US20120093817A1

US20120093817A1 - USE OF THE GENES IN THE HOG, Ras AND cAMP PATHWAY FOR TREATMENT OF FUNGAL INFECTION

Info

Publication number: US20120093817A1
Application number: US13/143,921
Authority: US
Inventors: Yong-Sun Bahn; Young-Joon Ko; Shin-Ae Maeng; Kwang Woo Jung; Gyu Bum Kim
Original assignee: Industry Academic Cooperation Foundation of Yonsei University
Current assignee: Nutrex Tech Co Ltd
Priority date: 2009-01-09
Filing date: 2010-01-08
Publication date: 2012-04-19
Also published as: WO2010079998A3; WO2010079998A2; US20130237446A1; US9017956B2

Abstract

Provided herein are uses of genes for HOG, Ras and cAMP signal transduction pathways to treat fungal infection. To regulate the HOG pathway of Cryptococcus neoformans, roles of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 genes were investigated to find that a biosynthesis level of ergosterol is increased when these genes are inhibited. When the genes are inhibited, a large amount of ergosterol is distributed on a fungal cell membrane. Accordingly, since there are many working points of an ergosterol-binding antifungal agent, an efficiency of the ergosterol-binding antifungal agent can be considerably improved. To regulate the Ras and cAMP pathways of Cryptococcus neoformans, roles of RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 and HSP122 genes were investigated to find that a sensitivity to a polyene- or azole-based drug is increased when these genes are inhibited. Therefore, an antifungal pharmaceutical composition including an inhibitor against the gene or protein encoded by the same can be used as an excellent combined antifungal agent which can reduce a conventional amount of an antifungal agent used and increase an efficiency.

Description

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of Korean Patent Applications No. 10-2009-0001947, filed on Jan. 9, 2009, and No. 10-2009-0127206, filed on Dec. 18, 2009, and all the benefits accruing therefrom under 35 U.S.C. §119 which is hereby incorporated by reference as if fully set forth herein.

BACKGROUND

1. Field
The present invention relates to uses of genes for HOG, Ras and cAMP signal transduction pathways to treat fungal infection.
2. Description of the Related Art
The existence and proliferation of an organism in a specific environment is usually determined by an ability to react and adapt to various environmental stresses and maintain cell homeosis. Cells regulate a key process by performing a series of combined signal networks. Among these, a p38/Hog1 mitogen-activated protein kinase (MAPK) dependent signal pathway plays an important role to regulate a wide range of stress reactions in eukaryotes, for example, from yeasts to humans.
A stress-activated p38 MAPK in a mammal induces various stress-related signals limiting change in osmosis, UV radiation, programmed apoptosis, and adaptation to an immune response by generation of cytokine and control of inflammation. Similar stress-sensitive signal transduction systems have been discovered in other species. Fungi have p38-like MAPKs regulating various stress-related responses. In the budding yeast, Saccharomyces cerevisiae (S. cerevisiae), HogI MAPK regulates a stress-related response to osmotic shock, oxidative damage and heavy metal damage. The fission yeast, Schizosaccharomyces pombe (S. pombe), also has a homolog of HogI, Sty1 (also known as Spc1 and Phh1), which is associated with adaptation to various stresses including osmotic shock, heat shock, oxidative damage and heavy metal damage, carbon deficiency and UV radiation. Interestingly, Sty1 is also associated with growth control, reproduction and differentiation. Hog1 MAPK orthologs are also found in other ascomycete pathogenic fungi including Candida albicans (Hog1) and Aspergillus fumigatus (SakA), and known to mediate reactions induced by various environmental causes including osmotic shock, UV radiation, oxidative damage and high temperature.
A common molecular mechanism of the p38/HogI MAPK signal transduction network is highly conserved in many eukaryotic cells. While the p38/Hog1 MAPK is non-phosphorylated under normal growth conditions, it is activated by double phosphorylation of Thr and Tyr residues at a TGY motif using a MAPK kinase (MAPKK) activated through phosphorylation by a MAPKK kinase (MAPKKK) in a higher signal system in response to a specific environmental stress. Subsequently, the phosphorylated p38/Hog1 MAPKs are transferred to a nucleus after a dimer is formed to trigger activation of a transcription regulatory factor and induce overproduction of stress-preventing genes resistant to external stress conditions.
In spite of the conserved regulatory mechanism of the p38/Hog1 MAPK, fungi and mammals have developed a distinctive set of a higher regulatory systems. Particularly, fungi use a two-component-like phosphorelay system, which is not present in mammals, but found only in bacteria, fungi and plants. The fungal phosphorelay system is composed of three components including a hybrid sensor kinase, histidine-containing phosphotransfer protein (HPt), and a response regulator. The three components have not been observed in mammals, and thus are considered a good target for an antifungal agent.
Basidiomycetous, Cryptococcus neoformans (C. neoformans), also uses a stress-activated Hog1 MAPK system to adapt to various environmental stresses including osmotic shock, UV radiation, heat shock, oxidative damage, toxic metabolites and antifungal agents. C. neoformans is a human pathogenic fungus found everywhere in the world, causing cryptococcal disease in the skin and lungs and cryptococcal encephalomeningitis in immunocompromised patients. While C. neoformans var. grubii (antigen-type A) is the most frequently found (>90% of environmental and clinical strains), C. neoformans var. neoformans (antigen-type D) is common only in a specific region in Europe, but not frequently found (<10%). However, it has been confirmed that C. gattii, known as C. neoformans of antigen-types B and C, are primary pathogens attacking normal people who have no immune problems.
However, it is inferred that, compared with other fungal Hog1 MAPK systems, the Hog1 MAPK pathway in C. neoformans is not characteristically developed only to correspond to various environmental stresses, but also to regulate production of two pathogenic factors such as an antiphagocytic capsule and an antioxidant melanine and sexual differentiation, and thus plays a critical role as an important signal transduction regulator in C. neoformans cross-talking to another signal transduction pathway. Recently, the inventors found that most Hog1 MAPKs in many C. neoformans strains are always phosphorylated under non-stress conditions, and rapidly dephosphorylated to activate the Hog1 MAPKs in response to the osmotic shock and treatment of an antifungal agent, fluodioxonyl, which clearly contrasts with Hog1 MAPK systems in other fungi. Double phosphorylation at the TGY motif of Hog1 needs Pbs2 MAPKK. A fungus-specific phosphorelay system which is in a higher level of a Pbs2-Hog1 pathway is also found only in C. neoformans. The C. neoformans phosphorelay system includes 7 different sensor hybrid histidine kinases (TcoI-7), a Ypd1 phosphotransfer protein, and two reaction regulators (Ssk1 and Skn7). The Pbs2-Hog1 pathway is generally regulated by Ssk1, not by Skn7. Among the 7 Tco proteins, Tco1 and Tco2 play distinctive and overlapping roles to activate the Ssk1 and the Pbs2-Hog1 MAPK pathway. However, the Tco1 and Tco2 regulate some Ssk1 and Hog1-related phenotypes, and therefore other higher receptor or sensor proteins should be discovered. More recently, a protein, Ssk2 MAPKKK, serving as a linker between the phosphorelay system and the Pbs2-Hog1 MAPK pathway was identified by comparative analysis of a meiotic map between antigen-type D f1 brother strains, B3510 and B3502, showing different phosphorylation patterns of Hog1. The most noticeable fact is that interchange of Ssk2 alleles between two C. neoformans strains showing different Hog1 phosphorylation patterns changes a phenotype controlled by constitutive Hog1 phosphorylation. Unlike S. cerevisiae and S. pombe, C. neoformans has single MAPKKK and Ssk2 regulating the Hog1 MAPK. While a downstream signal transduction network of the Hog1 MAPK pathway in C. neoformans has yet to be discovered, identification and characterization of the downstream signal transduction network of the Hog1 MAPK are needed to develop a target for a new antifungal agent.
In the past, fungal infections were mainly local infections such as athlete's foot, jock itch, or oral thrush, and rarely systemic infections. However, recently, systemic infections have become as frequent, coming in fourth in frequency among total infections occurring in hospitals.
The antifungal agents which have been developed so far may be classified into two major groups: those having an azole structure and those not having an azole structure. The azole-based antifungal agents include ketoconazole, fluconazole, itraconazole and voriconazole, while the non-azole-based antifungal agents include terbinafine, flucytosine, amphotericin B and caspofungin.
The ketoconazole, fluconazole, itraconazole and voriconazole having an azole structure have similar mechanisms to allylamine-based naftifine and terbinafine. These two different antifungal agents serve to inhibit enzymes required for the conversion of lanosterol into ergosterol, which is a main component of a fungal cell membrane. The azole-based antifungal agents inhibit a microsomal enzyme, and the acrylamine-based antifungal agents inhibit a squalene epoxidase, both having a similar effect to the above-mentioned antifungal agents. Flucytocin (5-FC) is a metabolic antagonist inhibiting the synthesis of a nucleic acid, which has an antifungal reaction by non-competitively antagonizing the cause of miscoding a fungal RNA and DNA synthesis. Amphotericin B having a polyene structure has an antifungal reaction by binding to ergosterol in the fungal cell membrane to induce depolarization of the cell membrane and generating a hole to induce loss of the cell contents. An echinocandin-based antifungal agent, caspofungin, has a reaction reversibly inhibiting the formation of a fungal cell wall, and is different from those acting on the cell membrane described above.
The azole-based drug may lead to death caused by infection when being used on a patient having hypofunction of the liver, and thus a liver function test should precede administration. It is reported that flutocytosin has a dose-dependent bone marrow inhibiting action, liver toxicity, and can cause enterocolitis. Since such side effects are increased when renal insufficiency occurs, monitoring of a renal function is very important to a patient. In addition, flutocytosin is contraindicated for pregnant woman. A major toxicity of amphotericin B is a glomerulus renal toxicity induced by renal artery vasoconstriction, which is dose dependent. Therefore, when a lifetime cumulative dose is 4 to 5 g or more, a rate of permanent loss of the renal function is increased. Furthermore, the renal toxicities such as excessive loss of potassium, magnesium and bicarbonate due to toxicity of a renal tube and low production of erithropoietins may be generated. Moreover, as acute responses, symptoms such as thrombophlebitis, chills, shivering, and hyperpnea may be shown. Since the conventionally developed antifungal agents show various side effects according to kinds of drugs, development of a new therapy which can reduce such side effects and increase an antifungal effect is demanded.
Meanwhile, in pathogenic fungi distributed in the world, including Aspergillus fumigatus, Candida albicans (C. albicans) and C. neoformans, Ras- and cAMP-signal transduction pathways are evolutionarily conserved, and significantly functional and structural differences are still being found (Pukkila-Worley & Alspaugh, 2004, Rolland et al., 2002, Wong & Heitman, 1999, Thevelein & de Winde, 1999, Alspaugh et al., 1998, Lengeler et al., 2000, and Bahn et al, 2007). In C. neoformans causing fatal fungal encephalomeningitis, the cAMP-signal transduction pathway is important in producing and differentiating pathogenic factors (Idnurm et al., 2005). Like S. cerevisiae and C. albicans, it was confirmed that two major higher signal transduction regulators of adenylyl cyclase (Cac1), adenylyl cyclase-associated protein 1 (Aca1) and Gα subunit protein (Gpa1) regulate a cAMP-signal transduction pathway of C. neoformans (Bahn et al., 2004 and Alspaugh et al., 1997). The disruption of GPA1 genes leads to multiple phenotypes of cells, which include incomplete production of core pathogenic factors, melanin and a capsule, essential for survival and proliferation of C. neoformans in a host, and a decrease in mating, which is important in distribution of infectious spores (Alspaugh et al., 1997). Aca1 physically interacts with a Cac1 adenylyl cyclase, and does not regulate a basic level of cAMP but dominates most cAMP-dependent phenotypes by regulating the induction of cAMP (Bahn et al., 2004). A deletion mutant of CAC1 produces a phenotype more defected than a deletion mutant of gpalΔ or acalΔ, and gpalΔ acalΔ double deletion mutants are equivalent to the cac1Δ deletion mutant in phenotype (Bahn et al., 2004). This indicates that Cal1 is activated by both of Aca1 and Gpa1. In a lower signal system of the Cac1 of C. neoformans, two catalytic subunits of a protein kinase A (PKA), Pka1 and Pka2, and a regulatory subunit, Pkr1, are included. While Pka1 plays a dominant role for cAMP signal transduction in a background of an antigen-type A C. neoformans H99 strain, Pka2 also plays the same role in an antigen-type D C. neoformans JEC21 strain (Hicks et al., 2004). Nevertheless, a pka1Δ-pka2Δ double deletion mutant shows a phenotype the same as the cac1Δ deletion mutant, and the cAMP signal transduction from Cac1 is split into two PKA catalytic subunits (Bahn et al., 2004). Interestingly, the deletion of PDE1, not PDE2, repairs some phenotypes including the depletion of a melanin of the gpalΔ deletion mutant, which indicates that different phosphodiesterases act in various fungi (Hicks et al., 2005).
It is revealed that two Ras proteins, Ras1 and Ras2, are found in Cryptococcus, and play common and distinctive roles (Alspaugh et al., 2000, D'Souza et al., 2001, and Waugh et al., 2002). Among these proteins, Ras1 is a major C. neoformans Ras protein supporting high-temperature growth and invasive growth essential for survival and growth in a host and stimulating sexual differentiation (Alspaugh et al., 2000). Though the ras2Δ deletion mutant does not have a recognizable phenotype, the overexpression of RAS2 somewhat inhibits most of the ras1 mutation phenotypes (Waugh et al., 2002). Like S. cerevisiae, disruption of the RAS1 and RAS2 genes affects cell viability at every temperature, which indicates that the Ras protein is essential for the growth of cells in general. Among various Ras-related phenotypes, only invasive growth and mating are cAMP-dependent, but high-temperature growth is cAMP-independent and a Ras1-specific phenotype (Alspaugh et al., 2000, Waugh et al., 2003). Interestingly, Cac1 does not bear a Leucine-rich repeat (LRR) domain, which is a binding site to a GTP-binding Ras in S. cerevisiae (Shima et al., 1997). Since an adenylyl cyclase/cyclase-related protein complex can provide a secondary Ras-binding site to activate the protein complex as shown in S. cerevisiae (Shima et al., 2000), Ras1 can still interact with an Aca1/Cac1 complex for activating the Ras1 in C. neoformans. Recently, it has been reported that a GEF protein, Cdc24, is a Ras-effecter protein, and regulates the growth of C. neoformans at high temperature in a lower system of Ras1 and a higher system of Rho-like GTPase Cdc42 (Nichols et al., 2007). Consequently, C. neoformans cAMP-signal transduction pathway is regulated by three different higher signal regulators, Ras1, Gpa1 and Aca1.
Despite the presence of the common higher signal regulators (Ras1, Aca1 and Gpa1) of Cac1, functional correlation between the components and target gene regulated by each regulator in C. neoformans remains still unclear.

