KR20110124941A - Use of tco1, mbp1, atf1 or ras1 for treatment of fungal infection - Google Patents

Abstract

PURPOSE: An inhibitor of Tco1, Mbp1, Atf1 or Ras1 for manufacturing an antifungal agent is provided to remarkably increase the efficiency of an antifungal agent. CONSTITUTION: An antifungal pharmaceutical composition contains an inhibitor of Tco1, Mbp1, Atf1 or Ras1 of Cryptococcus neoformans. The inhibitor is administered together with nucleic acid synthesis suppressing antifungal agent successively or simultaneously. The antifungal agent is pyrimidine analog, flucytisine. An antifungal complex agent contains the pharmaceutical composition and antifungal agent for suppressing nucleic acid suppression. A composition for screening antifungal agents contains Tco1, Mbp1, Atf1 or Ras1.

Description

Use of Tco1, Mbp1, Atf1 or Ras1 for treatment of fungal infection}

The present invention relates to the use for the treatment of fungal infections of Tco1, Mbp1, Atf1 or Ras1, one of the major hybrid sensor kinase acting on HOG signaling pathway.

Fungal infections have been a major health care problem worldwide for decades as the number of immunocompromised patients due to AIDS, chemotherapy and organ transplantation has increased. Despite worldwide attention, and the emergence of clinical the limited number of antifungal agents that are available for use with the drug-resistant strains of Candida (Candida), Aspergillus (Aspergillus), Fusarium (Fusarium), non-cor (Mucor) and It is a major obstacle in overcoming and treating various fungal infections caused by Cryptococcus .

The main reason for the low number of antifungal agents is the difficulty in finding new fungal specific targets, primarily because fungi have eukaryotic structures similar to mammalian cells.

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 inducing antifungal transfer of fungal RNA and uncompetitively antagonizing DNA synthesis, and having amphotericin B having a polyene structure. Γ exhibits antifungal action by binding to ergosterol inside the fungal cell membrane, causing depolarization of the cell membrane, and forming pores to cause loss of intracellular contents. 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.

The limited number of antifungal agents available has led researchers to develop new antifungal targets or to try different combination therapies with multiple drugs to have synergistic antifungal effects. Major benefits for synergistic combinations of antifungal agents include inherent toxicity and dose reduction for each drug, potential reduction in the number of drug resistant strains, and expansion of the antifungal spectrum. Various antifungal combinations have thus been tested in vitro and in vivo. For example, the combination of fluconazole and amphotericin B, which are opposing combinations, is actually more effective in vivo than treatment separately with each drug. Combinations with drugs targeting nucleic acids such as amphotericin B or fluconazole with fluoroquinolone or rifabutin have also been found to have a synergistic effect against Candida or Aspergillus infections. Furthermore, the addition of human cytokines often has a synergistic effect as in the case of macrophage colony-stimulating factor (M-CSF) -amphotericin B combination therapy. Similarly, human lactoferrin has also been shown to have a synergistic effect with fluconazole.

Recently, it has been shown that simultaneous fluctuations in fungal signaling pathways associated with azole or polyene treatments have synergistic effects. For example, the Ca ²⁺ -calcineurin signaling pathway by application of cyclosporin A and tacrolimus (FK506) that inhibit calcineurin or mutations in genes encoding the catalytic or regulatory subunits of calcineurin when associated with azoles. Inhibition has a synergistic antifungal effect against Cryptococcus neoformes, azole-resistant Candida albicans and other Candida species.

We have previously shown that the combined treatment between the antifungal agent Fludioxonyl and Tacrolimus shows a surprising synergistic effect on Cryptococcus neoformus. Fludioxosonyl treatment inhibits normal cell growth following cell swelling and mitosis by activating the HOG-signaling pathway and accumulating intracellular glycerol, and defects of such cells in the calcineurin pathway that regulate cell wall integrity Accelerated by inhibition. More recently, inhibition of HOG signaling, such as mutations in the SSK1 , SSK2 , PBS2 and HOG1 genes, has been shown to make Cryptococcus neoforms very sensitive to amphotericin B, for the treatment of Cryptococcosis. Another novel antifungal therapy has been proposed (KR10-2009-0001947).

The present invention aims to discover new target genes for the development of antifungal agents that can provide synergistic antifungal effects by investigating the hypersensitivity of genes in the HOG pathway to known antifungal agents.

The present invention relates to the use of an inhibitor against Tco1, Mbp1, Atf1 or Ras1 protein or gene of Cryptococcus neoformus for the preparation of an antifungal agent, an antifungal pharmaceutical composition comprising the inhibitor, and an effective amount of the inhibitor to the subject. Provided are methods for treating fungal infections, including.

In another aspect, the present invention is the use of Tco1, Mbp1, Atf1 or Ras1 of Cryptococcus neoformus for screening antifungal agent, the composition for screening an antifungal agent comprising the protein or gene, a candidate material and contacting the protein or gene, It provides a method for screening an antifungal agent comprising determining whether a candidate substance inhibits or promotes the activity of the protein or gene.

In the present invention, Cryptococcal kusu neo irradiated with hypersensitivity to known genes in the HOG path of satiety's antifungal agents, TCO1, MBP1, when ATF1 or Ras1 gene is suppressed and sensitivity to the flu Saito God inhibition of nucleic acid synthesis sex antifungal dramatically It is newly found to increase. Inhibition of the TCO1, MBP1, ATF1 or Ras1 genes can significantly increase the efficacy of nucleic acid synthesis inhibitory antifungal agents, thus inhibiting the TCO1, MBP1, ATF1 or Ras1 genes or proteins encoded by Cryptococcus neoformes. Antifungal pharmaceutical composition comprising a can be used as an excellent complex antimicrobial therapeutic agent that can increase the efficacy while reducing the use capacity of the nucleic acid synthesis inhibitory antifungal agent.

1 is an analysis showing that flu cytosine exhibits a synergistic or antagonistic antifungal effect by inhibition of the HOG pathway.
2 is an analysis showing that the combination of amphotericin B and flu cytosine by the inhibition of the HOG pathway shows a synergistic or antagonistic antifungal effect.
Figure 3 shows the results of DNA microarray analysis performed to characterize the role of the hybrid sensor kinase and the function of hog1 for flu cytosine.
4 and 5 show 59 known genes out of 194 individual flucytosine dependent genes identified by the DNA microarray analysis.
Figures 6 and 7 tabulate 122 of the unknown genes among 194 individual flucytosine dependent genes identified by the DNA microarray analysis.
FIG. 8 is an analysis showing that inhibition of the Mbp1 transcriptional regulator, which is expected to be affected by major hybrid sensor kinase acting on HOG signaling pathway, shows synergistic antifungal effect of flu cytosine.
9 is an analysis showing that inhibition of atf1, which is expected to be a lower transcription factor of the HOG pathway, shows synergistic or antagonistic antifungal effects of flucytosine and amphotericin B.

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

The present invention provides an antifungal pharmaceutical composition comprising an inhibitor against a Tco1, Mbp1, Atf1 or Ras1 protein or gene of Cryptococcus neoformus for the production of an antifungal agent.

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, which are not present in mammals in the HOG1 pathway. Because of the use of a two-component-like phosphorelay system consisting of three elements, the present inventors are well known for the mutation of genes involved in the HOG signaling pathway for the development of new antifungal targets. We tested the hypersensitivity to antifungal agents. As a result, it was surprisingly found that the inhibition of Tco1 in the regulation of HOG pathway of Cryptococcus neoforms significantly enhanced the fungal hypersensitivity to flu cytosine. As can be seen in the examples below, hog1Δ ( Hog1 MAPK mutant), pbs2Δ ( Pbs2 MAPKK mutant), ssk2Δ ( Ssk2 MAPKKK mutant), and ssk1Δ ( response regulator mutant) mutants are directed to flucytosine than the wild type. In contrast to slightly higher resistance to mutations, mutations in the TCO1 gene, one of the major hybrid sensor kinases acting on the HOG signaling pathway, dramatically improved cell susceptibility to flu cytosine. In addition, carrying out two kinds of transcription factors in the mbp1 atf1 △ △ mutant strain the gene is destroyed encoding Atf1 and Mbp1 in the HOG pathway as shown in the examples also showed a significantly increased sensitivity flu cytosine. In addition, the ras1Δ mutant also showed very increased flu cytosine sensitivity. Therefore, an antifungal pharmaceutical composition comprising an inhibitor against the protein or gene of Cryptococcus neoformus Tco1, Mbp1, Atf1 or Ras1 can increase the efficacy while reducing the dose of a nucleic acid synthesis inhibitory antifungal agent such as flu cytosine. It can be used as an excellent combination antibacterial agent.

Therefore, the present invention provides the use of an inhibitor against a protein or gene of Cryptococcus neoformus Tco1, Atf1 or Mbp1 for the preparation of an antifungal agent, an antifungal pharmaceutical composition comprising the inhibitor, and an effective amount of the inhibitor to the subject. It provides a method for treating a fungal infection comprising the same.

In the present invention, Tco1, Mbp1, Atf1 or Ras1, which is used as a target for blocking the HOG1 signaling system, means a Tco1, Mbp1, Atf1 or Ras1 protein or is interpreted to mean a TCO1, MBP1, ATF1 or Ras1 gene. Thus, a Tco1, Mbp1, Atf1 or Ras1 inhibitor is interpreted to include both inhibitors on Tco1, Mbp1, Atf1 or Ras1 proteins or inhibitors on TCO1, MBP1, ATF1 or Ras1 genes.

In one embodiment, the inhibitor of Tco1, Mbp1, Atf1 or Ras1 protein of Cryptococcus neoforms may be an inhibitor that blocks signaling by binding to the protein and inhibiting its activity. In another embodiment, the inhibitor of the TCO1, MBP1, ATF1 or Ras1 gene of Cryptococcus neoforms may be an inhibitor that inhibits signaling by inhibiting the expression of the gene. In the present invention, the TCO1, MBP1, ATF1 or Ras1 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 Tco1, Mbp1, Atf1 or Ras1 protein may have an amino acid sequence of SEQ ID NO: 1 to 4, respectively, wherein the TCO1, MBP1, ATF1 or Ras1 gene is a nucleic acid of SEQ ID NO: 5 to 8, respectively It may have a sequence or cDNA sequence of SEQ ID NOs: 9 to 12, which is merely illustrative of the sequence of the Cryptococcus neoformans antigen type A H99 strain, the sequence of Tco1, Mbp1, Atf1 or Ras1 of the present invention is limited thereto. It doesn't happen.

In the present invention, the protein or gene of Tco1, Mbp1, Atf1 or Ras1 is interpreted to include variants or fragments thereof having substantially the same activity.

Antifungal activity by inhibition of Tco1, Mbp1, Atf1 or Ras1 proteins or genes can treat Cryptococcosis and meningitis caused by infection with Cryptococcus neoformes.

Thus, in one embodiment of the present invention, the use of a Tco1, Mbp1, Atf1 or Ras1 inhibitor, a Tco1, Mbp1, Atf1 or Ras1 inhibitor for the manufacture of a medicament for the treatment of diseases such as Cryptococcosis and meningitis Provided is a pharmaceutical composition for treating diseases such as nephropathy and meningitis, and a method for treating diseases such as Cryptococcosis and meningitis comprising administering to a subject an effective amount of an inhibitor of Tco1, Mbp1, Atf1 or Ras1.

In addition to Cryptococcosis and meningitis exemplified herein, diseases caused by fungal infections are well known in the art. In the present invention, as long as the inhibition of Tco1, Mbp1, Atf1 or Ras1 protein or gene enhances the efficacy of nucleic acid synthesis inhibitory antifungal agent, those skilled in the art will be able to inhibit the protein or gene for the prevention or treatment of diseases associated with fungal infection. Could be.

In the present invention, 'inhibitors of Tco1, Mbp1, Atf1 or Ras1 protein' used to block the HOG1 signaling system includes all inhibitors that bind to Tco1, Mbp1, Atf1 or Ras1 protein to block signaling. For example, such inhibitors can be peptides or compounds that bind to Tco1, Mbp1, Atf1 or Ras1 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 or monoclonal antibody against a protein of Tco1, Mbp1, Atf1 or Ras1 of Cryptococcus neoformus. Such polyclonal antibodies or monoclonal antibodies can be prepared using antibody production methods known in the art.

In the present invention, the HOG1 signaling pathway, inhibitors of TCO1, MBP1, ATF1 or Ras1 gene "is used to block includes both inhibitors to block the signal transduction by inhibiting the expression of TCO1, MBP1, ATF1 or Ras1 gene . 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 may be an antisense oligonucleotide, siRNA, shRNA, miRNA or a vector comprising the gene of TCO1, MBP1, ATF1 or Ras1 of Cryptococcus neoformus . 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 nucleic acid synthesis inhibitory antifungal agent through complex treatment with the nucleic acid synthesis inhibitory antifungal agent. Therefore, the antifungal pharmaceutical composition of the present invention is characterized in that it is administered sequentially or simultaneously with a nucleic acid synthesis inhibitory antifungal agent. Nucleic acid synthesis inhibitory antifungal agent means an antifungal agent that targets the enzyme involved in the nucleic acid synthesis of the fungus or has a structure similar to pyrimidine to inhibit the nucleic acid synthesis of the fungus. Such nucleic acid synthesis inhibitory antifungal agents are known in the art, and any nucleic acid synthesis inhibitory 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 nucleic acid synthesis inhibitory antifungal agent may be a pyrimidine analog. For example, the pyrimidine analog can be flucytosine.

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 nucleic acid synthesis inhibitory 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 Tco1, Mbp1, Atf1 or Ras1 protein of Cryptococcus neoforms for screening antifungal agent, the composition for screening an antifungal agent comprising the protein, a candidate and contacting the protein, the candidate is It provides a method for screening an antifungal agent comprising determining whether to inhibit or promote the activity of the protein.

