may have a nucleic acid sequence of 10 to 10 or a cDNA sequence of SEQ ID NOs: 11 to 15, respectively, which is merely illustrative of the sequence of Cryptococcus neoformus antigen type A H99 strain, SSK1, TCO2, SSK2, PBS2 of the present invention Or the sequence of HOG1 is not limited thereto.

In the present invention, the SSK1, TCO2, SSK2, PBS2 or HOG1 protein, the SSK1, TCO2, SSK2, PBS2 or HOG1 gene and the like are interpreted to include variants or fragments thereof having substantially the same activity as these.

In one embodiment, the inhibitor may be an inhibitor for the SSK1 protein or gene. SSK1 is not only an important upstream response regulator of HOG1, but also a gene not found in mammals, so it can be an excellent target for the development of antifungal agents. Therefore, the SSK1 inhibitor may improve the efficacy of the ergosterol-binding antifungal agent by increasing the amount of biosynthesis of the ergosterol fungus while lowering the possibility of any side effects caused by the inhibition of SSK1 when administered in vivo.

Inhibition of SSK1, TCO2, SSK2, PBS2 or HOG1 proteins or genes enhances the biosynthesis of ergosterol, increasing the distribution of ergosterol on fungal cell membranes. Therefore, the binding target of the ergosterol-binding antifungal agent which binds to ergosterol and destroys the fungal cell membrane increases, so that the killing efficacy of the ergosterol-binding antifungal agent can be increased while reducing the effective amount of the ergosterol-binding antifungal agent. Will be. Antifungal activity by inhibition of SSK1, TCO2, SSK2, PBS2, or HOG1 can treat Cryptococcosis and meningitis due to infection with Cryptococcus neoformes.

Accordingly, one embodiment of the present invention includes the use of an SSK1, TCO2, SSK2, PBS2 or HOG1 inhibitor, SSK1, TCO2, SSK2, PBS2 or HOG1 inhibitor for the manufacture of a medicament for the treatment of diseases such as Cryptococcosis and Meningitis It provides a pharmaceutical composition for treating diseases such as Cryptococcosis and meningitis, and a method for treating diseases such as Cryptococcosis and meningitis comprising administering to the subject an effective amount of an SSK1, TCO2, SSK2, PBS2 or HOG1 inhibitor do.

In addition to Cryptococcosis and meningitis exemplified herein, diseases caused by fungal infections are well known in the art. In the present invention, the inhibition of the SSK1, TCO2, SSK2, PBS2 or HOG1 protein or gene enhances the biosynthesis of ergosterol to increase the distribution of ergosterol on the cell membrane of the fungus to enhance the efficacy of the ergosterol-binding antifungal agent As described above, those skilled in the art will be able to inhibit the proteins or genes for the prevention or treatment of diseases associated with fungal infections.

In the present invention, 'inhibitors of SSK1, TCO2, SSK2, PBS2 or HOG1 protein' used to block HOG1 signaling system are all inhibitors that bind to SSK1, TCO2, SSK2, PBS2 or HOG1 protein to block signaling. Include. For example, such inhibitors may be peptides or compounds that bind to SSK1, TCO2, SSK2, PBS2, or HOG1 proteins, and the like. Such inhibitors may be selected through screening methods exemplified below, such as protein structure analysis, and may be designed using methods known in the art. In one embodiment, the inhibitor may be a polyclonal antibody or monoclonal antibody against one or more proteins selected from the group consisting of SSK1, TCO2, SSK2, PBS2 and HOG1 of Cryptococcus neoformus. Such polyclonal antibodies or monoclonal antibodies can be prepared using antibody production methods known in the art.

In the present invention, 'inhibitors of the SSK1, TCO2, SSK2, PBS2 or HOG1 genes used to block the HOG1 signaling system' are inhibitors that block signaling by inhibiting the expression of the SSK1, TCO2, SSK2, PBS2 or HOG1 genes. Include all of them. For example, such inhibitor may be a peptide, nucleic acid or compound that binds to the gene. Such inhibitors may be selected through screening methods exemplified below, such as cell based screening, and may be designed using methods known in the art. In one embodiment, the inhibitor is an antisense oligonucleotide, siRNA, shRNA, miRNA or a vector comprising at least one gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2 and HOG1 of Cryptococcus neoformus. Can be. Such antisense oligonucleotides, siRNAs, shRNAs, miRNAs or vectors comprising them can be prepared using methods known in the art. In the present invention, the 'vector' refers to a gene construct comprising an external DNA inserted into the genome encoding the polypeptide. A vector related to the present invention is a vector in which the nucleic acid sequence that inhibits the gene is inserted into the genome, and examples of the vector include a DNA vector, a plasmid vector, a cosmid vector, a bacteriophage vector, a yeast vector, or a viral vector.

The antifungal pharmaceutical composition of the present invention does not exhibit antifungal activity alone, but enhances the fungal killing ability of the ergosterol-binding antifungal agent through a combination treatment with the ergosterol-binding antifungal agent. Therefore, the antifungal pharmaceutical composition of the present invention is characterized in that it is administered sequentially or simultaneously with ergosterol-binding antifungal agent. Ergosterol-binding antifungal means an antifungal agent that binds to ergosterol on fungal cell membranes to cause depolarization of the cell membranes, form pores, and cause loss of intracellular contents to kill fungi. Such ergosterol-binding antifungal agents are known in the art, and any ergosterol-binding antifungal agent will show a marked increase in antifungal effect when used with the antifungal pharmaceutical composition of the present invention. In one embodiment, the ergosterol-binding antifungal agent may be a polyene-based antifungal agent. In one aspect, the polyene-based antifungal agent may be one or more antifungal agents selected from the group consisting of amphotericin B, natamycin, limosidine, philippines, nystatin and candiesine. In a preferred embodiment, the polyene-based antifungal agent may be amphotericin B.

In this aspect, the present invention also provides an antifungal pharmaceutical composition comprising the inhibitor of the present invention and an antifungal complex preparation comprising a known ergosterol-binding antifungal agent.

The antifungal pharmaceutical composition or antifungal complex preparation of the present invention may be prepared using a pharmaceutically acceptable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant may include excipients, disintegrants, sweeteners, binders, coatings, Solubilizers such as swelling agents, lubricants, lubricants or flavoring agents can be used.

The antifungal pharmaceutical composition of the present invention can be preferably formulated into a pharmaceutical composition comprising one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration.

Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable solutions, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Further, it can be suitably formulated according to each disease or ingredient, using the method disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA as an appropriate method in the field.

Pharmaceutical formulation forms of the pharmaceutical compositions of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and sustained release formulations of the active compounds, and the like. Can be.

The pharmaceutical compositions of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, sternum, transdermal, nasal, inhalation, topical, rectal, oral, intraocular or intradermal routes. Can be administered.

An effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required to prevent or treat a disease or to achieve a bone growth inducing effect. Thus, the type of disease, the severity of the disease, the type and amount of the active and other ingredients contained in the composition, the type of formulation and the age, weight, general health, sex and diet, sex and diet, time of administration, route of administration and composition of the patient. It can be adjusted according to various factors including the rate of secretion, the duration of treatment, and the drug used concurrently. For example, in adults, when administered once or several times a day, the inhibitor of the present invention is administered once or several times a day, when the compound is 0.1ng / kg to 10g / kg, a polypeptide, In the case of protein or antibody, 0.1ng / kg ~ 10g / kg, antisense oligonucleotide, siRNA, shRNAi, miRNA can be administered at a dose of 0.01ng / kg ~ 10g / kg.

In the present invention, the 'object' includes, but is not limited to, humans, orangutans, chimpanzees, mice, rats, dogs, cattle, chickens, pigs, goats, sheep, and the like.

In another aspect, the present invention is the use of one or more proteins selected from the group consisting of SSK1, TCO2, SSK2, PBS2 and HOG1 of Cryptococcus neoformes for screening antifungal agents, compositions for screening antifungal agents comprising the protein, candidates and A method for screening an antifungal agent comprising contacting the protein and determining whether the candidate inhibits or promotes the activity of the protein.

Similarly, the present invention also provides the use of one or more genes selected from the group consisting of SSK1, TCO2, SSK2, PBS2 and HOG1 of Cryptococcus neoformes for screening antifungal agents, compositions for antifungal screening comprising said genes, candidates A method for screening an antifungal agent comprising contacting a substance with the gene and determining whether the candidate substance inhibits or promotes expression of the gene.

The present invention also provides an antifungal screening method by a yeast two-hybrid system capable of monitoring physical contact between SSK1 and SSK2 of Cryptococcus neoformes, between SSK1 and YPD1, or between YPD1 and TCO2 proteins for the screening of antifungal agents. Provide the law. This method has the advantage of screening large amounts of candidates in a short time.

As mentioned above, inhibition of SSK1, TCO2, SSK2, PBS2, or HOG1 of Cryptococcus neoforms blocks the HOG1 signaling system, thereby enhancing the biosynthesis of ergosterol. Thus, substances screened to inhibit the protein or gene can be used as an antifungal agent to enhance fungal killing ability in combination with ergosterol-binding antifungal agents.

Confirmation of the reaction between the protein or the gene and the candidate is used to confirm the reaction between the protein-protein, protein-compound, DNA-DNA, DNA-RNA, DNA-protein, DNA-compound, RNA-protein, RNA-compound. Conventional methods can be used. For example, a hybridization test for confirming the binding between the gene and the candidate substance in vitro, reaction of mammalian cells with the test substance, and then Northern analysis, quantitative PCR, quantitative real-time PCR, etc. A method of measuring the expression rate of a gene, or a method of connecting a reporter gene to the gene and introducing it into a cell, reacting with a test substance and measuring the expression rate of a reporter protein, a method of measuring activity after reacting the protein and a candidate substance, Yeast two-hybrid, search for phage-displayed peptide clones that bind to Idbf protein, high throughput screening (HTS) using drug and natural chemical libraries Screening using a hit hit HTS, cell-based screening, or DNA array The can be used.

In addition to the protein or gene, the composition for screening may include distilled water or a buffer to stably maintain the structure of the nucleic acid or protein. In addition, the composition for screening may include a cell expressing the protein or gene, or a cell containing a plasmid expressing the gene under a promoter capable of controlling transcription rate for in vivo experiments. .

In the screening methods of the present invention, individual nucleic acids, proteins, peptides, and other extracts that have been suspected of having potential as a medicament to inhibit signal transduction by the HOG1 signaling system or randomly selected according to conventional selection methods. Or natural products, compounds, and the like.

Matters related to genetic engineering in the present invention are described in Sambrook et al. (Sambrook, et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY (2001)) and Frederick et al. M. Ausubel et al., Current protocols in molecular biology volumes 1, 2, 3, John Wiley & Sons, Inc. (1994)).

Advantages and features of the present invention and methods for achieving them will be apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various forms. It is provided to fully inform the person having the scope of the invention, which is defined only by the scope of the claims.

[Example]

Experimental Method

Strains and Culture Conditions

Cryptococcus neoformance used in this example is shown in Table 1 [Bahn YS, Geunes-Boyer S, Heitman J (2007) Eukaryot Cell 6: 2278-2289 .; Bahn YS, Kojima K, Cox GM, Heitman J (2005) Mol Biol Cell 16: 2285-2300 .; Bahn YS, Kojima K, Cox GM, Heitman J (2006) Mol Biol Cell 17: 3122-3135 .; Perfect JR, Ketabchi N, Cox GM, Ingram CW, Beiser CL (1993) J Clin Microbiol 31: 3305-3309; Kwon-Chung KJ, Edman JC, Wickes BL (1992) Genetic association of mating types and virulence in Cryptococcus neoformans . Infect Immun 60: 602-605.]. Cryptococcus neoformes was cultured in yeast extract-peptone-dextrose (YPD) medium unless otherwise indicated.

For isolation of total RNA used for DNA microarrays, wild type H99, hog1 mutant (YSB64), ssk1 mutant (YSB261), skn7 mutant (YSB349) strains were incubated for 16 hours at 30 ° C. in 50 ml YPD medium. Thereafter, 5 ml of overnight culture was inoculated with 100 ml of fresh YPD medium and further incubated at 30 ° C. for 4-5 hours at ₆₀₀ ° C. until absorbance (OD) reached approximately 1.0 (OD _{600 nm} = 1.0). For zero-time samples, 50 ml of culture in 100 ml was sampled and rapidly cooled in liquid nitrogen. 50 ml of YDP containing 2 M NaCl, 40 μg / ml fludioxosonyl (PESTANAL, Sigma, 100 mg / ml stock in dimethylsulfoxide), or 5 mM hydrogen peroxide (H ₂ O ₂ ) Was added (final concentration 1 M NaCl, 20 μg / ml fludioxonil, 2.5 mM H ₂ O _{2, respectively} ) _. During incubation in each stress-inducing medium, 50 ml of culture was sampled at 30 and 60 minutes to precipitate in a tabletop centrifuge and cooled with liquid nitrogen to lyophilze overnight. Lyophilized cells were then used for total RNA isolation. As biological replicates for DNA microarrays, three independent cultures for each strain and culture conditions were prepared for total RNA isolation.

TABLE 1

Strain Genotype Parent Serotype A H99 MAT a YSB64 MAT a hog1D :: NAT-STM # 177 H99 YSB123 MAT a pbs2D :: NAT-STM # 213 H99 YSB261 MAT a ssk1D :: NAT-STM # 205 H99 YSB264 MAT a ssk2D :: NAT-STM # 210 H99 YSB349 MAT a skn7D :: NAT-STM # 201 H99 YSB278 MAT a tco1D :: NAT-STM # 102 H99 YSB281 MAT a tco2D :: NAT-STM # 116 H99 YSB324 MAT a tco1D :: NAT-STM # 102 tco2D :: NEO YSB278 YSB284 MAT a tco3D :: NAT-STM # 119 H99 YSB417 MAT a tco4D :: NAT-STM # 123 H99 YSB286 MAT a tco5D :: NAT-STM # 125 H99 YSB348 MAT a tco7D :: NAT-STM # 209 H99 Serotype D JEC21 MAT a B-3501 MAT a YSB267 MAT a pbs2D :: NAT-STM # 213 JEC21 YSB139 MAT a hog1D :: NAT-STM # 177 JEC21 YSB338 MAT a ssk2D :: NAT-STM # 210 JEC21 YSB340 MAT a ssk2D :: NAT-STM # 210 B-3501

Total RNA Manufacturing

For isolation of total RNA, 3 ml of sterile 3 mm glass beads were added to the lyophilized cell particles (SIGMUND LINDER), shaken, and homogenized. 4 ml of TRizol was added thereto and incubated at room temperature for 5 minutes. Then, 800 μl of chloroform was added and incubated for 3 minutes at room temperature, and transferred to a 15 ml round bottom tube (SPL) and centrifuged at 10,000 rpm for 15 minutes at 4 ° C. (Sorvall SS-34 rotor). 2 ml of supernatant was transferred to a new round bottom tube, inverted several times by adding 2 ml isopropanol and incubated for 10 minutes at room temperature. The mixture was then recentrifuged at 10,000 rpm for 10 minutes at 4 ° C. and the precipitate was washed with 4 ml of 75% ethanol diluted with diethylpyrocarbonate (DEPC) treated water. The precipitate was dried at room temperature and washed again with 500 μl DEPC treated water. The concentration and purity of the total RNA samples were measured by OD _{260 nm} and gel electrophoresis, respectively. For control total RNA (for Cy3 labeling), all total RNAs prepared from wild type, hog1, ssk1, skn7 mutant cells cultured under the conditions described above were pooled. (pooled reference RNAs)

cDNA Synthesis and Cy3 / Cy5 Labeling

For cDNA synthesis, total RNA concentration was adjusted to 1 μg / μl with DEPC treated water, 15 μl total RNA (15 μg) and 1 μl 5 μg / μl oligo dT (5'-TTTTTTTTTTTTTTTTTTTTV-3 ') / pdN6 (Amersham) (10 μg / μl of 1: 1 mixture, respectively) was added and incubated at 70 ° C. for 10 minutes, and then placed on ice for 10 minutes. Then 15 μl cDNA synthesis mixture {3 μl 0.1 M DTT, 0.5 RNasin [Promega], 0.6 μl aa-dUTP (5- (3-aminoallyl) -2'-deoxyuridine 5'-triphosphate) / dNTPs [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) mixture], 1.5 μl AffinityScript reverse transcription Enzyme (Stratagene), 3 μl AffinityScript buffer, 7 μl water] were added and incubated at 42 ° C. for 2 hours. Then 10 μl of 1N NaOG and 10 μl of 0.5M EDTA (pH 8.0) were added and incubated at 65 ° C. for 15 minutes. After incubation, 25 μl of 1M HEPES buffer (pH 8.0) and 450 μl of DEPE-treated water were added, and the entire mixture was concentrated through a Microcon 30 filter (Millipore) and vacuum dried for 1 hour.

For Cy3 and Cy5 labeling of the prepared cDNA, Cy3 and Cy5 were dissolved in 10 μl DMSO and 1.25 μl of each dye and divided into separate tubes. CDNA prepared as described above and 9 μl of 0.05M Na-bicarbonate (pH 8.0) were added and incubated for 15 minutes at room temperature. cDNAs prepared from pooled reference RNAs were mixed with Cy3 as a control and cDNAs prepared from each test RNA (each experimental condition) were mixed with Cy5. Each mixture was incubated for one more hour at dark room temperature and purified by QIAquick PCR Purification Kit (QIAGEN).

Microarray Hybridization and Washing

Cryptococcus neoformance D 70-mer microarray slide (Duke University) containing 7,936 spots was pre-hydridization buffer [42.4 ml sterile distilled water, 2 ml 30% BSA (Sigma), 600 at 42 ° C. 10 μl 10% SDS, 15 ml 20 × SSC], washed with distilled water and isopropanol and centrifuged briefly (110xg, 2 min). Cy3 and Cy5-labeled cDNA samples were combined and passed through a Microcon 30 filter. Concentrated and vacuum dried. Dried cDNA samples were retreated with hybridization buffer [250 μl 50% formamide, 125 μl 20x SSC, 5 μl 10% SDS, 120 μl dH ₂ O, 500 μl total] and 1 μl poly-A tail DNA (Sigma) After the addition was further incubated for 3 minutes at 100 ℃ and cooled for 5 minutes at room temperature. Microarray slides were aligned with a hybridization chamber (DieTech), dust was removed and covered with Lifterslips (Erie Scientific). Cy3 / Cy5-labeled cDNA samples were applied between Lifterslips and slides. To prevent the slides from drying, 10 μl of 3 × SSC buffer was applied to the slides and the slides were then incubated at 42 ° C. for 16 hours. After incubation, the microarray slides were washed in three different washing buffers (washing buffer 1 (10 ml 20x SSC, 600 μl 10% SDS, 189.4 ml dH ₂ O, 42 ° C.), washing buffer 2 (3.5) in an orbital stirrer. ml 20x SSC, 346.5 ml dH ₂ 0), washing buffer 3 (0.88 ml 20x SSC, 349.12 ml dH ₂ 0)], respectively.

For each total RNA sample, three independent DNA microarrays were performed with three independent biological replicates, including 1-dye-swap experiments.