SUMMARY

The present invention provides to finding a new target gene to develop an antifungal agent by investigating a signal transduction network of HOG, Ras and cAMP pathways.
In one aspect, a use of an inhibitor against at least one protein or a gene coding for the same selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans to prepare an antifungal agent, an antifungal pharmaceutical composition including the inhibitor, and a method of treating fungal infection including injecting an effective amount of the inhibitor into a subject are provided.
In another aspect, a use of at least one protein or a gene coding for the same selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans to screen an antifungal agent, a composition for screening an antifungal agent including the protein or gene, and a method of screening an antifungal agent including contacting the protein or gene with a candidate material and determining whether the candidate material inhibits or stimulates an activity of the protein or gene are provided.
In still another aspect, a use of an inhibitor against at least one protein or a gene coding for the same selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of C. neoformans to prepare an antifungal agent, an antifungal pharmaceutical composition including the inhibitor, and a method of treating fungal infection including injecting an effective amount of the inhibitor into a subject are provided.
In yet another aspect, a use of at least one protein or a gene coding for the same selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of C. neoformans to screen an antifungal agent, a composition for screening an antifungal agent including the protein or gene, and a method of screening an antifungal agent including contacting the protein or gene with a candidate material and determining whether the candidate material inhibits or stimulates an activity of the protein or gene are provided.
In the present invention, to regulate a HOG pathway of C. neoformans, roles of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 genes are investigated to reveal that a biosynthesis level of ergosterol is increased when these genes are inhibited. Since a large amount of ergosterol is distributed on a fungal cell membrane when the genes are inhibited, an efficiency of an ergosterol-binding antifungal agent can be considerably increased due to many working points of the ergosterol-binding antifungal agents. In addition, in the present invention, to regulate Ras and cAMP pathways of C. neoformans, roles of RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 and HSP122 genes were investigated to reveal that sensitivity to a polyene- or azole-based drug is increased when these genes are inhibited. Thus, an antifungal pharmaceutical composition including an inhibitor against the gene or protein encoded by the same can be used as an excellent combined antibacterial drug which can reduce an amount of a conventional antifungal agent used and increase efficiency.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other aspects of the inventive concept will become more readily apparent by describing in further detail exemplary embodiments thereof which reference to the accompanying drawings, in which:

FIG. 1 shows identification of genes whose expression is regulated by Hog, Ssk1 and Skn7 of C. neoformans under normal conditions with no stress on the genome level (fold change is expressed by color);

FIG. 2 shows analysis results for induction of ergosterol biosynthesis genes by disturbance of a HOG signal transduction pathway and an ergosterol content in a cell;

FIG. 3 shows analysis results showing that the inhibition of the HOG pathway gives an elevated antifungal effect with amphotericin B in C. neoformans;

FIG. 4 shows analysis results showing that the inhibition of the HOG pathway gives an antagonistic antifungal effect with respect to some azole drugs in C. neoformans;

FIG. 5 shows analysis results showing that gene coding for an efflux pump of Na⁺ and K⁺, ENA1 and NHA1, are lower-system target genes regulated by the HOG pathway, and the inhibition of these genes gives high sensitivity to polyene-based drugs and azole-based drugs;

FIG. 6 shows analysis results for transcripts of ras1Δ, aca1Δ, gpa1Δ, cac1Δ, and pka1Δ pka2Δ deletion mutants of C. neoformans (fold change is expressed by color);

FIG. 7 shows functional categories of genes differently regulated by the ras1Δ, aca1Δ, gpa1Δ, cac1Δ, and pka1Δ pka2Δ deletion mutants of C. neoformans;

FIG. 8 shows regulation of a significant ratio of Ras- and cAMP-dependent genes by environmental stress;

FIG. 9 shows an identification result of a cAMP-signal transduction pathway dependent gene in C. neoformans;

FIG. 10 shows analysis results showing that the inhibition of the Ras- and cAMP-signal transduction pathways increases a sensitivity to polyene-based or azole-based (itraconazole) antifungal agent, independent of ergosterol biosynthesis; and

FIG. 11 shows analysis results showing that the expression of HSP12 and HSP122 is up-regulated by the cAMP- and HOG-signal transduction pathways, and increases sensitivity to polyene-based antifungal agents by hsp12Δ and hsp122Δ deletion mutants.