Similarly, the present invention also provides the use of a gene of TCO1, MBP1, ATF1 or Ras1 of Cryptococcus neoforms for screening an antifungal agent, a composition for screening an antifungal agent comprising the gene, a candidate and contacting the gene, It provides a method for screening an antifungal agent comprising determining whether a 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 Tco1, Mbp1, Atf1 or Ras1 protein of Cryptococcus neoformus and YPD1 for the screening of antifungal agents. This method has the advantage of screening large amounts of candidates in a short time.

As mentioned above, inhibition of Tco1, Mbp1, Atf1 or Ras1 of Cryptococcus neoforms can dramatically improve fungal hypersensitivity to nucleic acid synthesis inhibitory antifungal agents. Therefore, the substance screened as inhibiting the protein or gene can be used as an antifungal agent to enhance fungal killing ability in combination with a nucleic acid synthesis inhibitory antifungal agent.

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 will be implemented in various forms, and only the embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the technical field to which the present invention pertains. It is provided to fully convey the scope of the invention to those skilled in the art, and the present invention 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.

Strain Genotype Parent Serotype A H99 MAT a YSB42 MATa cac1 △ :: NAT-STM # 159 H99 YSB51 MATa ras1 △ :: NAT-STM # 150 H99 YSB64 MAT a hog1 △ :: NAT-STM # 177 H99 YSB123 MAT a pbs2 △ :: NAT-STM # 213 H99 YSB261 MAT a ssk1 △ :: NAT-STM # 205 H99 YSB264 MAT a ssk2 △ :: NAT-STM # 210 H99 YSB349 MAT a skn7 △ :: NAT-STM # 201 H99 YSB278 MAT a tco1 △ :: NAT-STM # 102 H99 YSB281 MAT a tco2 △ :: NAT-STM # 116 H99 YSB324 MAT a tco1 △ :: NAT-STM # 102tco2 △ :: NEO YSB278 YSB284 MAT a tco3 △ :: NAT-STM # 119 H99 YSB417 MAT a tco4 △ :: NAT-STM # 123 H99 YSB286 MAT a tco5 △ :: NAT-STM # 125 H99 YSB348 MAT a tco7 △ :: NAT-STM # 209 H99 YSB488 MATa mbp1 △ :: NAT-STM # 150 H99 YSB489 MATa mbp1 △ :: NAT-STM # 150 H99 YSB676 MATa atf1 △ :: NAT-STM # 220 H99 YSB678 MATa atf1 △ :: NAT-STM # 220 H99

Antifungal Drug Sensitivity Testing

"Bahn YS, Kojima K, Cox GM, Heitman J (2005) Mol Biol Cell 16: 2285-2300." And dH ₂ O after washing each strain incubated at 30 ° C. overnight in YPD medium, as described in “Bahn YS, Kojima K, Cox GM, Heitman J (2006) Mol Biol Cell 17: 3122-3135”. Dilutions were sequentially (1 to 10 ⁴ dilutions) at and 2 μl of the dilutions were spotted with solid YPD medium containing the indicated concentrations of the antifungal agent. Cells were incubated at 30 ° C. for 72 hours and photographed.

Transcript Analysis by DNA Microarray

Total RNA was isolated for DNA microarray analysis as follows. Wild type H99 , tco1Δ, tco2Δ and hog1Δ mutant 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 600 nm for 4 hours at the same temperature until the absorbance reached approximately 1.0. Each was then divided into two 50 ml flasks, one of which was not treated with flu cytosine and the other flask was inoculated with 25 µg / ml of flu cytosine and incubated at 30 ° C. for 90 minutes. The culture was centrifuged at 3000 rpm for 5 minutes. The culture was then rapidly cooled in liquid nitrogen and lyopgilized overnight. As biological replicates for DNA microarrays, three independent cultures for each strain were prepared for total RNA isolation.

Total RNA Manufacturing

For isolation of total RNA, 3 ml volume of sterilized 3 mm glass beads (SIGMUNI LINDER) was added to the freeze-dried cell particles, shaken and homogenized. 4 ml TRizol was added thereto and incubated at room temperature for 5 minutes. Then, 800 μl of chloroform was added and incubated at room temperature for 5 minutes, 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 the supernatant was transferred to a new round-bottom tube and inverted several times to incubate with the addition of 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 its precipitate 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 absorbance at 260 nm and confirmed by gel electrophoresis. For control total RNA (for Cy3 labeling), all strains prepared from wild type tco1Δ, tco2Δ and hog1Δ mutant cells cultured under the conditions described above were pooled with total RNAs.

cDNA Synthesis and Cy3 / Cy5 Labeling

For cDNA synthesis, the 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 were added, followed by incubation at 70 ° C. for 10 minutes. , Reacted for 10 minutes in a box containing ice. Then 15 μl cDNA synthesis mixture {3 μl 0.1 M DTT, 0.5 RNasin [Promega], 0.6 μl aa-dUTP / dNTPs, 1.5 μl affinityScript, 3 μl affinityScript buffer, 7 μl DEPC} was added and 2 at 42 ° C. Incubated for hours. Then, 10 μl of 1N NaOH 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 whole mixture was concentrated through a Micron 30 filter 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 were mixed with Cy5. Each mixture was incubated for one more hour at dark room temperature and purified by QIAquick PCR purification kit (QIAGEN).

Washing of Microarray Hybridization

Cryptococcus neoformance antigenic D 70-mer microarray slides (provided by Duke University) containing 7,936 spots were pre-hybrdization buffer [42.4ml DEPC water, 2 ml 30% BSA (sigma) at 42 ° C. , 600 μl 10% SDS, 15 ml 20 × SSC], and immersed in DEPC water and isopropanol several times, washed, and centrifuged for 2 minutes. Cy3 and Cy5-labeled cDNA samples were combined and concentrated through a Microcon 20 filter to dry in vacuo. Dried cDNA samples were again treated with hybridization buffer [250 μl 50% formamide, 125 μl 20x SSC, 5 μl 10% SDS, 120 μl DEPC water, 500 μl total] and 1 μl poly A tail DNA (sigma) After incubation for 3 minutes at 100 ℃ and cooled for 5 minutes at room temperature. Microarray slides were aligned with a hybridization chamber (DieTech), dust removed and covered with liftslips (Erie Scienfic). Cy3 / Cy5-labeled cDNA samples were applied between slip and slide, and slides were then incubated at 42 ° C. for 16 hours. After incubation, the microarray slides were washed 2, 5 and 5 minutes with three different wash buffers in an orbital stirrer, respectively.

For each total RNA sample, three independent DNA microarrays were performed with three independent biological replicates.

Microarray Slide Scanning and Data Analysis

After hybridization and washing, the microarray slices 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.wust1.edu/activity/ma/ cneoformans ). Since we used whole 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. Using genotype A gene sequences, each S. cerevisiae gene name or ID listed in the table was identified by blastp irradiation (e value range: e-4).

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, ANOVA analysis (p Value <0.01).

Example 1 Analysis of the Effect of Antifungal Agents by Inhibition of Genes in the HOG Pathway of Cryptococcus Neoforms

The antifungal effect of flu cytosine by inhibition of genes in the HOG pathway of Cryptococcus neoforms was tested.

First, C. neoformans strains of [WT (H99), hog1 △ (YSB64), pbs2 △ (YSB123), ssk2 △ (YSB264), ssk1 △ (YSB261), skn7 △ (YSB349), tco1 △ (YSB278), tco2 Δ ( YSB281), tco3Δ ( YSB284), tco4Δ ( YSB417), tco5Δ ( YSB286), and tco7Δ ( YSB348)] were tested for sensitivity to flucytocin.

1 is an analysis showing that flu cytosine exhibits a synergistic or antagonistic antifungal effect by inhibition of the HOG pathway.

As can be seen from FIG. 1, hog1Δ ( Hog1 MAPK mutant), pbs2Δ ( Pbs2 MAPKK mutant), ssk2Δ ( Ssk2 MAPKKK mutant), and ssk1Δ (response regulator mutant) mutations are more directed against flucytosine than the wild type. Slightly higher resistance. In contrast, the skn7Δ mutations showed low hypersensitivity to flucytosine compared to the wild type. However, mutations in the TCO1 gene, one of the major hybrid sensor kinases acting on the HOG signaling pathway, dramatically improved cell susceptibility to flu cytosine. In contrast, another hybrid sensor kinase of the HOG pathway, Tco2, acted differently. Tco2 is known as a double hybrid sensor kinase because it has two histidine kinase domains and two reaction modulator domains in one polypeptide. tco2Δ mutations showed dramatic resistance to flucytosine . tco3 △, tco4 △, tco5 △ , △ and tco7 other hybrid sensor kinase mutants, including mutations showed a wild-type level of sensitivity to flu cytosine.

Recently, the most widely used antifungal therapy for the treatment of Cryptococcosis is amphotericin B treatment or a combination treatment of amphotericin B and flucytosine. Therefore, the sensitivity of various HOG mutations to the combination therapy of amphotericin B and flu cytosine was measured.

2 is an analysis showing that the combination of amphotericin B and flu cytosine by the inhibition of the HOG pathway shows a synergistic or antagonistic antifungal effect.

As can be seen from FIG. 2, the ssk1Δ , ssk2Δ , pbs2Δ and hog1Δ mutations all showed hypersensitivity to the combination treatment of amphotericin B and flucytosine . This is similar to the terry ampo ssk1 △ when new B-only treatment of, ssk2 △, pbs2 △ △ hog1 and sensitization of mutation research in our previous study. Our previous study, while there are more resistant to tco1 △ mutations amphotericin B than the other mutations tco △, △ tco2 mutations may bars shown represent the sensitization against amphotericin B. In the present study, on the other hand, we found that the tco1Δ mutation was slightly more susceptible to the combination of amphotericin B and flucytosine compared to the wild type (FIG. 2). Interestingly, when the tco2Δ mutation is treated with amphotericin B and plutocytosine, the hypersensitivity of the tco2Δ mutation to amphotericin B disappears and is more resistant to complex treatment than the wild type.

In conclusion, potential inhibitors of Tco1 can significantly increase the antifungal effect of flu cytosine. However, it is not desirable to use Tco1 inhibitors in combination with flucytosine and amphotericin B, since inhibition of Tco1 in a combination treatment of flucytosine and amphotericin B increases the resistance to amphotericin B.

Example 2 Screening of Genome Levels of Cryptococcus Flucytocin Related Genes by Comparative Transcriptase Analysis Using a Microarray

To characterize the role of the hybrid sensor kinase and the function of Hog1 against flu cytosine, antigen-type A wild-type (WT, H99) strains with and without flu cytosine using the DNA microarray analysis described above Transcript comparison analysis of the tco1Δ , tcoΔ , and hog1Δ mutations was performed. Of the 6894 genes monitored by this DNA microarray, the expression levels of 200 genes were found to be statistically significant when treated with flu cytosine in wild-type strains (ANOVA test, p <0.05). FIG. 3 is a clustering image of 194 genes whose expression levels differ by more than two times, and also shows the expression levels of these flucytosine-related genes in tco1 , tco2 , and hog1 variants. As shown in the DNA microarray analysis results, the expression of flu cytosine-related genes of Cryptococcus neoforms is greatly influenced by HOG signaling pathway.

Figures 4 and 5 tabulate 59 genes whose similar functions exist in yeast and whose function is known among the 194 genes dependent on individual flu cytosine identified by transcript analysis. It has been found that most of these are very different from the flucytosine related genes reported in yeast, and thus Cryptococcus neoforms responds to flucytosine in a very unique manner. In particular, MBP1, whose ortholog was found in S. cerevisiae among the evolutionarily conserved flucytocin-related genes, was remarkable, showing similar pattern with array data.

Figures 6 and 7 show 122 genes of unknown function of individual flucytosine related genes 194 identified by transcript analysis (except 13 genes without serotype A gene ID). Of the 194 flucytosine dependent genes selected, many (122, 64%) had no orthologs from other fungi, suggesting that Cryptococcus neoformes contains a unique set of flucytocin dependent genes.

Example 3 Analysis of Antifungal Effect of Flucytosine by Inhibition of Genes Controlled by HOG of Cryptococcus Neoforms

C. neoformans strains (WT ( H99 ), hog1 △ ( YSB64), ssk1 △ ( YSB261), skn7 △ ( YSB349), mbp1 △ ( YSB488, YSB489)] were incubated at 30 ° C. overnight in YPD liquid medium and washed. Diluted sequentially in dH ₂ O (1-10 ⁴ dilution) and 3 μl of the dilutions were spotted with solid YPD medium containing the indicated concentrations of antifungal agent to confirm antifungal drug sensitivity. Cells were incubated at 30 ° C. for 72 hours and photographed.

FIG. 8 is an analysis showing that inhibition of the Mbp1 transcriptional regulator, which is expected to be affected by major hybrid sensor kinase acting on HOG signaling pathway, shows synergistic antifungal effect of flu cytosine.

Of the 194 genes regulated by the Tco1 gene obtained through DNA microarrays of the Tco1 gene, we found that the Mbp1 gene, which encodes an APSES protein known to be involved in differentiation and morphological changes, is regulated by the Tco1 gene. As can be seen from FIG. 4 , in the tco1Δ, tco2Δ, and hog1Δ mutations, the expression level of the Mbp1 transcription factor gene was significantly decreased by the flu cytosine treatment compared with the wild type.

Based on these results, it was expected that the Tco1 gene and the Mbp1 transcription factor gene would be correlated, and experimented with whether the inhibition of the Mbp1 gene regulated by the Tco1 gene showed susceptibility to nucleic acid synthesis inhibitors such as flu cytosine.