Microarray Slide Scanning and Data Analysis

After hybridization and washing, the microarray slides were scanned with a GenePix 4000B scanner (Axon Instrument) and the signals were passed to GenePix Pro (version 4.0) and to a gal file ( http://genome.wustl.edu/activity/ma/cneoformans ). Since we used the total RNAs isolated from the antigenic type A Cryptococcus neoformus strain, we used antigenic D cryptocurrency using a blastp irradiation (e value range: e-4) to find the corresponding antigenic type A gene ID. The 70-mer oligonucleotide sequence printed on the Cous neoformus slide was examined against the antigenic A Cryptococcus neoformus genomic database. Each S. cerevisiae was identified by blastp irradiation (e value range: e-4) for each S. cerevisiae using antigenic A gene sequence.

For hierarchical and statistical analysis, data transferred from GenePix software was analyzed by GeneSpring (Agilent) using LOWESS standardization, reliable gene filtering, clustering (standard correlation and mean linkage), zero-transformation, and ANOVA analysis (P Value <0.01).

Ergosterol analysis

The ergosterol content is described in "Arthington-Skaggs BA, Jradi H, Desai T, Morrison CJ (1999) Quantitation of ergosterol content: novel method for determination of fluconazole susceptibility of Candida albicans. J Clin Microbiol 37: 3332-3337" Was measured with slight deformation. Briefly, each Cryptococcus neoformman strain was incubated for 24 hours at 30 ° C. in 100 ml YPD medium. 100 ml cultures were separated into two 50 ml cultures for duplicate measurements, precipitated in a tabletop centrifuge and washed with distilled water. The cell precipitate was cooled in liquid nitrogen and lyophilized overnight. The dried cell precipitate was weighed for standardization of the ergosterol content, 5 ml of 25% alcoholic potassium hydroxide was added and transferred to a sterile rod silicic acid glass screw-cap tube. The cells were then incubated at 80 ° C. for 1 hour and cooled at room temperature. Then 1 ml of distilled water and 3 ml of heptane were added and stirred for 3 minutes. 200 μl of heptene layer was sampled and mixed with 800 μl of 100% ethanol, and its optical density (OD) was measured at both 281.5 nm and 230 nm. The ergosterol content was calculated as follows:% ergosterol = [(OD _{281.5 nm} / 290) x F] / cell precipitate weight-[(OD _{230 nm} / 518) x F] / cell precipitate weight), where F Is the ethanol dilution factor and 290 and 518 are the E values (percentage / centimeter) that determine crystalline ergosterol and 24 (28) dehydroergosterol, respectively.

Stress sensitivity test

"Bahn YS, Kojima K, Cox GM, Heitman J (2005) Mol Biol Cell 16: 2285-2300." And: the method as described in "Bahn YS, Kojima K, Cox GM, Heitman J (2006) Mol Biol Cell 17 3122-3135", was washed over to each strain cultured overnight in YPD medium at 30 ℃ dH ₂ Diluted sequentially in O (1-10 ⁴ dilutions) and spotted with solid YPD medium containing the indicated concentration of stress-inducing reagent or antifungal agent. To determine antifungal drug sensitivity, cells were treated with amphotericin B (0.05, 0.1, 0.3, 0.4, 0.5, 0.6, 0.8 or 1.0 μg / ml) (Sigma), fluconazole (16 or 18 μg / ml) ( Sigma), itraconazole (0.5 μg / ml) (Simga) and ketoconazole (0.2 μg / ml) (Sigma) were spotted on solid YPD medium.

Example 1 DNA Microarray Analysis of Cryptococcus Neoforms hog1, ssk1, skn7 Mutations

To investigate the HOG1 signaling network of Cryptococcus neoforms, comparative analysis of transcripts of antigen-type A wild-type strains (WT, H99), hog1, ssk1, skn7 mutants was performed using DNA microarray analysis. It was performed under stress conditions such as over osmotic shock (1 M NaCl), oxidative shock (2.5 mM H ₂ O ₂ ), and antifungal drug fludioxonil (40 μg / ml). Total RNAs were isolated from cells grown at each stress condition after 0 min (stress free), 30 min and 60 min incubation. Three independent RNA samples were obtained under each condition as biological replicates for DNA microarray analysis. Reference RNAs pooled from all RNA samples were used as control RNAs for Cy3 labeling. We used a 70-mer antigenic type D Cryptococcus neoformans DNA microarray chip containing 7,936 spots based on information obtained from the Cryptococcus neoformance genome database.

The expression levels of HOG1, SSK1, SKN7 genes and known Hog1-associated genes in our array data such as GPP1 (glycerol-3-phosphatase) and GPD1 (glycerol-3-phosphate dehydrogenase) were investigated. .

Figure 1 shows the results of the identification of the genome level of genes regulated by C. neoformans Hog, Ssk1, Skn7 in normal growth conditions without stress (Fold change is expressed in color). 1A shows the relative expression levels of SKN7, SSK1, and HOG1 genes compared to wild-type strain (H99) in hog1 (YSB64), ssk1 (YSB261), and skn7 (YSB349) mutant backgrounds, respectively, and FIG. 1B shows WT, hog1, ssk1 , Conditional tree analysis of a wide range of expression levels in skn7 mutants is shown. FIG. 1C shows the results of a hierarchical clustering analysis of 950 genes showing significantly different expression patterns (ANOVA test, P <0.01) in at least one mutant strain under normal growth conditions (YDP, 30 ° C.), FIG. 1D A venn diagram showing Hog1, Ssk1, Skn7-dependent genes is shown. Genes that exhibit at least two-fold significant upregulation or downregulation in each mutant strain.

As can be seen in FIG. 1, as expected, the comparative expression levels of HOG1, SSK1, SKN7 genes in each corresponding mutation were very low compared to wild type strains. In addition, in other fungi, the expression of the homologous genes GPD1 (CNAG_01745) and GPP1 (CNAG_01744), known as Hog1-related stress defense genes, was greatly reduced (4.5- and 2.5-fold, respectively) in hog1 and ssk1 mutations, reducing the quality of our array data. Support it.

We investigated how HOG1, SSK1, and SKN7 mutations affect gene expression patterns in Cryptococcus neoforms. Of the 7,936 spots examined, 3,858 spots were found to be reliable based on the cross-gin error model (cutoff 10). As supporting previous findings, the transcription levels of hog1 mutations were significantly similar to ssk1 mutations based on condition tree analysis (FIG. 1B). In the hog1, ssk1, skn7 mutations, all of the 950 genes of highly reliable genes showed significantly different expression patterns compared to the wild type (ANOVA test, P <0.01) (FIG. 1C), which indicates the overall Cryptococcus neoformus About 15% of genes are transcriptionally affected by fluctuations of the two-component system and HOG signaling pathways, even under stress-free conditions. 559 of these genes showed more than two-fold induction in at least one mutation (FIG. 1D). Some key findings have been made by: First, only 51 genes (9%) were regulated by Skn7, while the majority of genes (555 genes, 99%) increased or decreased expression by Ssk1 or Hog1 under stress-free conditions. Only four of the Skn7-dependent genes were found to be Skn7-specific (FIG. 1D). Thus, it seems clear that HOG1 and SSK1 mutations alter transcription patterns at the genome level under normal conditions. Second, there is much overlap between Ssk1- and Hog1-dependent genes (422 out of 555, 76%) than between Skn7- and Hog1-dependent genes (45 out of 467 genes, 10%). Many exist, further verifying that Ssk1 is the regulator of Hog1 MAPK's major superstructure. Third, regardless of the significant overlap of genes regulated by Ssk1 and Hog1, there were some Ssk1-specific (90 genes), Hog1-specific (40 genes) genes, and Ssk1 and Hog1 It is strongly suggested that each may have different target (s) or supervisory regulators that are not exactly in the linear path (FIG. 1D). This explains why the ssk1 mutant has a slightly different phenotype (ie higher hydrogen peroxide sensitivity) than the hog1 mutant, and why Hog1 can still be phosphorylated without Ssk1 response modulators when exposed to NaCl.

The genes regulated by Hog1 and Ssk1 cover a variety of functional categories, including energy production and conversion, amino acid / carbohydrate / fat transport and metabolism, translation and protein biosynthesis, post-translational modifications, signal transduction, and stress-defense mechanisms. It is shown that remodeling in various aspects of function can be simply caused by the fluctuation of the HOG pathway without external stress. In addition, it should be noted that more than one-third of Hog1- and Ssk1-dependent genes do not have any functional orthologs from other organisms, indicating that Cryptococcus neoforms generates Cryptococcus-specific Hog1 / Ssk1-dependent genes. It appears to be.

Among the Ssk1- and Hog1-regulatory genes identified in our array analysis, several groups of genes have provided new insights into the pathogenesis of Cryptococcus neoforms and the possible mechanisms of the HOG pathway that regulates sexual reproduction. First, one group of genes involved in iron transport and regulation was found to be more induced in ssk1 and hog1 mutants than in wild-type strains. These genes include SIT1 (CNAG_00815 and CNAG_07138), which encodes a siderophore, CFO1 (CNAG_06241) and CFO2 (CNAG_02958), which encodes peroxidase, and CFT1 (CNAG_06242), which encode Fe transporters. Cryptococcus neoformus Sit1 is homologous to S. cerevisiae Arn3 / Sit1, which has a high affinity for hydroxyxamate siderophore perioxamine, and Cryptococcus neoformus Cfo1 / Cfo2 and Cft1 in S. cerevisiae Is homologous to the HIV / multicopper peroxidase complex (Ftr1-Fet3). Since iron transport regulation and melanin synthesis appear to be highly associated with Cryptococcus neoformes, the increased melanin synthesis observed in both hog1 and ssk1 mutations may be correlated with increased expression of a group of genes involved in iron transport. .

Second, the GPA2 gene (CNAG_00179), which encodes a G-protein subunit in the pheromone-responsive MAPK pathway, is significantly upregulated in ssk1 or hog1 mutations (12.1 fold 13.3 fold increased, respectively). These findings indicate that the increased pheromone production and sexual reproduction found in ssk1 and hog1 mutations may have resulted from increased expression of Gpa2 induced during mating and may enhance the mating process of Cryptococcus neoforms.

Third, several genes involved in oxidative stress defense were differentially regulated by HOG1 and SSK1 mutations. As expected from previous findings that the hog1 and ssk1 mutants show hypersensitivity to hydrogen peroxide, the two genes (CNAG_04981 and CNAG_00575) that are homologous to the CTA1 gene, encoding catalase A, which translates H ₂ O ₂ into H ₂ O Significantly down-regulated at. In addition, basal expression levels of the SOD2 gene (mitochondrial superoxide dismutase) decreased in both hog1 and ssk1 mutations, further verifying the role of the HOG pathway in oxidative stresstm regulation. Interestingly, however, several genes involved in the oxidative stress response (TRR1 (thioredoxin reductase), TSA1 (thioredoxin peroxidase), GRX5 (glutathione-dependent oxidoreductase), CCP1 (mitochondrial cytochrome-c peroxidase)) The basal expression level of increased more than twofold in hog1 mutations (3.8-fold, 3.1-fold, 2.1-fold, 9.5-fold changes, respectively) but not in the ssk1 mutant. The SRX1 gene (sulpyredoxin), which is also involved in the oxidative stress response, was also reduced in the ssk1 mutant (4.2 fold) than in the hog1 mutant (1.3 fold). These results may explain why hog1 mutations are relatively more resistant to H ₂ O ₂ than ssk1 mutations.

Example 2 Expression Analysis of Ergosterol Biosynthesis Genes

Of the genes upregulated by HOG1 and SSK1 gene mutations, it was noteworthy that homologous genes for ERG28 (CNAG_07208) play a key role in fungal ergosterol biosynthesis. Previous microarray analysis of S. cerevisiae revealed that ERG28 expression was tightly correlated with other ergosterol biosynthetic genes. Erg28 is an endoplasmic reticulum (ER) membrane backbone protein and is essential for yeast erosteroster biosynthesis by interacting strongly with Erg27, Erg25, Erg11, Erg6 and weakly with Erg26, Erg1. This discovery led to the identification of expression patterns of other ergosterol biosynthetic genes in the array data.

2A shows the relative expression levels of ergosterol biosynthesis in hog1, ssk1, skn7 mutations compared to wild type. Fold changes are expressed in color, and the exact number of each gene is shown in a table to the right of the hierarchical clustering diagram. 2B shows cellular ergosterol content in WT (H99), skn7 (YSB349), ssk1 (YSB261), ssk2 (YSB264), hog1 (YSB64) mutations. The left and right graphs show each strain and the relatively increased percentage of ergosterol in the ergosterol content compared to wild type, respectively. Each bar represents the mean from four independent experiments and error bars represent standard deviations. (*): Ssk1, ssk2, pbs2, and hog1 mutations have significantly higher levels of ergosterol compared to wild type (P <0.05, Bonferroni multiple comparison test).

As can be seen in FIG. 2A, most of the ergosterol biosynthesis genes were upregulated in hog1 and ssk1 mutants compared to wild-type strains, but not skn7 mutants. Genes such as ERG27, ERG13, ERG26, ERG10, IDI1, HMG1, and ERG8 are upregulated in ssk1 mutations, whereas genes such as ERG11, ERG6, MVD1, ERG5, ERG25, ERG20, ERG4 are up-regulated in both ssk1 and hog1 mutations. It was regulated. In contrast, none of the genes in the skn7 mutant were significantly upregulated, and in fact some genes, including the ERG13, ERG1, ERG3, ERG7 and ERG2 genes, were downregulated in the skn7 mutant.

To validate the microarray data, it was tested whether the increased expression levels of several ergosterol biosynthetic genes in hog1 and ssk1 mutants actually affected the cell's ergosterol content. As a result, as can be seen from FIG. 2B, the ergosterol content of cells was much higher in hog1 and ssk1 mutants than in wild type and skn7 mutants, indicating that increased expression of some ergosterol biosynthetic genes resulted in increased ergosterol in the cell. Indicates that it caused production. The ssk2 (MAPKKK) and pbs2 (MAPKK) mutations in the HOG pathway appear to have significantly higher cellular ergosterol levels than wild type and skn7 mutations (FIG. 2B), which further validates our array data.

This finding stimulated the investigation of the susceptibility of mutations to ergosterol biosynthetic genes in the two-component system and the HOG pathway or to antifungal drugs targeting ergosterol itself. First, the susceptibility to ssk1, skn7, ssk2, pbs2, hog1 mutants made against the antigenic A H99 strain background against the polyene antifungal drug, amphotericin B, was tested. Ampoterisin B ultimately causes death by binding to ergosterol in the fungal cell membrane and disrupting membrane integrity. It was assumed that the increased ergosterol content observed in ssk1, ssk2, pbs2, and hog1 mutations could make them hypersensitive to amphotericin B because of the increased drug target number.

Figure 3 is an analysis showing that inhibition of the HOG pathway shows an elevated antifungal effect with amphotericin B in C. neoformans. 3A to 3B show photographs obtained by culturing each C. neoformans strain spotted on YPD solid medium containing amphotericin B at the indicated concentrations at 30 ° C. for 72 hours, and FIG. 3C shows the cancer cells at the indicated concentrations. C. neoformans antigen-type A strains and antigen-type D strains spotted on YPD solid medium containing erysin B are photographed by incubation at 30 ° C. for 72 hours.

As can be seen from FIG. 3, as the hypothesis tested, ssk1, ssk2, pbs2, hog1 mutants showed significant hypersensitivity to amphotericin B treatment compared to wild type (FIG. 3A), indicating that the ergosterol content was higher than that of wild type HOG. Consistent with the finding that it was much higher in pathway mutations (FIG. 2B). In contrast, skn7 mutations showed wild-type levels of resistance to amphotericin B (FIG. 3A), which is also illustrated by previous data that the ergosterol content of cells in skn7 mutations was similar to wild-type (FIG. 2B).

We also examined the amphotericin B-sensitivity of Cryptococcus neoformas strains with mutant hybrid sensor kinase (Tco1, Tco2, Tco3, Tco4, Tco5, Tco7) that act on top of the Ssk1 response modulator. We have shown in previous studies that Tco1 and Tco2 play a role of differentiation and differentiation in regulating some of the Hog1-dependent phenotypes. Here we found that Tco1 and Tco2 each play a role in detecting and responding to amphotericin B. Of the Tco proteins, only Tco2, a double hybrid sensor kinase with two response modulator regions and two histidine kinase regions in one polypeptide, showed hypersensitivity to amphotericin B (FIG. 3B), indicating that Tco2 is responsible for the HOG pathway. Through the detection and response of amphotericin B to give drug resistance. However, the fact that the degree of hypersensitivity observed in the tco2 mutation is less than that of the ssk1 mutation suggests other possibilities. One possibility is that other unknown receptors / sensors may be present to respond to amphotericin B. Another possibility is that since Ssk1, Ssk2, and Pbs2 (except Tco2) are all related to the structural phosphorylation level of Hog1, structurally phosphorylated Hog1 inhibits the ergosterol biosynthetic pathway under normal conditions regardless of the presence of receptors / sensors. You can do it.

To test the hypothesis, amphotericin B sensitivity of other Cryptococcus neoforms strains such as JEC21, B3501-A was tested and exhibited differential Hog1 phosphorylation levels. In the validation of the second hypothesis, the JEC21 strain in which Hog1 was not always phosphorylated showed hypersensitivity to amphotericin B, even more than the ssk2 mutation in the H99 strain background (FIG. 3C). In the JEC21 strain background, mutations in the SSK2, PBS2, HOG1 genes did not affect the sensitivity to amphotericin B (FIG. 3C). In contrast, in the B3501 strain that Hog1 was constantly phosphorylated, it showed higher resistance to amphotericin B than JEC21, although to a lesser extent than in the H99 strain (FIG. 3C). Similar to the H99 strain, SSK2 MAPKKK mutants that disrupt Hog1 phosphorylation increased amphotericin B sensitivity (FIG. 3C). All these data indicate that constantly phosphorylated Hog1 inhibits the ergosterol biosynthetic pathway under normal conditions.

To validate this finding, we included triazoles (fluconazole and itraconazole) and imidazole (ketoconazole) that inhibit the fungal cytochrome P450 enzyme 14-dimethylase and ultimately prevent the conversion of lanosterol to ergosterol. The susceptibility of mutations to azole compounds was tested.

FIG. 4 is an analysis showing that inhibition of the HOG pathway in C. neoformans shows antagonistic antifungal effects on some azole drugs, with each C. neoformans strain YPD containing fluconazole, ketoconazole, itraconazole at the indicated concentrations. Spotted on a solid medium and then incubated for 72 hours at 30 ℃ shows a photograph taken.

We anticipated that ssk1 and hog1 mutations, particularly those with increased expression of many ergosterol biosynthetic genes, including ERG11, would be highly resistant to azole compounds. As can be seen from Figure 4, all of the ssk1, ssk2, pbs2, hog1 mutations showed sensitive resistance to fluconazole and ketoconazole, but not itraconazole. Interestingly, skn7 mutations also showed higher resistance to fluconazole and ketoconazole than the wild type. Of the hybrid sensor kinase, only Tco1 and Tco2 show differential sensitivity to azole compounds. Although smaller than the HOG mutation, the tco2 mutation showed higher resistance to fluconazole and ketoconazole than the wild type. Conversely, tco1 mutations are hypersensitive to all azole drugs, indicating that Tco1 may regulate the HOG pathway in contrast to Tco2 in Cryptococcus neoforms. As a result, inactivation of the HOG pathway increases ergosterol content by induction of ergosterol biosynthesis genes and thus a synergistic effect on amphotericin B treatment, but an antagonistic effect on fluconazole and ketoconazole.