DETAILED DESCRIPTION

Hereinafter, the present invention will be described in detail.
An antifungal pharmaceutical composition including an inhibitor against at least one protein or gene coding for the same selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans to prepare an antifungal agent is provided.
A HOG1 pathway is a cell signal transduction system regulating responses induced by various stresses. Particularly, since fungi use a two-element-like phosphorelay system composed of three elements such as a hybrid sensor kinase, a histidine-containing phosphotransfer protein (HPt) and a response regulator, which are not present in mammals, the inventors carried on an investigation of roles of genes involved in the HOG1 pathway to develop a target for a new antifungal agent. As a result, surprisingly, it was found that, in the regulation of the HOG pathway in C. neoformans, a biosynthesis level of ergosterol is increased when SSK1, TCO2, SSK2, PBS2, and HOG1 genes are inhibited. As will be confirmed in the following embodiments, when the genes are inhibited, a large amount of ergosterol is distributed on a fungal cell membrane and working points of the ergosterol-binding antifungal agent are also increased. Therefore, an efficiency of the ergosterol-binding antifungal agent can be considerably increased. In addition, when ENA1 and NHA1 genes, the expression of which is known to be regulated by the HOG signal transduction pathway, are inhibited, it is confirmed that, regardless of the change in ergosterol level, a sensitivity to polyene-based drugs such as amphotericin B and azole-based drugs are considerably increased. Thus, the antifungal pharmaceutical composition including an inhibitor against at least one protein selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans may be used as an excellent combined antibacterial drug which can reduce an amount of the conventional ergosterol-binding antifungal agent or azole-based antifungal agent used and increase efficiency.
Accordingly, a use of an inhibitor against at least one protein or gene coding for the same selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans to prepare an antifungal agent, an antifungal pharmaceutical composition including the inhibitor, and a method of treating fungal infection including injecting an effective amount of the inhibitor to a subject are provided.
In the specification, it is understood that SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 used as a target to interrupt a HOG1 signal transduction system indicates an Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 protein, or an SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 gene. Thus, it is understood that an SSK1, TCO2, SSK2, PBS2, HOG 1, ENA1 or NHA1 inhibitor includes either an inhibitor against an Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 protein or an inhibitor against an SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 gene.
In one exemplary embodiment, the inhibitor against at least one protein selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans may bind to the protein to inhibit an activity thereof, thereby interrupting signal transduction. In another exemplary embodiment, the inhibitor against at least one gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of C. neoformans may inhibit expression of the gene to interrupt signal transduction. In the specification, the SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 gene may be a DNA coding for the gene or mRNA transcripted therefrom. Thus, the inhibitor against the gene may bind to the gene itself to disturb transcription or bind to the mRNA transcripted from the gene to disturb translation of the mRNA.
In one exemplary embodiment, the Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 protein may have an amino acid sequence of SEQ ID NOs: 1-7 respectively, and the SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 gene may have a nucleic acid sequence of SEQ ID NOs: 8-14 respectively or a cDNA sequence of SEQ ID NOs:15-21 respectively. However, this is merely an example of a sequence of C. neoformans antigen-type A H99 strain, and the sequence of the SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 is not limited thereto.
In the specification, it is understood that the Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 protein or the SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 gene includes a variant or fragment thereof having substantially the same activity as the protein or gene.
In one exemplary embodiment, the antifungal pharmaceutical composition may include at least one inhibitor against at least one protein selected from the group consisting of Ssk1, Ena1 and Nha1. SSK1 may be a good target to develop an antifungal agent because it is not only an important upstream reaction regulator of HOG1, but also a gene which is not found in mammals. Therefore, the SSK1 inhibitor may reduce a possibility of generating certain side effects and increase a biosynthesis level of ergosterol in a fungus, thereby improving the efficiency of an ergosterol-binding antifungal agent. Meanwhile, ENA1 and NHA1 are defined as lower-system target genes regulated by a HOG pathway. When these genes are inhibited, sensitivity to an azole-based drug such as fluconazole, ketoconazole and itraconazole is also considerably increased as well as that to a polyene-based drug such as amphotericin B. Therefore, the inhibitors simultaneously or independently inhibiting these genes may exhibit very high antifungal activities when used in combination with the polyene- or azole-based drug.
The inhibition of the Ssk1, Tco2, Ssk2, Pbs2 or Hog1 protein or gene improves the biosynthesis of ergosterol and increases the distribution of ergosterol on a fungal cell membrane. Thus, since binding targets of the ergosterol-binding antifungal agent disrupting the fungal cell membrane by being bound to ergosterol are increased, an effective amount of the ergosterol-binding antifungal agent may be reduced and a killing ability of the ergosterol-binding antifungal agent may be increased. In addition, the inhibitors against ENA1 and NHA1 considerably increase drug sensitivities to the azole-based antifungal agent as well as the polyene-based antifungal agent, and thus amounts of these drugs used can be reduced and a killing ability may be improved. Such an antifungal activity induced by the inhibition of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 may treat cryptococcal disease and encephalomeningitis by infection of C. neoformans.
Thus, in one exemplary embodiment, use of an Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 inhibitor to prepare a drug for treating a disease such as cryptococcal disease or encephalomeningitis, a pharmaceutical composition for treating a disease such as cryptococcal disease or encephalomeningitis including the Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 inhibitor, and a method of treating a disease such as cryptococcal disease or encephalomeningitis including injecting an effective amount of the Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 inhibitor to a subject are provided.
Other than cryptococcal disease or encephalomeningitis exemplified in the specification, diseases induced by fungal infection are well known in the art. In the specification, the inhibition of the Ssk1, Tco2, Ssk2, Pbs2, Hog1 protein or gene is revealed to improve the efficiency of the ergosterol-binding antifungal agent or azole-based antifungal agent, and thus those of ordinary skill in the art may inhibit the protein or gene to prevent or treat a disease induced by fungal infection.
In the specification, the “inhibitor of the Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 protein” used to interrupt a HOG1 signal transduction system includes all inhibitors binding to the Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 protein to interrupt signal transduction. For example, such an inhibitor may be a peptide or compound binding to the Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 protein. Such an inhibitor may be selected by a screening method to be described below in analysis of a protein structure, and may be designed using a known method in the art. In one exemplary embodiment, the inhibitor may be a polyclonal or monoclonal antibody with respect to at least one protein selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 or Nha1 of C. neoformans. Such a polyclonal or monoclonal antibody may be prepared using a method of preparing an antibody known in the art.
In the present invention, the “inhibitor against the SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 gene” used to interrupt a HOG1 signal transduction system includes every inhibitor inhibiting the expression of the SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 or NHA1 gene to interrupt signal transduction. For example, such an inhibitor may be a peptide, nucleic acid or compound binding to the gene. The inhibitor may be selected by a screening method shown below in cell-based screening, and may be designed using a known method in the art. In one exemplary embodiment, the inhibitor may be an antisense oligonucleotide, siRNA, shRNA, miRNA or vector including the same with respect to at least one gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of C. neoformans. The antisense oligonucleotide, siRNA, shRNA, miRNA or vector including the same may be prepared using a known method in the art. In the specification, the “vector” refers to a gene construct including foreign DNA inserted into a genome coding for a polypeptide. The vector used herein is a vector in which a nucleic acid sequence inhibiting the gene is inserted into a genome, and may include a DNA vector, a plasmid vector, a cosmid vector, a bacteriophage vector, a yeast vector, or a viral vector.
A pharmaceutical antifungal pharmaceutical composition including an inhibitor against at least one protein or gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of C. neoformans does not exhibit an antifungal activity alone, but increases a fungal killing ability of an antifungal agent in combination with the ergosterol-binding antifungal agent or azole-based antifungal agent. Therefore, the antifungal pharmaceutical composition may be sequentially or simultaneously injected with the ergosterol-binding antifungal agent or azole-based antifungal agent.
The ergosterol-binding antifungal agent refers to an antifungal agent binding to ergosterol on a fungal cell membrane to induce depolarization of the cell membrane and forming a hole to induce the loss of contents in a cell, thereby killing fungi. Such an ergosterol-binding antifungal agent is known in the art, and any ergosterol-binding antifungal agent considerably increases the antifungal effect when used with the antifungal pharmaceutical composition. In one exemplary embodiment, the ergosterol-binding antifungal agent may be a polyene-based antifungal agent. In one aspect, the polyene-based antifungal agent may be at least one antifungal agent selected from the group consisting of amphotericin B, natamycin, rimocidin, filipin, nystatin and candicin. In the preferable embodiment, the polyene-based antifungal agent is amphotericin B. Meanwhile, the azole-based antifungal agent may be at least one antifungal agent selected from the group consisting of ketoconazole, fluconazole, itraconazole and voriconazole.
In this aspect, an antifungal combined formulation including the antifungal pharmaceutical composition including the inhibitor of the present invention; and a known ergosterol-binding antifungal agent or azole-based antifungal agent are provided.
An antifungal pharmaceutical composition including an inhibitor against at least one protein or gene coding for the same selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of C. neoformans to prepare an antifungal agent is also provided.
The inventors performed investigation on, rather than simply the genes involved in Ras and cAMP pathways, the roles of the genes involved in Ras and cAMP pathways, to develop a target for a new antifungal agent. The result newly revealed that, surprisingly, in the Ras and cAMP pathways of C. neoformans, when a RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene is inhibited, a sensitivity to one of the polyene- or azole-based drugs, an itraconazole antifungal agent, is increased. As will be confirmed in the following exemplary embodiment, when the genes are inhibited, the sensitivity to the polyene- or itraconazole antifungal agent in a fungus may be considerably increased. Thus, the antifungal pharmaceutical composition including an inhibitor against at least one protein or gene coding for the same selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of C. neoformans may be used as an excellent combined antibacterial drug which can reduce an amount of a conventional polyene-based or itraconazole antifungal agent used and improve an efficiency.
Therefore, use of an inhibitor against at least one protein or gene coding for the same selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of C. neoformans to prepare an antifungal agent, an antifungal pharmaceutical composition including the inhibitor, and a method of treating fungal infection including injecting an effective amount of the inhibitor into a subject are provided.
In the present invention, it is construed that RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 used as a target to interrupt the Ras- and cAMP signal transduction systems is a Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein, or a RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene. Accordingly, it is construed that a RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 inhibitor includes every inhibitor against the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein or inhibitor against the RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene.
In one exemplary embodiment, an inhibitor against at least one protein selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of C. neoformans may be an inhibitor that binds to the protein to inhibit an activity, thereby interrupting signal transduction. In another exemplary embodiment, an inhibitor against at least one gene selected from the group consisting of RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 and HSP122 of C. neoformans may be an inhibitor inhibiting expression of the gene, thereby interrupting signal transduction. In the present invention, the RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene may be DNA coding for the gene or mRNA transcripted therefrom. Therefore, the inhibitor against the gene may bind to the gene to interrupt transcription or bind to mRNA transcripted from the gene to interrupt translation of the mRNA.
In one exemplary embodiment of the present invention, the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein may have an amino acid sequence of one of SEQ ID NOs: 16-24 respectively, and the RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene may have a nucleic acid or cDNA sequence corresponding to the protein. However, the sequence just shows a sequence of a C. neoformans antigen-type A H99 strain, and thus the sequence of the RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 is not limited thereto.
In the present invention, it is construed that the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein, or the RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene includes a variant or fragment thereof having substantially the same activity as the protein or gene.
In one exemplary embodiment, the inhibitor may be an inhibitor against a Cac1 or Pka1 protein or gene.
Inhibition of the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein or gene coding for the same may increase a sensitivity to a polyene- or azole-based antifungal agent, and thus an effective amount of the polyene- or azole-based antifungal agent may be reduced and a killing ability of the antifungal agent may be increased. An antifungal activity caused by the inhibition of the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 may treat cryptococcal disease and encephalomeningitis induced by infection of C. neoformans.
Thus, in one exemplary embodiment of the present invention, use of a Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 inhibitor to prepare a drug for treating diseases such as cryptococcal disease and encephalomeningitis, a pharmaceutical composition for treating diseases such as cryptococcal disease and encephalomeningitis including the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 inhibitor and a method of treating diseases such as cryptococcal disease and encephalomeningitis including injecting an effective amount of the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 inhibitor into a subject are provided.
Other than the cryptococcal disease and encephalomeningitis stated herein, diseases induced by fungal infection are well known in the art. In the present invention, it is revealed that the inhibition of the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein or gene coding for the same increases the sensitivity to a polyene- or azole-based itraconazole antifungal agent, thereby increasing the efficiency of the antifungal agent. Therefore, those of ordinary skill in the art can inhibit the protein or genes to prevent or treat a disease induced by the fungal infection.
In the present invention, the “inhibitor of the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein” used to interrupt the RAS or cAMP signal transduction system includes every inhibitor binding to the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein to interrupt signal transduction. For example, such an inhibitor may be a peptide or compound binding to the Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 or Hsp122 protein. The inhibitor may be selected by a screening method to be described below in analysis of a protein structure, and may be designed using a method known in the art. In one exemplary embodiment, the inhibitor may be a polyclonal or monoclonal antibody with respect to at least one protein selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of C. neoformans. The polyclonal or monoclonal antibody may be constructed using a known method of constructing an antibody in the art.
In the present invention, the “inhibitor of the RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene” used to interrupt the RAS or cAMP signal transduction system includes every inhibitor inhibiting the expression of the RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 gene to interrupt signal transduction. For example, such an inhibitor may be a peptide, nucleic acid or compound binding to the gene. The inhibitor may be selected by a screening method to be described below in analysis of a protein structure, and may be designed using a method known in the art. In one exemplary embodiment, the inhibitor may be an antisense oligonucleotide, siRNA, shRNA, miRNA or a vector including the same with respect to at least one gene selected from the group consisting of RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 and HSP122 of C. neoformans. Such an antisense oligonucleotide, siRNA, shRNA, miRNA or a vector including the same may be constructed using a known method in the art. In the present invention, the “vector” is a gene construct including foreign DNA inserted into a genome coding for a polypeptide. The vector related to the present invention may be a vector formed by inserting a nucleic acid sequence inhibiting the gene into the genome, which may be a DNA vector, plasmid vector, cosmid vector, bacteriophage vector, yeast vector or viral vector.
The antifungal pharmaceutical composition of the present invention including the inhibitor against at least one protein or gene coding for the same selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1 , Hsp12 and Hsp122 of C. neoformans increases a fungal killing ability of the antifungal agent in combination with a polyene- or azole-based antifungal agent. Thus, the antifungal pharmaceutical composition of the present invention is sequentially or simultaneously injected with the polyene- or azole-based antifungal agent. The polyene- or azole-based antifungal agent is known in the art, and any one of the polyene- or azole-based antifungal agent significantly increases the antifungal effect when used with the antifungal pharmaceutical composition of the present invention. In one aspect, the polyene-based antifungal agent may be at least one of amphotericin B, natamycin, rimocidin, filipin, nystatin and candicin. In a preferable embodiment, the polyene-based antifungal agent may be amphotericin B. In one aspect, the azole-based antifungal agent may be at least one selected from the group consisting of ketoconazole, fluconazole, itraconazole and voriconazole.
In another exemplary embodiment, the antifungal pharmaceutical composition may be sequentially or simultaneously injected along with the inhibitor against at least one protein selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of C. neoformans. As will be confirmed from the following exemplary embodiment, when expression of HOG1 is inhibited as well as expression of CAC1 or PKA1 of C. neoformans, the sensitivity to amphotericin B is proportionally increased. This is because genes of the cAMP pathway increase the sensitivity to amphotericin B by a mechanism different from that increasing the sensitivity to amphotericin B due to the increase in biosynthesis of ergosterol when the genes of the HOG pathway described above are inhibited.
According to the aspect, the present invention also provides an antifungal combined formulation including the antifungal pharmaceutical composition including the inhibitor of the present invention; and at least one antifungal agent selected from the group consisting of a polyene-based antifungal agent, an azole-based antifungal agent and an inhibitor against at least one protein or gene coding for the same selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans. Preferably, the antifungal combined formulation may include the antifungal pharmaceutical composition including the inhibitor of the present invention, a polyene-based antifungal agent, and at least one antifungal agent selected from the group consisting of inhibitors each against at least one protein or gene coding for the same selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of C. neoformans.
The antifungal pharmaceutical composition or antifungal combined formulation of the present invention may be prepared using a pharmaceutically suitable and physiologically available adjuvant, wherein the adjuvant may be a solubilizer such as a diluting agent, a dispersing agent, a sweetening agent, a binding agent, a coating agent, a blowing agent, a lubricant, a gliding agent or a flavoring agent.
The antifungal pharmaceutical composition of the present invention may be formulated into a pharmaceutical composition including at least one pharmaceutically available carrier other than an active component for administration.
In the composition formulated in a liquid-phase solution, a pharmaceutically available carrier may be suitable for sterilization and living organisms, and may be saline, sterilized water, Ringer's solution, buffered saline, albumine injection, dextrose solution, maltodextrin solution, glycerol, ethanol or a mixture of at least one thereof. When necessary, another conventional additive such as an antioxidant, buffer or bacteriostatic agent may be added. In addition, a diluting agent, a dispersing agent, a surfactant, a binding agent or a lubricant may be added, and thus the composition may be formulated in the form of an injectable formulation such as an aqueous solution, a suspension or an emulsion, a pill, a capsule, a granule or a tablet. Furthermore, the composition may be formulated using a suitable method in the art, which is disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton, Pa. according to diseases or components.
Types of a pharmaceutical formulation of the pharmaceutical composition of the present invention may include a granule, an acida, a coated tablet, a tablet, a capsule, a suppository, a syrup, a juice, a suspension, an emulsion, a drop or injectable liquid and a sustained-release formulation of an active compound.
The pharmaceutical composition of the present invention may be injected by a conventional method via an intravenous, intraarterial, abdominal, sternal, percutaneous, nasal, inhaling, local, rectal, oral, intraocular or intradermal route.
An effective amount of the active component of the pharmaceutical composition of the present invention indicates an amount required for preventing or treating a disease, or achieving an effect of inducing bone growth. Accordingly, the effective amount may vary depending on various factors such as kinds of a disease, severity of a disease, kinds and contents of the active component and other components contained in the composition, kinds of dosage forms and patient's age, weight, health, sex and dietary habits, injection times and routes, release rates of the composition, duration of treatment, and co-injected drugs. For adults, when the composition is injected one or more times a day, the dosages may be, but not limited to, 0.1 ng/kg to 10 g/kg for a compound, 0.1 ng/kg to 10 g/kg for a polypeptide, protein or antibody, and 0.01 ng/kg to 10 g/kg for an antisense oligonucleotide, siRNA, shRNAi or miRNA, respectively.
In the present invention, the “subject” may be, but not limited to, a human, orangutan, chimpanzee, mouse, rat, dog, cow, chicken, pig, goat or sheep.
Furthermore, the present invention provides a use of at least one protein selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, and Nha1 of C. neoformans to screen an antifungal agent, a composition for screening an antifungal agent including the protein, and a method of screening an antifungal agent including contacting the protein with a candidate material and determining whether the candidate material inhibits or stimulates an activity of the protein.
The present invention also provides a use of at least one gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of C. neoformans to screen an antifungal agent, a composition for screening an antifungal agent including the gene, and a method of screening an antifungal agent including contacting the gene with a candidate material and determining whether the candidate material inhibits or stimulates expression of the gene.
The present invention also provides a method of screening an antifungal agent by a yeast two-hybrid system capable of monitoring physical contact between SSK1 and SSK2, SSK1 and YPD1 or YPD1 and TCO2 proteins of C. neoformans. When this method is used, a large amount of the candidate materials can be screened quickly.
As described above, when SSK1, TCO2, SSK2, PBS2 or HOG1 of C. neoformans is inhibited, the HOG1 signal transduction system is interrupted, and thus biosynthesis of ergosterol is improved. Therefore, the material screened to inhibit the protein or gene may be used as an antifungal agent improving a fungal killing ability, along with an ergosterol-binding antifungal agent. The material screened to inhibit ENA1 or NHA1 may be used as an antifungal agent improving a fungal killing ability when used with an ergosterol-binding antifungal agent or azole-based antifungal agent.
The present invention also provides a use of at least one protein selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1 , Pka1, Hsp12 and Hsp122 of C. neoformans to screen an antifungal agent, a composition for screening an antifungal agent including the protein, and a method of screening an antifungal agent including contacting the protein with a candidate material and determining whether the candidate material inhibits or stimulates an activity of the protein.
The present invention also provides a use of at least one gene selected from the group consisting of RAS1, RAS2, CDC24, GPA1, ACA1, PKA1, HSP12 and HSP122 of C. neoformans to screen an antifungal agent, a composition for screening an antifungal agent including the gene, and a method of screening an antifungal agent including contacting the gene with a candidate material and determining whether the candidate material inhibits or stimulates expression of the gene.
The present invention also provides a method of screening an antifungal agent by a yeast two-hybrid system capable of monitoring physical contact between Gpa1 and Cac1 , Cac1 and Aca1, Ras1 and Cdc24 or Ras2 and Cdc24 proteins of C. neoformans. When this method is used, a large amount of the candidate materials can be screened quickly.
As described above, when RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 or HSP122 of C. neoformans is inhibited, the RAS or cAMP signal transduction system is interrupted, thereby increasing a sensitivity to a polyene- or azole-based antifungal agent. Thus, the material screened to inhibit the protein or gene may be used as an antifungal agent improving a fungal killing ability when used with the polyene- or azole-based antifungal agent.
Confirmation of the reaction between the protein or gene and the candidate material may be performed by a conventional method of confirming the reaction between a protein and a protein, a protein and a compound, DNA and DNA, DNA and RNA, DNA and a protein, DNA and a compound, RNA and a protein, or RNA and a compound. For example, a hybrid test for confirming a bond between the gene and a candidate material in vitro, a method of measuring an expression level of the gene through northern blotting, quantitative PCR or quantitative real time PCR after reaction of mammalian cell and a test material, a method of connecting a reporter gene to the gene to introduce the gene into a cell, reacting the cell with a test material and measuring an expression level of a reporter protein, a method of reacting the protein with a candidate material and measuring an activity, a yeast two-hybrid, searching for a phage-displayed peptide clone binding to an Idbf protein, high throughput screening (HTS) using a natural substance and a chemical library, drug hit HTS, cell-based screening or a screening method using a DNA array may be used.
The screening composition may include distilled water or a buffer stably maintaining the structure of a nucleic acid or protein, other than the protein or gene. In addition, the screening composition may include a cell expressing the protein or gene, or a cell containing a plasmid expressing the gene in the presence of a promoter regulating a transcription rate for an in vivo test.
In the screening method of the present invention, a test material may be individually a nucleic acid, a protein, a peptide, a different extract or natural substance or a compound assumed to have possibility as a drug inhibiting signal transduction through a HOG1 signal transduction system according to a conventional screening method or randomly selected.
The matters related to a genetic engineering technique in the present invention are made more clear by the literatures disclosed by Sambrook et al. [Molecular Cloning, A Laboratory Manual, Cold Spring Harbor laboratory Press, Cold Spring Harbor, N.Y. (2001)] and Frederick M. Ausubel et al. [Current protocols in molecular biology volume 1, 2, 3, John Wiley & Sons, Inc. (1994)].
While exemplary embodiments have been disclosed herein, it should be understood that other variations may be possible. Such variations are not to be regarded as a departure from the spirit and scope of exemplary embodiments of the present application, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