As a result, as can be seen in Figure 8, the mutation of the Mbp1 transcription factor gene increased the cell sensitivity to flu cytosine. Therefore, the antifungal pharmaceutical composition comprising an inhibitor of Tco1 as well as the Mbp1 gene can be used as an excellent antimicrobial therapeutic agent that can increase the efficacy while reducing the use amount of a nucleic acid synthesis inhibitory antifungal agent.

Under the premise that transcription factor genes that are directly or indirectly related to HOG signaling pathways are also available as excellent antimicrobial therapeutics, they inhibit the Atf1 gene among known transcription factors of HOG signaling pathways and thus inhibit the synthesis of nucleic acid synthesis inhibitors such as flu cytosine. The response pattern was investigated.

Transcription factors that regulate gene expression directly related to HOG signaling pathways are involved in the response mechanisms to various external stimuli, as already identified in Saccharomyces cerevisiae and Schizosaccharomyces pombe . In the case of C. neoformans , a bioinformatics approach was performed to search for the transcription factor Atf1 gene that is directly related to HOG signaling pathway and found that the inhibition of Atf1 gene increased the sensitivity to nucleic acid synthesis inhibitors such as flu cytosine.

As can be seen from FIG. 9, hog1Δ ( Hog1 MAPK mutant) mutations showed slightly lower hypersensitivity to flucytosine than the wild type, but ras1Δ mutations showed very increased flucytocin sensitivity. The cac1Δ mutations showed wild type levels of sensitivity to flucytosine . Atf1 transcription factor mutations directly affected by the HOG gene showed more hypersensitivity to flu cytosine than hog1Δ mutations. However, polyene-based antifungal agents such as amphotericin B showed increased resistance, unlike hog1 △ mutants.