Figure 1 shows the results of the identification of the genome level of genes regulated by C. neoformans Hog, Ssk1, Skn7 in normal growth conditions without stress (Fold change is expressed in color).

Figure 2 shows the results of analysis of the induction of ergosterol biosynthesis genes and cellular ergosterol content by the shaking of the HOG signaling pathway.

Figure 3 is an analysis showing that inhibition of the HOG pathway shows an elevated antifungal effect with amphotericin B in C. neoformans.

4 is The analysis shows that inhibition of the HOG pathway in C. neoformans has antagonistic antifungal effects on some azole drugs.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Use of the genes in the HOG pathway for treatment of fungal          infection <130> P08925 <160> 15 <170> KopatentIn 1.71 <210> 1 <211> 1309 <212> PRT <213> Cryptococcus neoformans serotype A H99 strain <220> <221> PEPTIDE (1) .. (1309) <223> amino acid sequence of SSK1 <400> 1 Met Trp Gly Ser Asn Ala Ser Ile Ala Ala Ser Glu Ser Thr Asp Ser   1 5 10 15 Leu Ser Pro Ala Pro Ser Gln Ser Ala Ala Val Glu Phe Pro Leu Pro              20 25 30 Val Ser Ser Arg Pro Ser Leu Thr Ser Ala Ala His Pro Ser Gln Met          35 40 45 Ser Ala Ser Ser Ser Ser Thr Ser Ser Gln Pro Leu Phe Asp Trp Arg      50 55 60 Ile Pro Lys Pro Thr Ser Pro Arg Thr Arg Met Asp Pro Phe Asp Thr  65 70 75 80 Phe Asp Pro Val Ser Ser Ser Ser Glu Asp Asp Pro Val Pro Gln Glu                  85 90 95 Ser Arg Arg Ala Gly His Gln Arg Ser Val Thr Asp Pro Leu Leu Arg             100 105 110 Asp Gly Gln Pro Leu Asp Met Glu Phe Thr Thr Ala Gly Pro Pro Ile         115 120 125 Gln Ser Tyr Asp Phe Glu Gln Pro Pro Thr Phe Ser Arg Thr Leu Ser     130 135 140 Ser Pro Leu Pro Ala Lys Val Gly Ser Leu Arg His Pro Met Pro Phe 145 150 155 160 Thr Ile Asp Asp Leu Ser Ser Arg Asn Val Asn Ser Thr His Arg Pro                 165 170 175 Gln Pro Thr Thr Pro Leu His Ser Ile Ser Val Glu Leu Ala Asp Ser             180 185 190 Leu Gln Ser Ala Ile Gln Thr Leu Leu His Leu Ser Pro Pro His Leu         195 200 205 Leu Asp Asn Ala Lys Glu Gln Tyr Ser Gly Cys Thr Val Gln Ile Pro     210 215 220 Ala Thr Ser Leu Ser Ala Leu Leu Thr Ser Met Arg Gly Leu Asn Phe 225 230 235 240 Leu Ser Ala His Ala Glu Glu Leu Val Asp Met Ser Ala Arg Gly Asp                 245 250 255 Pro Pro Val Leu His Gln Glu Asp Phe Asp Val Gly Glu Leu Leu Gln             260 265 270 Asn Val Ala Asp Met Leu Ser Gly Glu Ala Ala Glu Lys Arg Ile Asp         275 280 285 Phe Val Leu Phe His Gly Asp Val Ala Met Arg His Val Ser Val Tyr     290 295 300 Gly Asp Ser Asp Gly Ile Ser Tyr Thr Leu Ser His Val Ile Arg Gln 305 310 315 320 Ile Leu Ala Val Ala Asn Tyr Asp Asp Thr Ile Glu Leu Gly Leu Gln                 325 330 335 Val Ile Pro Gln Ser Pro Ser Leu Ala Ser Ala Val Gly Leu Pro Leu             340 345 350 Thr Ser Ala Asp Val Ser Gly Gly Gly Gly Val Lys Ser Ala Ser Thr         355 360 365 Ser Arg Ser Gly Ser Pro Asn Asn Ser Leu Ser Arg Ser Asn Ser Val     370 375 380 His Asp Gly Pro Leu Leu Cys Val Phe Glu Ile Val His Asn Ile Tyr 385 390 395 400 Gln Pro Pro Pro Ser Ser Ala Ser Ala Thr Pro Lys Ala Glu Leu Asn                 405 410 415 Pro Phe Thr His Leu Ala Glu Glu Thr Glu Ala Leu Lys Pro Arg Leu             420 425 430 Asp Thr Ala Phe Cys Lys Asn Leu Leu His Arg Gln Asn Ala Val Leu         435 440 445 Lys Val Asp Val Gln Pro Ser Ser Pro Leu Gly Ser Gly Met Pro Arg     450 455 460 Arg Ala Tyr Ala Leu Ser Val Leu Leu Pro Arg Gly Lys Pro Ile Thr 465 470 475 480 Glu Pro Ala Ile Leu Ser Lys Glu Glu Gln Glu Val Arg Gln Pro Phe                 485 490 495 Ser Ser His Val Leu Ala Arg Glu Pro Thr Leu Asn Glu Leu Ser Glu             500 505 510 Phe Ala Glu Ser Leu Arg Gly Arg Lys Val Phe Ile His Ala Asn Leu         515 520 525 Ser Ser Val Phe Ala Arg His Leu Thr Ser Tyr Leu Ala Ala Trp Gly     530 535 540 Met Asp Ile Ser His Leu Pro Thr Asp Gly Asp Glu Ala Asp Lys Leu 545 550 555 560 Lys Asp Val Ala Ala Lys His Asp Ser Ala Tyr Thr Gly Ser Met Gly                 565 570 575 Val Ser Gly Gly Thr Thr Ser Ser Ala Glu Thr Pro Tyr Ser Ile Lys             580 585 590 Pro Thr Gly Val Thr Ala Val Gln Pro Gly His Phe Val Ile Ile Asp         595 600 605 Asp Asp Val Ala Val Leu Arg Arg Glu Leu Val Arg Ile Arg Ser Glu     610 615 620 Leu Leu Pro Ile Leu Phe Lys Pro Arg Leu Ser Lys Arg Pro Thr Met 625 630 635 640 Thr Ser Arg Thr Arg Ser Thr Pro Ser Leu Arg Gln Val Pro Pro Arg                 645 650 655 Ser Ser Ser Gly Ser Val Leu Ile His Phe Thr Ser Leu Ala Asn Tyr             660 665 670 Asn Arg Val Arg Asp Ala Ile Ala Ser Phe Val Gly Ala Pro Gly Leu         675 680 685 Thr Asn Pro Glu Thr Tyr Val Gln Pro Glu Val Ile Val Ile Pro Lys     690 695 700 Pro Val Gly Pro Arg Arg Phe Leu Thr Ala Leu His Thr Ala Val Lys 705 710 715 720 Gln Pro Met Val Asp Pro Phe Phe Ser Pro Ile Ala Thr Ser Pro Arg                 725 730 735 Ser Pro Gly Gly Gly Tyr Phe Gly Gly Leu Arg Thr Pro Thr Glu Arg             740 745 750 Glu Ser Gly Phe Phe Asp Ser Val Ala Glu Glu Pro His Glu Glu Ala         755 760 765 Asp Ser Arg Pro Asp Tyr Ala Thr Val Gln Lys Ala Arg Ser Pro Leu     770 775 780 Gly Glu Phe Pro Pro Ser Ala Ala Gln Ile Val Arg Thr Asn Gln Gly 785 790 795 800 Leu His Leu Ser Leu Pro Thr Pro Asn Glu Ile Met Thr Thr Pro Ala                 805 810 815 Pro Glu Tyr Phe Ser Gly Ser Ser Lys Ser Pro Ser Ser Gly Ala Ser             820 825 830 Gly Val Val Met Gln Ser Pro Asp Gly Arg Pro Phe Gly Met Phe Phe         835 840 845 Glu Pro Pro Ile Lys Asn Glu Arg Arg Gly Ser Thr His Arg Thr Pro     850 855 860 Ser Asp Ser Ile Arg Arg Lys Gln Ala Asn Arg Arg Ala Ser Thr Ser 865 870 875 880 Asp Glu Pro Phe Ser Ser Pro Ser Thr Ala Leu Pro Pro Arg Arg Ser                 885 890 895 Ser Thr Ile Ser Thr Thr Gly Asn Glu Glu His Arg Ser Ser Pro Ile             900 905 910 Ala Asn Val Thr Asp Arg Pro Thr His Ser Arg Val Asn Ser Arg Arg         915 920 925 Lys Asn Asn Leu Pro Ala Ala Glu Gln Pro Ile Leu Ala Val Gly Arg     930 935 940 Ala Lys Gly Arg Glu Arg Ser Glu Thr Val Thr Lys Gly Gly Asp Leu 945 950 955 960 Gly Ser Arg Lys Gly Thr Pro Ala Ala Ser Pro Arg Ile Glu Glu Lys                 965 970 975 Lys Glu Leu Glu Arg Gly Glu Lys Thr Lys Ser Leu Ala Pro Ser Thr             980 985 990 Ala Pro Thr Lys Lys Asn Ala Lys Val Asp Val Val Val Pro Pro Ile         995 1000 1005 Asn Val Leu Ile Val Glu Asp Asn Pro Ile Asn Gln Asn Ile Leu Ser    1010 1015 1020 Met Phe Leu Arg Lys Lys Lys Ile Lys Asn Ser Ser Ala Lys Asp Gly 1025 1030 1035 1040 Ala Glu Ala Val Glu Lys Trp Arg Thr Gly Gly Phe His Leu Ile Leu                1045 1050 1055 Met Asp Ile Gln Leu Pro Val Met Asp Gly Ile Ala Ala Thr Lys Glu            1060 1065 1070 Ile Arg Arg Leu Glu Arg His Asn Asn Ile Gly Val Phe Pro Ser Thr        1075 1080 1085 Pro Ala Ala Glu Leu Pro Arg Gly Gln Asn Val Ala Asp Ser Pro Pro    1090 1095 1100 Pro Ser Ser Pro Phe Arg Ser Ser Val Ile Val Ala Leu Thr Ala 1105 1110 1115 1120 Ser Ser Leu Gln Ser Asp Arg Val Ala Ala Leu Ala Ala Gly Cys Asn                1125 1130 1135 Asp Phe Leu Thr Lys Pro Val Ser Leu Lys Trp Leu Asp Lys Lys Ile            1140 1145 1150 Val Glu Trp Gly Cys Met Gln Ala Leu Ile Asp Phe Asp Gly Trp Arg        1155 1160 1165 Arg Trp Lys Ser Ser Asp Thr Lys Asn Pro Ser Glu Thr Lys Gln Gly    1170 1175 1180 Phe Ser Val Gly Pro Gln Gln Ala Ala Arg Ser Leu Ala Ser Arg Leu 1185 1190 1195 1200 Arg Ile Glu Arg Lys Gly Ser Arg Ser Pro Ala Ala Pro Val Ser Thr                1205 1210 1215 Pro Arg Leu Asn Leu Gln Ser Ala Thr Pro Asp Arg Pro Glu Thr Pro            1220 1225 1230 Pro Asp Ser Thr Ser Gln Met Pro Lys Ala Pro Pro Val Ala Ala Ser        1235 1240 1245 Asp Pro Pro Leu Ser Pro Lys Ser Leu Asn Lys Thr Val Asn Asp Val    1250 1255 1260 Phe Glu Gln Ala Asp Ala Arg Leu Glu Asn Ala Arg Glu Glu Gln Gly 1265 1270 1275 1280 Val Ser Ser Gln Lys Glu Asn Thr Ser Leu Thr Asp Ser Thr Asn Thr                1285 1290 1295 Thr Ile Thr Pro Ser Lys Thr Tyr Pro Ala Pro Pro Pro            1300 1305 <210> 2 <211> 1691 <212> PRT <213> Cryptococcus neoformans serotype A H99 strain <220> <221> PEPTIDE (222) (1) .. (1691) <223> amino acid sequence of TCO2 <400> 2 Met Ile Leu Gly Thr Asp Ile Asp Leu Ser Ser Ile Pro Thr Ala Phe   1 5 10 15 Leu Glu Ala Tyr Pro Phe Pro Ala Val Val Phe Val Ile Asp Ser Pro              20 25 30 Pro Ser Pro Arg Pro Arg Leu His Ser Arg Asn Thr Asp Thr Thr Ile          35 40 45 Arg Arg Thr Asp Gly Gln Ile Ser Pro Leu Thr Gly Pro Pro Val Gln      50 55 60 Gln Phe Ala Ser Ala Pro Val Val Trp Gly Asn Gln Arg Trp His Glu  65 70 75 80 Leu Ala Gln Gly Lys Thr Ile Ala Glu Cys Val Asp Val Ala Ser Gln                  85 90 95 Asn Lys Leu Gln Thr Trp Val Glu Asn Asp Thr Gly Asp Lys Ser Glu             100 105 110 Ser Leu Ala Leu Asp Leu Lys Val Pro Gln Gly Val Thr Leu His Leu         115 120 125 Ala Lys Thr Ile Leu Pro Leu Ser Pro Pro Ser Ser Ser Gln Ser Leu     130 135 140 Cys Ile Leu Ile Ser Gln Tyr Ile Asp Lys Pro Glu Ser Phe Ala Pro 145 150 155 160 Pro Ile Ser Ser Gly Asp Ile Leu Phe Ser Ser Leu Ser Arg Leu Ser                 165 170 175 Gln Thr Phe Ser Arg Ser Ser Ser Phe Ser Ser Asn Pro Arg Lys Ser             180 185 190 Ile Asp Val Pro Ala Ser Leu Ser Glu His Arg Gly Ser Ala Thr Ser         195 200 205 Thr Ser Ser Asn Leu Arg Ser Ser Ile Asp Leu Thr Ser Pro Asn Ser     210 215 220 Gln Pro Ser Pro Leu Asn Arg Glu Gln Ser Thr Tyr Phe Thr His Gly 225 230 235 240 Ser Ala Thr Arg Glu Glu Arg Pro Ser Val Arg Arg Arg Arg Ser Pro                 245 250 255 Pro Ile Ser Met Thr Arg Pro Lys Pro Leu Glu Ser His Ala Gln Glu             260 265 270 Cys Trp Asp Leu Val Glu Asn Phe Asp Trp Ser Lys Thr Ala Leu Gly         275 280 285 Pro Arg Glu Gln Trp Met Asp Ala Leu Asp Pro Val Leu Ala Ile Thr     290 295 300 Phe Glu Ser Arg Thr Ala Asp Cys Ala Trp Leu Gly Pro Asp Leu Glu 305 310 315 320 Leu Val Tyr Asn Lys Ala Tyr Gln Glu Leu Val Asp His Pro Asn Ala                 325 330 335 Phe Gly Lys Pro Ala Arg Gln Val Trp Ala Thr Asn Trp Asp Tyr Leu             340 345 350 Glu Pro Leu Val Lys Arg Cys Leu Ser Gly Thr Pro Val Tyr Lys Asp         355 360 365 Asn Asp Pro Leu Phe Trp Arg Arg Tyr Gly Asn Gly Arg Leu Leu Glu     370 375 380 His Tyr His Thr Trp Arg Tyr Val Pro Ile Thr Gly Lys Asp Gly Ser 385 390 395 400 Val Leu Gly Ile Phe Asn Gln Ser Ile Glu Val Thr Asp Ser Val Leu                 405 410 415 Leu Glu Arg Arg Met Gly Thr Thr Arg Glu Leu Ser Glu His Met Ser             420 425 430 Phe Ile Arg Thr Thr Glu Asp Phe Phe Ser Ser Val Ala Asp Val Phe         435 440 445 Ser Gln Asn Pro Thr Asp Ile Pro Phe Ala Leu Cys Tyr Arg Val Arg     450 455 460 Gln Val Asp Thr Asp Gly Thr Phe Val His Leu Asp Val Ser Leu Gln 465 470 475 480 Ser Ser Val Gly Val Pro Glu Gly His Pro Ser Ala Pro Asp Gln Ile                 485 490 495 Pro Val Ser Phe Leu Asn Gly Asn Pro Tyr Pro Ser Asn Val Glu Arg             500 505 510 Ser Phe Ser Pro Ala Phe Ser Ile Val Ser Ile His Ser Ser Ser Ser         515 520 525 His Arg Val Cys His Val Ser Glu Asp Thr Thr Gln Trp Pro Ile Ala     530 535 540 Lys Ala Leu Gln Arg Arg Gln Cys Val Ile Ile Glu Glu Cys Ser Gln 545 550 555 560 Leu Ile Glu Gly Tyr Pro Ile Arg Arg Trp Asp Gly Leu Pro Phe Ser                 565 570 575 Ala Ile Val Val Pro Ile Cys Ser Glu Gly Ser Pro Glu Ile Pro Asp             580 585 590 Ala Val Val Ile Leu Gly Leu Asn Val Arg Arg Cys Phe Asp His Glu         595 600 605 Tyr Asp Ser Trp Ile His Ser Ile Arg Ser Gln Leu Ser Ser Ala Leu     610 615 620 Val Met Val Lys Ala Arg Glu Ala Glu Gln Lys Met Val Glu Glu Ser 625 630 635 640 Ala Arg Met Glu Lys Ala Lys Val Ala Trp Phe Arg Gly Ala Ala His                 645 650 655 Asp Leu Arg Ser Pro Leu Thr Leu Val Ala Gly Pro Leu Ala Asp Val             660 665 670 Leu Asp Ser Asp Leu Asn Ser Ser Gln Arg Thr Ala Leu Thr Val Ala         675 680 685 Gln Arg Asn Leu Asp Arg Leu Val Arg Leu Val Asn Ala Leu Met Asp     690 695 700 Phe Ser Arg Val Glu Ala Gly Arg Met Glu Gly Arg Phe Val Pro Thr 705 710 715 720 Asn Leu Ser Gln Phe Val Thr Gln Leu Ala Alu Leu Phe Lys Pro Ala                 725 730 735 Ile Glu Arg Leu Gly Leu Glu Tyr Val Leu Asp Val Gln Pro Ser Glu             740 745 750 Glu Leu Val Phe Ile Asp Pro Val Leu Phe Glu Thr Val Val Ser Asn         755 760 765 Leu Ile Gly Asn Ala Leu Lys Tyr Thr Glu Thr Gly Ser Ile Thr Val     770 775 780 Arg Val Gln Tyr Thr Asp Tyr Ala Glu Val Ser Val Ile Asp Thr Gly 785 790 795 800 Val Gly Ile Pro Lys Asn Glu Leu Ala Leu Val Thr Glu Trp Phe His                 805 810 815 Arg Ala Ser Thr Ala Ile His Ser Gly Thr Gln Gly Thr Gly Leu Gly             820 825 830 Leu Ala Leu Ala Lys Glu Leu Leu Lys Leu His Lys Gly Glu Leu Leu         835 840 845 Val Glu Ser Gln Thr Ala Asn Glu Ser Gly Gly Pro His Gly Ser Ile     850 855 860 Phe Thr Ala Lys Ile Pro Leu Asp Phe Lys Pro Ser Pro Ser Ala His 865 870 875 880 Ile Ile Pro Ser Val Glu Ser His Lys Thr Phe Gly Lys Tyr Ser Lys                 885 890 895 Ala Val Ala Asp Glu Ala Met Arg Trp Val Gly Asp Ser Asp Ala Ala             900 905 910 Ser Glu Ala Tyr Asp Met Ser Ser Gly Thr Gly Val Ser Ser Ala Gly         915 920 925 Ser Gly Ser Gly Asn Thr Thr Thr Phe Gly Pro Lys Phe Ala Asp Ala     930 935 940 Phe Leu Phe Asp Lys Asn Asp Ile Val Leu Ile Val Glu Asp Asn Val 945 950 955 960 Asp Met Arg Glu Tyr Ile Arg Gln Leu Phe Ala Pro Tyr Cys Thr Val                 965 970 975 Leu Glu Ala Ser Asn Gly Glu Gln Ala Tyr Asn Met Ala Thr Gln Asn             980 985 990 Pro Pro Asn Leu Ile Leu Ser Asp Val Leu Met Pro Lys Leu Ser Gly         995 1000 1005 Met Glu Leu Leu Gln Arg Ile Arg Ser His Pro Asp Thr Arg Ile Val    1010 1015 1020 Pro Met Val Leu Ile Ser Ala Ile Ala Gly Asp Glu Ser Arg Val Glu 1025 1030 1035 1040 Ala Leu Leu Asn Gly Ala Asp Asp Tyr Leu Ala Lys Pro Phe Lys Pro                1045 1050 1055 Lys Glu Leu Ile Ala Arg Val His Leu His Met Gln Val Gly Lys Lys            1060 1065 1070 Arg Ala Lys Leu Glu Ala Leu Tyr Ala Gln Arg Glu Thr Glu Leu Thr        1075 1080 1085 Ala Leu Ser Asp Tyr Cys Pro Ile Gly Ile Phe Arg Gly Asp Lys Tyr    1090 1095 1100 Gly His Ile Val Tyr Ala Asn Ala Ala Trp Arg Ala Gln Ser Gly Leu 1105 1110 1115 1120 Leu Val Gly Asp Pro Asn Asp Trp Ala Ser Tyr Val His Pro Asp Ser                1125 1130 1135 Lys Ala Gln Leu Leu Glu Gln Trp Asn Gln Trp Leu Arg Gly Asp Leu            1140 1145 1150 Lys Glu Phe Arg Ala Ala Trp Arg Trp Ser Asn Gly Ile Pro Val Arg        1155 1160 1165 Ser Ile Leu Val Arg Leu Asp Asp Val Lys Glu Gly Phe Ser Gly Leu    1170 1175 1180 Ile Gly Cys Val Val Asp Val Ser His Glu Glu Arg Arg Leu Ile Glu 1185 1190 1195 1200 Ala Glu Glu Arg Arg Lys Glu Ala Glu Glu Ser Lys His Gln Gln Glu                1205 1210 