EXAMPLES

Experimental Procedures

Strains and Growth Conditions

The C. neoformans strains used in this examples are listed in Table 1[Bahn Y S, Geunes-Boyer S, Heitman J (2007) Eukaryot Cell 6: 2278-2289.; Bahn Y S, Kojima K, Cox G M, Heitman J (2005) Mol Biol Cell 16: 2285-2300.; Bahn Y S, Kojima K, Cox G M, Heitman J (2006) Mol Biol Cell 17: 3122-3135.; Perfect J R, Ketabchi N, Cox G M, Ingram C W, Beiser C L (1993) J Clin Microbiol 31: 3305-3309; Kwon-Chung K J, Edman J C, Wickes B L (1992) Genetic association of mating types and virulence in Cryptococcus neoformans. Infect Immun 60: 602-605.].
The C. neoformans strains were cultured in YPD (yeast extract-peptone-dextrose) medium unless indicated separately.

TABLE 1

Strain	Genotype	Parent

Serotype A
H99	MATα
KN99	MATa
CBN45	MATα ras1Δ::NEO	H99
CBN64	MATα ras1Δ::NEO RAS1::NAT	CBN45
MWC12	MATα ras2Δ::URA5	H99
CBN32	MATα cdc24Δ::NEO	H99
CBN33	MATα cdc24Δ::NEO CDC24::NAT	CBN32
YSB6	MATα aca1Δ::NAT-STM#43	H99
YSB51	MATα ras1Δ::NAT-STM#150	H99
YSB53	MATα ras1Δ::NAT-STM#150	H99
YSB64	MATα hog1Δ::NAT-STM#177	H99
YSB123	MATα pbs2Δ::NAT-STM#213	H99
YSB261	MATα ssk1Δ::NAT-STM#205	H99
YSB264	MATα ssk2Δ::NAT-STM#210	H99
YSB349	MATα skn7Δ::NAT-STM#201	H99
YSB278	MATα tco1Δ::NAT-STM#102	H99
YSB281	MATα tco2Δ::NAT-STM#116	H99
YSB324	MATα tco1Δ::NAT-STM#102 tco2D::NEO	YSB278
YSB284	MATα tco3Δ::NAT-STM#119	H99
YSB417	MATα tco4Δ::NAT-STM#123	H99
YSB286	MATα tco5Δ::NAT-STM#125	H99
YSB348	MATα tco7Δ::NAT-STM#209	H99
YSB73	MATα ras1Δ::NEO	H99
YSB42	MATα cac1Δ::NAT-STM#159	H99
YSB83	MATα gpa1Δ::NAT	H99
YSB188	MATα pka1Δ::NAT	H99
YSB194	MATα pka2Δ::NAT-STM#205	H99
YSB200	MATα pka1Δ::NAT pka2Δ::NEO	YSB188
YSB174	MATα aca1Δ::NAT-STM#43 ras1::NEO	YSB278
YSB182	MATα cac1Δ::NAT-STM#159 ras1::NEO	H99
YSB156	MATα hog1Δ::NAT-STM#177 cac1::NEO	H99
YSB112	MATα ura5 pka1::URA5 hog1::NATSTM#177	H99
YSB58	MATa aca1Δ::NEO	KN99
YSB79	MATa cac1Δ::NEO	KN99
YSB81	MATa hog1Δ::NEO	KN99
YSB175	MATα aca1Δ::NEO ras1Δ::NATSTM#150	YSB58
YSB187	MATα cac1Δ::NEO ras1Δ::NATSTM#150	YSB79
YSB606	MATα gre2Δ::NAT-STM#224	H99
YSB607	MATα gre2Δ::NAT-STM#224	H99
YSB609	MATα pkp1Δ::NAT-STM#224	H99
YSB610	MATα pkp1Δ::NAT-STM#224	H99
YSB599	MATα hsp12Δ::NAT-STM#224	H99
YSB600	MATα hsp12Δ::NAT-STM#224	H99
YSB603	MATα hsp122Δ::NAT-STM#224	H99
YSB604	MATα hsp122Δ::NAT-STM#224	H99
YSB590	MATα ena1Δ::NAT nha1::NEO	AI167
YSB591	MATα ena1Δ::NAT nha1::NEO	AI167
YSB586	MATα nha1Δ::NEO	H99
YSB587	MATα nha1Δ::NEO	H99
YSB588	MATα nha1Δ::NEO	H99
Serotype D
JEC21	MATα
B-3501	MATα
YSB267	MATα pbs2Δ::NAT-STM#213	JEC21
YSB139	MATα hog1Δ::NAT-STM#177	JEC21
YSB338	MATα ssk2Δ::NAT-STM#210	JEC21
YSB340	MATα ssk2Δ::NAT-STM#210	B-3501

Each NAT-STM# indicates the Nat^rmarker with a unique signature tag.

DNA Microarray Array Analysis

For total RNA isolation used in DNA microarray, the wild-type H99, hog1Δ (YSB64), ssk1Δ (YSB261), and skn7Δ (YSB349), ras1Δ (YSB51), aca1Δ (YSB6), gpa1Δ (YSB83), cac1Δ (YSB42) and pka1Δ pka2Δ (YSB200) mutant strains were grown in 50 ml YPD medium at 30° C. for 16 hr. Then 5 ml of the overnight culture was inoculated into a 100 ml of fresh YPD medium and further incubated for 4-5 hr at 30° C. until it approximately reaches to the 1.0 of optical density (OD) at 600 nm (OD600 nm=1.0). For zero-time samples, 50 ml out of the 100 ml culture was sampled and rapidly frozen in liquid nitrogen. To the remaining 50 ml culture, 50 ml of YPD containing 2 M NaCl, 40 μg/ml fludioxonil (PESTANAL, Sigma, 100 mg/ml stock solution in dimethylsulfoxide), or 5 mM H2O2 was added (final concentration of 1 M NaCl, 20 μg/ml fludioxonil, or 2.5 mM H2O2, respectively). During incubation in each stress-inducing medium, 50 ml of the culture was sampled at 30 and 60 min, pelleted in a tabletop centrifuge, frozen in liquid nitrogen, and lyophilized overnight. The lyophilized cells were subsequently used for total RNA isolation. As biological replicates for DNA microarray, 3 independent cultures for each strain and growth condition were prepared for total RNA isolation.