Therefore, the antifungal pharmaceutical composition comprising inhibitors against Ras1 and Atf1, including Tco1 and Mbp1 of Cryptococcus neoformus, is an excellent complex antimicrobial therapeutic agent that can increase the efficacy while reducing the use capacity of a nucleic acid synthesis inhibitor. Can be used.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Use of Tco1, Atf1 or Mbp1 for treatment of fungal infection <130> P08978 <160> 12 <170> KopatentIn 1.71 <210> 1 <211> 1383 <212> PRT <213> Cryptococcus neoformans serotype A H99 strain <220> <221> PEPTIDE (222) (1) .. (1383) <223> amino acid sequence of Tco1 <400> 1 Met Ser Leu Pro Asp Ala Tyr Pro Pro Val Ile Ala Thr Glu Gly Ser   1 5 10 15 Thr Ala Phe Gln Ser His Ile Leu Ser Leu Leu Ser Val Ile Ser Gln              20 25 30 His Pro Ser Pro Asp Tyr Pro Phe Pro Thr Ser Gly Asp Ser Thr Leu          35 40 45 Gln Asp Asn Ala Ser Val Ala Ala Lys Lys Ser Ala Thr Leu Ser Thr      50 55 60 Phe Pro Gly Lys Lys Thr Pro Ala Glu Asp Ala Ile Glu Arg Ala Ile  65 70 75 80 Ile Ala Leu Gly Asp Arg Val Trp Thr Ser Glu Arg Thr Gln Ala His                  85 90 95 Leu Val Lys Asp Thr Lys Arg Asp Val Pro Ala Gln Pro Gly Ser Asp             100 105 110 Leu Leu Thr Pro Asp Trp Thr Pro Pro Val Pro Thr Leu Ser Asp Ser         115 120 125 Ser Pro Asp Pro Val Cys Pro Thr Cys Ala Arg Pro Val Ser Ser Thr     130 135 140 Glu Ser Ser Ser Phe Thr Pro Gln Gln Ile Ala Ser Phe Tyr His Pro 145 150 155 160 Phe Thr Ala Ala Leu Ser Thr Pro Pro Pro Leu Pro Glu Gln Met                 165 170 175 Phe Phe Pro Gln Ser Val Leu Ala Ala Tyr Val Ser Ala Thr Ser Asp             180 185 190 Ser Glu Ser Ala Gly Asp Ser Gly Met Ser Ala Glu Lys Glu Leu Glu         195 200 205 Leu Leu Lys Ala Gln Val Gln Asp Ile Ala Arg Val Cys Lys Ala Val     210 215 220 Ala Thr Gly Asp Leu Thr Gln Lys Ile Ile Val Pro Val Lys Gly Gln 225 230 235 240 Thr Met Thr Glu Leu Lys Asn Ile Ile Asn Ala Met Val Asp Arg Leu                 245 250 255 Gln Thr Phe Ala Val Glu Val Glu Arg Val Ser Leu Glu Val Gly Thr             260 265 270 Glu Gly Lys Leu Gly Gly Gln Ala Val Val Pro Asn Val Glu Gly Thr         275 280 285 Trp Lys Val Leu Thr Ala Val Val Asn Lys Leu Ala Ala Asn Leu Thr     290 295 300 Asn Gln Val Arg Ser Ile Ala Lys Val Thr Lys Ala Val Ala Lys Gly 305 310 315 320 Asp Leu Ser Glu Thr Ile Asp Val Glu Ala Ser Gly Glu Ile Ala Glu                 325 330 335 Leu Lys Thr Thr Val Asn Gly Met Val Met Ser Leu Arg Thr Leu Ala             340 345 350 Asp Glu Val Ser Arg Val Ser Leu Glu Val Gly Ser Gln Gly Lys Leu         355 360 365 Gly Gly Gln Ala Asn Val Pro Asp Val Glu Gly Val Trp Lys Asp Leu     370 375 380 Thr Val Asn Val Asn Arg Met Cys Glu Ser Leu Thr Thr Gln Val Arg 385 390 395 400 Ser Ile Gly Ser Val Thr Thr Ala Val Ala Arg Gly Asp Leu Ser Lys                 405 410 415 Met Ile Glu Ile Glu Ala Glu Gly Glu Met Ala Val Leu Lys Asn Thr             420 425 430 Val Asn Ser Met Val Arg Gln Leu Thr Val Phe Ala Lys Glu Val Thr         435 440 445 Arg Val Ala Leu Glu Val Gly Thr His Gly Gln Leu Gly Gly Gln Ala     450 455 460 Val Val Pro Gly Val Glu Gly Val Trp Asp Asp Leu Thr Thr Asn Val 465 470 475 480 Asn Lys Met Ala Arg Asn Leu Thr Asp Gln Val Arg Glu Ile Ala Met                 485 490 495 Val Thr Lys Ala Val Ala Arg Gly Asp Leu Ser Lys Thr Val Asn Ala             500 505 510 Asp Val Gln Gly Glu Ile Leu Asp Leu Lys Cys Thr Val Asn Glu Met         515 520 525 Val Ala Gln Leu Thr Val Phe Ala Ala Glu Val Thr Arg Val Ser Leu     530 535 540 Glu Val Gly Thr Glu Gly Lys Leu Gly Gly Gln Ala Val Val Pro Asn 545 550 555 560 Val Glu Gly Thr Trp Met Val Leu Thr Asp Asn Val Asn Leu Met Ala                 565 570 575 Leu Asn Leu Thr Thr Gln Val Arg Ser Val Ala Glu Val Thr Thr Ala             580 585 590 Val Ala Ala Gly Asp Leu Ser Lys Lys Ile Asn Val Ala Ala Phe Gly         595 600 605 Glu Ile Leu Glu Leu Lys Asn Thr Val Asn Asn Met Val Asp Ser Leu     610 615 620 Arg Ser Phe Ser Ser Glu Val Thr Arg Val Ala Arg Glu Val Gly Thr 625 630 635 640 Asp Gly Arg Leu Gly Gly Gln Ala Lys Val Pro Gly Val Ala Gly Thr                 645 650 655 Trp Lys Asp Leu Thr Asp Cys Val Asn Ile Met Ala Ala Asn Leu Thr             660 665 670 Glu Gln Val Arg Thr Ile Ala His Ala Thr Thr Ala Val Ala Arg Gly         675 680 685 Asp Leu Thr Gln Lys Val Val Gly Val Lys Val Ser Gly Glu Ile Leu     690 695 700 Asp Leu Val Asn Thr Ile Asn Asn Met Ile Asp Gln Leu Ala Ile Phe 705 710 715 720 Ala Ala Glu Val Thr Arg Val Ala Arg Glu Val Gly Thr Glu Gly Lys                 725 730 735 Leu Gly Val Gln Ala Glu Val Glu Asn Ile Glu Gly Thr Trp Gln Glu             740 745 750 Ile Thr Ser Asn Val Asn Thr Met Ala Ser Asn Leu Thr Ser Gln Val         755 760 765 Arg Ala Phe Ala Gln Ile Ser Ala Ala Ala Thr Asp Gly Asp Phe Thr     770 775 780 Arg Phe Ile Thr Val Glu Ala Ser Gly Glu Met Asp Ser Leu Lys Thr 785 790 795 800 Lys Ile Asn Gln Met Val Tyr Asn Leu Arg Glu Ser Ile Glu Lys Asn                 805 810 815 Thr Lys Ala Arg Gln Glu Ala Glu Met Ala Asn Arg Ser Lys Ser Glu             820 825 830 Phe Leu Ala Asn Met Ser His Glu Ile Arg Thr Pro Met Asn Gly Ile         835 840 845 Ile Gly Met Thr Val Leu Thr Leu Glu Ser Glu Leu Thr Arg Gln Gln     850 855 860 Arg Glu Asn Leu Met Ile Val Ser Ser Leu Ala Gly Ser Leu Leu Thr 865 870 875 880 Ile Ile Asp Asp Ile Leu Asp Ile Ser Lys Ile Glu Ala Gly Arg Met                 885 890 895 Thr Met Glu Gln Ile Pro Phe Ser Leu Arg Val Ala Val Phe Ser Val             900 905 910 Leu Lys Thr Leu Cys Val Lys Ala Ala Gln Asn Lys Leu Asp Leu Ile         915 920 925 Phe Asp Val Asp Pro Thr Met Pro Asp Gln Leu Ile Gly Asp Pro Leu     930 935 940 Arg Leu Arg Gln Val Ile Thr Asn Leu Ile Gly Asn Ala Val Lys Phe 945 950 955 960 Thr Thr Lys Gly Gln Val Ala Leu Ser Cys Arg Val Lys Arg Tyr Val                 965 970 975 Ala Val Ala Asp Ala Val Glu Leu Glu Phe Cys Val Ala Asp Thr Gly             980 985 990 Ile Gly Ile Lys Gln Asp Lys Leu Asp Val Ile Phe Asp Thr Phe Ala         995 1000 1005 Gln Ala Asp Gly Ser Thr Thr Arg Lys Tyr Gly Gly Thr Gly Leu Gly    1010 1015 1020 Leu Thr Ile Ser Lys Arg Leu Val Asn Leu Met Ser Gly Asp Leu Trp 1025 1030 1035 1040 Val Glu Ser Glu Tyr Gly Gln Gly Ser Arg Phe Tyr Phe Thr Thr Val                1045 1050 1055 Ala Glu Met Thr Ser Val Pro Arg Asp Gln Ile Ile Glu Arg Leu Ser            1060 1065 1070 Pro Trp Ser Gly Arg Ser Val Leu Phe Ile Asp Thr Leu Gly Asp Gln        1075 1080 1085 Thr Gly Ile Val Glu Met Leu Gln Glu Leu Gly Leu Lys Pro Ile Val    1090 1095 1100 Ile His Ser Val Asn Asp Val Tyr Leu Leu Gln Asp Gln Gly Leu Thr 1105 1110 1115 1120 Val Phe Asp Thr Met Ile Val Asp Ser Leu Lys Ala Ala Gly Glu Leu                1125 1130 1135 Arg Gly Ile Glu Tyr Leu Arg Tyr Ile Pro Ile Val Leu Leu Ala Pro            1140 1145 1150 Ser Asn Lys Pro Ser Gly Pro Ser Asn Pro Ser Phe Ile Ser Leu Pro        1155 1160 1165 Glu Ser Arg Arg Arg Leu Leu Ala Leu Pro Ser Ala Ser Glu Gln Leu    1170 1175 1180 Leu Ser Pro Ile Pro Val Lys Asp Cys Leu Glu Met Gly Ile Asn Thr 1185 1190 1195 1200 Tyr Tyr Thr Thr Pro Leu Ser Leu Gln Glu Leu Ser Asn Ala Ile Val                1205 1210 1215 Pro Ala Leu Glu Ser His Gln Ile Gln Pro Gly Asp Thr Val Arg Asp            1220 1225 1230 Thr Ile Leu Ser Val Leu Leu Ala Glu Asp Asn Leu Val Asn Gln Lys        1235 1240 1245 Leu Ala Val Lys Leu Met Glu Val Ala Gly His Lys Ile Glu Val Ala    1250 1255 1260 Asp Asn Gly Glu Ile Ala Leu Glu Lys Tyr Lys Arg Arg Gln Leu Ala 1265 1270 1275 1280 Arg Thr Pro Phe Asp Val Ile Leu Met Asp Val Ser Met Pro Val Met                1285 1290 1295 Gly Gly Met Glu Ala Thr Gly Leu Ile Arg Glu Phe Glu Ala Asn Glu            1300 1305 1310 Gly Val Pro Arg Thr Pro Ile Ila Ala Leu Thr Ala His Ala Met Ile        1315 1320 1325 Gly Asp Lys Glu Arg Cys Leu Ala Ala Gly Met Asp Glu Tyr Val Thr    1330 1335 1340 Lys Pro Leu Arg Arg Gly Asp Leu Leu Ala Ser Ile Ala Lys Val Leu 1345 1350 1355 1360 Ala Pro Lys Gln Ser Ala Ser Gly Leu Glu Met Pro Ala Val Ser Ile                1365 1370 1375 Pro Ala Asn Ile Tyr Gly Arg            1380 <210> 2 <211> 766 <212> PRT <213> Cryptococcus neoformans serotype A H99 strain <220> <221> PEPTIDE (222) (1) .. (766) <223> amino acid sequence of Mbp1 <400> 2 Met Gly Lys Lys Val Ile Ala Ser Gly Gly Asp Asn Gly Pro Asn Thr   1 5 10 15 Ile Tyr Lys Ala Thr Tyr Arg Tyr Ala Thr Phe Leu Tyr Phe Ala Pro              20 25 30 Arg Thr Ala Arg Met Pro Val Tyr Glu Met Val Cys Arg Asp Val Ala          35 40 45 Val Met Arg Arg Arg Ser Asp Ala Tyr Leu Asn Ala Thr Gln Ile Leu      50 55 60 Lys Val Ala Gly Phe Asp Lys Pro Gln Arg Thr Arg Val Leu Glu Arg  65 70 75 80 Glu Val Gln Lys Gly Glu His Glu Lys Val Gln Gly Gly Tyr Gly Lys                  85 90 95 Tyr Gln Gly Thr Trp Ile Pro Ile Glu Arg Gly Leu Ala Leu Ala Lys             100 105 110 Gln Tyr Gly Val Glu Asp Ile Leu Arg Pro Ile Ile Asp Tyr Val Pro         115 120 125 Thr Ser Val Ser Pro Pro Pro Ala Pro Lys His Ser Val Ala Pro Pro     130 135 140 Ser Lys Ala Arg Arg Asp Lys Glu Lys Glu Thr Gly Arg Thr Lys Ala 145 150 155 160 Thr Pro Ser Arg Thr Gly Pro Thr Ser Ala Ala Ala Leu Gln Ala Gln                 165 170 175 Ala Gln Leu Asn Arg Ala Lys Met His Asp Ser Thr Pro Asp Ala Asp             180 185 190 Ala Ser Phe Arg Ser Phe Glu Glu Arg Val Ser Leu Thr Pro Glu Asp         195 200 205 Asp Ser Ser Ser Asp Thr Pro Ser Pro Val Ala Ser Val Met Thr Asp     210 215 220 Gln Asp Met Glu Val Asp Lys Met Gly Met His Met Asn Met Pro Asn 225 230 235 240 Val Thr Leu Ser Gln Asn Met Glu Glu Leu Gly Ala Gly Ser Arg Lys                 245 250 255 Arg Ser Ala Ala Met Met Met Glu Asp Glu Asp Gln Phe Gly His Leu             260 265 270 Arg Ser Val Arg Gly Asn Ser Ala Val His Thr Pro His Gly Thr Pro         275 280 285 Arg His Leu Gly Ile Gly Met Pro Pro Glu Pro Ile Gly Pro Glu Gln     290 295 300 Tyr Thr Asp Ile Ile Leu Asn Tyr Phe Val Ser Glu Thr Ser Gln Ile 305 310 315 320 Pro Ser Ile Leu Val Ser Pro Pro His Asp Phe Asp Pro Asn Ala Pro                 325 330 335 Ile Asp Asp Asp Gly His Thr Ala Leu His Trp Ala Cys Ala Met Gly             340 345 350 Arg Val Arg Val Val Lys Leu Leu Leu Thr Ala Gly Ala Ser Ile Phe         355 360 365 Ala Gly Asn Asn Ala Glu Gln Thr Pro Leu Met Arg Ser Val Met Phe     370 375 380 Ser Asn Asn Tyr Asp Met Arg Lys Phe Pro Glu Leu Tyr Glu Leu Leu 385 390 395 400 His Arg Ser Thr Leu Asn Ile Asp Lys Gln Asn Arg Thr Val Phe His                 405 410 415 His Ile Ala Asn Leu Ala Leu Thr Lys Gly Lys Thr His Ala Ala Lys             420 425 430 Tyr Tyr Met Glu Thr Ile Leu Ala Arg Leu Ala Asp Tyr Pro Gln Glu         435 440 445 Leu Ala Asp Val Ile Asn Phe Gln Asp Glu Glu Gly Glu Thr Ala Leu     450 455 460 Thr Ile Ala Ala Arg Ala Arg Ser Arg Arg Leu Val Lys Ala Leu Leu 465 470 475 480 Asp His Gly Ala Asn Pro Lys Ile Lys Asn Arg Asp Ser Arg Ser Ala                 485 490 495 Glu Asp Tyr Ile Leu Glu Asp Glu Arg Phe Arg Ser Ser Pro Val Pro             500 505 510 Ala Pro Asn Gly Gly Val Gly Lys Val Ser Thr Ser Ala Ala Ala Glu         515 520 525 Lys Pro Ile Phe Ala Pro Gln Leu Tyr Phe Ser Glu Ala Ala Arg Leu     530 535 540 Cys Gly Gly Gln Ala Leu Thr Asp Ile Thr Ser His Met Gln Ser Leu 545 550 555 560 Ala Arg Ser Phe Asp Ala Glu Leu Gln Gly Lys Glu Arg Asp Ile Leu                 565 570 575 Gln Ala Lys Ala Leu Leu Thr Asn Ile His Thr Glu Val Thr Glu Asn             580 585 590 Gly Arg Ser Ile Thr Ala Ile Thr Asn Gln Ala Ala Pro Leu Glu Glu         595 600 605 Lys Arg His Glu Leu Glu Ser Leu Gln Ala Ser Leu Lys Thr Lys Val     610 615 620 Lys Asp Ala Leu Lys Lys Gly Tyr Ile Gly Trp Leu Glu Gly Glu Leu 625 630 635 640 Ile Arg Glu Gln Arg Trp Glu Lys Gly Glu Leu Glu Gly Asn Glu Glu                 645 650 655 Glu Lys Ala Ala Val Gln Ala Leu Arg Asp Val Pro Thr Gly Gly Gln             660 665 670 Glu Val Val Gln Ala Glu Glu Glu Lys Leu Arg Trp Glu Ile Glu Glu         675 680 685 Lys Arg Lys Arg Arg Ala Met Phe Val Glu Lys Phe Val Arg Ala Gln     690 695 700 Ala Glu Ala Gly Thr Ser Glu Gln Ile Ala Lys Tyr Arg Lys Leu Val 705 710 715 720 Ser Ala Gly Leu Gly Gly Val Ser Thr Asn Glu Val Asp Glu Leu Met                 725 730 735 Asn Gln Leu Leu Glu Gly Leu Glu Glu Glu Asn Asp Asn Gln Val Tyr             740 745 750 Asn Thr Ser Ala Gly Glu Ser Gly Pro Ser Ser Trp Val Gln         755 760 765 <210> 3 <211> 618 <212> PRT <213> Cryptococcus neoformans serotype A H99 strain <220> <221> PEPTIDE (222) (1) .. (618) <223> amino acid sequence of Atf 1 <400> 3 Met Ala Ala Val Ala Gln Ala Pro Pro Ser Met Pro His Ser Ser Ile   1 5 10 15 Glu Asn Ser Val Thr Arg Pro Ser Ser Ser Ala Ser Asn Ser Lys Arg              20 25 30 Gln His Ser Glu Ala Pro Ala Ser Arg Gly Ser Glu Glu Asn Ser Asn          35 40 45 Leu Ala Ala Thr Leu Thr Val Thr Glu Pro Thr Pro Val Glu Asp Lys      50 55 60 Glu Ser Lys Glu Ser Gly Asn Ala Gln Ala