1215 Leu Leu Ile Asp Leu Thr Ser His Glu Ile Arg Thr Pro Val Ser Ala            1220 1225 1230 Ile Leu Gln Cys Ser Asp Leu Val Lys Glu Asn Leu Val Ala Leu Lys        1235 1240 1245 Asp Gln Leu Arg Gly Ala Gly Pro Lys Gly Phe Val Pro Ser Gln Glu    1250 1255 1260 Leu Leu Ala Asp Leu Glu Gln Asp Val Glu Ala Leu Glu Ser Ile Tyr 1265 1270 1275 1280 Gln Cys Gly Leu Val Gln Glu Arg Ile Ala Gly Asp Val Leu Ser Leu                1285 1290 1295 Ala Arg Ile Gln Leu Asp Met Leu Ser Leu His Asp Ile Asp Val Asn            1300 1305 1310 Leu Arg Arg Glu Gly Arg Lys Val Ser Ser Ile Phe Ala Ser Glu Ala        1315 1320 1325 Lys Met Lys Asp Ile Asp Leu Gln Leu Glu Phe Gly Pro Thr Ile Glu    1330 1335 1340 Gln Ser Lys Val Leu Ala Ile Lys Thr Asp Pro Val Arg Leu Gly Gln 1345 1350 1355 1360 Val Val Thr Asn Leu Ile Ser Asn Ala Ile Arg Phe Thr Ser Ser Ser                1365 1370 1375 Asp Val Arg Lys Ile Thr Ile Gln Tyr Asp Val Ser Phe Val Pro Pro            1380 1385 1390 Ala Asp Asp Ser Cys Ala Leu Pro Ser Ser Val Gly Leu Pro Asp Ile        1395 1400 1405 Leu Pro Val Lys Glu Asn Thr Pro Leu Trp Leu Phe Val Ser Val Thr    1410 1415 1420 Asp Ser Gly Pro Gly Met Thr Glu Gln Glu Leu Ser Val Leu Phe Gln 1425 1430 1435 1440 Arg Phe Ala Gln Gly Asn Lys Met Ile His Thr Lys Tyr Gly Gly Ser                1445 1450 1455 Gly Leu Gly Leu Phe Ile Cys Arg Lys Ile Thr Glu Leu Leu Gly Gly            1460 1465 1470 Arg Ile Glu Val Leu Ser Gln Val Gly His Gly Ser Val Phe Arg Phe        1475 1480 1485 Phe Ile Lys Thr Arg Ala Val Ala Pro Pro Ser Ala Ile Ala Ala Leu    1490 1495 1500 Val Glu Ser Ser Pro Leu Lys Pro Val Ser Ala Thr Ser Pro Ser Ser 1505 1510 1515 1520 Ser Leu Ala Met Ser Arg Ser Ser Ser Arg Ser Thr Asn Val Thr Thr                1525 1530 1535 Pro Ile Glu Gly Gly Gly Thr Glu His Val Leu Ile Val Glu Asp Asn            1540 1545 1550 Leu Ile Asn Gln Thr Val Leu Lys Arg Gln Leu Val Lys Ala Gly Leu        1555 1560 1565 Ser Cys Asn Val Ala Ser Asn Gly Leu Glu Ala Leu Asn Val Ile Arg    1570 1575 1580 Glu Val His Arg Gln His Arg Arg Gly Gly Pro Asn Arg Lys Arg Leu 1585 1590 1595 1600 Phe Asp Val Val Leu Met Asp Leu Glu Met Pro Val Met Asp Gly Ile                1605 1610 1615 Thr Ala Val Arg Glu Ile Arg Gln Ser Glu Ala Ala Gly Thr Leu Gly            1620 1625 1630 Arg Asn Met Val Ile Ala Leu Thr Gly Asn Ala Arg Gln Gly Gln Ile        1635 1640 1645 Asp His Ala Leu Ala Ser Gly Phe Asp Asp Val Val Ile Lys Pro Tyr    1650 1655 1660 Ile Leu Val Asp Leu Leu Asn Lys Ile Lys Ser Met Lys Val Arg Lys 1665 1670 1675 1680 Leu Glu Leu Glu Thr Ala Lys Ala Gln Glu Glu                1685 1690 <210> 3 <211> 1417 <212> PRT <213> Cryptococcus neoformans serotype A H99 strain <220> <221> PEPTIDE (222) (1) .. (1417) <223> amino acid sequence of SSK2 <400> 3 Met Ser Asn Pro Thr Ser Pro Ser Asn Pro Ser Asp Thr Gly Pro Ser   1 5 10 15 Ser Ala Ser Asn Val Thr Ser Ser Ser Ser Lys Thr Gly Arg Arg Ser              20 25 30 Val Arg Leu Phe Ala Pro Asp Glu Glu Asp Ser Ser Asp Glu Asp Gly          35 40 45 Gly Leu Ile Gly Val Pro Ala Glu Thr Thr Phe Lys Asp Asp Glu Ile      50 55 60 Pro Pro Ser Asn Pro Arg Ser Ala Ser Tyr Pro Gly Pro Pro Ala His  65 70 75 80 Thr Ser Pro Thr Ser Lys Ile Ser Thr Ile Val Ser Ser Ala Ser Ala                  85 90 95 Ala Gln Pro Lys Leu Ala Arg Ser Ile Thr Tyr Val Ala Pro Asn Ala             100 105 110 Val Ser Ser Arg Pro Ala Tyr Pro Leu Asn Pro Ala Gly Ser Glu Thr         115 120 125 Leu His Ala Ser Gly Arg Ser Tyr Thr Asp Pro Asp Ile Gly Tyr Phe     130 135 140 Ser His Asp Ala Gly Asp Asp Gly Trp Gly Ser Asp Asp Asp Asp Glu 145 150 155 160 Leu Arg Ser Pro Gly Trp Gly Ile Ser His His Asn Met Asp Ser Gly                 165 170 175 Gly Lys Thr Asn Gly Ser Pro Gln Leu Pro Ile Lys Pro Ala Asp Val             180 185 190 Thr Glu Asp Glu Gly Gln Glu Arg Leu Asp Trp Gln Gly Met Leu Glu         195 200 205 Ser Val Leu Asn Ser Asp Val Leu Lys Val Glu Glu Gln Arg Ile Tyr     210 215 220 Asn Ser Met Pro Thr Asp Ser Phe Arg Glu Glu Ile Gly Lys Thr Leu 225 230 235 240 Trp Trp Gln Ile Arg Ala Lys Leu Arg Gly Arg Thr Glu Ala Glu Glu                 245 250 255 Lys Lys Arg Val Gln Glu Arg Arg Ala Arg Val Val Asp Pro Val Leu             260 265 270 Glu Glu Ile Asn Glu Phe Lys Tyr Asp Pro Lys Asn Asn Pro Glu Gly         275 280 285 Glu Glu Asp Ser Asp Gly Asp Pro Gln Asp Ala Thr Ser Thr Ala Ala     290 295 300 Pro Gln Ser Lys Ala Leu Asn Gln Val Asn Thr Val Leu Ala Lys Leu 305 310 315 320 His Ala Ile Lys Gly Leu Tyr Pro Asn Leu Ala Ala Met Arg Ala Asp                 325 330 335 Lys Val Leu Tyr Thr Asp Glu Asn Phe Arg Lys Arg Ala Asp Ala Leu             340 345 350 Thr Ser Trp Ser Ile Ile Val Ser Ser Leu Gln Thr Gln Leu Lys Leu         355 360 365 Leu Gln Lys Trp Thr Gly Ser Asp Glu Leu Asp Ile Thr Lys Pro Asn     370 375 380 Thr Thr His Glu Lys Ala Leu Val Gly Lys Tyr Lys Tyr His Ser Ile 385 390 395 400 Asp Ser Lys Gly Thr Pro Gly Arg Asp Ala Ala Asp Asp Ser Ser Phe                 405 410 415 Leu Asp Arg Val Ile Lys Glu Asp Asn Leu Gln Arg Thr Phe Glu Arg             420 425 430 Arg Ala Phe Val Asp Met Ile Asn Leu Val Arg Asn Ala Lys Glu Thr         435 440 445 Val Ile Ser Tyr Leu Pro Gln Phe Gln Glu Gln Asn Leu Pro Asp Phe     450 455 460 Gln Tyr Glu Ile Val Arg Leu Ile Gly Phe Pro Gly Arg Leu Ile Ile 465 470 475 480 Glu Ala Val Lys Val Arg Leu Asp Ala Ala Ser Arg Leu Leu Asp Pro                 485 490 495 Asn Pro Met Val Val Glu Asp Phe Ile Glu Asn Leu Arg Leu Ser Ile             500 505 510 Ser Leu Ala Val Leu Ile Arg Lys Gln Tyr Asp Glu Ile Met Ala Pro         515 520 525 Asp Ala Glu Gly Arg Trp Lys Ile Pro His Cys Leu Pro Thr Glu Tyr     530 535 540 Asn Asp Val Leu Leu Asp Ala Leu Arg Thr Phe Phe Lys Leu Leu His 545 550 555 560 Trp Arg Leu Arg Gly Val Gly Lys Ala Ser Tyr Tyr Lys Glu Thr Glu                 565 570 575 Val Leu Glu Glu Glu Ala Pro Phe Leu Tyr Glu Ala Ala Glu Ala Ile             580 585 590 Val Gly Gly Asp Met Val Val Ala Glu Gln Tyr Cys Ala Leu Ser Asn         595 600 605 Lys Leu Leu Ile Arg Ser Ala Asn Tyr Leu Asp Gln Gln Leu Arg Val     610 615 620 Pro Ile His Ser Pro Ser Arg Asp Lys Glu Arg Gly Asp Lys Glu Arg 625 630 635 640 Asp Gly Ser Ser Ser Ser Gln Arg Asn Arg Asp Gly Arg Asp Ser Ser                 645 650 655 Leu Pro Gly Pro Pro Lys His Met Lys Val Glu Glu Leu Phe Ser Trp             660 665 670 Tyr Ser Lys Leu Leu Asp Ser Ala Arg Met Arg His Arg Lys Thr Gln         675 680 685 Arg Phe Cys Arg Lys Leu Thr Gln Arg Phe Asp Asn Ser Ala Glu Tyr     690 695 700 Ser Ile Glu Glu Thr Glu Met Asp Met Leu Val Glu Thr Leu Gln Asp 705 710 715 720 Thr Gly His Phe Leu Val Tyr Thr Gly Lys Phe Glu Ala Asn Gly Thr                 725 730 735 Tyr Ile Val Ala Asp Gly Ser Leu Trp Gly Gln Pro Asp Asp Val Arg             740 745 750 His Leu Leu Lys Arg Val Phe Ser Val Thr Ile Pro Gly Ser Arg Val         755 760 765 Arg Pro Arg Gln Thr Thr Ser Gln Val Ser Val Gly Gly Ala Ser Pro     770 775 780 Ser Asn Gly Gln Val Ala Ala Gln His Asp Pro Ala Asp Pro Tyr Pro 785 790 795 800 Glu Ala Asp Asp Phe Asp Asp Glu Ala Leu Ala Ala Tyr Ile Leu Leu                 805 810 815 Ile Ser Pro Arg Gln Ser Phe Val Trp Ser Gly Ala Val Met Thr Leu             820 825 830 Asp Val Asp Tyr Ile Asp Tyr Glu Leu Pro Asp Asn Arg Val Arg Leu         835 840 845 Ile Ala Asp Gly Pro Thr Lys Arg Leu Ala Leu Cys Lys Leu Tyr Phe     850 855 860 Lys Gln Ala Leu Ile His Pro Asp Thr Gly Glu Thr Ile Asp Leu Pro 865 870 875 880 Cys Val Val Glu Ala Gln Ala His Leu Pro Thr Ile Gln Lys Gln Leu                 885 890 895 Val Lys Ile Ala Lys Ser Ser Tyr Arg Leu Ser Glu Cys Ile Val Gln             900 905 910 Ser Ala Pro Leu Val Arg Asn Ala Phe Arg Gly Lys Pro Gly Ser Gln         915 920 925 Glu Leu Val Glu Asn Trp Tyr Ser Phe Ala Thr Glu His Gly Thr Arg     930 935 940 Val Leu Ile His Ile Glu Pro Ser Val Trp Glu Arg Phe Asn Arg Leu 945 950 955 960 Leu Met Arg Leu Ala Ile Ser Trp Ile Ser Phe Ile Ser Gln Glu Cys                 965 970 975 Asn Pro Thr Asp Arg Lys Thr Phe Arg Trp Thr Val Ala Ala Leu Thr             980 985 990 Tyr Ala Phe Asn Met Thr Arg Gly Ser Asn Ile Leu Ala Leu Asp Arg         995 1000 1005 Ser Glu Phe Ser Leu Leu Arg Arg Ser Ser Met Glu Ala Lys Lys Glu    1010 1015 1020 Ala Asp Arg Ile Glu Ala Met Arg Arg Leu Gln Arg Leu Gln Glu Asn 1025 1030 1035 1040 Leu Asp Asp Glu Phe Leu Pro Arg Thr Pro Thr Glu Ser Gly Asp Gln                1045 1050 1055 Pro Arg Ile Asp Arg Ser Ile Arg Leu Thr Val Glu Glu Arg Leu Arg            1060 1065 1070 Leu Ile Ala Glu Leu Glu Ala Arg Arg Asp Glu Leu Ala Pro Ala Pro        1075 1080 1085 Val Gly Gln Val Leu Asp Glu Glu Val Ser Glu Asp Arg Ala Leu Val    1090 1095 1100 Phe Leu Ala Ala Ser Lys Ser Asn Ile Ser Met Arg Trp Gln Gln Gly 1105 1110 1115 1120 Ala Tyr Ile Gly Gly Gly Ala Ser Gly Ser Val Tyr Leu Gly Tyr Ser                1125 1130 1135 Leu Gln Asp Asn Thr Val Phe Ala Val Lys Ile Leu Pro Thr Val Asp            1140 1145 1150 Leu Gln Ser Ser Pro Ala Leu Tyr Glu Ser Ile Lys Arg Glu Ser Asp        1155 1160 1165 Val Met Ser Leu Leu Ser His Pro Asn Ile Val Gly Phe Leu Gly Leu    1170 1175 1180 Glu Val His Arg Asn Arg Val Cys Leu Phe Gln Glu Tyr Cys Glu Gly 1185 1190 1195 1200 Gly Ser Leu Ala Gly Met Leu Glu Tyr Gly Lys Ile Asp Asp Glu Glu                1205 1210 1215 Val Val Gly Ala Phe Thr Ile Gln Leu Leu Arg Gly Leu Glu Tyr Leu            1220 1225 1230 His Thr Asn Arg Ile Glu His Arg Asp Leu Lys Pro Glu Asn Ile Leu        1235 1240 1245 Ile Gly Ala Asn Ser Val Leu Lys Leu Ala Asp Phe Gly Thr Ala Lys    1250 1255 1260 Ile Ile Lys Ser Asn Lys Thr Leu Ala Arg Thr Arg Gly Gly Ala His 1265 1270 1275 1280 Ala Lys Met Glu Gly Leu Glu Gly Thr Pro Met Tyr Met Ala Pro Glu                1285 1290 1295 Met Ile Lys Asn Gln Arg Thr Gly Lys Leu Gly Ala Cys Asp Ile Trp            1300 1305 1310 Gly Leu Gly Cys Ile Val Leu Gln Met Ile Thr Gly Arg Lys Pro Trp        1315 1320 1325 Ser Phe Leu Asp Phe Asp Asn Glu Trp Ala Ile Met Phe His Leu Gly    1330 1335 1340 Ala Thr Lys Glu Pro Pro Pro Leu Pro Asp Pro Asn Glu Met Ser Asp 1345 1350 1355 1360 Gln Gly Ile Glu Phe Ile Asp Gln Cys Leu Ser Leu Asp Pro Glu Ala                1365 1370 1375 Arg Pro Val Ala Ser Glu Leu Leu Gln Asp Glu Trp Leu Val Pro Met            1380 1385 1390 Leu Glu Gln Met Val Ser Cys Leu Ser Cys Arg Ala Gly Ala Arg Ile        1395 1400 1405 Pro Arg Tyr Ile Gly Asp Gly Pro Lys    1410 1415 <210> 4 <211> 609 <212> PRT <213> Cryptococcus neoformans serotype A H99 strain <220> <221> PEPTIDE (222) (1) .. (609) <223> amino acid sequence of PBS2 <400> 4 Met Thr Asp Pro Thr Pro Pro Ala Leu Asp Ser Leu Ser Leu Ala Asp   1 5 10 15 Lys Ala Pro Thr Pro Glu Glu Ser Pro Glu Asp Ala Ala Glu Gln Pro              20 25 30 Lys Pro Ala Ala Ser Pro Ser Ala Gly Thr Pro Gly His Asp Ala Gln          35 40 45 Ser Ser Ser Thr Ser Pro Pro Gln Arg Pro Gln Ser Met Gln Thr Asn      50 55 60 Asp Lys Ala Pro Asp Thr Ser Ala Pro Ala Ser Arg Pro Gln Pro Gln  65 70 75 80 His Val Pro Ala Ser Ala Pro Ala Leu Pro Ser Thr Asn Pro Val Arg                  85 90 95 Pro Gln Pro Gly Ala Arg Pro Gly Ala Ala Arg Gly Met Pro Ala Pro             100 105 110 Met Gly Met Arg Ala Gln Ala Gly Arg Gly Ala Gly Gly Pro Gln Met         115 120 125 Gln Thr Lys Met Leu Pro Ser Leu Gln Ala Lys Met Asp Lys Ile Ala     130 135 140 Ala Ser Arg Gln Gly Pro Pro Pro Ser Ser Gly Met His Asp Pro Asn 145 150 155 160 Ala Thr Ser Met Gly Ala Leu Leu Arg Ser Gln Ala Leu Arg Ala Pro                 165 170 175 Gly Ala Ser Gln Ala Pro Pro Gly Pro Gly Pro Ala Ser Gly Pro Phe             180 185 190 Gly Leu Ala Ala Arg Arg Ala Ala Ala Gly Gly Pro Pro Arg Pro Asn         195 200 205 Leu Gly Met Met Gly Met Gly Ala Ser Ala Pro Gly Ala Val Gly Arg     210 215 220 Gly Ser Gly Leu Ala Gly Arg Arg Gly Pro Pro Gly Gly Leu Thr Leu 225 230 235 240 Ser Gly Met Lys Gly Ala Ile Lys Asp Glu Gly Asn Lys Phe Ser Asp                 245 250 255 Phe Gln Gly Val Met Asp Pro Ser Gly Ser Leu Arg Phe Ser Lys Lys             260 265 270 Ala Val Leu His Ala Lys Gly Val Asp Phe Glu Asp Gly Gln Ser Phe         275 280 285 Lys Ile Asn Met Asp Glu Ile Glu Val Leu Gly Glu Leu Gly Lys Gly     290 295 300 Asn Tyr Gly Ser Val His Lys Val Phe His Arg Pro Thr Gly Val Thr 305 310 315 320 Met Ala Met Lys Glu Ile Arg Leu Glu Leu Asp Asp Ser Lys Leu Asn                 325 330 335 Gly Ile Ile Met Glu Leu Asp Ile Leu His Arg Ala Val Ala Pro Glu             340 345 350 Ile Val Glu Phe Tyr Gly Ala Phe Thr Ile Glu Ser Cys Val Tyr Tyr         355 360 365 Cys Met Glu Tyr Met Asp Ala Gly Ser Leu Asp Ser Leu Thr Gly Gly     370 375 380 Gly Val Ala Ala Lys Asp Gln Thr Lys Asp Glu Glu Asn Asp Ala Thr 385 390 395 400 Lys Arg Val Pro Glu Asp Val Leu Arg Arg Ile Thr Ala Arg Ile Val                 405 410 415 Lys Gly Leu Arg Phe Leu Lys Asp Glu Leu Gln Ile Ile His Arg Asp             420 425 430 Val Lys Pro Thr Asn Val Leu Ile Asn Gly Lys Gly Glu Val Lys Met         435 440 445 Cys Asp Phe Gly Val Ser Gly Gln Leu Glu Lys Ser Leu Ala Lys Thr     450 455 460 Asn Ile Gly Cys Gln Ser Tyr Met Ala Pro Glu Arg Ile Lys Ser Glu 465 470 475 480 Thr Ala Asn Gln Asn Pro Thr Tyr Thr Val Ser Ser Asp Val Trp Ser                 485 490 495 Val Gly Leu Ser Ile Val Glu Leu Ala Lys Gly Cys Tyr Pro Tyr Pro             500 505 510 Pro Glu Thr Tyr Ala Asn Val Phe Ala Gln Leu Gln Ala Ile Val His         515 520 525 Gly Thr Pro Pro Thr Leu Pro Pro Gly Tyr Ser Asp Asn Ala Asn Asp     530 535 540 Phe Val Ala Lys Cys Leu Glu Lys Asp Pro Asn Arg Arg Pro Thr Tyr 545 550 555 560 Ala Gln Leu Leu Glu His Pro Phe Leu Val Ala Asp Lys Gly Ala Glu                 565 570 575 Val Asp Met Val Gly Trp Val Glu Gly Ala Leu Lys Arg Lys Ala Glu             580 585 590 Arg Gly Ile Ala Ser Leu Asn Pro Ile Gln Pro Pro Val Pro Leu Glu         595 600 605 Pro     <210> 5 <211> 365 <212> PRT <213> Cryptococcus neoformans serotype A H99 strain <220> <221> PEPTIDE (222) (1) .. (365) <223> amino acid sequence of HOG1 <400> 5 Met Ala Asp Phe Val Lys Leu Ser Ile Phe Gly Thr Val Phe Glu Val   1 5 10 15 Thr Thr Arg Tyr Val Asp Leu Gln Pro Val Gly Met Gly Ala Phe Gly              20 25 30 Leu Val Cys Ser Ala Lys Asp Gln Leu Ser Gly Thr Ser Val Ala Ile          35 40 45 Lys Lys Ile Met Lys Pro Phe Ser Thr Pro Val Leu Ser Lys Arg Thr      50 55 60 Tyr Arg Glu Leu Lys Leu Leu Lys His Leu Arg His Glu Asn Ile Ile  65 70 75 80 Ser Leu Ser Asp Ile Phe Ile Ser Pro Leu Glu Asp Ile Tyr Phe Val                  85 90 95 Thr Glu Leu Leu Gly Thr Asp Leu His Arg Leu Leu Thr Ser Arg Pro             100 105 110 Leu Glu Lys Gln Phe Ile Gln Tyr Phe Leu Tyr Gln Ile Leu Arg Gly         115 120 125 Leu Lys Tyr Val His Ser Ala Gly Val Val His