Total RNA Preparation

For total RNA isolation, the lyophilized cell pellets were added with 3 ml volume of sterile 3 mm glass bead (SIGMUND LINDER), homogenized by shaking, added with 4 ml of TRizol reagent (Tri reagent, Molecular Research Center), and allowed to incubate at room temperature for 5 min. Then 800 μl of chloroform was added, incubated for 3 min at room temperature, transferred to the 15 ml of the round-bottom tube (SPL), and centrifuged by 10,000 rpm at 4° C. for 15 min (Sorvall SS-34 rotor). Two milliliter of the supernatant was transferred to the new round-bottom tube, added with 2 ml isopropanol, inverted several times, and allowed to incubate for 10 min at room temperature. Then the mixture was re-centrifuged by 10,000 rpm at 4° C. for 10 min, and its pellet was washed with 4 ml of 75% ethanol diluted with diethylpyrocarbonate (DEPC) treated water and centrifuged by 8,000 rpm at 4° C. for 5 min. The pellet was dried at room temperature and resuspended with 500 μl DEPC-treated water. Concentration and purity of total RNA sample were calculated by measuring OD260 nm and gel electrophoresis, respectively. For control total RNA (for Cy3 labeling), all of total RNAs prepared from wild-type, hog1Δ, ssk1Δ, skn7Δ, ras1Δ, aca1Δ, gpa1Δ, cac1Δ and pka1Δ pka2Δ mutant cells grown in conditions described above were pooled (pooled reference RNAs).

cDNA Synthesis and Cy3/Cy5 Labeling

For cDNA synthesis, total RNA concentration was adjusted to 1 μg/μl with DEPC-treated water, and 15 μl of the total RNA (15 μg) was added with 1 μl of 5 μg/μl oligo dT (5′-TTTTTTTTTTTTTTTTTTTTV-3′)/pdN6 (Amersham)(1:1 mixture of 10 μg/μl, respectively), incubated at 70° C. for 10 min, and place on ice for 10 min. Then 15 μl of cDNA synthesis mixture {3 μl 0.1 M DTT, 0.5 μl RNasin [Promega], 0.6 μl aa-dUTP (5-(3-aminoallyl)-2′-deoxyuridine 5′-triphosphate)/dNTPs [a mixture of 6 μl dTTP (100 mM), 4 μl aa-dUTP (100 mM), 10 μl dATP (100 mM), 10 μl dCTP (100 mM), 10 μl dGTP (100 mM)], 1.5 μl AffinityScript reverse transcriptase (Stratagene), 3 μl AffinityScript buffer, 7 μl water] was added and incubated at 42° C. for 2 hrs. Then 10 μl of 1 N NaOH and 10 μl of 0.5 M EDTA (pH 8.0) were added and incubated at 65° C. for 15 min. After incubation, 25 μl of 1 M HEPES buffer (pH 8.0) and 450 μl of DEPE-treated water were added, and the whole mixture was concentrated through Microcon30 filter (Milipore) and vacuum-dried for 1 hr.
For Cy3 and Cy5 (Amersham) labeling of the prepared cDNA, Cy3 and Cy5 were dissolved in 10 μl DMSO and 1.25 μl of each dye was aliquoted into separate tubes. The cDNAs prepared as described above were added with 9 μl of 0.05 M Na-bicarbonate (pH 8.0) and incubate at room temperature for 15 min. The cDNAs prepared from pooled reference RNAs were mixed with Cy3 as a control and the cDNAs prepared from each test RNA (each experimental condition) were mixed with Cy5. Each mixture was further incubated at room temperature for 1 hr in the dark and purified by QIAquick PCR purification kit (QIAGEN).

Microarray Hybridization and Washing

C. neoformans serotype D 70-mer microarray slide containing 7,936 spots (Duke University) was pre-hybridized at 42° C. in 60 ml of pre-hybridization buffer [42.4 ml sterile distilled water, 2 ml 30% BSA (Sigma), 600 μl 10% SDS, 15 ml 20×SSC], washed with distilled water and isopropanol, and dried by brief centrifugation (110×g, 2 min). The Cy3- and Cy5-labeled cDNA samples were combined, concentrated through Microcon30 filter, and vaccum-dried. The dried cDNA samples were resuspended with 24 μl of 1× hybridization buffer [250 μl 50% formamide, 125 μl 20×SSC, 5 μl 10% SDS, 120 μl dH2O, total 500 μl], added with 1 μl polyA tail DNA (Sigma), further incubated at 100° C. for 3 min and allowed to cool for 5 min at room temperature. The microarray slides were aligned into the hybridization chamber (DieTech), removed of any dusts, and covered by Lifterslips (Erie Scientific). The Cy3/Cy5-labeled cDNA samples were applied in between Lifterslips and slides. To prevent slides from being dried, 10 μl of 3× SSC buffer was applied onto the slides, which were subsequently incubated for 16 hr at 42° C. After incubation, the microarray slides were washed with three different washing buffers [wash buffer 1 (10 ml 20×SSC, 600 μl 10% SDS, 189.4 ml dH2O, preheated at 42° C.), wash buffer 2 (3.5 ml 20×SSC, 346.5 ml dH2O), wash buffer 3 (0.88 ml 20×SSC, 349.12 ml dH2O)] for 2, 5, and 5 min, respectively, on the orbital shaker.
For each total RNA sample, 3 independent DNA microarray with 3 independent biological replicates were performed, including one-dye swap experiment.

Microarray Slide Scanning and Data Analysis

After hybridization and washing, the microarray slides were scanned by GenePix 4000B scanner (Axon Instrument) and the signals were analyzed with GenePix Pro (Ver. 4.0) and gal file (http://genome.wustl.edu/activity/ma/cneoformans). Since we used total RNAs isolated from serotype A C. neoformans strains, 70-mer oligonucleotide sequence printed on the serotype D C. neoformans slides was queried against serotype A C. neoformans genome database by blastp search (e-value cut-off: e-4) to find the corresponding serotype A gene ID. Using the serotype A gene sequence, each S. cerevisiae gene name or ID listed in the Tables was identified by blastp search (e-value cut-off: e-4).
For further hierarchical and statistical analysis, data transported from GenePix software were analyzed with GeneSpring (Agilent) by employing LOWESS normalization, reliable gene filtering, clustering (standard correlation and average linkage) and zero-transformation, and ANOVA analysis (P<0.01).

Ergosterol Assay

Ergosterol contents were measured as previously described in “Arthington-Skaggs B A, Jradi H, Desai T, Morrison C J (1999) Quantitation of ergosterol content: novel method for determination of fluconazole susceptibility of Candida albicans. J Clin Microbial 37: 3332-3337”, but with slight modification. Briefly, each C. neoformans strain was grown in 100 ml YPD medium for 24 h at 30° C. The 100 ml culture was splitted into two 50 ml cultures for duplicate measurement, pelleted in a tabletop centrifuge, and washed with sterile water. The cell pellet was frozen in liquid nitrogen and lyophilized overnight. The dried cell pellet was weighed for normalization of ergosterol contents, added with 5 ml of 25% alcoholic potassium hydroxide, and transferred to a sterile borosilicated glass screw-cap tube. Subsequently, the cells were incubated at 80° C. for 1 h and allowed to cool to room temperature. Then 1 ml of sterile water and 3 ml of heptane were added and vortexed for 3 min. Then 200 μl of the heptane layer is sampled and mixed with 800 μl of 100% ethanol, and its optical density (OD) was measured at both 281.5 nm and 230 nm. Ergosterol contents were calculated as the following: % ergosterol=[(OD281.5 nm/290)×F]/pellet weight−[(OD230 nm/518)×F]/pellet weight, where F is the ethanol dilution factor and 290 and 518 are the E values (in percentages per centimeter) determined for crystalline ergosterol and 24(28)dehydroergosterol, respectively.

Stress Sensitivity Test

Each strain was incubated overnight at 30° C. in YPD medium, washed, serially diluted (1 to 10⁴dilutions) in dH₂O, and spotted (3 μl) onto solid YPD medium containing indicated concentrations of stress-inducing agents or antifungal drugs as previously described in “Bahn Y S, Kojima K, Cox G M, Heitman J (2005) Mol Biol Cell 16: 2285-2300.” and “Bahn Y S, Kojima K, Cox G M, Heitman J (2006) Mol Biol Cell 17: 3122-3135”. To examine antifungal drug sensitivity, the cells were spotted on agar-solid YPD media containing amphotericin B(Sigma), fluconazole(Sigma), itraconazole(Sigma), ketoconazole (Sigma) and fludioxonil. Then spotted cells were incubated at 30° C. for 2-4 days and photographed.

Disruption of CAMP-Signaling Dependent Genes

For gene disruption, information of genomic DNA structure (exon and intron) for each gene was obtained from serotype A C. neoformans genome database (http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans/MultiHome.ht ml). The GRE2 (CNAG_—02182.2), HSP12 (CNAG_—03143.2), HSP122 (CNAG_—01446.2) and PKP1 (CNAG_—00396.2) genes were deleted by overlap PCR or double joint PCR PCR) with split markers and biolistic transformation in the C. neoformans serotype A H99 strain as previously described (Bahn et al., 2005, Davidson et al., 2002). Primers for generation of the 5′ and 3′ flanking regions of each gene and dominant selectable nourseothricin resistant marker (NAT, nourseothricin acteryltransferase) were described in the supplemental table 1. Gold microcarriers beads (0.8˜1.2 μm [Bioworld Inc] or 0.6 -μm [BioRad]) were coated with gel-extracted deletion cassettes produced by overlap PCR and biolistically transformed into the strain H99. Stable transformants selected on YPD medium containing nourseothricin or G418 were subject to the first screening by diagnostic PCR with primers listed in Table 2. Positive mutants were further confirmed by Southern blot analysis using gene-specific probes prepared by primers listed in Table 2.

TABLE 2

Primer Name	Sequence	Description

B79	TGTGGATGTCTGGCGGAGGATA	Screening primer on ACT promtre

B1026	GTAAAACGACGGCCAGTGAGC	M13 forward (extended)

B1027	CAGGAAACAGCTATGACCATG	M13 reverse (extended)