Gly Ser Ser Asp Lys Asp  65 70 75 80 Lys Pro Ala Asn Gln Ala Ser Glu Val Gln Arg Pro Pro Leu Arg Pro                  85 90 95 Asn Ala Val Ala Arg Ser Arg Leu Glu Gln Glu Pro Asn Pro Phe Glu             100 105 110 Gln Ser Phe Arg Asp Ser His Gly Pro Gly Thr Ser Asp Arg Asp Thr         115 120 125 Pro Pro Arg Gly Thr Asp Ala Thr Ser Thr Arg His Asn Ala Leu Pro     130 135 140 Pro Leu Ser Ser Leu Thr Ser Pro Ala Ala Ala Asp Pro Thr Gln Phe 145 150 155 160 Pro Trp Leu Ala Asn Gln Ser Leu Arg Ser Gly Pro Leu Ser Pro Ala                 165 170 175 Met Leu Thr Gly Pro Gln Gly Ala Asn Gln Ser Ser Thr Ser Asn Asn             180 185 190 Leu Arg Ser Asn Glu Gly Asn Ala Glu Gly Gly Phe Glu Ser Ser Gly         195 200 205 Phe Arg Thr Gly Phe Thr Pro Gly Thr Gly Ser Gly Phe Thr Pro Gly     210 215 220 Tyr Asn Ser Phe Ile Asn Ser Ser Leu Asn Ser Leu Pro Ile Pro Ser 225 230 235 240 Pro Asn Thr Ala Ala Phe Leu Asn Ser Ile Thr Asn Val Thr Pro Leu                 245 250 255 Gly Glu Ser Ser Glu Ile Ala Gln Ala Ala Ala Ala Gln Ala Gln Ala             260 265 270 Gln Gly Gln Thr Ala Ser Asn Ala Ser Ser Ala Ser Gln Asn Ser Gln         275 280 285 Ser Gln Ser Ser Thr Glu Pro Leu Gln Pro Pro Ser Ala Ile Pro Pro     290 295 300 His Met Gln Pro His Pro His Gln Pro His Pro Leu Ser His Ala His 305 310 315 320 Ser Pro Ala Asn Asn His Ser Ala His Pro Gly Gln Asp Thr Ile Thr                 325 330 335 Pro Asn Thr Leu Asn Ala Leu Thr Gly Val Phe Gly Asp Met Ser Arg             340 345 350 Ala Pro Ala Gly Ala Pro Gly Gln Asn Gln Phe Phe Ala Pro Ala Met         355 360 365 Ser Ala His Pro Gly His Pro Gly His Pro Gly His Pro His Thr Ala     370 375 380 His Pro Gly Ile Pro Gly Val Ala Gly Val Pro Gly Val His Gly Val 385 390 395 400 Pro Gly His Pro Gly Val Gln Leu Pro Met Gly Tyr Val Asp Tyr Ala                 405 410 415 Gln His Asn Ala Asn Ala Ala Ser Gln Ala Ala Asn Gly Leu Phe Leu             420 425 430 Leu Ser Gln Ala His Gln Glu Leu Ser Lys Arg Glu Glu Ala Glu Gly         435 440 445 Thr Pro Gly Ser Ala Val Gly Gly Pro Thr Gly Arg Ser Ser Arg Gly     450 455 460 Gly Lys Gly Ser Asn Thr Ser Thr Pro Gln Thr Gly Ser Lys Arg Lys 465 470 475 480 Ser Asp Gly Ala Ala Arg Gly Asn Ala Lys Lys Gly Lys Lys Ala Thr                 485 490 495 Gly Ala Ala Ala Ile Ala Ala Ser Ile Pro Val Gln Lys Lys Gly Gly             500 505 510 Ser Ser Ser Gly Ser Gly Asn Gly Ser Glu Asp Glu Asp Asp Gly Asn         515 520 525 Asp Arg Lys Glu Gln Lys Phe Glu Thr Glu Glu Glu Lys Arg Lys Asn     530 535 540 Phe Leu Glu Arg Asn Arg Gln Ala Ala Leu Lys Cys Arg Gln Arg Lys 545 550 555 560 Lys Ala Trp Leu Asn Glu Leu Gln Ser Lys Val Glu Gly Leu Thr Ile                 565 570 575 Glu Asn Glu Arg Leu Gln Gln Thr Val Gln Gln Met His Asp Glu Val             580 585 590 Gly Arg Leu Thr Ala Ile Leu Met Gln His Arg Asp Cys Gly Leu Gly         595 600 605 Ile Pro Ala Pro Tyr Gly Gly Arg Leu Arg     610 615 <210> 4 <211> 170 <212> PRT <213> Cryptococcus neoformans serotype A H99 strain <220> <221> PEPTIDE (222) (1) .. (170) <223> amino acid sequence of Ras 1 <400> 4 Met Ser Gly Asn Gly His Tyr Arg Arg Asp Gln Arg Leu Val Val Val   1 5 10 15 Gly Cys Gly Ala Phe Arg Glu Tyr Asn Pro Thr Ile Glu Asp Ser Tyr              20 25 30 Arg Lys Gln Val Val Val Asp Asn Glu Ala Thr Thr Leu Glu Ile Leu          35 40 45 Asp Thr Ala Gly Gln Glu Glu Tyr Ala Ala Met Ala Asp Gln Trp Tyr      50 55 60 Thr Phe Gly Ser Gly Phe Leu Leu Val Tyr Ser Leu Thr Asp Arg Ser  65 70 75 80 Ser Phe Glu Glu Ile Gln Asn Phe His Arg Glu Ile Leu Arg Val Lys                  85 90 95 Asp Arg Asp Tyr Val Pro Cys Val Ile Ile Cys Asn Lys Cys Asp Leu             100 105 110 Gln Lys Tyr Arg Ser Val Gly Gln Leu Glu Gly Arg Glu Leu Ala Arg         115 120 125 Ser Val His Ala Pro Phe Ile Glu Cys Ser Ala Ala Glu Arg Val Asn     130 135 140 Val Asp Val Ala Phe Asn Glu Leu Val Lys Leu Val Arg Lys Asp Glu 145 150 155 160 Arg Val Arg Ile Asn Tyr Asp Ile Ala Phe                 165 170 <210> 5 <211> 5498 <212> DNA <213> Cryptococcus neoformans serotype A H99 strain <220> <221> gene (222) (1) .. (5498) <223> genomic DNA sequence of TCO1 <400> 5 ggtcagcttc tgtttctgct tccacctcac cccgctttca catcttcctt tcatccccag 60 ccacgtgcac agcctagcct cctgccacaa gccgtacagc ccacattttc ggccttcttt 120 ccgactgaac aaggcgtctg tcgatctaga gctagagtag agcgtctcct cttgctagcg 180 tattcagata ccgaagcaaa ggtgactaaa ggtgggtcgc cactcatctc gttgcaaaga 240 cgaaactccc cggctttcgt cagcgcagct tttgcgatgg cggccgctct ttcgtctttg 300 ccacacctct tgccgtcccg tgtcccccaa tcaaagctga catatccctt tagactcgtt 360 gcccctgctg tacttctccc cgccccttcc gcacacaccc accatcaccg accgatctcc 420 tccccacaaa cgggcaaggt tggccgccat gtccctcccc gatgcctacc ctccggtcat 480 agccaccgag gggagcacgg cgttccagtc gcacattctg tccctcttgt cagtgatatc 540 ccaacatcca tctcccgatt atccattccc aacgagtggc gattccactc tgcaagacaa 600 cgcttctgtg gcggcaaaaa agagtgctac cctttcaacc tttcccggca aaaagacccc 660 cgctgaggat gcgattgagc gagctatcat tgctctcggt gatcgcgttt ggacttctga 720 gaggactcag gcgcatctgg taaaggacac caaacgcgac gttcctgccc aacctggttc 780 agatctgctt actcctgatt ggactcctcc agtccccacc ctcagcgact cctctcccga 840 tcctgtttgt cctacctgtg cccggcccgt ttcttccacc gaaagctctt ccttcactcc 900 gcagcagatt gcatctttct atcatccttt tacggctgcc ctgtcaacac caccgcctcc 960 tctccccgaa caaatgttct tcccacagag tgttcttgct gcttatgtct ctgctacttc 1020 cgacagcgaa tcagctggcg attctggcat gtctgccgaa aaggaacttg aacttctcaa 1080 agctcaagtt caggacatcg cccgtgtgtg taaagctgtc gccactggtg atcttactca 1140 gaagattatt gtacccgtta aaggtcagac catgactgaa ctcaaaaaca tcattaatgc 1200 aatggtggat cgtctgcaaa cttttgccgt ggaagtcgag cgtgtgtctt tggaagttgg 1260 tactgaagga aagctcggtg gacaggccgt cgtacccaat gtcgaaggca cgtggaaggt 1320 tctcaccgct gttgtgaaca aacttgccgc caatttgact aaccaagtgc gaagcatcgc 1380 aaaagtcacg aaagccgtag caaagggtga tttatcggaa accatcgacg tggaggcgag 1440 tggtgaaatt gcagaattaa agactacagt caatggaatg gtcatgtctc tcagaacatt 1500 ggcagacgag gtcagcaggg tgtctttgga agtcggtagt caaggaaagc tgggaggtca 1560 agccaatgtg ccggatgtgg agggagtatg gaaagatcta actgtcaatg taagcaggcc 1620 ccaatataac aaggcatcat tgacactatg caggtgaatc gaatgtgtga aagtttaacg 1680 acgcaagtgc gatcaatcgg cagtgttacc acagccgttg ctagagtcag tatttccctt 1740 tttctgcttt aatcgcacta atactttcta tctagggtga cttaagcaag atgattgaga 1800 ttgaagcaga gggagaaatg gccgtcttga agaatgtagg taaaaaatca tactaagatg 1860 agtatgcttc taatcgcact tatcattata gaccgtcaat tcaatggttc ggcaactaac 1920 tgtattcgcc aaagaagtga ctcgagttgc gttagaggtc ggtacccatg gccagctggg 1980 cggtcaagcc gtcgttcccg gcgttgaagg cgtatgggac gatcttacaa ccaatgtcaa 2040 caagatggcc aggaatttga cagatcaagt gcgggagatt gccatggtcg taagctgatc 2100 gaaacattta taagatagta gattaacatc tcttcttgca gacaaaagct gtcgctcggg 2160 gtgacttatc caagacagtc aatgccgacg tacaaggtga aattcttgac ttgaagtgta 2220 ctgtcaatga aatggtgagt acggattaca gaaaccttca aacacaaaag cttaccaacg 2280 tgccatgtaa ggttgcccaa ctgacagtat tcgctgccga ggttactcgt gtatctcttg 2340 aagttggaac agaggggaaa ttgggtggac aagctgtcgt gccgaacgtg gaaggcactt 2400 ggatggtcct tacggacaat gttaacctaa tggtatggct atttcactgg cattacatga 2460 tgatcttgat atcttatgcg cacataggcc ctaaacttga ctactcaagt gcgttccgtt 2520 gctgaagtgt aggtgttcgt gattcagagg aaggcatcag cttactccgt ctctgcagta 2580 caacggccgt cgctgctggt gatcttagca agaaaattaa cgtcgccgcg ttcggcgaaa 2640 tcctcgagct taaaaacact gtcaataaca tggtggattc tctgcgttcg ttctcatccg 2700 aagtcacccg tgtcgcgcga gaagtcggaa ccgatgggcg actaggtggt caggccaagg 2760 ttccgggagt ggccggtact tggaaggact tgacagattg tgttaacatc atggcggcca 2820 atttgactga gcaggtccga accatcgcgc atgccactgt aagtgttctt gaacccaatt 2880 agcctttgcg actgctgacc aacttgtcag actgcagtag ctcgtggtga tttgactcaa 2940 aaggttgtcg gcgtcaaagt atctggagag atccttgatc tggtgaatac gatcaataat 3000 atgattgacc aattagctat attcgcggca gaagtgacgc gtgtcgctag ggaagttggt 3060 acagagggaa agttaggagt gcaggccgaa gttgaaaaca ttgaaggaac ctggcaagag 3120 atcacgtaag gcgtcacctt tacttccagt caagcgcgta attgacttgt ttcgcagatc 3180 caacgtgaac accatggcct caaacctgac atcccaagtt cgagcctttg ctcaaatttc 3240 cgccgctgca acagatggtg acttcacaag gttcatcact gttgaagcgt ctggtgaaat 3300 ggatagccta aaaacgaaga ttaatcagat ggtgtacaat ctcagagagt ccatcgagaa 3360 aaacaccaag gcgagacagg aagctgaaat ggccaacaga agtaaatccg agtttttggc 3420 caatatgtcc catgaaatcc ggacgccaat gaacggtatt atcggtatga cagtattgac 3480 actggagagc gagttgacta ggcaacaacg agagaatttg atgattgtct ccagtttggc 3540 cggatccctg ttaactatca tcgacgatat cctcgatatt tctaagagtg agtgagagtg 3600 tgctgaacat caatgtgaat ataatgctga tttgatgcat agttgaggcg ggccgcatga 3660 ccatggaaca aatccccttc tccttacgtg tagccgtctt ctcggtcctc aaaaccctat 3720 gtgttaaggc agcccagaat aagctggatc tcatctttga cgttgatccc acgatgcctg 3780 atcaacttat tggtgatcca cttcgtcttc gccaagtcat taccaacctc atcggtaacg 3840 ctgtgaagtt cactaccaaa ggtcaagttg ctctttcttg ccgagtcaaa cgctacgttg 3900 ctgtcgccga cgctgttgag cttgaattct gcgtggcaga cactggtatc ggtatcaagc 3960 aggataagct tgatgtcatc tttgacacct tcgcgcaagc ggatggatct accactcgca 4020 agtacggtgg gactggcctg ggattgacca tctccaaacg acttgtgaac ctgatgagtg 4080 gtgacctttg ggttgagtca gaatatgggc agggaagtag attctatttc actactgttg 4140 cagagatgac cagcgtgccg agggatcaaa taatagaacg gctatcgcct tggtccggca 4200 gaagcgtgtt gtttattgac acacttggtg atcaaacggg aatcgtggaa atgctccaag 4260 aacttggttt aaagcctatc gttatccatt ctgtgaacga tgtgtacctt ttacaggatc 4320 aaggcttgac tgtctttgac accatgatcg ttgattctct taaagctgtg agtccgagtc 4380 gttcaaagtg gtacagtatt attgatgtta tttccaggcg ggagaactga gaggcataga 4440 atatcttcgc tacatcccca ttgttctcct tgcaccatcg aacaaaccaa gtggaccttc 4500 taatccatct ttcatctcac ttcccgaatc ccgccgccgt cttcttgccc ttccttccgc 4560 ctccgaacaa cttctctcac ctatacccgt caaggattgc ttagaaatgg gtatcaacac 4620 atactatacc acccctctct cgttacaaga gttatccaac gcgatagtcc ccgcgctgga 4680 atctcatcag atccaaccag gcgacactgt tagggacacc atcctcagcg tgttacttgc 4740 ggaagacaac ctagtgaatc agaaactggc tgttaaattg atggaggtcg ctggtcataa 4800 gattgaagtt gcagacaatg gtgaaattgc gttagaaaag tacaagaggc gacagctggc 4860 tagaacgcca tttgacgtca ttctggtgag tcgttgatag cttgtaaatc tatattctga 4920 ctgaccaaaa tgattagatg gatgtttcaa tgcctgtgat gggaggcatg gaagccacgg 4980 gtctgattcg cgagtttgaa gcgaatgagg gcgtacctcg cacacctatt atcgctctta 5040 cggcccacgc gatgatcggt gacaaggaga gatgtcttgc ggctggcatg gacgaatacg 5100 tcacgaagcc tctgaggaga ggcgatttat tggcatcaat agccaaagta ttagctccca 5160 agcagtctgc cagcggcttg gaaatgccgg ccgtatctat tcccgcaaat atctatggta 5220 gataaccctt ctctcgttcg cccgattgtc gacctccatt cgagattatc tcgtcaatat 5280 ctcataccct attcaactag atgacggcct ccatgctgtc gcatcgcaat gtttataccc 5340 cttctagccc ctcattttcc tttcaaaatg tttcctttta ttttattctg ctttctttct 5400 aggtttattc gctaaacaca ccctctaaac gttaccacag aggtgtacct cggattgctt 5460 ggaatcttgg gttgtctatg taccactttt gggtctcg 5498 <210> 6 <211> 6650 <212> DNA <213> Cryptococcus neoformans serotype A H99 strain <220> <221> gene (222) (1) .. (6650) <223> genomic DNA sequence of MBP1 <400> 6 aaatgataga gtagtacgag tagtacgtag atgccgataa gatggaaaat cgcatgcccg 60 agcgcaccaa ggaacatcaa taaataattg agcttatccc gcgtctccgc gcgtcgcccg 120 ccgatcactc ggccttcttc tatcttgttg ctgcggatgt catctcatca cacatgcact 180 gctcagcatc aaatatggcc gatggcctat atacagtaac agattcctgg gctaagattt 240 ctgacggaca cactacagtc ggcctatcag atcctgtctt gacttatgtc gccaagctat 300 tacggatctt tgcgacttcg gatacttaag aatgtgggtt tcaattacag cggactccgt 360 cccacccatg atggaacgtc atatatgtcg tagaacatgg actaaatcgt gccaatttga 420 cacccacgtt tcgactacat ccattatgaa atgcgactcg tgaatagctt ggattcaaga 480 gagtggtcgt acgtgaccga agagagacat gaatcagtcg aagagttgtc gactcatgta 540 tggcgatcgc cttgcaacat ggagactgat ccttgcttct tgaaagatta aagttgacct 600 caaaattcta gtttcaaaaa ggaggggttt gttcagttgg cgagggatgg taacacaaca 660 ggtatgcata tgctgggtat agatcgttac agttcgttac tttgtcgctc ttgactccat 720 cactccccct attagtccat atctccatct tgttcgtcat ctactccacc ttctcttcca 780 gtctcgtgga taaactcttc ctcctgcatc atagccatct cttccatctc agcatcggca 840 ttgtcaagat atactgggtc ataagcctcc tcattggaaa cggcgcccgc ctggttgggc 900 atgtagggaa gaagttcgtc gggcggggaa gtaaggaggg tattgtagat ttggttgcca 960 atctcatcca gcttcccaga ggcatcagga tagaaacgcg tatccgcctc ttctattatc 1020 tgtaccaggc cattaaatgg catattggtt ttttgagaca tgtaaagact tacagagtag 1080 agagtgactg gttctccagg tgcaaggtta catagctgaa ggacttcggc ctttgtaagt 1140 tgatgatctt gcagctcgtt cgccagttga gcaacaccct ctggtgtttg acgggaagtc 1200 ggtaccaagt cagaacaaag atatttgata acctgtacat aggtcagtat accgtcatgg 1260 agttggaaca gccagactca cctcgttctg aatccagacc agttcgtcac ttaaccctct 1320 tctctctgcc accttcaact ccacttcctt ctccgcactg agaggttcaa ataaactagg 1380 atcatccttt ttgtccggat catccctttc taacggatac aacttccttg ctgccgccga 1440 atccctggcc ttttttagag cgattgcctc ccccaagcga tcggtctctt gcttgagaga 1500 gataaaatga tccaagactt cacgattcgc cagatgttgt ggtgctgtgc tagagaactg 1560 gcctttttca gcttaaatcg tggaagtata ccttaccgac ataccttcat tgtcagctgt 1620 atatttgtat gctacttttc ttgcagaata tggcattccc ttctgcaact tgtcgaaccc 1680 aacaggcgac ctccactgcc