Arg Asp Leu Lys Pro     130 135 140 Ser Asn Ile Leu Val Asn Glu Asn Cys Asp Leu Lys Ile Cys Asp Phe 145 150 155 160 Gly Leu Ala Arg Ile Gln Asp Pro Gln Met Thr Gly Tyr Val Ser Thr                 165 170 175 Arg Tyr Tyr Arg Ala Pro Glu Ile Met Leu Thr Trp Gln Lys Tyr Asp             180 185 190 Val Ala Val Asp Ile Trp Ser Thr Gly Cys Ile Phe Ala Glu Met Leu         195 200 205 Glu Gly Lys Pro Leu Phe Pro Gly Lys Asp His Val Asn Gln Phe Ser     210 215 220 Ile Ile Thr Glu Leu Leu Gly Thr Pro Pro Asp Asp Val Ile Gln Thr 225 230 235 240 Ile Ala Ser Glu Asn Thr Leu Arg Phe Val Gln Ser Leu Pro Lys Arg                 245 250 255 Glu Lys Val Pro Phe Ser Thr Lys Phe Pro Asn Ala Asp Pro Val Ser             260 265 270 Leu Asp Leu Leu Glu Lys Met Leu Val Phe Asp Pro Arg Thr Arg Ile         275 280 285 Ser Ala Ala Glu Gly Leu Ala His Glu Tyr Leu Ala Pro Tyr His Asp     290 295 300 Pro Thr Asp Glu Pro Val Ala Ala Glu Val Phe Asp Trp Ser Phe Asn 305 310 315 320 Asp Ala Asp Leu Pro Val Asp Thr Trp Lys Val Met Met Tyr Ser Glu                 325 330 335 Ile Leu Asp Phe His Asn Leu Gly Asp Ile Ser Gln Asn Glu Ala Glu             340 345 350 Gly Pro Val Thr Gly Glu Val Pro Ala Ala Pro Ala Ser         355 360 365 <210> 6 <211> 4121 <212> DNA <213> Cryptococcus neoformans serotype A H99 strain <220> <221> gene (222) (1) .. (4121) <223> genomic DNA sequence of SSK1 <400> 6 atgtggggct caaatgcttc catcgccgcc tcggagtcga ccgactccct ttcccccgcc 60 ccctcccagt ctgccgccgt agagttcccc ctgcccgtaa gctcccgccc gtctctcact 120 tccgccgctc acccctccca gatgtccgct tcctcctcct ccacctcctc ccagcctctc 180 tttgattggc gcattcccaa gcccacctca ccccgcacac gcatggaccc attcgacact 240 tttgatcctg tatcctcaag ctcagaggat gatccggtcc ctcaagagag ccgccgtgct 300 ggtcatcagc gctccgtaac agatcctctt ttacgagatg gccaacccct cgatatggag 360 ttcactactg ccgggccgcc tatacagagc tacgactttg aacaaccgcc cacgtttagc 420 agaacgttat cttcccctct tccagctaaa gtcggctcgc ttagacaccc tatgccattc 480 accattgacg atttaagctc tcgcaatgtg aattcaaccc atcgtccgca gccgactaca 540 cctctgcatt ccatatccgt tgagttagcc gattccttgc aatcggccat tcaaacatta 600 ctgcatctgt ccccacctca cctactcgat aacgcaaaag aacagtactc tggatgcacg 660 gtacagatac ctgctacctc gctttcggcc cttttgacct ctatgagagg cctcaacttc 720 ttgtcggcgc atgctgaaga actggtcgac atgagtgcac gtggagatcc acctgtactc 780 catcaagaag acttcgatgt aggagaactt ttgcaaaacg ttgcggatat gttgagtgga 840 gaagcagcag aaaaacggat tgatttcgtt ttgttccatg gcgacgtagc gatgaggcat 900 gtcagtgtgt atggagattc tgatggaatt agctatacct tgagtcatgt aagtcctaca 960 tgcacctgct gcatactgac aaacaggtta ttcgacaaat actggcagta gccaattacg 1020 atgataccat agaactcggc cttcaagtca ttcctcaaag tccatcttta gcttccgccg 1080 tcggacttcc tctaacctct gccgatgtta gtggaggagg tggtgtcaag tcagcgtcca 1140 catctcggtc aggctccccc aataacagtc tctctcgatc taattctgtc catgacgggc 1200 cccttctctg tgtgttcgaa atagtacata acatctatca gccaccacca agctcggcat 1260 ccgccactcc taaagccgag ctgaaccctt tcactcatct tgctgaagaa accgaagcct 1320 tgaaaccaag attggataca gcattttgca aaaacctgct tcatcggcaa aatgctgtcc 1380 tcaaagttga tgtgcagcct tcatctcctt taggatccgg gatgccccgt agagcttacg 1440 cgttatcagt gctcctacca agaggtaaac ccatcactga gcctgcaata ctttctaaag 1500 aggaacaaga agttcgtcaa ccattttcat cccacgtact tgcacgagaa cccaccctca 1560 atgagctctc ggaatttgct gaatcattac gaggaagaaa ggtgtttatc catgccaatt 1620 tgagtagtgt tttcgcgaga cacctcacga gctacctagc tgcatgggga atggatatat 1680 cgcatctacc gacagatggc gatgaggctg ataaattgaa ggatgtcgcg gccaaacatg 1740 actcggctta tactggatct atgggtgtgt caggcggcac tacttccagc gcagaaacgc 1800 cctattcaat taaaccgacc ggcgtgactg ctgttcaacc tggacacttt gtcattatcg 1860 acgatgatgt tgcggtcttg cgccgtgaac tcgtgcgcat ccgttcagaa ttacttccca 1920 ttctctttaa acctagactg tcaaagcgtc ccactatgac ttctcgaacc cgttccaccc 1980 cttcattgcg acaggtcccc ccaaggtcat catcgggttc tgtacttata cactttacct 2040 ctcttgccaa ttataaccga gttcgagacg cgattgcgag ctttgtgggg gcgccagggt 2100 taaccaatcc ggaaacttat gttcagccgg aggtgatagt gatacccaag cctgttggac 2160 cacgaagatt tttgactgct ctgcataccg ctgtgaaaca gcctatggtt gacccatttt 2220 tctcccctat cgccacatct cctagatcac caggcggagg ttactttggt ggtttgcgga 2280 ctccgacgga gagagaatca ggattctttg attctgttgc agaagaacca catgaagagg 2340 cggattcgcg accagattat gccacggtgc agaaagccag atctccttta ggagaatttc 2400 cgccttctgc ggcacagatc gttcgtacca accaaggctt gcatctttcg cttcccactc 2460 caaatgaaat tatgacaacg cctgctccag aatatttttc tgggtcttcc aagtctccta 2520 gctctggtgc gtccggagtc gtgatgcaga gccctgatgg tcgtcctttc ggaatgtttt 2580 tcgaaccgcc cataaaaaat gagcgccgcg gatctactca caggacgcct tccgattcca 2640 tcaggaggaa acaagcgaac cgccgtgcgt ctacaagtga tgaacccttt tcttcacctt 2700 ctaccgccct acctccccgt cgctcgtcca caatttctac gactggcaat gaggaacacc 2760 gcagttcacc tatcgctaac gtcacagacc gtcctaccca ttcaagggta aattcaagaa 2820 ggaagaacaa tcttccggcg gcggagcaac ctattttggc tgtgggcagg gcaaaaggca 2880 gggaaagatc ggagactgtc acgaagggag gggacctcgg gtcgagaaaa ggtacaccag 2940 cggcaagccc acgtatagag gagaagaagg aattggaaag aggcgagaag actaaaagcc 3000 tggctccttc aactgctcct acgaagaaga atgctaaagt cgatgttgtg gtgccgccca 3060 tcaacgtgct gattgttgaa ggtaaatctt ccattcaaat gatttgttca aacaccgact 3120 gacagataac tagacaaccc catcaatcaa aacattttga gtatgttcct gagaaaaaag 3180 aagataaaga attcctcggc caaggatggc gcagaagctg ttgaaaagtg gaggactgga 3240 ggcttccatc tgattctggt aggctatgat ctctttcttt gattcgtgca gtacttattg 3300 gacccttcgc agatggatat ccaattgccc gtcatggatg gcatagctgc taccaaagag 3360 attcgtcgac ttgaacgtca caataacatt ggcgtttttc catcgactcc agcggccgaa 3420 cttcctcggg gtcaaaatgt tgcggattct ccaccaccat cttctccatt tcgctcgtca 3480 gttatcattg ttgccctgac agcctcgtcc ttgcaaagcg acagagtagc tgctttggct 3540 gctggctgta atgacttctt gaccaagcct gtgtctttga aatggttgga caaaaagatt 3600 gtggaatggg gttgtatgca ggttagtgat ctcttctttt tttttgatta tagctgatct 3660 aaatataggc attgattgat tttgacggct ggcgacgatg gaagagctcc gataccaaga 3720 atcctagcga aactaagcag ggcttctcag tgggccctca acaggctgct aggtcgcttg 3780 ctagcagact acgtattgaa cgcaaaggat ctcgatctcc ggcagctcca gtatcaaccc 3840 cgcgactcaa tttgcagtcg gcaaccccag ataggccaga aaccccccca gattccacgt 3900 cacaaatgcc aaaggccccg cccgttgcag cctctgaccc cccgttatct cccaagtcgc 3960 tgaacaagac agttaatgat gtcttcgagc aagcagacgc tagactcgaa aatgcgcggg 4020 aggaacaagg agtatcaagt caaaaggaaa acacaagctt aacagattca acaaacacca 4080 ccattacgcc ctcaaagacc tatccggctc ctcccccatg a 4121 <210> 7 <211> 5666 <212> DNA <213> Cryptococcus neoformans serotype A H99 strain <220> <221> gene (222) (1) ... (5666) <223> genomic DNA sequence of TCO2 <400> 7 atgatcttag gaaccgacat cgacctgtcg tctataccaa cggcgtttct cgaggtgtgt 60 atcgctgtcc ctttcgagtt gcaccatgaa gctgaccatg agaacgcatt cgccgccaac 120 ttccaaatct gttctgggct tgtcttttgg acaaccatct aggcttatcc cttcccagcg 180 gttgtgttcg tgatcgattc tccccctagc ccaagaccgc ggctccattc cagaaataca 240 gacacgacca ttcggcgaac ggatggccaa atatcgcctc ttacaggtcc tccagtgcaa 300 cagttcgcgt cagcgcccgt ggtatggggc aatcaacgat ggcacgagct ggctcagggg 360 aaaacaattg cagagtgcgt ggatgtggcg tcacagaaca agctgcaaac ttgggtggaa 420 aatgacaccg gtgacaagtc ggagagtttg gccctggacc tgaaggtgcc gcaaggcgtg 480 actcttcatc tggcaaagac catattgcca ttaagtccac cgtcctcctc tcagtcactt 540 tgcatcctta tatcgcaata tatcgataag ccggaaagtt tcgcgccacc aatctcatct 600 ggagatattc ttttctcttc tctatcgcga ctctcccaga ctttttctcg gtcatcctct 660 ttttcatcca accctagaaa atcgattgat gtccctgcgt cactatccga acaccggggt 720 tctgctacat cgacaagtag caatctgcgc tcttcgatcg atttgacttc ccctaattct 780 caaccctctc cactaaaccg tgaacaaagc acgtacttca cccatggctc cgcgaccaga 840 gaagagcgac cctcagtaag gcgtagacgg tcaccgccaa tctcaatgac gaggcccaag 900 cctcttgaga gccatgctca agaatgctgg gacttggtag agaatttcga ctggtcaaaa 960 acagcattag ggccgagaga acagtggatg gatgcgttag atcctgttct ggcaatcaca 1020 tttgaatcca gaacggcaga ttgtgcctgg ttagggcctg atctagagct agtttagtga 1080 gatactatcc gtcttgcaaa aataatggcg actaactttt atgatcagca ataaggcgta 1140 tcaagagctg gttgaccatc ccaatgcttt tggaaaacct gcaagacaag tttgggctac 1200 caattgggac tacttggaac ccctggtcaa gcgatgtctc agtgggaccc cggtctacaa 1260 ggacaacgac ccgcttttct ggcgtcgata cggcaatggt cgacttctgg aacattacca 1320 cacttggcga tatgtcccga taacgggcaa agatggctca gtgcttggca tcttcaacca 1380 gtcaattgag gtcaccgact cagtactgct agagaggcga atgggcacga ccagggaact 1440 ttcggaacac atgtcgttta ttcgtacaac tgaggacttt tttagctcgg ttgccgacgt 1500 ctttagtcag aaccctactg acataccgtt cgcactttgt taccgggtcc gacaagttga 1560 caccgatggg acatttgtcc atttggacgt ctcgcttcag tcgtccgtcg gtgtacccga 1620 aggccatccg tctgctccag atcaaattcc cgtcagcttc ttaaatggta acccttaccc 1680 tagcaatgtc gagcgatcat tttctcctgc tttctcaatc gtttcaatcc actcttcgag 1740 cagtcatcga gtctgtcacg tctctgaaga cactacacaa tggcccatcg ccaaagccct 1800 acaaaggcgg caatgtgtca tcatcgaaga atgttcgcaa ttaatagaag gatatcctat 1860 ccgtcgctgg gatgggcttc cattctcagc cattgtcgtg cccatatgct ctgaagggtc 1920 tcccgaaatc cctgacgccg ttgttattct tggtctcaat gtgcgacgtt gttttgacca 1980 tgaatacgat tcctggattc actctattcg gtcacaacta tcttcggccc tcgtgatggt 2040 caaggcgcgt gaagctgaac aaaagatggt tgaggaaagc gcacgtatgg agaaagcaaa 2100 agtcgcttgg ttcagaggag ccgcgcacga ccttcgtagt ccattaaccc tcgtcgctgg 2160 accgcttgcc gatgtgcttg attcggattt gaactcgagt cagcgcacgg ctttgaccgt 2220 tgcgcaacgc aatcttgatc gtttagtgcg cttggtcaac gccctcatgg atttctcgag 2280 ggtggaagct ggacgaatgg aaggacgatt tgttccgacg aacttgagtc aattcgtgac 2340 acagttggca gctcttttca agcctgcaat agaaagattg gggttagaat acgtactaga 2400 tgtccagcca agcgaggagc ttgttttcat cgatcctgtt ctgtttgaga ccgtggtatc 2460 aaaccttatt ggcaatgcgc tcaaatacac tgaaacgggt tctatcactg ttcgggtgca 2520 atacacggat tacgcagagg tctcggtcat cgataccggt gtgggtatac cgaaaaatga 2580 gctggcactg gtgaccgaat ggttccacag ggcaagtact gccattcact cgggaaccca 2640 gggaacagga ttgggactgg ctttggccaa ggaattgctc aagttgcata aaggagaatt 2700 gcttgtcgag tctcaaaccg ccaatgagtc aggaggtcct catgggtcca tttttacagc 2760 gaaaattcct cttgatttca agccctctcc atcggctcat atcattccgt ccgtcgaatc 2820 tcacaagacg tttggcaaat acagtaaagc cgtcgcagac gaagccatgc gctgggttgg 2880 ggactcagat gccgctagtg aggcgtacga catgtcgagc ggtaccggag tctcaagcgc 2940 tggtagtggc tctggaaaca cgaccacctt cggacccaag tttgcagatg cctttttgtt 3000 tgataagaac gacattgtgc ttattgtgga agacaatgtc gacatgcgtg aatacatacg 3060 acagcttttc gccccttatt gtaccgtact cgaagcttcc aatggtgaac aggcttacaa 3120 tatggctacc caaaaccctc ccaacctcat tttgtcggac gtgctcatgc ccaaattatc 3180 tggtatggag ctactacaaa ggatcagatc ccatcctgac actcgcattg tgcctatggt 3240 ccttatttcg gctattgctg gtgatgagtc tagggttgag gctctgctaa acggcgctga 3300 tgactatctt gccaagcctt tcaaacccaa ggaactcatc gcgcgtgttc acctgcacat 3360 gcaagttggc aagaaacgtg ccaagctcga agcgctatac gcccaacgcg aaacagaatt 3420 gacagctcta tctgactatt gtccgatcgg tatcttccga ggagacaaat atggccatat 3480 tgtttatgcg aacgcagctt ggcgtgcgca gagcggcctt ttggtgggtg accctaacga 3540 ttgggcatct tatgtgcacc cggattcgaa agcgcagctc ttggaacaat ggaatcagtg 3600 gttgaggggg gatttgaagg agttccgagc ggcttggaga tggtctaatg gcatccctgt 3660 caggagcatc ttggtccggt tagatgacgt caaggaaggg ttttctgggt taattgggtg 3720 cgtagtggat gtgtctcatg aagagagacg attaatcgaa gctgaggaaa gaagaaaaga 3780 ggcggaagag agtaaacatc agcaagaact ccttattgac ttgacaagtc atgaaattag 3840 gaccccggtg tcagcaatcc tgcagtgctc agatcttgtt aaagagaatc ttgtagctct 3900 gaaggaccag ttgagaggag cggggccaaa gggctttgtg ccgagtcaag aattactggc 3960 tgatcttgag caggatgtgg aagctttgga aagtaattca ccatcccctt tcatgctaat 4020 ttcgaaacta acagtgattt tgtaaggtat ttatcagtgc ggtcttgtgc aggaacgcat 4080 tgccggagat gttctttcgc tggctcgtat ccaactcgat atgctgagtt tgcacgacat 4140 tgacgtcaac ttgcgccgag aaggcaggaa agtttcgtcc atctttgcat cggaagccaa 4200 gatgaaggat atcgacctcc aattggaatt tggacctact atcgaacagt ccaaagtgct 4260 ggccatcaag acagatcccg tgagattagg ccaggtggta acaaatctca tttccaacgc 4320 cattcggttt acatcttcga gtggtgagtt tcatccaacc cataatgtgc tatagtgata 4380 ctgaattgta attcatttag atgtccgaaa gattactatc caatacgacg tatcgtttgt 4440 ccctcctgcc gatgactctt gcgccctccc ttcatctgtt ggcttgcccg acatacttcc 4500 tgtgaaagag aatactccac tatggctgtt tgtcagtgtt accgattctg gacctggtat 4560 gacagagcaa gagttatctg ttttgttcca aaggtttgcc cgtaagtcct aagcccattc 4620 tctttgtcga ggttacgttg acgaatcttg atatctagag ggcaataaga tgattcatac 4680 taagtatggc ggaagcggtt tgggactgtt catctgtcga agtgagtgat tggaggaggt 4740 tttttttttt ttttggtggg gggatacatg atgctgaaac catctacaga gattacagag 4800 cttcttggcg gtcgtatcga agtgctcagc caagtcgggc acggtagtgg tgagtgcccg 4860 tgtgctctgt tatccattgc atgctgctga atcgagtgcc tttcccaagt tttccgattc 4920 ttcattaaaa cgcgcgctgt cgctcctccg tccgccatcg ctgctctcgt agaatcctct 4980 cctctcaaac cggtatccgc cacttcacct tcttcctcgt tagccatgag ccgatcgtct 5040 tctcggagca caaacgtcac tacacctata gagggtggtg ggaccgagca cgtgttgatc 5100 gtggaagata acctaatcaa tcagactgtc ctgaagcgac agctcgtcaa ggcgggttta 5160 tcgtgcaacg gtgagttctc catcccatcc tctgtgattc ccacatttat cttgattgct 5220 gttgtatagt cgcgagtaat ggccttgaag ctcttaatgt catccgtgaa gtccatcggc 5280 aacaccgacg cggtgggccg aaccgtaaaa ggctatttga cgtggtattg atggatctcg 5340 agatgccagt gatggatggt atcaccgccg tacgagagat acgacagtcc gaggccgcgg 5400 ggacattggg caggaatatg gtgattgccc tgacggggaa cgcgagacaa ggacagattg 5460 atcatgcctt ggcttctgga tttgacgatg gtgagttggg aagacccttt tttgggggct 5520 cagaggacga tgtggattat gaacttttct gacacttgat tgtagtcgtc atcaaaccgt 5580 atatcctggt agatttgctg aacaagatca aatctatgaa agttagaaaa ttggagttgg 5640 aaactgcgaa agctcaagaa gagtga 5666 <210> 8 <211> 4744 <212> DNA <213> Cryptococcus neoformans serotype A H99 strain <220> <221> gene (222) (1) .. (4744) <223> genomic DNA sequence of SSK2 <400> 8 atgtccaacc caacctcccc ctcaaacccc tcagacaccg gcccgtcctc ggcgtccaac 60 gtcacatcct cgtcctcaaa gaccggacgc agatcagtgc gcctctttgc cccagacgag 120 gaggacagct cagacgagga cggcggcctc atcggcgtgc ccgcagagac cacattcaag 180 gacgacgaga gtgagtagat acgcaggaac caaatgcagc gcatactcac actccacagt 240 ccctccttcc aacccacgtt ccgcctccta ccccgggcca ccggcacaca cctcccccac 300 ctctaaaatc tcaaccatcg tttcgtccgc ttctgcagcc cagccaaaac ttgcacgttc 360 aataacatac gtcgcaccca atgccgtctc ctcccggccg gcatatcccc tcaatcccgc 420 agggtcagaa accttacacg cctcgtacga gacatggcga aaaacgcgat ataccctcga 480 gtcattcggc agaggatcca aaccagacgg gaagaatacg gcacaaagag gaaggtcgta 540 tactgacccc gatataggat actttagcca cgatgcagga gatgatggtt ggggctcaga 600 tgatgacgac gaattgagat cccctggctg gggcatatcc catcataaca tggactctgg 660 aggcaagacg aacgggtcac cacagttgcc tataaagccc gccgatgtca ccgaggatga 720 aggacaggaa cgtttagatt ggcaaggcat gctggaaagt gtcctcaact cggatgttct 780 caaggtggag gaacaacgta tctacaattc catgccgaca gattcattca gagaagagat 840 tggaaagacc ctttggtggc aaatccgtgc caaactgcgt gggaggacag aggcggagga 900 gaagaaacgg gtgcaagagc gacgagcgag agtggtggac ccggtgctgg aagagataaa 960 cgagttcaag tacgacccaa aaaataaccc agaaggcgaa gaagacagtg atggcgatcc 1020 gcaagacgcg acttcgactg ctgcacccca atccaaagct ctcaatcaag tcaacaccgt 1080 tctcgccaaa cttcatgcaa tcaaaggtct ttatcccaac