B1614	TGTTTAGCACCAGCGGAGTC	HSP12-5′ screening primer

B1615	CACGATGAAAGTGCGTTGAAG	HSP12-left flanking primer 1

B1616	GCTCACTGGCCGTCGTTTTACACTGTCGGTGAAAGATTGC	HSP12-left flanking primer 2

B1617	CATGGTCATAGCTGTTTCCTGAGAACGACAACCAGGAGTC	HSP12-right flanking primer 1

B1618	GCTCTGTGCTGACATTATCTGC	HSP12-right flanking primer 2

B1707	GAAAGTGCGTTGAAGTGATG	HSP12-probe primer 1

B1708	AGTAGAAGCAGCGGACTAAAG	HPS12-probe primer 2

B1619	GCGTAGTGGAGATTGGTTTC	GRE2-5′ screening primer

B1620	ATCCCCTCCACTTTACCTCC	GRE2-left flanking primer 1

B1621	GCTCACTGGCCGTCGTTTTACAAGTCTCCCTTAGCGATAG	GRE2-left flanking primer 2

B1622	CATGGTCATAGCTGTTTCCTGACCACACCCCTGAAGAAAC	GRE2-right flanking primer 1

B1623	AACTGTTTCGTCTTGTGTGTC	GRE2-right flanking primer 2

B1705	ATAGCAACTTCTTCCGTCG	GRE2-probe primer 1

B1706	TGTTGCCTGTGCTCACTTG	GRE2-probe primer 2

B1629	CCTCTGACAGCCACATACTG	PKP1-5′ screening primer

B1630	AATGAAGTTCCTGCGACAG	PKP1-left flanking primer 1

B1631	GCTCACTGGCCGTCGTTTTACAATGGGATGAGAACGCAC	PKP1-left flanking primer 2

B1632	CATGGTCATAGCTGTTTCCTGAGCATTTTCCAGCATCAGC	PKP1-right flanking primer 1

B1633	GGTGTGGAACATCTTTTGAG	PKP1-right flanking primer 2

B1711	CTGGTTCATCTTGGGTGTC	PKP1-probe primer 1

B1712	TCTGAGCATACCACTCCTTTAC	PKP1-probe primer 2

B1666	TCTCATTCGCATCCTCTG	HSP122-5′ screening primer

B1667	GTTGGGCAGATAATGTTTGTG	HPS122-left flanking primer 1

B1668	GCTCACTGGCCGTCGTTTTACACGGCGTCAGACATTGTG	HSP122-left flanking primer 2

B1669	CATGGTCATAGCTGTTTCCTGACAAGAGAAGTCCACTACTCAG	HPS122-right flanking primer 1

B1670	GCAAGGTAATGATGAGCG	HSP122-right flanking primer 2

B1709	GCGACTGAGATGTAGACCAAC	HSP122-probe primer 1

B1710	CTCGGAACGACATAATAAGC	HSP122-probe primer 2

B1673	CACACCTGGTAAGAGATAGCG	NHA1-left flanking primer 1

B1674	GCTCACTGGCCGTCGTTTTACAGTGGTAGAAGTAGGGCAGC	NHA1-left flanking primer 2

B1675	CATGGTCATAGCTGTTTCCTGACAGGGTCCAACAAGGATG	NHA1-right flanking primer 1

B1676	TGCTACGATTGTGGTCAGCC	NHA1-right flanking primer 2

B1677	GGACGAGACGAGTTATCAAAC	NHA1-screening primer

B1698	CTTCATCAACTTGCGTGC	NHA1-probe primer

Example 1

DNA Microarray Analysis of C. neoformans hog1Δ, ssk1Δ, and skn7Δ Mutants

To investigate Hog1 signaling pathway in C. neoformans, we performed comparative transcriptome analysis of serotype A wild-type (WT, H99) strain, hog1Δ, ssk1Δ, and skn7Δ mutants under both normal growth conditions and stressed conditions, such as in the presence of osmotic shock (1 M NaCl), oxidative stress (2.5 mM H2O2), and antifungal drug fludioxonil (40 μg/ml), by using DNA microarray analysis. We isolated total RNAs from cells growing in each stress condition after zero (non-stress condition), 30, and 60 min incubation. We prepared 3 independent RNA samples for each condition as biological replicates for DNA microarray analysis. As a control RNA for common Cy3 labeling, we used reference RNAs that were pooled from all RNA samples prepared in this study. We used 70-mer serotype D C. neoformans DNA microarray chips containing total 7,936 spots, based on information from the C. neoformans genome database.
For basic validation of our array quality, we monitored expression levels of HOG1, SSK1, and SKN7 genes, and known Hog1-regulated genes, such as GPP1 (Glycerol-3-phosphatase) and GPD1 (Glycerol-3-phosphate dehydrogenase), in our array data.
FIG. 1 shows identification of genes whose expression is regulated by Hog, Ssk1 and Skn7 of C. neoformans under normal conditions with no stress on the genome level (fold change is expressed by color). FIG. 1A shows relative expression levels of HOG1, SSK1, and SKN7 genes in each corresponding mutant compared to WT strain. FIG. 1B shows condition tree analysis result in WT, hog1Δ, ssk1Δ, skn7Δ mutant. FIG. 1C shows clustering analysis result of 950 genes which are exhibited significantly different expression patterns in hog1Δ, ssk1Δ, or skn7Δ mutants compared to WT (ANOVA test, P<0.01) under normal growth condition(YDP, 30□). FIG. 1D shows Venn diagram presenting HOG1, SSK1, and SKN7-dependent genes that include genes up- or down-regulated over 2 folds.
As expected, relative expression levels of HOG1, SSK1, and SKN7 genes in each corresponding mutant compared to WT strain were very low(FIG. 1A). In addition, expression of GPD1 (glycerol-3-phosphate dehydrogenase, CNAG_—01745) and GPP1 (DL-glycerol-3-phosphatase, CNAG_—01744) homologous genes, which are well-known Hog1-regulated stress defense genes in other fungi, was highly reduced (4.5-fold and 2.5-fold reductions, respectively) in hog1Δ and ssk1Δ mutants, further supporting the quality of our array data.
We monitored how HOG1, SSK1, and SKN7 mutations affect gene expression patterns in C. neoformans under unperturbed normal conditions. Among 7,936 spots monitored, 3,858 spots were found to be reliable based on Cross-gene error model (cutoff 10). Supporting the previous finding, the transcription profile of the hog1Δ mutant was considerably similar to that of the ssk1Δ mutant, based on the condition tree analysis (FIG. 1B). A total of 950 genes in the reliable genes exhibited significantly different expression patterns in hog1Δ, ssk1Δ, or skn7Δ mutants compared to WT (ANOVA test, P<0.01) (FIG. 1C), indicating that about 15% of the whole C. neoformans genes could be transcriptionally affected by perturbation of the two-component system and HOG signaling pathways even under unstressed, normal conditions. Among them, 559 genes exhibited more than 2-fold induction in at least one of the mutants (FIG. 1D). Several key findings were made as the following. First, a majority of the genes (555 genes, 99%) were up- or down-regulated by either Ssk1 or Hog1 under unstressed conditions while only 51 genes (9%) were regulated by Skn7. Among the Skn7-dependent genes, only 4 genes were found to be Skn7-specific (FIG. 1D). Thus it appears to be clear that HOG1 and SSK1 mutations alter genome-wide transcription profiles under normal conditions in a greater scale than the SKN7 mutation (FIG. 1D). Second, there exist significantly higher overlaps between Ssk1- and Hog1-dependent genes (422 out of 555 genes, 76%) than between Skn7- and Hog1-dependent genes (45 out of 467 genes, 10%), further corroborating that Ssk1 is the major upstream regulator of the Hog1 MAPK. Third, regardless of the significant overlap in genes regulated by Ssk1 and Hog1, there were a number of Ssk1-specific (90 genes) and Hog1-specific (40 genes) genes, strongly suggesting that Ssk1 and Hog1 are not strictly in the linear pathway and could have other target(s) or upstream regulators, respectively (FIG. 1D). This explains why the ssk1Δ mutant exhibits slightly different phenotypes (i.e. higher sensitivity to hydrogen peroxide) compared to hog1Δ mutants and Hog1 can still be phosphorylated in the absence of Ssk1 response regulator under exposure to NaCl.
Genes regulated by Hog1 and Ssk1 cover a wide variety of functional categories, including energy production and conversion, amino acid/carbohydrate/lipid transport and metabolism, translational and protein biosynthesis, post-translational modification, signal transduction, stress-defense mechanisms, and others (Supplementary table 2), indicating that active remodeling of various aspects of cellular functions could occur simply by perturbation of the HOG pathway even without external stresses. Furthermore it should be noticed that more than one third of Hog1 and Ssk1-dependent genes do not have any functional orthologs in other organisms, indicating that C. neoformans appears to develop many cryptococcus-specific Hog1/Ssk1-dependent genes.
Among Ssk1- and Hog1-regulated genes identified by our array analysis, several groups of genes provided novel insights into the potential mechanism of the HOG pathway in controlling virulence factor and sexual reproduction of C. neoformans. First, a group of genes involved in iron transport and regulation were found to be highly induced in the ssk1Δ and hog1Δ mutants compared to the wild-type strain. These genes include SIT1 (CNAG_—00815 and CNAG_—07138) encoding siderophore-transporters, CFO1 (CNAG_—06241) and CFO2 (CNAG_—02958) encoding ferroxidases, and CFT1 (CNAG_—06242) encoding Fe transporter. The C. neoformans Sit1 are homologous to the S. cerevisiae Arn3/Sit1 having high affinity for the hydroxamate siderophore ferrioxamine and C. neoformans Cfo1/Cfo2 and Cft1 are homologous to high-affinity iron permease/multicopper ferroxidase complex (Ftr1-Fet3) in S. cerevisiae. Since iron transport regulation and melanin synthesis seem to be closely related in C. neoformans, increased melanin synthesis observed in both hog1Δ and ssk1Δ mutants could be correlated with increased expression of a group of genes involved in iron transport.
Second, the GPA2 gene (CNAG_—00179), encoding a G-protein α-subunit in the pheromone responsive MAPK pathway, is dramatically upregulated upon ssk1Δ or hog1Δ mutation (12.1- and 13.3-fold increases, respectively). This finding suggests that increased pheromone production and sexual reproduction found in ssk1Δ and hog1Δ mutants may result from enhanced expression of Gpa2 that is induced during mating and promotes the mating process of C. neoformans.
Third, several genes involved in oxidative stress defense were differentially regulated by HOG1 and SSK1 mutation. As expected from the previous finding that the hog1Δ and ssk1Δ mutants exhibit hypersensitivity to hydrogen peroxide, two genes (CNAG _—04981 and CNAG_—00575), which are homologous to the CTA1 gene encoding catalase A that detoxifies H2O2 to H2O, was drastically downregulated in both mutants (Supplementary Table 1). Furthermore, basal expression levels of the SOD2 gene (mitochondrial superoxide dismutase) were decreased in both hog1Δ and ssk1Δ mutants, further corroborating the role of the HOG pathway in oxidative stress response. Interestingly, however, basal expression levels of some genes involved in oxidative stress response [TRR1 (thioredoxin reductase), TSA1 (thioredoxin peroxidase), GRX5 (glutathione-dependent oxidoreductase), CCP1 (mitochondrial cytochrome-c peroxidase)] were more than 2-fold increased (3.8, 3.1, 2.1, and 9.5 fold changes, respectively) in the hog1Δ mutant, but not in the ssk1Δ mutant (Supplementary Table 2). The SRX1 gene (sulfiredoxin) also involved in oxidative stress response was more reduced in the ssk1Δ mutant (4.2-fold reduction) than the hog1Δ mutants (1.3-fold reductions). These results may explain why the hog1Δ mutants are relatively more resistant to H2O2 than the ssk1Δ mutant.

Example 2

Ergosterol Biosynthesis Genes are Transcriptionally Upregulated by Perturbation of the HOG Signaling Pathway