acacggaaac acgcgggaaa aaacagagaa aaatgtaaaa 1740 aaataaatcg gccctccacc acgcgcgcgt ctgtcgtttt ccggccacaa cccagccgcg 1800 tcgcctcatt cgcgctttct ccaaatacaa gtattccgca aaacatcctc catcttctct 1860 tgccatctct cttttgcctc atatacatca cagagtgagg aagaagcatc tctcagccac 1920 aattggcgac acagatttac tagtcgcctc tttttccgcc ctcttctacc ttcaaatctc 1980 accccgagac acgttccgcc atgggcaaga aggtcatcgc ctctggtgga gataatgggc 2040 ccaataccat ctacaaggtg cgcatttaca ccaatactat tcggatggca taacagattg 2100 ctttaaaagc attgctaact caatgtcccg ttctacaggc gacttacagg tacgccacat 2160 tcctttattt cgcgccgcgc accgcccgaa tgtatgtggt atgtttgcat gtttgctaac 2220 cttgaaatca ctttctacga tagtggagtg tgcgtaccat atccttcctt tgtttacctg 2280 cttctagcta acaatatttc taggcctgta tacgagatgg tctgtcgcga tgtcgcggtt 2340 atgcgcagac gttcggacgc gtatctgaac gcgactcaaa tcctgaaagt agccggtttc 2400 gacaagcctc agcgaacacg agttctggag agggaagtgc aaaagggaga gcatgaaaag 2460 gtccagggtg gctacggcaa ataccaaggt cagattactt ttttttcctt caatcaccca 2520 tcccgcaagc tgactcgatg taggtacctg gattcctatc gagcgtggtc ttgctcttgc 2580 caagcaatat ggtgttgaag acattctccg gcctatcatc gactacgtcc ccacctctgt 2640 atcacctcct cctgctccta aacattccgt tgcgcctcca tcaaaagccc ggagggacaa 2700 ggaaaaagaa actggtcgaa ccaaggctac cccttcaaga accggtccaa catcagccgc 2760 tgctcttcaa gctcaagcac aacttaatcg agccaagatg catgattcca ctcctgacgc 2820 cgatgctagc ttccgctctt ttgaggagag agtcagctta acacctgaag atgactcgag 2880 cagtgacaca ccgagccccg tcgcgagtgt tatgactgac caggacatgg aagtcgataa 2940 gatggggatg cacatgaaca tgcccaacgt gacgctgtcc caaaatatgg aggaattggg 3000 agctggctca agaaaacgta gcgccgcaat gatgatggag gatgaagatc aatttggtca 3060 tctccggtca gtcaggggta atagcgctgt ccacactcct catggtactc ctcgacacct 3120 tggtatcggt atgcctccag aaccaatcgg ccccgagcaa tacaccgaca tcatacttaa 3180 ctactttgtc tctgaaacct ctcaaatacc gtccatcctt gtcagccctc ctcacgactt 3240 cgatcctaat gctcccattg acgatgacgg tcataccgca cttcactggg cttgtgctat 3300 gggtcgagta cgcgtcgtca agctgcttct caccgcaggc gcgtcaatct ttgccggtaa 3360 caatgccgaa caaactcctc ttatgcgcag tgtcatgttc tcaaataact atgacatgcg 3420 taaattcccg gagctgtacg aacttcttca ccgatctacc cttaacattg acaaacaaaa 3480 tcgaaccgtt ttccaccaca tcgccaacct tgctcttaca aaagggaaaa ctcatgccgc 3540 caagtactac atggagacta ttctcgctcg tttggctgac taccctcaag aacttgccga 3600 cgtgatcaac tttcaagatg aagaaggtga aactgcctta actattgctg cgcgagccag 3660 aagccgccga ctggtgaaag ctctgctcga ccacggtgcc aaccccaaga tcaagaaccg 3720 tgactctcgt tcagcggaag attacatcct tgaggatgag cgattccgtt catctcctgt 3780 tccagctccc aatggtggtg tcggtaaagt tagtacctct gctgccgccg aaaaacctat 3840 ctttgctcct cagttgtact tctccgaggc ggccaggtta tgtggcggcc aagcattaac 3900 cgatattact tcccacatgc agtcgctcgc tcgatctttc gacgctgaat tgcaaggcaa 3960 ggaacgagac attctccaag ccaaggctct tcttaccaac atccatactg aggtcacaga 4020 aaatggtcga tcaatcactg ctatcaccaa tcaagcggct cctctcgaag aaaagcgaca 4080 tgagcttgag tctctacaag cgtctctgaa aacaaaagta aaggacgctt tgaagaaagg 4140 ttatattggg tggcttgagg gcgaactgat aagggaacaa cgatgggaga agggtgagct 4200 cgagggaaac gaagaggaga aggcggctgt tcaggctttg agggatgttc ccaccggtgg 4260 tcaggaggtt gttcaggccg aggaggaaaa gttaagatgg gagattgagg aaaagagaaa 4320 gcgaagggct atgtttgtgg aaaagtttgt cagagcacag gccgaagtaa gtttctgggg 4380 aatgttcaag taaagcaatg ctcataattt gcaggctggt acaagtgagc agattgccaa 4440 gtacaggaaa ctggtatccg ctgggctcgg aggcgtttca acaaatgaag tagatgagtt 4500 gatgaaccag ttattagagg taggtcctgc attctatcgg cctaatgaga gctcaactga 4560 tcttttttcc agggtctcga agaggagaat gataatcaag tgtataacac atcggctgga 4620 gaatcaggtc cttcatcatg ggtgcagtaa tatggtcatt gggaatgaag gaaaggaagg 4680 aatcatgtgg tcaataattg ggagttctca gatctctatt ctgtattacc aaaaggtttc 4740 tgcgcatgat gtgacttggt cttgggtctc ttaaatggtt tttacttcta gtaactatgc 4800 gaatgcaaaa tgcatctatc tcttggttat gctgctatga gagaatcgaa taatctaata 4860 tccctttctg tatgctcata cccaacagct ctattgcctt cgtccgtgtc gtacttagcg 4920 gtccaagccc attttccgcc tctgcctctc cagcctgatg ggacatgtct acctctatct 4980 ctatctctta atgccatcgc tcttgcttca tgagcaacta agccgccagg ttcatcacct 5040 gacccatcaa acgcgaggac aaacgccctc ctttccgtga ctccccacac cctcccacct 5100 tccccttgca gcgagtacac cggagagccc ttgccaccgg gggagaagca gctccaacca 5160 gttttactat tacgtatatc aaagaaggcg acagttccat ggcgagcgcc gccgcccgcg 5220 atgtagtgtt cgccggtgaa acatgtcgag tagaatgcag agtcgtccgc ccacgtgaac 5280 ggatcttgga aggtcaagat cggggaaaga gagggagatc gaagatcgtg tatgcgaaga 5340 tgggagtcat accatgccga gaggagtaac gatgggtggt gagaagagga aggcgagggg 5400 agacgaaggt cgtatgggga agacgtctgg gggtactccg gaccttggag aagacgggag 5460 ggtgtaggag aaatgcccga gggtaaaatg gagtggatag atatggcggt cttggtaccc 5520 aacatggccg taggcgataa agtcggatgg gaagtaatga ggagactgga ccaagccact 5580 ttcccctcca agctcattgc cgtcggctca atccatggtg accgagtgtg caagagcgag 5640 accaccccat cccaagaagt ggtcatcatc agctcaccct tctctgagcc agtcactttg 5700 cgtaacctgg tagaccttgt actctcgtgc ggataccgag ctgtgctctt tgggatggta 5760 ggtccataat ctggtagaat gacccgttct atttggccgt cgaactttgc gatggcgaaa 5820 gaaccttgac cgtcgtgcag gtcgacaatt gatacgacgt ccgatttgct ccctgagccg 5880 ggggtacgca agacgaaacc tctccctgga ccaaccacct tgccgccata cctaggctct 5940 ccgtaatacc ctcttggtgc atatagggaa tgaacaataa cttcgccacc tgcaccgata 6000 acaatccttc ctttatcctc atccacataa attgtgggta tggcgctttg ccgccatgtc 6060 ctgcccattt gtaacgcatg ccaggtatgc gactccagag atttgttgat acggtagtta 6120 tgctttgcta aaagaaatgg cggccattgt gatggaggag gagagagagt ccgatgatac 6180 tggcgatggg ttatgaggta taacggtatt ccaaggctat ccacctgatg ctgtctcaat 6240 aatctaatca acgtgtgaag catataagac acttactcta cttcgcatta gctttcctgc 6300 ctgtcgacat ttcagcagat cctggattgt caacttttta aagatcggcc caaggacatc 6360 gtctggcaat gtatctaatc gatgtggagc agtggagcta tagtgttccc tcatcatctg 6420 tcgagcagat atatgcgcca tatttttgtg ctagttctgt aaagggattg atattatccc 6480 tgtcaaagaa cgcacagatg gaaatttgcg aaaattgcga taatgcaagt acaaagaatt 6540 gatagacact tgtatatgaa tgagtggcga gcccatgtgg aggtccctcc accgcgccaa 6600 ttgtttaatt tggattgtct tacttgccaa taggtattct atatatcaaa 6650 <210> 7 <211> 6183 <212> DNA <213> Cryptococcus neoformans serotype A H99 strain <220> <221> gene (222) (1) .. (6183) <223> genomic DNA sequence of ATF1 <400> 7 cgcgagctct tgcaagccga atgataaaaa ctatcagtca gccaggcatt acacacgccg 60 agaaagggta tctagatttt tgcataatac cttttaaacg tctaaaaacg tccaaaaagc 120 cgccgctcat tgcgtgccca ttttctacgc cattctctag agtttgaaca ttgtagagtt 180 tgaccgaaaa acaagagatg gaaaactttt tcttatatac aacaccggcc gtttctcctg 240 tcacatccat ctcgcatcct tcctcttgac cgcctgattc tcaagtgaca ggcccaacac 300 tcgcactcca ctccctccgc tttccgctta aaactgctga atcttttgcg acaccctatc 360 acgcagggat gtgtacgcat tcatgagcgt ttgctcatta atgacgatat ctgtcgtatc 420 aggtcgaggg cgggagacgc gagaggcaat tgctgcagcg tctgtttcca gaatgatcct 480 gcgtttttgg gtttgtttgt tagcttggct ctagtttagg agatggctct agtttaggag 540 atggctctag tttaggagat ggctctagtt taggagatgg ctctagttta ggacgatggg 600 agatggacga cgagtagagg gaaaaaagac aacctaccca tcatccaccg tctcgtccgc 660 agccaacaag accagatcca agctctccaa tacgttcctc ttctcgactg aacctctcaa 720 aagcaagttg acggcatcaa agaatgcgga aagggtttga ttgagcatca gctcgttgga 780 tgtcgagagg gggccgacga tgatgaatgt gaggtcgacg acggagcggg tgaggataat 840 gtgcggtgga agcgggtgta tctcgcctat catcgtaaat tattcaatca ctctccatga 900 ggggaggacg taccggcacc acgacgtatc ttgtcaaaca cactcttctc aaacgtcttt 960 tgctccttga aaccgaccag cccgcccatt cctggccctc cagcgccaac accaagctca 1020 atggggattc cagtcgcagg aacagcttgg tgcggggggt tatagtactt tgcaaagacc 1080 cgttggccgt cagagtcgag gatgagtaga gcggtgacgg tgtagaggga aagatttgac 1140 tgtaaaggat aatacaggga cgtatgttag cgctggcgat attggaatgg gcccgagtgc 1200 agagggatac ttgccatggt gatggatgga tgaggagtag aggagtggat tggggatcgt 1260 actgtataag gggatctgga tattcggttc aagcctcgct caacaggtcg tgattgattg 1320 cccgacgcga gaggataggc gtggtgggag tggatatttt ataggcatgc ccaccaacaa 1380 ggtaagaccc gcgcctttgg ttttgggaga aaagcgataa agtaaagcca taaataatat 1440 cctttatttt tttggtcaag tgttgtttgc gtcatcgaat ggggggggtt ttctcagtgt 1500 cgtcatttcc atcttgttgg tgtagttttg gttccttctt tccactcacc cttccgtcgt 1560 cactcacatt aattgattcc agcagacccc catccataat catattctcc cacacatcgt 1620 cataaccatc tgcccaactt tggcactgct cattacagtt atacacctgt gagttcatca 1680 ctccttttat tttttcatca cttctcccct ctccgccttt gctttaataa tcaatccgcc 1740 ttccgcccat ctccacccat cttctccatc ttctcttgtc atgtctttat catgtaccca 1800 aatgctgact acctgtccac catgaaatgt attacagtac aactgtttaa agtcaatcgc 1860 aggctatttg ccactccatt gtagcattaa aacgctccac tcttcttttc cacgcgtgca 1920 ttcgtcgccc acacaaaaaa agaacaaaaa aaaagtgtat tactagaaac agacatacat 1980 attcttgctc tgcttgaaca atggctgcag ttgcacaagc acctccttca atgcctcact 2040 cgtctattga gaactctggt acgtgaactt cttttacaaa ttcgtcaccc cccccctccc 2100 tctcttcaca tatcctccac gcgtacgcga ttagtcgtgt ccggcggcat tatctattca 2160 aattacaaaa ctattttatt ctcgccccac aatgacatca cataacgctg acattactat 2220 tcaatcgtcc ccgttatttc tcaaatggtt ttctagtgac ccgaccttca tcttctgctt 2280 cgaattcgaa acgccagcac tctgaagctc ctgcgtcaag aggcagtgaa gaaaactcaa 2340 acttggctgc gactctgact gtcacagaac cgacaccggt cgaggataag gagagcaagg 2400 aaagcgggaa tgcacaaggt atgttggtct gttgcctttt gtctcaatcg ccgccctcat 2460 tttgacgcct ccaacagccg gttcttccga caaagacaaa cctgccaacc aagcttctga 2520 agtacagaga cctcctcttc gacctaatgc tgtggcgcgc tcccgactcg aacaagaacc 2580 caacccattc gaacagtcat tccgcgactc tcacggtcct ggaacatctg accgcgacac 2640 ccctcctcgt ggtacagacg cgacctctac ccgccacaac gctttaccac ctctttcatc 2700 actcaccagc cctgctgctg cggaccctac tcagttcccc tggcttgcca atcaatctct 2760 tcgttcgggc cctttgagcc ctgctatgct cactggtccc cagggcgcaa atcaatcttc 2820 aacttctaat aatctcaggt ccaacgaagg gaacgctgaa ggcggattcg agtcttcagg 2880 ttttaggact gggtttacac ctggtacagg atcgggtttt acccctggtt acaactcgtt 2940 tataaactcg tcgctcaatt ctttgcctat tccttcgccg aacacggccg ctttcctcaa 3000 ctcaattaca aacgtcactc ctcttggaga gagtagcgag attgctcaag ccgctgctgc 3060 acaagcacaa gcacaaggcc aaacagcgtc caacgcttcc tctgcctcgc aaaattctca 3120 gagccagtct tctaccgaac cgctccaacc tccttccgct attccgcctc atatgcaacc 3180 tcatccccat caaccccacc ccctctcaca cgctcactca ccagcaaaca accattctgc 3240 ccacccgggc caagatacaa ttacgcccaa cacactcaac gcgcttaccg gtgtttttgg 3300 cgatatgtcc cgagctcctg ccggtgcgcc tggccaaaac cagtttttcg cgccagccat 3360 gtctgcccat cccggtcatc ccggtcaccc aggtcatcca cacactgcac atccaggtat 3420 ccctggcgta gccggcgtgc caggcgtcca tggtgtccca ggccatccag gagtccaatt 3480 gccgatggga tatgtagact acgcacaaca taatgctaat gcggcttcac aggctgcaaa 3540 cggtcttttc cttctcagtc aagcacatca agagttgtca aagcgagaag aagcagaggg 3600 gacgccaggg agtgcggtcg ggggacctac aggtcgatca tcacgtggag gaaaaggttc 3660 caacacctct acgccccaga ccgggtcaaa gaggaaatct gatggagcgg ctagagggaa 3720 tgccaagaag gggaaaaagg ctaccggtgc cgccgccatt gctgcttcta taccggtcca 3780 gaagaagggg ggaagttcga gtgggagtgg gaatggaagt gaagatgagg atgatggaaa 3840 tgataggaaa gaacaaaagt ttgagacaga ggaggagaag cggaagaatt tcttggagag 3900 gaatcgacaa ggcaagtgtg catttcccct cccttttctt ttaaaaggct aacctgctcg 3960 gtttttttca cactaaacag ccgccctcaa atgccgtcag cgcaagaaag catggctcaa 4020 cgagctccaa tccaaagtcg aaggacttac catcgagaat gaacgtctcc agcaaacggt 4080 gcagcagatg catgacgaag ttggcaggct tactgctatc ttgatgcaac atcgtgattg 4140 cgggctgggt atccctgcgc cttatggggg aaggttgcga tgagcgggta atgtcctcgc 4200 cgtcatctat ttttcccact tcatttccca cgcactcaac acgatgaatt atgtatagtc 4260 ccacgcctct tgcagcaaac acaccgcaac acaacccctt tcttatcaat acacactacg 4320 gcctgtatca atcatctcgc aaccaaactg caggaagatg gatggaatgg atagatgaaa 4380 aggatagtcg ggagatattt ggtcatggtg tgatcattct tcaaaaatgg cgtttggtgt 4440 ttttcattca accacttgta tcagtagaca ctctccactt tttttcatat ttgctctgaa 4500 ttttcttacc ttctgcttga cttgtctatg ccttacttgc cttttcgtgg tcatatagtc 4560 tttggacctc ttatcgctac atttatatta tgaatacact gttgcgatct ttattaaaga 4620 attttttttc tctttcttca tctgttttga cgttcgcatg tcgttcgtca gctctcttcc 4680 tcatctttcg tcgtcatcat tctttttttt tatcagtgta attcgttaag aagctcgatt 4740 tatcgatcag ctcttctttt ctttttcttg aatatgcaag cataatacgc aacaaacttg 4800 gcattataaa ggaagggaag aagagacgga ggaaagggaa tggtgaacaa ggcaccgggt 4860 gccgtttcag gaatttatac accggcttct ccgctctttc cgtccggtcc tgatcaggta 4920 aatctaaacg atgtaacaaa tagcaaaaga ggatctaaac tactaaacta ctgacccagt 4980 atgtagttcc tctttctaaa cttcctaaac cttcagatca gccatcagta tgtttacttt 5040 gtcaattaca ggcagatgcg cgtgcgtatg aaacggcatg atatcatcta aaaagtctat 5100 ttagtgattt agcgttatgt acctttttct aaatttatga tggttcctca gcgcatttat 5160 agaccggttt tcggtgtttc aagttttgcc cttggtctgt tcctgctttt ccatgatccg 5220 tgtctgcgat tctagcgtgc agcgtgtttg attttccgag ttatcatcat cgctagcgtt 5280 atttggatac actattgcag ctgtgctttt tcctttctat tttcgcgttt tcttgcctcg 5340 cgaccaggca gggtatatat atacatccag cgtctttctg ttcactttcg atctaatctc 5400 cctccttctc tttcacttca tcccttcctg accttccttt ctcaacctag cttgtcgacc 5460 ggcaaaaacg caagattaga caacgaaggc acgatgaaat ctagcttgat caccaccatc 5520 ctcctccctc ttgtcctctc tgcccctttt ccagcccggc gagatgacca ctcccaggcc 5580 gatatacgcg cagctcactt gcatgcttac gctgaagcga gaggtgtcag tgattctacc 5640 agtgtaatca gcttgaacga cgcgtacgtt gctattaccc cttgtctttt