ctcgcagcca tgcgagccga 1140 caaggttctc tataccgatg aaaatttccg caaacgcgcc gacgcattga cctcttggtc 1200 catcatcgtt tcatccctcc aaacccagct caaactcttg caaaaatgga caggttccga 1260 tgagcttgac atcaccaagc ccaacacgac ccacgagaaa gcattggtcg gcaagtacaa 1320 gtatcactct atcgacagca agggtacgcc cggcagggat gcagccgatg actcgagttt 1380 cctcgatcgt gtgataaaag aagataacct tcaacggaca ttcgagcgtc gagcgtttgt 1440 agacatgatc aacctcgtgc gcaacgccaa ggagacggtc atcagctatc tcccccagtt 1500 ccaagaacaa aatcttcccg atttccagta cgaaatcgtt cgtcttattg gtttccccgg 1560 tcgacttatc attgaagctg tcaaggttcg tttggatgct gcatcccgac tacttgaccc 1620 gaaccctatg gtcgtcgaag actttatcga aaaccttcgt ctatccattt cgctcgccgt 1680 gctaatccgg aaacaatacg acgaaatcat ggcacccgat gccgagggga gatggaaaat 1740 cccgcattgc ttgccgacag agtacaatga tgttctgctc gatgcgctga ggacattttt 1800 caaattgttg cattggagat tacgaggagt ggggaaagcg agttattaca aggaaacaga 1860 agtgttggaa gaagaggcgc cgttcttgta tgaagcggcg gaggctattg taggcggtga 1920 tatggttgtt gcagagcagt attggtgagt ttgaaatcgt atcatcctgg caaggagctt 1980 agtgctaagt atcgatgtaa atagcgcgtt atccaacaag ctccttatac gttcagcaaa 2040 ttatcttgac cagcaacttc gggtaccaat acattccccg tctcgcgaca aggaacgtgg 2100 tgacaaggag cgcgatggct cttcgtcttc tcaacgtaac cgtgacggcc gtgatagctc 2160 gctgcccggc ccaccgaaac acatgaaagt cgaagaactc ttctcatggt actccaaact 2220 ccttgattcc gctcgtatgc gacaccgtaa aacccaacgt ttctgtcgta aactcaccca 2280 acgattcgat aattccgccg aatattcaat cgaggagacg gagatggaca tgctggtgga 2340 gacattgcaa gatactggtc atttcttggt atataccggg aaatttgagg cgaatgggac 2400 gtatatcgtt gcggatggga gtctctgggg tcagccggac gatgtgagac atctgttgaa 2460 gagggtgttt tcagtgacga ttcctggatc tcgagtccgt ccaaggcaga caacctcgca 2520 agtatctgtc ggaggtgcga gcccgtccaa tggtcaagtc gcggcgcaac atgatcctgc 2580 agatccgtac cccgaggcag acgattttga cgacgaagcg ctcgcggctt acatcctcct 2640 catctcccca cgccaaagtt ttgtatggtc cggagcggtc atgacgctgg atgtggatta 2700 catcgactat gaactacctg ataaccgagt cagattgatc gctgacggtc ccaccaagcg 2760 gttagcgctg tgcaaacttt atttcaagca agcgctcatt caccctgata cgggcgaaac 2820 aatcgacttg ccatgtgtgg ttgaggctca agcgcattta ccgaccattc agaaacaact 2880 tgtcaagatt gctaaatcga gttatcgtct ttcagagtgc attgtccagt ctgcaccact 2940 cgtccgcaat gcgttcaggg gcaaaccggg atcacaagag ttggtggaga attggtacag 3000 ttttgcgaca gagcatggga cgagagtgtt gatccatatt gagcctagtg tatgggagcg 3060 attcaatcgg ttgttgatgc gtctggcgat cagttggatt agctttatca gtcaagagtg 3120 taaccccaca gaccgcaaga cgttccgatg gactgtggca gctttgacct atgcgttcaa 3180 catgacgaga gggagtaaca ttctcgcgct tgatcgatca gaattttcgc ttttgaggag 3240 gtatgttggt gtttgtgtgt cactgttggt tagccacttt gatatcctcg gcgcaaggtc 3300 gagtatggag gccaaaaagg aggcagacag gattgaggcg atgaggaggt tacaacggct 3360 tcaagaaaac ctggacgacg aattcctgcc ccggactccg acagagtctg gcgatcaacc 3420 acgtatcgac cgctctataa ggctcacagt cgaagaacgt ctccgtctca ttgccgagct 3480 cgaagctcgt cgtgacgagt tggcacccgc acccgtcggt caagtccttg atgaagaagt 3540 ctctgaagac cgtgcgttgg tgttccttgc agcttccaaa tccaacattt ctatgcgatg 3600 gcagcaaggc gcgtacatcg gtggaggtgc atcgggaagc gtgtacttgg gatactcgtt 3660 gcaggataac actgtgtttg ctgtcaagat cttgccaacg gtggatctgc agagtagtcc 3720 ggcgttgtac gaaagtatca agcgagaatc ggatgtgatg agcttgttga gtcatccgaa 3780 tatcgttggt ttccttgggt tggaagtgca taggaacaga gtttgtcttt tccaagaggt 3840 aagtgcttgt tgttgtttcc atttgtgttg ggagggtgtg gtgccaaagc tgatgttcgt 3900 gattttagta ctgtgaagga gggtcgctgg caggtatgct cgaatatggc aaaattgacg 3960 atgaggaagt cgttggagcg tttacgatcc agctgttacg cggccttgag tatctgcaca 4020 ccaaccgcat cgaacaccga gatctcaaac cagaaagtaa gctgacaccc atcttttgat 4080 cctttccaac acacacacac taactcgtgt tctccacaga tattctcatc ggcgccaatt 4140 ctgtcctcaa gctggccgac tttggtaccg ccaaaatcat caaatccaac aagacgctcg 4200 cccgtacacg tggtggcgcg cacgccaaga tggagggtct tgagggtaca ccgatgtaca 4260 tggcgccaga gatgatcaag aaccagagga ctggcaagct gggtgcttgt gatatctggg 4320 gtttaggatg tatcgttttg cagatgatca ctggtaggaa gccatggagc ttcttggact 4380 ttgataatga atggtacgtc ttttcttgca atgatgtttt ccgcgtaggg agttatgagc 4440 tgataatatg attagggcaa tcatgttcca tcttggtgcg acaaaggagc cacctcctct 4500 acccgatccc aacgagatgt ccgaccaagg tatcgaattc attgatcaat gtctttcttt 4560 ggatccggaa gcgaggccgg tggccagcga gttattgcaa gatgaatggc tggttccaat 4620 gttggagcag atggtgagtt gtctttcatg tatgtgtaaa aaatggtcag aagcttatct 4680 gctttgcaaa acaggccgag ctggagcaag aataccccga tatattggcg atgggccaaa 4740 gtga 4744 <210> 9 <211> 2202 <212> DNA <213> Cryptococcus neoformans serotype A H99 strain <220> <221> gene (222) (1) .. (2202) <223> genomic DNA of PBS2 <400> 9 atgacagacc ctacgccccc cgccctggac agtctctccc tggcagacaa ggcgcctact 60 cccgaagaaa gtcccgaaga cgccgctgaa cagcccaagc ccgcggcctc accgtccgca 120 ggcacacccg gccatgacgc ccaaagctca tccacctcgc ccccgcaacg ccctcagtcc 180 atgcagacaa atgacaaggc gccagataca tctgctccgg cttccaggcc ccaaccgcaa 240 catgtccctg catcggcacc tgcgcttccc tctaccaacc ccgtccgtcc acagccgggc 300 gcccgtcctg gagcggcgag gggtatgccc gcgcccatgg gtatgcgggc gcaagcaggc 360 cgaggcgctg gcggccccca gatgcagacc aagatgctgc ccagtttgca ggctaaaatg 420 gacaaggtgt gtatcgctcc atcatttatc ccgctgcata ctcatccaga ggctgtgctg 480 acaaaccaca ctatgctatc attagatcgc ggcgtctcgg caagggccac ctccctcctc 540 tggcatgcat gatccgaatg ccacatccat gggcgccctc ttacgctccc aagccctccg 600 cgcccccggc gcatcgcaag ctcctcccgg ccccggaccg gcttcaggcc ctttcggtct 660 cgccgctcgg cgcgcagctg ctgggggccc tccgagaccg aatttgggta tgatgggtat 720 gggtgcaagt gcgccgggtg cggttggacg gggatcaggt ctggcgggta gacgggggcc 780 ccctggagga ctgacactga gtgggatgaa gggtgcgatc aaggatgagg gaaacaagtt 840 ttcagacttt cagggtgtca tgtgggttca gcagactcct tttccatgac tgtgggctga 900 tctcaagtac agggacccgt ctggatcgct gagattctca aagaaggctg tcctgcatgc 960 aaagggcgtg gactttgagg atgggcaaag tttcaagatc aatatggatg agatcgaggt 1020 gcttggagaa ttaggaaagg gcaattacgg ttctgtgcac aaagtcttcc accgtccgac 1080 aggcgtcacc atggccatga aggtgatctt attctttctt gcgtcgcttc tggtccagta 1140 actaacaaac acgacaggaa atccggttag aacttgacga ttccaagctc aacggcatca 1200 ttatggaact cgacatccta caccgggccg ttgctcccga aatagtcgaa ttctacggtg 1260 cattcaccat tgaatcatgc gtctactact gtatggagta catggatgcc ggttcactcg 1320 actctctcac cggtggcggt gtggcggcca aagatcaaac aaaggatgaa gaaaacgatg 1380 cgacaaaacg agtgccggag gatgtattga ggaggattac agcgagaatc gtgaaagggt 1440 tgaggttctt gaaggatgaa ttgcagatca tccatcgagg tgagttttcc atgtgcaatg 1500 aaaacgggag gaaatgtgct gatatgatgt agacgtcaaa cccacaaatg tgttaatcaa 1560 tggcaaggga gaggtcaaga tgtgtgactt tggcgtttca ggtcagctcg aaaagagttt 1620 ggccaagacc aatatcggtt gtcaatccta catggctgta cgtctttccc tctcctccat 1680 ctcaaagagc ctcccagcta acccgattcc ctctctttct ttagcccgaa cgtatcaagt 1740 ctgaaactgc caaccagaat cctacatata ctgtctcttc agacgtctgg tctgtcggtc 1800 tgtccattgt cgagcttgcc aaggggtgtt acccctaccc accggagacg tatgcgaatg 1860 tgtttgcgca gttgcaggcg attgtgcatg gcactccgcc aacgttgcca cctgggtaca 1920 gcgataatgc gaatgatttc gttgccaagt ggtacgtctc tcaccccttt ctcttcgtgt 1980 ttgaatttga caatgctgat aatgagcgca atctttagtc ttgagaaaga tcccaaccga 2040 cgaccgactt atgctcagct cttagaacat cctttcttgg tagcggacaa gggcgcagaa 2100 gttgacatgg ttggatgggt ggaaggggcg ttgaagcgca aggcagagag ggggattgcg 2160 agcctgaatc ctatccaacc acctgtccct ttggaaccat aa 2202 <210> 10 <211> 1620 <212> DNA <213> Cryptococcus neoformans serotype A H99 strain <220> <221> gene (222) (1) .. (1620) <223> genomic DNA sequence of HOG1 <400> 10 atggccgatt ttgtcaagct ctccatcttt ggaaccgtat gtttctttta ttgctctttc 60 tcttttccca ccaccgtcat gatctgctct tccaaccaac caacctacga acacgcggcg 120 tttgtttttt ccgttggcca ctggatcata tcgtgttgat tctgtccata cgccggatgg 180 aggagatctg taaaggcaag gccgcggacg ctgatggatg ggctttctcc atggataggt 240 ttttgaggtt accacgcgtt atgtcgacct ccaacctgtc ggtatgggcg ctttcggtct 300 cgtctggtga gtcttgtttt tctcaagcaa ctatcctttc atctggtttt tcaacccagc 360 gtcgaaacag gtcgtccgac ctttgcatgt cgatgtagag atgtgaactg acaaaaccat 420 cttgtttgat gcagttccgc caaggatcag ctgtctggaa cttctgtggc tatcaagaag 480 attatgaagc ccttttcaac ccctgttctt tccaagagga cttaccgaga gctcaagctt 540 cttaagcact tgagacatga gaacattatc tctcttagtg acattttcat ctctcctctc 600 gaagatatgt gagttttgct caatagttgc atatcaaaga aggggggagg gggcctgctg 660 acatttatcc aatagctact ttgtcaccga gctgctcggt actgaccttc atcgactcct 720 tacctctcgc cctcttgaga agcaattcat ccaatacttc ctttatcaaa tcctccgtgg 780 tctcaagtat gtccactctg ccggtgtagt ccatcgagac ttgaagcctt caaacattct 840 cgtcaacgag aactgtgact tgaagatttg cgatttcggc cttgcgagga tccaagaccc 900 tcagatgact ggttatgttt ctacgaggta ctaccgagca cccgagatca tgttgacatg 960 gcaaaagtat gatgtcgcgg gtgagtttca agttttacgt ttgggggtgg tcttttaatt 1020 ggcgatccat gctgaccacg caaaaaatca gttgacattt ggagtaccgg ctgtatcttt 1080 gcggagatgc tggagggcaa gccattattc cccggaaagg accacgtgaa ccaattctca 1140 atcatcaccg aattgctcgg tactccgccg gacgatgtca ttcaaactat cgcctctgaa 1200 aacactctcc gtttcgtcca gagtctgccc aagcgcgaaa aggtcccatt ctccaccaag 1260 ttccccaacg ccgaccctgt gtctcttgat ttgttagaga agatgctcgt gtttgaccct 1320 cgtacccgta tatccgccgc tgaaggtctc gcgcacgagt atcttgcgcc ttaccatgat 1380 cctaccgatg agcctgttgc cgccgaggtg tttgattgga gttttaacga tgcggatttg 1440 ccggtggata cttggaaggt gatgatgtat agtgaaattc ttggtaagtc tctgtgcctt 1500 gccttttttt gggtattata ctaacgtcgg actttagact tccacaacct cggagatatt 1560 tcacagaacg aagcagaggg acccgttact ggcgaagtcc ccgctgctcc tgccagctaa 1620                                                                         1620 <210> 11 <211> 3930 <212> DNA <213> Cryptococcus neoformans serotype A H99 strain <220> <221> C_region (222) (1) .. (3930) <223> coding region sequence of SSK1 <400> 11 atgtggggct caaatgcttc catcgccgcc tcggagtcga ccgactccct ttcccccgcc 60 ccctcccagt ctgccgccgt agagttcccc ctgcccgtaa gctcccgccc gtctctcact 120 tccgccgctc acccctccca gatgtccgct tcctcctcct ccacctcctc ccagcctctc 180 tttgattggc gcattcccaa gcccacctca ccccgcacac gcatggaccc attcgacact 240 tttgatcctg tatcctcaag ctcagaggat gatccggtcc ctcaagagag ccgccgtgct 300 ggtcatcagc gctccgtaac agatcctctt ttacgagatg gccaacccct cgatatggag 360 ttcactactg ccgggccgcc tatacagagc tacgactttg aacaaccgcc cacgtttagc 420 agaacgttat cttcccctct tccagctaaa gtcggctcgc ttagacaccc tatgccattc 480 accattgacg atttaagctc tcgcaatgtg aattcaaccc atcgtccgca gccgactaca 540 cctctgcatt ccatatccgt tgagttagcc gattccttgc aatcggccat tcaaacatta 600 ctgcatctgt ccccacctca cctactcgat aacgcaaaag aacagtactc tggatgcacg 660 gtacagatac ctgctacctc gctttcggcc cttttgacct ctatgagagg cctcaacttc 720 ttgtcggcgc atgctgaaga actggtcgac atgagtgcac gtggagatcc acctgtactc 780 catcaagaag acttcgatgt aggagaactt ttgcaaaacg ttgcggatat gttgagtgga 840 gaagcagcag aaaaacggat tgatttcgtt ttgttccatg gcgacgtagc gatgaggcat 900 gtcagtgtgt atggagattc tgatggaatt agctatacct tgagtcatgt tattcgacaa 960 atactggcag tagccaatta cgatgatacc atagaactcg gccttcaagt cattcctcaa 1020 agtccatctt tagcttccgc cgtcggactt cctctaacct ctgccgatgt tagtggagga 1080 ggtggtgtca agtcagcgtc cacatctcgg tcaggctccc ccaataacag tctctctcga 1140 tctaattctg tccatgacgg gccccttctc tgtgtgttcg aaatagtaca taacatctat 1200 cagccaccac caagctcggc atccgccact cctaaagccg agctgaaccc tttcactcat 1260 cttgctgaag aaaccgaagc cttgaaacca agattggata cagcattttg caaaaacctg 1320 cttcatcggc aaaatgctgt cctcaaagtt gatgtgcagc cttcatctcc tttaggatcc 1380 gggatgcccc gtagagctta cgcgttatca gtgctcctac caagaggtaa acccatcact 1440 gagcctgcaa tactttctaa agaggaacaa gaagttcgtc aaccattttc atcccacgta 1500 cttgcacgag aacccaccct caatgagctc tcggaatttg ctgaatcatt acgaggaaga 1560 aaggtgttta tccatgccaa tttgagtagt gttttcgcga gacacctcac gagctaccta 1620 gctgcatggg gaatggatat atcgcatcta ccgacagatg gcgatgaggc tgataaattg 1680 aaggatgtcg cggccaaaca tgactcggct tatactggat ctatgggtgt gtcaggcggc 1740 actacttcca gcgcagaaac gccctattca attaaaccga ccggcgtgac tgctgttcaa 1800 cctggacact ttgtcattat cgacgatgat gttgcggtct tgcgccgtga actcgtgcgc 1860 atccgttcag aattacttcc cattctcttt aaacctagac tgtcaaagcg tcccactatg 1920 acttctcgaa cccgttccac cccttcattg cgacaggtcc ccccaaggtc atcatcgggt 1980 tctgtactta tacactttac ctctcttgcc aattataacc gagttcgaga cgcgattgcg 2040 agctttgtgg gggcgccagg gttaaccaat ccggaaactt atgttcagcc ggaggtgata 2100 gtgataccca agcctgttgg accacgaaga tttttgactg ctctgcatac cgctgtgaaa 2160 cagcctatgg ttgacccatt tttctcccct atcgccacat ctcctagatc accaggcgga 2220 ggttactttg gtggtttgcg gactccgacg gagagagaat caggattctt tgattctgtt 2280 gcagaagaac cacatgaaga ggcggattcg cgaccagatt atgccacggt gcagaaagcc 2340 agatctcctt taggagaatt tccgccttct gcggcacaga tcgttcgtac caaccaaggc 2400 ttgcatcttt cgcttcccac tccaaatgaa attatgacaa cgcctgctcc agaatatttt 2460 tctgggtctt ccaagtctcc tagctctggt gcgtccggag tcgtgatgca gagccctgat 2520 ggtcgtcctt tcggaatgtt tttcgaaccg cccataaaaa atgagcgccg cggatctact 2580 cacaggacgc cttccgattc catcaggagg aaacaagcga accgccgtgc gtctacaagt 2640 gatgaaccct tttcttcacc ttctaccgcc ctacctcccc gtcgctcgtc cacaatttct 2700 acgactggca atgaggaaca ccgcagttca cctatcgcta acgtcacaga ccgtcctacc 2760 cattcaaggg taaattcaag aaggaagaac aatcttccgg cggcggagca acctattttg 2820 gctgtgggca gggcaaaagg cagggaaaga tcggagactg tcacgaaggg aggggacctc 2880 gggtcgagaa aaggtacacc agcggcaagc ccacgtatag aggagaagaa ggaattggaa 2940 agaggcgaga agactaaaag cctggctcct tcaactgctc ctacgaagaa gaatgctaaa 3000 gtcgatgttg tggtgccgcc catcaacgtg ctgattgttg aagacaaccc catcaatcaa 3060 aacattttga gtatgttcct gagaaaaaag aagataaaga attcctcggc caaggatggc 3120 gcagaagctg ttgaaaagtg gaggactgga ggcttccatc tgattctgat ggatatccaa 3180 ttgcccgtca tggatggcat agctgctacc aaagagattc gtcgacttga acgtcacaat 3240 aacattggcg tttttccatc gactccagcg gccgaacttc ctcggggtca aaatgttgcg 3300 gattctccac caccatcttc tccatttcgc tcgtcagtta tcattgttgc cctgacagcc 3360 tcgtccttgc aaagcgacag agtagctgct ttggctgctg gctgtaatga cttcttgacc 3420 aagcctgtgt ctttgaaatg gttggacaaa aagattgtgg aatggggttg tatgcaggca 3480 ttgattgatt ttgacggctg gcgacgatgg aagagctccg ataccaagaa tcctagcgaa 3540 actaagcagg gcttctcagt gggccctcaa caggctgcta ggtcgcttgc tagcagacta 3600 cgtattgaac gcaaaggatc tcgatctccg gcagctccag tatcaacccc gcgactcaat 3660 ttgcagtcgg caaccccaga taggccagaa acccccccag attccacgtc acaaatgcca 3720 aaggccccgc ccgttgcagc ctctgacccc ccgttatctc ccaagtcgct gaacaagaca 3780 gttaatgatg tcttcgagca agcagacgct agactcgaaa atgcgcggga ggaacaagga 3840 gtatcaagtc aaaaggaaaa cacaagctta acagattcaa caaacaccac cattacgccc 3900 tcaaagacct atccggctcc tcccccatga 3930 <210> 12 <211> 5076 <212> DNA <213> Cryptococcus neoformans serotype A H99 strain <220> <221> C_region (222) (1) .. (5076) <223> coding region sequence of TCO2 <400> 12 atgatcttag gaaccgacat cgacctgtcg tctataccaa cggcgtttct cgaggcttat 60 cccttcccag cggttgtgtt cgtgatcgat tctcccccta gcccaagacc gcggctccat 120 tccagaaata cagacacgac cattcggcga acggatggcc aaatatcgcc tcttacaggt 180 cctccagtgc aacagttcgc gtcagcgccc gtggtatggg gcaatcaacg atggcacgag 240 ctggctcagg ggaaaacaat tgcagagtgc gtggatgtgg cgtcacagaa caagctgcaa 300 acttgggtgg aaaatgacac cggtgacaag tcggagagtt tggccctgga