Among genes upregulated by mutation of HOG1 and SSK1 genes, a gene homologous to ERG28 (CNAG_—07208) was noticeable since it plays a key role in the fungal sterol biosynthesis. Previous microarray analysis performed in S. cerevisiae revealed that expression of ERG28 is tightly correlated with other ergosterol biosynthetic genes. Erg28 is an endoplasmic reticulum (ER) transmembrane scaffold, protein, which is essential for the yeast sterol biosynthesis by interacting strongly with Erg27, Erg25, Erg11, and Erg6 and weakly with Erg26 and Erg1. This finding led us to monitor expression patterns of other sterol biosynthetic genes in our array data.
FIG. 2A shows the relative expression profiles of ergosterol biosynthesis genes in hog1Δ, ssk1Δ, and skn7Δ mutants compared to WT strain. The fold change is illustrated by a color (see color bar scale) and exact value for each gene was indicated in the table placed right side of the hierarchical clustering diagram. FIG. 2B shows cellular ergosterol contents in WT (H99), skn7Δ (YSB349), ssk1Δ (YSB261), ssk2A (YSB264), and hog1Δ (YSB64) mutants. Left and right graphs demonstrate % ergosterol in each strain and relative increase of ergosterol contents compared to WT, respectively. Each bar presents the average from four independent experiments and error bar indicates the standard deviation. Asterisks (*): The ssk1Δ, ssk2Δ, pbs2Δ, and hog1Δ mutants contain significantly higher ergosterol levels compared to the WT (P<0.05, as analyzed by using the Bonferroni multiple comparison test).
Interestingly, a majority of the ergosterol biosynthetic genes were upregulated in hog1Δ and ssk1Δ mutants, but not in the skn7Δ mutant, compared to the wild-type strain (FIG. 2A). Genes, such as ERG11, ERG6, MVD1, ERG5, ERG25, ERG20, and ERG4, were upregulated in both ssk1Δ and hog1Δ mutants while genes, such as ERG27, ERG13, ERG26, ERG10, IDI1, HMG1, and ERG8, were upregulated only in the ssk1Δ mutant (FIG. 2A). In contrast, none of genes were significantly upregulated in the skn7 mutant and indeed some of genes, including ERG13, ERG1, ERG3, ERG7, and ERG2 genes, were downregulated in the skn7Δ mutant (FIG. 2A).
To verify our microarray data, we examined whether increased expression levels of some of the ergosterol biosynthesis genes indeed affect cellular ergosterol contents in the hog1Δ and ssk1Δ mutants (FIG. 2B). In accordance with our microarray data, cellular ergosterol contents were much higher in the hog1Δ and ssk1Δ mutants than WT and skn7Δ mutants (FIG. 2B), suggesting that increased expression of some of ergosterol biosynthetic genes leads to enhanced production of cellular ergosterol. The ssk2Δ (MAPKKK) and pbs2Δ (MAPKK) mutants in the HOG pathway were also found to contain significantly higher levels of cellular ergosterol than WT and skn7Δ mutants (FIG. 2B), further corroborating our array data.
This finding prompted us to investigate the susceptibility of the mutants in the two-component system and the HOG pathway to antifungal drugs that are targeted to the ergosterol biosysnthetic genes or ergosterol itself. First we have examined the susceptibility of the ssk1Δ, skn7Δ, ssk2Δ, pbs2Δ, and hog1Δ mutants made in the serotype A H99 strain background to the polyen antifungal drug, amphotericin B, which binds to ergosterol in the fungal cell membrane and ultimately causes lethality by disrupting the membrane integrity. We hypothesized that increased ergosterol contents observed in ssk1Δ, ssk2Δ, pbs2Δ, and hog1Δ mutants could render them to be hypersensitive to amphotericin B due to the increased number of drug targets.
FIG. 3 shows analysis results showing that the inhibition of the HOG pathway confers synergistic antifungal effects with amphotericin B in C. neoformans. FIG. 3A-3B show pictures photographed after incubation at 30° C. for 72 h of each C. neoformans strain spotted on YPD agar containing indicated concentrations of amphotericin B. FIG. 3C shows pictures photographed after incubation at 30° C. for 72 h of C. neoformans serotype A strains and serotype D strains spotted on YPD agar containing indicated concentrations of amphotericin B.
Confirming our hypothesis, the ssk1Δ, ssk2Δ, pbs2Δ, and hog1Δ mutants exhibited dramatic hypersensitivity to amphotericin B treatment compared to WT (FIG. 3A), which is in good agreement with the finding that ergosterol contents were significantly higher in the HOG pathway mutants than WT (FIG. 2B). In contrast, the skn7Δ mutant showed WT-levels of resistance to amphotericin B (FIG. 3A), which can be also explained by the previous data showing that cellular ergosterol contents in the skn7Δ mutants are similar to those of WT (FIG. 2B).
We also monitored amphotericin B-susceptibility of C. neoformans strains having mutation hybrid sensor kinases (Tco1, Tco2, Tco3, Tco4, Tco5, and Tco7), which act upstream of the Ssk1 response regulator. Previously we have shown that Tco1 and Tco2 play redundant and distinct roles in controlling a subset of Hog1-dependent phenotypes. Here we found that Tco1 and Tco2 play discrete roles in sensing and responding to amphotericin B. Among Tco proteins, only Tco2, which is double hybrid sensor kinases containing two response regulator domains and two histidine kinase domains in a single polypeptide, showed hypersensitivity to amphotericin B (FIG. 3B), indicating that Tco2 is involved in sensing and responding to amphotericin B for conferring the drug-resistance via the HOG pathway. However, the fact that the degree of hypersensitivity observed in the tco2Δ mutant is lesser than the ssk1Δ mutant suggests other possibilities. One possibility is that other unknown receptor/sensors may exist to respond to the amphotericin B. The other possibility is that constitutively phosphorylated Hog1 may repress ergosterol biosynthetic pathway under normal conditions hypersensitivity regardless of the presence of receptors/sensors since Ssk1, Ssk2, and Pbs2, but not Tco2 proteins, are all involved in constitutive phosphorylation levels of Hog1.
To test the hypothesis, we have also examined the amphotericin B sensitivity of other C. neoformans strains, such as JEC21 and B3501-A, showing differential Hog1 phosphorylation levels. To support our second hypothesis, the JEC21 strain where Hog1 is not constitutively phosphorylated exhibited hypersensitivity to amphotericin B even more than the ssk2Δ mutant in the H99 strain background (FIG. 3C). In the JEC21 strain background, mutation of SSK2, PBS2, and HOG1 genes did not affect sensitivity to amphotericin B (FIG. 3C). In contrast, the B3501 strain where Hog1 is constitutively phosphorylated, albeit to a lesser extent than in the H99 strain, exhibited higher resistance to amphotericin B than the JEC21 (FIG. 3C). Similar to the H99 strain, mutation of the SSK2 MAPKKK that abolishes the Hog1 phosphorylations increased the amphotericin B sensitivity (FIG. 3C). All these data strongly indicate that constitutively phosphorylated Hog1 represses ergosterol biosynthetic pathway under normal conditions.
To further support this finding, we also examined the susceptibility of the mutants to azole compounds, including triazoles (fluconazole and itraconzaole) and imidazole (ketoconazole), which inhibit the fungal cytochrome P450 enzyme 14α-demethylase and eventually prevent conversion of lanosterol to ergosterol.
FIG. 4 shows analysis results showing that the inhibition of the HOG pathway confers antagonistic antifungal effects with some azole drugs in C. neoformans. It shows pictures photographed after incubation at 30° C. for 72 h of each C. neoformans strain spotted on YPD agar containing indicated concentrations of fluconazole, ketoconazole, and itraconazole.
We had expected that the ssk1Δ and hog1Δ mutants having increased expression of many ergosterol biosynthesis genes, particularly including ERG11, should show higher resistance to azole compounds. The ssk1Δ, ssk2Δ, pbs2Δ, and hog1Δ mutants all exhibited hyper-resistance to fluconazole and ketoconazole, but not to itraconazole (FIG. 4). Interestingly, the skn7Δ mutants also showed higher resistance to fluconazole and ketoconazole than WT (FIG. 4). Among hybrid sensor kinases, only Tco1 and Tco2 display differential sensitivity to azole compounds. Although to a lesser extent than the HOG mutants, the tco2Δ mutant exhibited higher resistance to fluconazole and ketoconazole than WT (FIG. 4). In contrast, the tco1Δ mutant exhibits hypersensitivity to all azole drugs (FIG. 4), indicating that Tco1 may regulate the HOG pathway in C. neoformans in an opposite manner to Tco2. In conclusion, inactivation of the HOG pathway increases ergosterol contents by induction of ergosterol biosynthesis genes and therefore confers synergistic effects with amphotericin B treatment, but antagonistic effects with fluconazole and ketoconazole.

Example 3

Finding and Characterizing the Downstream Target Genes Controlled by the HOG Pathway

We found ENA1 (serotype A ID: CNAG_—00531.2) and NHA1 (serotype A ID: CNAG_01678.2) genes as the downstream target genes controlled by the HOG pathway and performed an additional experiment. Cells excrete H+(proton) out of cell membrane using H+-ATPase pump such as Pma1, thereby playing a role in maintaining membrane potential essential to cell growth in a normal condition. On the contrary, potassium ion(K+), an ion useful to cell growth, flows into cells using K+ influx pump such as Trk1/Trk2. Na+, unlike K+, is classified as a toxic ion. When high concentration of Na+ is present in a cell, it should be excreted via efflux pump. Since K+ also has toxicity when it presents in high concentration, an efflux pump is needed. These are Ena1 and Nha1 which play a role as an efflux pump for Na+and K+.
The result showed that the two genes coding for the two efflux pumps are controlled by the HOG pathway. As shown in FIG. 5A, when the WT, skn7Δ, ssk1Δ and hog1Δ mutant strains were exposed to osmotic stress, the expression level of ENA1 and NHA1 was dependent on the deletion mutant of the HOG pathway genes.
Thus, in order to identify a characteristic of two genes, we prepared deletion mutant of each gene and double mutant (ena1Δ nha1Δ) eliminating both two genes. And then, we examined sensitivity of the mutants to the polyene-based antifungal agent such as amphotericin B (AmpB), and the azole-based antifungal agent such as fluconazole, ketoconazole and itraconazole. The ena1Δ and nha1Δ mutants did not show high sensitivity to the AmpB. However, surprisingly, the ena1Δ nha1Δ double mutant showed considerably increased sensitivity to AmpB (FIG. 5B). Although lower sensitivity than hog1Δ, high AmpB sensitivity of the ena1Δ nha1Δ suggests that these two efflux pumps play an important role in the polyene-based drug resistance. It is more noteworthy that ena1Δ and ena1Δ nha1Δ mutants also show high sensitivity to the azole-based drugs (FIG. 5C). It is a distinguished from the hog1Δ mutant which has high resistance to the azole-based antifungal agents and verify that the inhibitors simultaneously or independently targeting Ena1 and Nha1 may exhibit very high antifungal activities when used in combination with the polyene- or azole-based antifungal agents.

Example 4

Comparative Transcriptome Analysis of C. neoformans ras1Δ, aca1Δ, gpa1Δ, cac1Δ, and pka1Δ pka2Δ Mutants

To compare the downstream signaling network of Ras1-, Aca1-, and Gpa1-dependent signaling pathways, we performed comparative transcriptome analysis of the serotype A wild-type (WT, H99) strain, ras1Δ, aca1Δ, gpa1Δ, cac1Δ, and pka1Δ pka1Δ mutants by employing DNA microarray analysis as described in Materials and Methods. For basic validation of our array quality, we checked expression levels of the RAS1, ACA1, GPA1, CAC1, PKA1, and PKA2 genes in our array data. The relative expression levels of RAS1, ACA1, GPA1, CAC1, PKA1, and PKA2 in each corresponding mutant were very low compared to those in the wild type strain (0.08, 0.03, 0.09, 0.06, 0.07, and 0.12, respectively) (FIG. 6A), which supported the quality of our array.
From total 7,936 genes monitored by this DNA microarray, 565 genes exhibited differential expression patterns in the Ras- and cAMP mutants at statistically significant levels compared to the wild type strain (ANOVA test, P<0.05) (FIG. 6B). The hierarchical clustering analysis of the Ras- or cAMP-dependent genes revealed several important facts. First, the transcriptome patterns governed by the Ras1-signaling pathway were distinct from those controlled by the cAMP/PKA-signaling pathway. The statistical analysis indicated that basal expression levels of total 400 genes changed significantly in the ras1Δ mutant compared to the WT, whereas expression levels of 132 genes changed significantly in the aca1Δ, gpa1Δ, cac1Δ, and pka1Δ pka2Δ mutants (FIGS. 6C and 6D). Besides the number of genes regulated, the expression patterns of a majority of the Ras1-dependent genes were also distinguished from those of the cAMP-dependent genes, which supported that the Ras1-signaling pathway is largely independent of the cAMP-signaling pathway in C. neoformans. Second, the aca1Δ and gpa1Δ mutants showed transcriptome patterns similar to those of the cac1Δ and pka1Δ pka2Δ mutants, indicating that Aca1Δ and Gpa1 are the two major signaling modulators of the cAMP-signaling pathway (FIG. 6D). However, there were a small group of genes whose expression is differentially regulated between the aca1Δ and gpa1Δ mutants. This indicates that Aca1 and Gpa1 could have other minor signaling branches (FIG. 6D). As expected, the cac1Δ mutant exhibited transcriptome patterns almost identical to that of the pka1Δ pka2Δ mutant, further suggesting that Pka1 and Pka2 are necessary and sufficient protein kinase downstream of the adenylyl cyclase in C. neoformans (FIG. 6D).
The genes regulated by the Ras- and cAMP-signaling pathways cover a wide variety of cellular functions (FIG. 7). The cAMP-signaling dependent genes were over-represented for those involved in signal transduction mechanisms (15.2%), carbohydrate transport and metabolism (9.6%), and amino acid transport and metabolism (8.0%). These findings were rather expected results since the cAMP-pathway is one of central signal transduction cascades that regulate growth, differentiation, and virulence of C. neoformans and is known to sense glucose and amino acids (Bahn et al., 2004, Xue et al., 2006). Similarly, genes involved in signal transduction mechanisms were most over-represented in the ras1Δ mutant (12.1%) (FIG. 7). In contrast to the cAMP-pathway, however, genes involved in cell wall/membrane/envelope biogenesis were over-represented (2.9%), which implies that Ras1 may be implicated in maintenance of cell wall integrity.
Among the Ras- and cAMP-dependent genes, a significant proportion of them were found to be environmental stress-regulated (FIG. 8). Our prior transcriptome analysis discovered a number of ESR (Environmental Stress Regulated) genes in C. neoformans (Ko et al., 2009). A total of 1,959 genes were found to be more than 2-fold up or downregulated in response to either of osmotic stress, oxidative stress, or antifungal drug (fludioxonil) treatment (Ko et al., 2009). Interestingly, our current array analysis revealed that a subset of the ESR genes (a total of 225 ESR genes) exhibited significant changes in expression levels in either the ras1Δ or cAMP mutants compared to the wild-type strain (ANOVA test, P<0.05) (FIG. 8). Among these, eighty-six ESR genes showed more than 2-fold induction or reduction in the mutants (FIG. 8). Furthermore, a total of 55 CSR (Common Stress Response) genes were found to be differentially regulated (ANOVA test, P<0.05) and 31 genes of them exhibited more than 2-fold induction or reduction in the mutants (FIG. 2B). The major proportion of the Ras- or cAMP-pathway-dependent ESR and CSR genes did not have any other homologs with significant homology (Table S6). Nevertheless, these results implied that the Ras-and cAMP-signaling pathways be implicated in diverse stress response of C. neoformans.