tttttgtcta 5700 caccaacaaa tcaagaatga aagctaaacc tctaaaacag atccacactc tcatccctcg 5760 tcgtgttcaa cccctccgcc acatactcct tcacccccca tgcctcagct tcatcagctg 5820 ctgctgcaca atcgaatgaa ctcctttctt caggtgaatc cctgaccttc aacccgacac 5880 ctactactac actgtcttta ctcggtaacg tcagtaccgc tgccacttct tcttctgggg 5940 gctcgtcggt atccggatca gtaacggggg tagcagctgc tgccaactct acctccaact 6000 ccacggacag tgataccggc agtaatggta gcagcggaga ctcaagtacg agtggggcgg 6060 acgctgtaag agggagatct aacctcaggt tgacgggtat tggtatgagc tttggagttt 6120 gtgcaattgg gcttggatgg ataggaggga tgagtctttt ctgagcaagg agaatacatc 6180 aag 6183 <210> 8 <211> 794 <212> DNA <213> Cryptococcus neoformans serotype A H99 strain <220> <221> gene <222> (1) .. (794) <223> genomic DNA sequence of RAS1 <400> 8 atgtctggaa atggacatta cagaagagac caaagactcg tggtagtggg atgtggcggt 60 aagttttaga ccacgtctca ggaccggtga tcgctcattc cttgggtttc ttcaggtgtt 120 ggcaaaactg ccattacaat ccgcttcatg acatcccagt tctacgatca gtaaggatga 180 ctttcttcct catcttattt ccttccttgc cttatagctt tcagagaata caatccaacc 240 atcgaagact catatagaaa gcaagtggta gttgataacg aagcaacaac cttggagatc 300 ctagacactg ccggccaagg ttggtctcgt tcctgacagt ttttgctttg attatacatg 360 cgttgacatg caggtgcatg gcagaggaat acgccgccat ggccgatcaa tggtacacct 420 tcggctcagg gtttctactc gtgtattctt tgacagaccg ctcatcattt gaggagatac 480 agaactttca cagggaaata ttaagagtga aagacagaga ttacgtgcca tgtgtaataa 540 tttgcaataa ggtaagtcca gtattttctc tcctttaatc cttatgatcg ctaagggaag 600 gacgtcagtg tgatctgcaa aagtatcgat cagttggaca gctcgagggc cgtgagctcg 660 cgcgatccgt acacgctcca tttatagagt gctcggcagc agaacgagtg aacgtggatg 720 tcgcattcaa cgagttggtc aagctcgtgc ggaaagatga acgagtaaga attaactacg 780 acatcgcttt ttga 794 <210> 9 <211> 4152 <212> DNA <213> Cryptococcus neoformans serotype A H99 strain <220> <221> C_region (222) (1) .. (4152) <223> coding region sequence of TCO1 <400> 9 atgtccctcc ccgatgccta ccctccggtc atagccaccg aggggagcac ggcgttccag 60 tcgcacattc tgtccctctt gtcagtgata tcccaacatc catctcccga ttatccattc 120 ccaacgagtg gcgattccac tctgcaagac aacgcttctg tggcggcaaa aaagagtgct 180 accctttcaa cctttcccgg caaaaagacc cccgctgagg atgcgattga gcgagctatc 240 attgctctcg gtgatcgcgt ttggacttct gagaggactc aggcgcatct ggtaaaggac 300 accaaacgcg acgttcctgc ccaacctggt tcagatctgc ttactcctga ttggactcct 360 ccagtcccca ccctcagcga ctcctctccc gatcctgttt gtcctacctg tgcccggccc 420 gtttcttcca ccgaaagctc ttccttcact ccgcagcaga ttgcatcttt ctatcatcct 480 tttacggctg ccctgtcaac accaccgcct cctctccccg aacaaatgtt cttcccacag 540 agtgttcttg ctgcttatgt ctctgctact tccgacagcg aatcagctgg cgattctggc 600 atgtctgccg aaaaggaact tgaacttctc aaagctcaag ttcaggacat cgcccgtgtg 660 tgtaaagctg tcgccactgg tgatcttact cagaagatta ttgtacccgt taaaggtcag 720 accatgactg aactcaaaaa catcattaat gcaatggtgg atcgtctgca aacttttgcc 780 gtggaagtcg agcgtgtgtc tttggaagtt ggtactgaag gaaagctcgg tggacaggcc 840 gtcgtaccca atgtcgaagg cacgtggaag gttctcaccg ctgttgtgaa caaacttgcc 900 gccaatttga ctaaccaagt gcgaagcatc gcaaaagtca cgaaagccgt agcaaagggt 960 gatttatcgg aaaccatcga cgtggaggcg agtggtgaaa ttgcagaatt aaagactaca 1020 gtcaatggaa tggtcatgtc tctcagaaca ttggcagacg aggtcagcag ggtgtctttg 1080 gaagtcggta gtcaaggaaa gctgggaggt caagccaatg tgccggatgt ggagggagta 1140 tggaaagatc taactgtcaa tgtgaatcga atgtgtgaaa gtttaacgac gcaagtgcga 1200 tcaatcggca gtgttaccac agccgttgct agaggtgact taagcaagat gattgagatt 1260 gaagcagagg gagaaatggc cgtcttgaag aataccgtca attcaatggt tcggcaacta 1320 actgtattcg ccaaagaagt gactcgagtt gcgttagagg tcggtaccca tggccagctg 1380 ggcggtcaag ccgtcgttcc cggcgttgaa ggcgtatggg acgatcttac aaccaatgtc 1440 aacaagatgg ccaggaattt gacagatcaa gtgcgggaga ttgccatggt cacaaaagct 1500 gtcgctcggg gtgacttatc caagacagtc aatgccgacg tacaaggtga aattcttgac 1560 ttgaagtgta ctgtcaatga aatggttgcc caactgacag tattcgctgc cgaggttact 1620 cgtgtatctc ttgaagttgg aacagagggg aaattgggtg gacaagctgt cgtgccgaac 1680 gtggaaggca cttggatggt ccttacggac aatgttaacc taatggccct aaacttgact 1740 actcaagtgc gttccgttgc tgaagttaca acggccgtcg ctgctggtga tcttagcaag 1800 aaaattaacg tcgccgcgtt cggcgaaatc ctcgagctta aaaacactgt caataacatg 1860 gtggattctc tgcgttcgtt ctcatccgaa gtcacccgtg tcgcgcgaga agtcggaacc 1920 gatgggcgac taggtggtca ggccaaggtt ccgggagtgg ccggtacttg gaaggacttg 1980 acagattgtg ttaacatcat ggcggccaat ttgactgagc aggtccgaac catcgcgcat 2040 gccactactg cagtagctcg tggtgatttg actcaaaagg ttgtcggcgt caaagtatct 2100 ggagagatcc ttgatctggt gaatacgatc aataatatga ttgaccaatt agctatattc 2160 gcggcagaag tgacgcgtgt cgctagggaa gttggtacag agggaaagtt aggagtgcag 2220 gccgaagttg aaaacattga aggaacctgg caagagatca catccaacgt gaacaccatg 2280 gcctcaaacc tgacatccca agttcgagcc tttgctcaaa tttccgccgc tgcaacagat 2340 ggtgacttca caaggttcat cactgttgaa gcgtctggtg aaatggatag cctaaaaacg 2400 aagattaatc agatggtgta caatctcaga gagtccatcg agaaaaacac caaggcgaga 2460 caggaagctg aaatggccaa cagaagtaaa tccgagtttt tggccaatat gtcccatgaa 2520 atccggacgc caatgaacgg tattatcggt atgacagtat tgacactgga gagcgagttg 2580 actaggcaac aacgagagaa tttgatgatt gtctccagtt tggccggatc cctgttaact 2640 atcatcgacg atatcctcga tatttctaag attgaggcgg gccgcatgac catggaacaa 2700 atccccttct ccttacgtgt agccgtcttc tcggtcctca aaaccctatg tgttaaggca 2760 gcccagaata agctggatct catctttgac gttgatccca cgatgcctga tcaacttatt 2820 ggtgatccac ttcgtcttcg ccaagtcatt accaacctca tcggtaacgc tgtgaagttc 2880 actaccaaag gtcaagttgc tctttcttgc cgagtcaaac gctacgttgc tgtcgccgac 2940 gctgttgagc ttgaattctg cgtggcagac actggtatcg gtatcaagca ggataagctt 3000 gatgtcatct ttgacacctt cgcgcaagcg gatggatcta ccactcgcaa gtacggtggg 3060 actggcctgg gattgaccat ctccaaacga cttgtgaacc tgatgagtgg tgacctttgg 3120 gttgagtcag aatatgggca gggaagtaga ttctatttca ctactgttgc agagatgacc 3180 agcgtgccga gggatcaaat aatagaacgg ctatcgcctt ggtccggcag aagcgtgttg 3240 tttattgaca cacttggtga tcaaacggga atcgtggaaa tgctccaaga acttggttta 3300 aagcctatcg ttatccattc tgtgaacgat gtgtaccttt tacaggatca aggcttgact 3360 gtctttgaca ccatgatcgt tgattctctt aaagctgcgg gagaactgag aggcatagaa 3420 tatcttcgct acatccccat tgttctcctt gcaccatcga acaaaccaag tggaccttct 3480 aatccatctt tcatctcact tcccgaatcc cgccgccgtc ttcttgccct tccttccgcc 3540 tccgaacaac ttctctcacc tatacccgtc aaggattgct tagaaatggg tatcaacaca 3600 tactatacca cccctctctc gttacaagag ttatccaacg cgatagtccc cgcgctggaa 3660 tctcatcaga tccaaccagg cgacactgtt agggacacca tcctcagcgt gttacttgcg 3720 gaagacaacc tagtgaatca gaaactggct gttaaattga tggaggtcgc tggtcataag 3780 attgaagttg cagacaatgg tgaaattgcg ttagaaaagt acaagaggcg acagctggct 3840 agaacgccat ttgacgtcat tctgatggat gtttcaatgc ctgtgatggg aggcatggaa 3900 gccacgggtc tgattcgcga gtttgaagcg aatgagggcg tacctcgcac acctattatc 3960 gctcttacgg cccacgcgat gatcggtgac aaggagagat gtcttgcggc tggcatggac 4020 gaatacgtca cgaagcctct gaggagaggc gatttattgg catcaatagc caaagtatta 4080 gctcccaagc agtctgccag cggcttggaa atgccggccg tatctattcc cgcaaatatc 4140 tatggtagat aa 4152 <210> 10 <211> 2301 <212> DNA <213> Cryptococcus neoformans serotype A H99 strain <220> <221> C_region (222) (1) .. (2301) <223> coding region sequence of MBP1 <400> 10 atgggcaaga aggtcatcgc ctctggtgga gataatgggc ccaataccat ctacaaggcg 60 acttacaggt acgccacatt cctttatttc gcgccgcgca ccgcccgaat gcctgtatac 120 gagatggtct gtcgcgatgt cgcggttatg cgcagacgtt cggacgcgta tctgaacgcg 180 actcaaatcc tgaaagtagc cggtttcgac aagcctcagc gaacacgagt tctggagagg 240 gaagtgcaaa agggagagca tgaaaaggtc cagggtggct acggcaaata ccaaggtacc 300 tggattccta tcgagcgtgg tcttgctctt gccaagcaat atggtgttga agacattctc 360 cggcctatca tcgactacgt ccccacctct gtatcacctc ctcctgctcc taaacattcc 420 gttgcgcctc catcaaaagc ccggagggac aaggaaaaag aaactggtcg aaccaaggct 480 accccttcaa gaaccggtcc aacatcagcc gctgctcttc aagctcaagc acaacttaat 540 cgagccaaga tgcatgattc cactcctgac gccgatgcta gcttccgctc ttttgaggag 600 agagtcagct taacacctga agatgactcg agcagtgaca caccgagccc cgtcgcgagt 660 gttatgactg accaggacat ggaagtcgat aagatgggga tgcacatgaa catgcccaac 720 gtgacgctgt cccaaaatat ggaggaattg ggagctggct caagaaaacg tagcgccgca 780 atgatgatgg aggatgaaga tcaatttggt catctccggt cagtcagggg taatagcgct 840 gtccacactc ctcatggtac tcctcgacac cttggtatcg gtatgcctcc agaaccaatc 900 ggccccgagc aatacaccga catcatactt aactactttg tctctgaaac ctctcaaata 960 ccgtccatcc ttgtcagccc tcctcacgac ttcgatccta atgctcccat tgacgatgac 1020 ggtcataccg cacttcactg ggcttgtgct atgggtcgag tacgcgtcgt caagctgctt 1080 ctcaccgcag gcgcgtcaat ctttgccggt aacaatgccg aacaaactcc tcttatgcgc 1140 agtgtcatgt tctcaaataa ctatgacatg cgtaaattcc cggagctgta cgaacttctt 1200 caccgatcta cccttaacat tgacaaacaa aatcgaaccg ttttccacca catcgccaac 1260 cttgctctta caaaagggaa aactcatgcc gccaagtact acatggagac tattctcgct 1320 cgtttggctg actaccctca agaacttgcc gacgtgatca actttcaaga tgaagaaggt 1380 gaaactgcct taactattgc tgcgcgagcc agaagccgcc gactggtgaa agctctgctc 1440 gaccacggtg ccaaccccaa gatcaagaac cgtgactctc gttcagcgga agattacatc 1500 cttgaggatg agcgattccg ttcatctcct gttccagctc ccaatggtgg tgtcggtaaa 1560 gttagtacct ctgctgccgc cgaaaaacct atctttgctc ctcagttgta cttctccgag 1620 gcggccaggt tatgtggcgg ccaagcatta accgatatta cttcccacat gcagtcgctc 1680 gctcgatctt tcgacgctga attgcaaggc aaggaacgag acattctcca agccaaggct 1740 cttcttacca acatccatac tgaggtcaca gaaaatggtc gatcaatcac tgctatcacc 1800 aatcaagcgg ctcctctcga agaaaagcga catgagcttg agtctctaca agcgtctctg 1860 aaaacaaaag taaaggacgc tttgaagaaa ggttatattg ggtggcttga gggcgaactg 1920 ataagggaac aacgatggga gaagggtgag ctcgagggaa acgaagagga gaaggcggct 1980 gttcaggctt tgagggatgt tcccaccggt ggtcaggagg ttgttcaggc cgaggaggaa 2040 aagttaagat gggagattga ggaaaagaga aagcgaaggg ctatgtttgt ggaaaagttt 2100 gtcagagcac aggccgaagc tggtacaagt gagcagattg ccaagtacag gaaactggta 2160 tccgctgggc tcggaggcgt ttcaacaaat gaagtagatg agttgatgaa ccagttatta 2220 gagggtctcg aagaggagaa tgataatcaa gtgtataaca catcggctgg agaatcaggt 2280 ccttcatcat gggtgcagta a 2301 <210> 11 <211> 1857 <212> DNA <213> Cryptococcus neoformans serotype A H99 strain <220> <221> C_region (222) (1) .. (1857) <223> coding region sequence of ATF1 <400> 11 atggctgcag ttgcacaagc acctccttca atgcctcact cgtctattga gaactctgtg 60 acccgacctt catcttctgc ttcgaattcg aaacgccagc actctgaagc tcctgcgtca 120 agaggcagtg aagaaaactc aaacttggct gcgactctga ctgtcacaga accgacaccg 180 gtcgaggata aggagagcaa ggaaagcggg aatgcacaag ccggttcttc cgacaaagac 240 aaacctgcca accaagcttc tgaagtacag agacctcctc ttcgacctaa tgctgtggcg 300 cgctcccgac tcgaacaaga acccaaccca ttcgaacagt cattccgcga ctctcacggt 360 cctggaacat ctgaccgcga cacccctcct cgtggtacag acgcgacctc tacccgccac 420 aacgctttac cacctctttc atcactcacc agccctgctg ctgcggaccc tactcagttc 480 ccctggcttg ccaatcaatc tcttcgttcg ggccctttga gccctgctat gctcactggt 540 ccccagggcg caaatcaatc ttcaacttct aataatctca ggtccaacga agggaacgct 600 gaaggcggat tcgagtcttc aggttttagg actgggttta cacctggtac aggatcgggt 660 tttacccctg gttacaactc gtttataaac tcgtcgctca attctttgcc tattccttcg 720 ccgaacacgg ccgctttcct caactcaatt acaaacgtca ctcctcttgg agagagtagc 780 gagattgctc aagccgctgc tgcacaagca caagcacaag gccaaacagc gtccaacgct 840 tcctctgcct cgcaaaattc tcagagccag tcttctaccg aaccgctcca acctccttcc 900 gctattccgc ctcatatgca acctcatccc catcaacccc accccctctc acacgctcac 960 tcaccagcaa acaaccattc tgcccacccg ggccaagata caattacgcc caacacactc 1020 aacgcgctta ccggtgtttt tggcgatatg tcccgagctc ctgccggtgc gcctggccaa 1080 aaccagtttt tcgcgccagc catgtctgcc catcccggtc atcccggtca cccaggtcat 1140 ccacacactg cacatccagg tatccctggc gtagccggcg tgccaggcgt ccatggtgtc 1200 ccaggccatc caggagtcca attgccgatg ggatatgtag actacgcaca acataatgct 1260 aatgcggctt cacaggctgc aaacggtctt ttccttctca gtcaagcaca tcaagagttg 1320 tcaaagcgag aagaagcaga ggggacgcca gggagtgcgg tcgggggacc tacaggtcga 1380 tcatcacgtg gaggaaaagg ttccaacacc tctacgcccc agaccgggtc aaagaggaaa 1440 tctgatggag cggctagagg gaatgccaag aaggggaaaa aggctaccgg tgccgccgcc 1500 attgctgctt ctataccggt ccagaagaag gggggaagtt cgagtgggag tgggaatgga 1560 agtgaagatg aggatgatgg aaatgatagg aaagaacaaa agtttgagac agaggaggag 1620 aagcggaaga atttcttgga gaggaatcga caagccgccc tcaaatgccg tcagcgcaag 1680 aaagcatggc tcaacgagct ccaatccaaa gtcgaaggac ttaccatcga gaatgaacgt 1740 ctccagcaaa cggtgcagca gatgcatgac gaagttggca ggcttactgc tatcttgatg 1800 caacatcgtg attgcgggct gggtatccct gcgccttatg ggggaaggtt gcgatga 1857 <210> 12 <211> 513 <212> DNA <213> Cryptococcus neoformans serotype A H99 strain <220> <221> C_region (222) (1) .. (513) <223> coding region sequence of RAS1 <400> 12 atgtctggaa atggacatta cagaagagac caaagactcg tggtagtggg atgtggcgct 60 ttcagagaat acaatccaac catcgaagac tcatatagaa agcaagtggt agttgataac 120 gaagcaacaa ccttggagat cctagacact gccggccaag aggaatacgc cgccatggcc 180 gatcaatggt acaccttcgg ctcagggttt ctactcgtgt attctttgac agaccgctca 240 tcatttgagg agatacagaa ctttcacagg gaaatattaa gagtgaaaga cagagattac 300 gtgccatgtg taataatttg caataagtgt gatctgcaaa agtatcgatc agttggacag 360 ctcgagggcc gtgagctcgc gcgatccgta cacgctccat ttatagagtg ctcggcagca 420 gaacgagtga acgtggatgt cgcattcaac gagttggtca agctcgtgcg gaaagatgaa 480 cgagtaagaa ttaactacga catcgctttt tga 513