cctgaaggtg 360 ccgcaaggcg tgactcttca tctggcaaag accatattgc cattaagtcc accgtcctcc 420 tctcagtcac tttgcatcct tatatcgcaa tatatcgata agccggaaag tttcgcgcca 480 ccaatctcat ctggagatat tcttttctct tctctatcgc gactctccca gactttttct 540 cggtcatcct ctttttcatc caaccctaga aaatcgattg atgtccctgc gtcactatcc 600 gaacaccggg gttctgctac atcgacaagt agcaatctgc gctcttcgat cgatttgact 660 tcccctaatt ctcaaccctc tccactaaac cgtgaacaaa gcacgtactt cacccatggc 720 tccgcgacca gagaagagcg accctcagta aggcgtagac ggtcaccgcc aatctcaatg 780 acgaggccca agcctcttga gagccatgct caagaatgct gggacttggt agagaatttc 840 gactggtcaa aaacagcatt agggccgaga gaacagtgga tggatgcgtt agatcctgtt 900 ctggcaatca catttgaatc cagaacggca gattgtgcct ggttagggcc tgatctagag 960 ctagtttaca ataaggcgta tcaagagctg gttgaccatc ccaatgcttt tggaaaacct 1020 gcaagacaag tttgggctac caattgggac tacttggaac ccctggtcaa gcgatgtctc 1080 agtgggaccc cggtctacaa ggacaacgac ccgcttttct ggcgtcgata cggcaatggt 1140 cgacttctgg aacattacca cacttggcga tatgtcccga taacgggcaa agatggctca 1200 gtgcttggca tcttcaacca gtcaattgag gtcaccgact cagtactgct agagaggcga 1260 atgggcacga ccagggaact ttcggaacac atgtcgttta ttcgtacaac tgaggacttt 1320 tttagctcgg ttgccgacgt ctttagtcag aaccctactg acataccgtt cgcactttgt 1380 taccgggtcc gacaagttga caccgatggg acatttgtcc atttggacgt ctcgcttcag 1440 tcgtccgtcg gtgtacccga aggccatccg tctgctccag atcaaattcc cgtcagcttc 1500 ttaaatggta acccttaccc tagcaatgtc gagcgatcat tttctcctgc tttctcaatc 1560 gtttcaatcc actcttcgag cagtcatcga gtctgtcacg tctctgaaga cactacacaa 1620 tggcccatcg ccaaagccct acaaaggcgg caatgtgtca tcatcgaaga atgttcgcaa 1680 ttaatagaag gatatcctat ccgtcgctgg gatgggcttc cattctcagc cattgtcgtg 1740 cccatatgct ctgaagggtc tcccgaaatc cctgacgccg ttgttattct tggtctcaat 1800 gtgcgacgtt gttttgacca tgaatacgat tcctggattc actctattcg gtcacaacta 1860 tcttcggccc tcgtgatggt caaggcgcgt gaagctgaac aaaagatggt tgaggaaagc 1920 gcacgtatgg agaaagcaaa agtcgcttgg ttcagaggag ccgcgcacga ccttcgtagt 1980 ccattaaccc tcgtcgctgg accgcttgcc gatgtgcttg attcggattt gaactcgagt 2040 cagcgcacgg ctttgaccgt tgcgcaacgc aatcttgatc gtttagtgcg cttggtcaac 2100 gccctcatgg atttctcgag ggtggaagct ggacgaatgg aaggacgatt tgttccgacg 2160 aacttgagtc aattcgtgac acagttggca gctcttttca agcctgcaat agaaagattg 2220 gggttagaat acgtactaga tgtccagcca agcgaggagc ttgttttcat cgatcctgtt 2280 ctgtttgaga ccgtggtatc aaaccttatt ggcaatgcgc tcaaatacac tgaaacgggt 2340 tctatcactg ttcgggtgca atacacggat tacgcagagg tctcggtcat cgataccggt 2400 gtgggtatac cgaaaaatga gctggcactg gtgaccgaat ggttccacag ggcaagtact 2460 gccattcact cgggaaccca gggaacagga ttgggactgg ctttggccaa ggaattgctc 2520 aagttgcata aaggagaatt gcttgtcgag tctcaaaccg ccaatgagtc aggaggtcct 2580 catgggtcca tttttacagc gaaaattcct cttgatttca agccctctcc atcggctcat 2640 atcattccgt ccgtcgaatc tcacaagacg tttggcaaat acagtaaagc cgtcgcagac 2700 gaagccatgc gctgggttgg ggactcagat gccgctagtg aggcgtacga catgtcgagc 2760 ggtaccggag tctcaagcgc tggtagtggc tctggaaaca cgaccacctt cggacccaag 2820 tttgcagatg cctttttgtt tgataagaac gacattgtgc ttattgtgga agacaatgtc 2880 gacatgcgtg aatacatacg acagcttttc gccccttatt gtaccgtact cgaagcttcc 2940 aatggtgaac aggcttacaa tatggctacc caaaaccctc ccaacctcat tttgtcggac 3000 gtgctcatgc ccaaattatc tggtatggag ctactacaaa ggatcagatc ccatcctgac 3060 actcgcattg tgcctatggt ccttatttcg gctattgctg gtgatgagtc tagggttgag 3120 gctctgctaa acggcgctga tgactatctt gccaagcctt tcaaacccaa ggaactcatc 3180 gcgcgtgttc acctgcacat gcaagttggc aagaaacgtg ccaagctcga agcgctatac 3240 gcccaacgcg aaacagaatt gacagctcta tctgactatt gtccgatcgg tatcttccga 3300 ggagacaaat atggccatat tgtttatgcg aacgcagctt ggcgtgcgca gagcggcctt 3360 ttggtgggtg accctaacga ttgggcatct tatgtgcacc cggattcgaa agcgcagctc 3420 ttggaacaat ggaatcagtg gttgaggggg gatttgaagg agttccgagc ggcttggaga 3480 tggtctaatg gcatccctgt caggagcatc ttggtccggt tagatgacgt caaggaaggg 3540 ttttctgggt taattgggtg cgtagtggat gtgtctcatg aagagagacg attaatcgaa 3600 gctgaggaaa gaagaaaaga ggcggaagag agtaaacatc agcaagaact ccttattgac 3660 ttgacaagtc atgaaattag gaccccggtg tcagcaatcc tgcagtgctc agatcttgtt 3720 aaagagaatc ttgtagctct gaaggaccag ttgagaggag cggggccaaa gggctttgtg 3780 ccgagtcaag aattactggc tgatcttgag caggatgtgg aagctttgga aagtatttat 3840 cagtgcggtc ttgtgcagga acgcattgcc ggagatgttc tttcgctggc tcgtatccaa 3900 ctcgatatgc tgagtttgca cgacattgac gtcaacttgc gccgagaagg caggaaagtt 3960 tcgtccatct ttgcatcgga agccaagatg aaggatatcg acctccaatt ggaatttgga 4020 cctactatcg aacagtccaa agtgctggcc atcaagacag atcccgtgag attaggccag 4080 gtggtaacaa atctcatttc caacgccatt cggtttacat cttcgagtga tgtccgaaag 4140 attactatcc aatacgacgt atcgtttgtc cctcctgccg atgactcttg cgccctccct 4200 tcatctgttg gcttgcccga catacttcct gtgaaagaga atactccact atggctgttt 4260 gtcagtgtta ccgattctgg acctggtatg acagagcaag agttatctgt tttgttccaa 4320 aggtttgccc agggcaataa gatgattcat actaagtatg gcggaagcgg tttgggactg 4380 ttcatctgtc gaaagattac agagcttctt ggcggtcgta tcgaagtgct cagccaagtc 4440 gggcacggta gtgttttccg attcttcatt aaaacgcgcg ctgtcgctcc tccgtccgcc 4500 atcgctgctc tcgtagaatc ctctcctctc aaaccggtat ccgccacttc accttcttcc 4560 tcgttagcca tgagccgatc gtcttctcgg agcacaaacg tcactacacc tatagagggt 4620 ggtgggaccg agcacgtgtt gatcgtggaa gataacctaa tcaatcagac tgtcctgaag 4680 cgacagctcg tcaaggcggg tttatcgtgc aacgtcgcga gtaatggcct tgaagctctt 4740 aatgtcatcc gtgaagtcca tcggcaacac cgacgcggtg ggccgaaccg taaaaggcta 4800 tttgacgtgg tattgatgga tctcgagatg ccagtgatgg atggtatcac cgccgtacga 4860 gagatacgac agtccgaggc cgcggggaca ttgggcagga atatggtgat tgccctgacg 4920 gggaacgcga gacaaggaca gattgatcat gccttggctt ctggatttga cgatgtcgtc 4980 atcaaaccgt atatcctggt agatttgctg aacaagatca aatctatgaa agttagaaaa 5040 ttggagttgg aaactgcgaa agctcaagaa gagtga 5076 <210> 13 <211> 4254 <212> DNA <213> Cryptococcus neoformans serotype A H99 strain <220> <221> C_region <222> (1) .. (4254) <223> coding region sequence of SSK2 <400> 13 atgtccaacc caacctcccc ctcaaacccc tcagacaccg gcccgtcctc ggcgtccaac 60 gtcacatcct cgtcctcaaa gaccggacgc agatcagtgc gcctctttgc cccagacgag 120 gaggacagct cagacgagga cggcggcctc atcggcgtgc ccgcagagac cacattcaag 180 gacgacgaga tccctccttc caacccacgt tccgcctcct accccgggcc accggcacac 240 acctccccca cctctaaaat ctcaaccatc gtttcgtccg cttctgcagc ccagccaaaa 300 cttgcacgtt caataacata cgtcgcaccc aatgccgtct cctcccggcc ggcatatccc 360 ctcaatcccg cagggtcaga aaccttacac gcctcaggaa ggtcgtatac tgaccccgat 420 ataggatact ttagccacga tgcaggagat gatggttggg gctcagatga tgacgacgaa 480 ttgagatccc ctggctgggg catatcccat cataacatgg actctggagg caagacgaac 540 gggtcaccac agttgcctat aaagcccgcc gatgtcaccg aggatgaagg acaggaacgt 600 ttagattggc aaggcatgct ggaaagtgtc ctcaactcgg atgttctcaa ggtggaggaa 660 caacgtatct acaattccat gccgacagat tcattcagag aagagattgg aaagaccctt 720 tggtggcaaa tccgtgccaa actgcgtggg aggacagagg cggaggagaa gaaacgggtg 780 caagagcgac gagcgagagt ggtggacccg gtgctggaag agataaacga gttcaagtac 840 gacccaaaaa ataacccaga aggcgaagaa gacagtgatg gcgatccgca agacgcgact 900 tcgactgctg caccccaatc caaagctctc aatcaagtca acaccgttct cgccaaactt 960 catgcaatca aaggtcttta tcccaacctc gcagccatgc gagccgacaa ggttctctat 1020 accgatgaaa atttccgcaa acgcgccgac gcattgacct cttggtccat catcgtttca 1080 tccctccaaa cccagctcaa actcttgcaa aaatggacag gttccgatga gcttgacatc 1140 accaagccca acacgaccca cgagaaagca ttggtcggca agtacaagta tcactctatc 1200 gacagcaagg gtacgcccgg cagggatgca gccgatgact cgagtttcct cgatcgtgtg 1260 ataaaagaag ataaccttca acggacattc gagcgtcgag cgtttgtaga catgatcaac 1320 ctcgtgcgca acgccaagga gacggtcatc agctatctcc cccagttcca agaacaaaat 1380 cttcccgatt tccagtacga aatcgttcgt cttattggtt tccccggtcg acttatcatt 1440 gaagctgtca aggttcgttt ggatgctgca tcccgactac ttgacccgaa ccctatggtc 1500 gtcgaagact ttatcgaaaa ccttcgtcta tccatttcgc tcgccgtgct aatccggaaa 1560 caatacgacg aaatcatggc acccgatgcc gaggggagat ggaaaatccc gcattgcttg 1620 ccgacagagt acaatgatgt tctgctcgat gcgctgagga catttttcaa attgttgcat 1680 tggagattac gaggagtggg gaaagcgagt tattacaagg aaacagaagt gttggaagaa 1740 gaggcgccgt tcttgtatga agcggcggag gctattgtag gcggtgatat ggttgttgca 1800 gagcagtatt gcgcgttatc caacaagctc cttatacgtt cagcaaatta tcttgaccag 1860 caacttcggg taccaataca ttccccgtct cgcgacaagg aacgtggtga caaggagcgc 1920 gatggctctt cgtcttctca acgtaaccgt gacggccgtg atagctcgct gcccggccca 1980 ccgaaacaca tgaaagtcga agaactcttc tcatggtact ccaaactcct tgattccgct 2040 cgtatgcgac accgtaaaac ccaacgtttc tgtcgtaaac tcacccaacg attcgataat 2100 tccgccgaat attcaatcga ggagacggag atggacatgc tggtggagac attgcaagat 2160 actggtcatt tcttggtata taccgggaaa tttgaggcga atgggacgta tatcgttgcg 2220 gatgggagtc tctggggtca gccggacgat gtgagacatc tgttgaagag ggtgttttca 2280 gtgacgattc ctggatctcg agtccgtcca aggcagacaa cctcgcaagt atctgtcgga 2340 ggtgcgagcc cgtccaatgg tcaagtcgcg gcgcaacatg atcctgcaga tccgtacccc 2400 gaggcagacg attttgacga cgaagcgctc gcggcttaca tcctcctcat ctccccacgc 2460 caaagttttg tatggtccgg agcggtcatg acgctggatg tggattacat cgactatgaa 2520 ctacctgata accgagtcag attgatcgct gacggtccca ccaagcggtt agcgctgtgc 2580 aaactttatt tcaagcaagc gctcattcac cctgatacgg gcgaaacaat cgacttgcca 2640 tgtgtggttg aggctcaagc gcatttaccg accattcaga aacaacttgt caagattgct 2700 aaatcgagtt atcgtctttc agagtgcatt gtccagtctg caccactcgt ccgcaatgcg 2760 ttcaggggca aaccgggatc acaagagttg gtggagaatt ggtacagttt tgcgacagag 2820 catgggacga gagtgttgat ccatattgag cctagtgtat gggagcgatt caatcggttg 2880 ttgatgcgtc tggcgatcag ttggattagc tttatcagtc aagagtgtaa ccccacagac 2940 cgcaagacgt tccgatggac tgtggcagct ttgacctatg cgttcaacat gacgagaggg 3000 agtaacattc tcgcgcttga tcgatcagaa ttttcgcttt tgaggaggtc gagtatggag 3060 gccaaaaagg aggcagacag gattgaggcg atgaggaggt tacaacggct tcaagaaaac 3120 ctggacgacg aattcctgcc ccggactccg acagagtctg gcgatcaacc acgtatcgac 3180 cgctctataa ggctcacagt cgaagaacgt ctccgtctca ttgccgagct cgaagctcgt 3240 cgtgacgagt tggcacccgc acccgtcggt caagtccttg atgaagaagt ctctgaagac 3300 cgtgcgttgg tgttccttgc agcttccaaa tccaacattt ctatgcgatg gcagcaaggc 3360 gcgtacatcg gtggaggtgc atcgggaagc gtgtacttgg gatactcgtt gcaggataac 3420 actgtgtttg ctgtcaagat cttgccaacg gtggatctgc agagtagtcc ggcgttgtac 3480 gaaagtatca agcgagaatc ggatgtgatg agcttgttga gtcatccgaa tatcgttggt 3540 ttccttgggt tggaagtgca taggaacaga gtttgtcttt tccaagagta ctgtgaagga 3600 gggtcgctgg caggtatgct cgaatatggc aaaattgacg atgaggaagt cgttggagcg 3660 tttacgatcc agctgttacg cggccttgag tatctgcaca ccaaccgcat cgaacaccga 3720 gatctcaaac cagaaaatat tctcatcggc gccaattctg tcctcaagct ggccgacttt 3780 ggtaccgcca aaatcatcaa atccaacaag acgctcgccc gtacacgtgg tggcgcgcac 3840 gccaagatgg agggtcttga gggtacaccg atgtacatgg cgccagagat gatcaagaac 3900 cagaggactg gcaagctggg tgcttgtgat atctggggtt taggatgtat cgttttgcag 3960 atgatcactg gtaggaagcc atggagcttc ttggactttg ataatgaatg ggcaatcatg 4020 ttccatcttg gtgcgacaaa ggagccacct cctctacccg atcccaacga gatgtccgac 4080 caaggtatcg aattcattga tcaatgtctt tctttggatc cggaagcgag gccggtggcc 4140 agcgagttat tgcaagatga atggctggtt ccaatgttgg agcagatggt gagttgtctt 4200 tcatgccgag ctggagcaag aataccccga tatattggcg atgggccaaa gtga 4254 <210> 14 <211> 1830 <212> DNA <213> Cryptococcus neoformans serotype A H99 strain <220> <221> C_region (222) (1) .. (1830) <223> coding region sequence of PBS2 <400> 14 atgacagacc ctacgccccc cgccctggac agtctctccc tggcagacaa ggcgcctact 60 cccgaagaaa gtcccgaaga cgccgctgaa cagcccaagc ccgcggcctc accgtccgca 120 ggcacacccg gccatgacgc ccaaagctca tccacctcgc ccccgcaacg ccctcagtcc 180 atgcagacaa atgacaaggc gccagataca tctgctccgg cttccaggcc ccaaccgcaa 240 catgtccctg catcggcacc tgcgcttccc tctaccaacc ccgtccgtcc acagccgggc 300 gcccgtcctg gagcggcgag gggtatgccc gcgcccatgg gtatgcgggc gcaagcaggc 360 cgaggcgctg gcggccccca gatgcagacc aagatgctgc ccagtttgca ggctaaaatg 420 gacaagatcg cggcgtctcg gcaagggcca cctccctcct ctggcatgca tgatccgaat 480 gccacatcca tgggcgccct cttacgctcc caagccctcc gcgcccccgg cgcatcgcaa 540 gctcctcccg gccccggacc ggcttcaggc cctttcggtc tcgccgctcg gcgcgcagct 600 gctgggggcc ctccgagacc gaatttgggt atgatgggta tgggtgcaag tgcgccgggt 660 gcggttggac ggggatcagg tctggcgggt agacgggggc cccctggagg actgacactg 720 agtgggatga agggtgcgat caaggatgag ggaaacaagt tttcagactt tcagggtgtc 780 atggacccgt ctggatcgct gagattctca aagaaggctg tcctgcatgc aaagggcgtg 840 gactttgagg atgggcaaag tttcaagatc aatatggatg agatcgaggt gcttggagaa 900 ttaggaaagg gcaattacgg ttctgtgcac aaagtcttcc accgtccgac aggcgtcacc 960 atggccatga aggaaatccg gttagaactt gacgattcca agctcaacgg catcattatg 1020 gaactcgaca tcctacaccg ggccgttgct cccgaaatag tcgaattcta cggtgcattc 1080 accattgaat catgcgtcta ctactgtatg gagtacatgg atgccggttc actcgactct 1140 ctcaccggtg gcggtgtggc ggccaaagat caaacaaagg atgaagaaaa cgatgcgaca 1200 aaacgagtgc cggaggatgt attgaggagg attacagcga gaatcgtgaa agggttgagg 1260 ttcttgaagg atgaattgca gatcatccat cgagacgtca aacccacaaa tgtgttaatc 1320 aatggcaagg gagaggtcaa gatgtgtgac tttggcgttt caggtcagct cgaaaagagt 1380 ttggccaaga ccaatatcgg ttgtcaatcc tacatggctc ccgaacgtat caagtctgaa 1440 actgccaacc agaatcctac atatactgtc tcttcagacg tctggtctgt cggtctgtcc 1500 attgtcgagc ttgccaaggg gtgttacccc tacccaccgg agacgtatgc gaatgtgttt 1560 gcgcagttgc aggcgattgt gcatggcact ccgccaacgt tgccacctgg gtacagcgat 1620 aatgcgaatg atttcgttgc caagtgtctt gagaaagatc ccaaccgacg accgacttat 1680 gctcagctct tagaacatcc tttcttggta gcggacaagg gcgcagaagt tgacatggtt 1740 ggatgggtgg aaggggcgtt gaagcgcaag gcagagaggg ggattgcgag cctgaatcct 1800 atccaaccac ctgtcccttt ggaaccataa 1830 <210> 15 <211> 1098 <212> DNA <213> Cryptococcus neoformans serotype A H99 strain <220> <221> C_region (222) (1) .. (1098) <223> coding region sequence of HOG1 <400> 15 atggccgatt ttgtcaagct ctccatcttt ggaaccgttt ttgaggttac cacgcgttat 60 gtcgacctcc aacctgtcgg tatgggcgct ttcggtctcg tctgttccgc caaggatcag 120 ctgtctggaa cttctgtggc tatcaagaag attatgaagc ccttttcaac ccctgttctt 180 tccaagagga cttaccgaga gctcaagctt cttaagcact tgagacatga gaacattatc 240 tctcttagtg acattttcat ctctcctctc gaagatatct actttgtcac cgagctgctc 300 ggtactgacc ttcatcgact ccttacctct cgccctcttg agaagcaatt catccaatac 360 ttcctttatc aaatcctccg tggtctcaag tatgtccact ctgccggtgt agtccatcga 420 gacttgaagc cttcaaacat tctcgtcaac gagaactgtg acttgaagat ttgcgatttc 480 ggccttgcga ggatccaaga ccctcagatg actggttatg tttctacgag gtactaccga 540 gcacccgaga tcatgttgac atggcaaaag tatgatgtcg cggttgacat ttggagtacc 600 ggctgtatct ttgcggagat gctggagggc aagccattat tccccggaaa ggaccacgtg 660 aaccaattct caatcatcac cgaattgctc ggtactccgc cggacgatgt cattcaaact 720 atcgcctctg aaaacactct ccgtttcgtc cagagtctgc ccaagcgcga aaaggtccca 780 ttctccacca agttccccaa cgccgaccct gtgtctcttg atttgttaga gaagatgctc 840 gtgtttgacc ctcgtacccg tatatccgcc gctgaaggtc tcgcgcacga gtatcttgcg 900 ccttaccatg atcctaccga tgagcctgtt gccgccgagg tgtttgattg gagttttaac 960 gatgcggatt tgccggtgga tacttggaag gtgatgatgt atagtgaaat tcttgacttc 1020 cacaacctcg gagatatttc acagaacgaa gcagagggac ccgttactgg cgaagtcccc 1080 gctgctcctg ccagctaa 1098  