Claims (11)

An antifungal pharmaceutical composition comprising an inhibitor against at least one protein selected from the group consisting of Tco1, Mbp1, Atf1 and Ras1 of Cryptococcus neoformans .
The method of claim 1,
The inhibitor is an antifungal pharmaceutical composition which is administered sequentially or simultaneously with a nucleic acid synthesis inhibitory antifungal agent.
The method of claim 2,
The nucleic acid synthesis inhibitory antifungal agent is a pyrimidine analogue anti-fungal pharmaceutical composition.
The method of claim 3,
The pyrimidine analog is flu cytosine, the pharmaceutical composition for antifungal.

An antifungal pharmaceutical composition comprising an inhibitor against at least one gene selected from the group consisting of TCO1 , MBP1 , ATF1, and Ras1 of Cryptococcus neoformans.
The method of claim 5,
The inhibitor is an antifungal pharmaceutical composition, characterized in that administered sequentially or simultaneously with a nucleic acid synthesis inhibitory antifungal agent.
The method of claim 6,
The nucleic acid synthesis inhibitory antifungal agent is a pyrimidine analogue anti-fungal pharmaceutical composition.
The method of claim 7, wherein
The pyrimidine analog is flu cytosine, the pharmaceutical composition for antifungal.
An antifungal complex preparation comprising the antifungal pharmaceutical composition of any one of claims 1 to 8 and a nucleic acid synthesis inhibitory antifungal agent.
An antifungal screening composition comprising one or more proteins selected from the group consisting of Tco1, Mbp1, Atf1, and Ras1 of Cryptococcus neoforms.
An antifungal screening composition comprising one or more genes selected from the group consisting of TCO1 , MBP1 , ATF1, and Ras1 of Cryptococcus neoforms .

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