Claims (17)

An inhibitor of at least one protein selected from the group consisting of SSK1, TCO2, SSK2, PBS2 and HOG1 of Cryptococcus neoformans, The inhibitor is an antibody against the protein, An antifungal pharmaceutical composition, which is administered sequentially or simultaneously with an ergosterol-binding antifungal agent. The method of claim 1, The inhibitor is an antifungal pharmaceutical composition that is an inhibitor for SSK1 protein. delete The method of claim 1, The ergosterol-binding antifungal agent is a polyene-based antifungal drug composition for antifungal. 5. The method of claim 4, The polyene-based antifungal agent is an antifungal pharmaceutical composition for at least one antifungal agent selected from the group consisting of amphotericin B, natamycin, limosidine, philippines, nystatin and candiesine. The method of claim 5, Said polyene antifungal agent is amphotericin B antifungal pharmaceutical composition. An inhibitor for one or more genes selected from the group consisting of SSK1, TCO2, SSK2, PBS2 and HOG1 of Cryptococcus neoformans, The inhibitor is an antisense oligonucleotide, siRNA, shRNA, miRNA or a vector comprising them for the gene, An antifungal pharmaceutical composition, which is administered sequentially or simultaneously with an ergosterol-binding antifungal agent. The method of claim 7, wherein The inhibitor is an antifungal pharmaceutical composition that is an inhibitor for the SSK1 gene. delete The method of claim 7, wherein The ergosterol-binding antifungal agent is a polyene-based antifungal drug composition for antifungal. The method of claim 10, The polyene-based antifungal agent is an antifungal pharmaceutical composition for at least one antifungal agent selected from the group consisting of amphotericin B, natamycin, limosidine, philippines, nystatin and candiesine. The method of claim 11, Said polyene antifungal agent is amphotericin B antifungal pharmaceutical composition. An antifungal complex preparation comprising an antifungal pharmaceutical composition according to any one of claims 1, 2, 4 to 8 and 10 to 12 and an ergosterol-binding antifungal agent. At least one protein selected from the group consisting of SSK1, TCO2, SSK2, PBS2 and HOG1 of Cryptococcus neoformans is contacted with a candidate outside the body, Determining whether the candidate inhibits the activity of the protein. A method of screening a medicament for increasing the susceptibility of fungi to ergosterol-binding antifungal agents. The method of claim 14, Said protein is an SSK1 protein. At least one gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2 and HOG1 of Cryptococcus neoformans is contacted with a candidate externally, Determining whether the candidate inhibits expression of the gene. A method of screening a medicament for increasing the susceptibility of fungi to ergosterol-binding antifungal agents. The method of claim 16, Said gene is an SSK1 gene.

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