Example 5

Identification of the Ras- or cAMP-Dependent Genes in C. neoformans

Next we further investigated individual Ras1- and cAMP-dependent genes identified by our transcriptome analysis.
Among the selected 161 Ras-dependent genes (2-fold cutoff, FIG. 6C), a majority of them (101 genes, 63%) do not have any orthologs in other fungi (Table S4), which indicated that C. neoformans contains a unique set of Ras-dependent genes. Among the evolutionary conserved Ras-dependent genes, three genes, PXL1, RDI1, and BEM3, whose orthologs are known to be involved in regulation of Rho-GTPase Cdc42 in S. cerevisiae, were notable since the Ras1-Cdc24 signaling pathway has been reported to be controlled by one of three Cdc42 homologues in C. neoformans (Nichols et al., 2007). RDI1 and BEM3 encode Rho-GDP dissociation inhibitor and Rho-GTPase activating protein, respectively (Price et al., 2008, Zheng et al., 1994). Notably, in a good agreement with the role of Ras1 in genotoxic stress response of C. neoformans (FIG. 4), a number of genes involved in regulation of DNA damage repair were identified as Ras-dependent genes. These include RNR2/RNR3 (Ribonucleotide-diphosphate reductase), RAD3 (DNA helicase, a subunit of nucleotide excision repair factor 3), RAD14 (a subunit of nucleotide excision repair factor 1), MSH6 (a protein required for mismatch repair), MND1 (a protein required for recombination and repair of DNA double strand breaks), and DNA2 (ATP-dependent nuclease). Finally, several genes, CHS1 (Chitin synthase 1), CDA2 (Chitin deacetylase), BGL2 (glucan 1,3-β-glucosidase), and GSC2 (Glucan synthase), involved in governing cell wall integrity were also identified as Ras-dependent genes 6, which further supported the role of Ras1 in maintaining cell wall integrity of C. neoformans.
The statistical comparison of transcriptome data obtained from the cAMP mutants (aca1Δ, gpa1Δ, cac1Δ, and pka1Δ pka2Δ) with that from the WT strain (ANOVA, P<0.05) identified 163 genes (FIG. 6C). Among these, 38 genes exhibited more than 2-fold induction or reduction in the cAMP mutants, except CAC1, ACA1, PKA1, and GPA1 (FIG. 9). A majority of the cAMP-dependent genes (31 genes, 81%) do not have any known function in C. neoformans or orthologs in S. cerevisiae, which indicated that C. neoformans contains a unique set of cAMP-dependent genes similarly to the Ras-dependent genes. This observation further corroborates that C. neoformans cAMP mutants have unique phenotypic characteristics that have not been observed in other fungi. Five cAMP-dependent genes (GRE2, ENA1, HSP12, CAT1, and PKP1) in C. neoformans appear to be evolutionarily conserved in other fungi. Interestingly, the GRE2, ENA1, and HSP12 genes are known to be transcriptionally regulated by environmental stress in S. cerevisiae. In C. neoformans, it has been recently reported that Ena1 not only controls osmotic stress under carbon starvation condition (Ko et al., 2009), but also is required for survival in alkaline pH and in vivo virulence (Idnurm et al., 2009). The GRE2 (genes de respuesta a estres, stress-responsive gene), a homolog of mammalian 3-β-hydroxysteroid dehydrogenase, is strongly induced in response to a variety of stresses, including osmotic and oxidative stress, upon binding of HOG-dependent Sko1 transcription factor to CRE (cAMP response element) in the promoter region in S. cerevisiae (Garay-Arroyo & Covarrubias, 1999, Rep et al., 2001). The heat shock protein HSP12 (03143) is a small hydrophilic protein whose expression is also induced by diverse stresses and regulated by both HOG and cAMP signaling pathways (Varela et al., 1995). Here we named this gene as HSC1 (HSP12-like C. neoformans gene 1, 03143).

EXAMPLE 6

Inhibition of the Ras and cAMP-Signaling Pathway Increased Polyene Sensitivity

Gre2 is involved in regulation of some of ergosterol biosynthesis genes, including ERG6, ERG10, and ERG19/MVD1 (Warringer & Blomberg, 2006). Furthermore, GRE2 is reported to be one of six genes whose expression increased with resistance to amphotericin B (AmpB) in S. cerevisiae (Anderson et al., 2009). Therefore, we examined whether the C. neoformans Ras- and cAMP-mutants are more susceptible to AmpB treatment than WT.
As shown in FIG. 10, the ras1Δ mutant showed higher susceptibility to AmpB than WT whereas the aca1Δ mutant exhibited slightly higher AmpB susceptibility (FIG. 10A). The ras1Δ aca1Δ double mutant exhibited higher AmpB-sensitivity than each single mutant (FIG. 10B), indicating that Ras1 and Aca1 redundantly or independently control AmpB sensitivity. Cdc24 appears to work downstream of Ras1 for regulation of the polyene drug resistance (FIG. 10C). Interestingly, the ras2Δ mutant was also slightly more sensitive to AmpB than WT, indicating that both Ras proteins control resistance to polyene drugs in C. neoformans.
Notably, the gpa1Δ and cac1Δ mutants showed much higher AmpB-sensitivity than WT and even than the ras1Δ or aca1Δ mutant (FIG. 10A). Downstream of the Cac1 adenylyl cyclase, the pka1Δ mutant, but not the pka2Δ mutant, showed increased susceptibility to AmpB (FIG. 10A), strongly indicating that the Gpa1-Cac1-Pka1 signaling cascade is one of signaling circuits to control the polyene drug sensitivity. The ras1Δ cac1Δ double mutant exhibited even higher AmpB susceptibility than each single mutant (FIG. 10B), indicating that the Ras- and Gpa1-Cac1-Pka1 pathways are independently involved in AmpB susceptibility. The ras1Δ and ras1Δ cac1Δ mutants generated in MATa background (KN99 strain) exhibited the same phenotypes (data not shown).
To address whether the involvement of the Ras- and cAMP-pathways in the polyene sensitivity is related to the levels of ergosterol biosynthesis, we checked expression levels of ergosterol biosynthesis genes in the mutants from our array data. Interestingly, none of ergosterol biosynthesis genes, except ERG3 and ERG25 (<less than 2-fold), exhibited significant expression changes in the ras1Δ, aca1Δ, gpa1Δ, cac1Δ, or pka1Δ pka2Δ mutants compared to WT (Table S2 and S3). Northern blot analysis showed that expression levels of the ERG3 and ERG25 genes in the mutants were not significantly different from those of WT (FIG. 10D). We also checked cellular ergosterol contents in the Ras- and cAMP-mutants and found that cellular ergosterol contents were not significantly increased in the Ras- and cAMP-mutants compared to WT whereas the hog1Δ mutant has increased ergosterol contents as previously reported (data not shown) (Ko et al., 2009). Furthermore, expression levels of ERG11 in the ras1Δ and cAMP mutants were not significantly different from those of WT (FIG. 10D). Supporting this finding, the gpa1Δ, cac1Δ, pka1Δ, pka2Δ, and pka1Δ pka2Δ mutants were nearly as resistant to fluconazole, which target to the fungal cytochrome P450 enzyme 14α-demethylase and inhibit conversion of lanosterol to ergosterol, as the WT strain (data not shown). All these data strongly implied that the Ras and cAMP-signaling pathway independently influence the polyene sensitivity without affecting ergosterol biosynthesis.
We have found in Examples 1 to 3 that the HOG pathway controls ergosterol biosynthesis of C. neoformans under unstressed conditions and the HOG pathways mutants are hyper-sensitive to AmpB, but hyper-resistance to fluconazole because of the increased cellular ergosterol contents in the mutants (Ko et al., 2009). Therefore, it is easily conceivable that the HOG and cAMP pathways influence the polyene sensitivity in different manners. Supporting this, we found that the hog1Δ cac1Δ and hog1Δ pka1Δ double mutants were even more sensitive to AmpB than the hog1Δ, cac1Δ, or pka1Δ single mutant (FIG. 10E). Unexpectedly, the hog1Δ cac1Δ double mutants also exhibited hypersensitivity to various azole drugs, such as fluconazole, ketoconazole, and itraconazole (FIG. 10F). Interestingly, the ras1Δ, aca1Δ, gpa1Δ, cac1Δ, and pka1Δ mutants all showed increased sensitivity to itraconazole (FIG. 10G). Particularly, both Ras1 and Ras2 appear to be involved in itraconazole susceptibility in a manner dependent of Cdc24 (FIG. 10H). Taken together, these date indicate that the HOG pathway and cAMP-signaling pathways independently control polyene and azole drug susceptibility.
One of key findings made by this study was that the Ras- and cAMP-signaling pathways controlled the polyene- and azole-based drug susceptibility in C. neoformans. Both Ras1 and Ras2 appeared to be involved in polyene susceptibility by using Cdc24 as a downstream effector. Interestingly, the ras1Δ aca1Δ mutant was also hypersensitive to amphotericin B, indicating that the Ras1 and Aca1 may play a minor role in susceptibility to the polyene drugs. It could be possible that perturbed action cytoskeleton regulation and cell wall integrity by ras1 and aca1 mutation makes cell more susceptible to the polyene drugs.
The cAMP-signaling pathway was even more significantly involved in polyene sensitivity than the Ras-signaling pathway. Mutation of the GPA1, CAC1, and PKA1, rendered C. neoformans cells to be hypersensitive to the polyene drugs, such as amphotericin B (AmpB). We recently reported that perturbation of the HOG pathway also renders C. neoformans cells to be hypersensitive to AmpB (Ko et al., 2009). However, the cAMP and HOG pathways appear to work differently for modulation of the polyene drug susceptibility. Inhibition of the HOG pathway, but not the cAMP pathway, increases ergosterol biosynthesis, which enhances the polyene drug susceptibility and azole drug resistance (Ko et al., 2009). Furthermore, the hog1Δ mutant exhibited higher sensitivity to AmpB than the cAMP mutants.

Example 7

Characterization of the cAMP-Dependent Genes in C. neoformans

We also addressed the role of the cAMP-dependent genes and in diverse stress response and antifungal drug susceptibility of C. neoformans due to the involvement of the cAMP-pathway in the process that we discovered in this study.
Hypersensitivity of the cAMP mutants to the polyene drug appeared to be partly contributed by decreased expression of the two heat shock proteins Hsp12 (H99 gene ID: CNAG_—03143.2), C. neoformans homologs of HSP12, and Hsp122(H99 gene ID: CNAG_—01446.2) (FIG. 11).
Interestingly, however, the hsp12Δ or hsp122Δ mutant exhibited slightly higher susceptibility to AmpB than WT, although the cac1Δ mutant was more sensitive to AmpB than the hsp12Δ or hsp122Δ mutant (FIG. 11A). Therefore, it was conceivable that decreased expression of HSP12 or HSP122 contributes to hypersensitivity of the cAMP mutants to AmpB.
To further characterize the regulatory mechanism of HSP12 and HSP122, we performed Northern blot analysis to confirm that the cAMP-signaling pathway modulated expression of the HSP12 and HSP122 genes. In S. cerevisiae, HSP12 is not expressed under unstressed, glucose-rich condition, but is induced in response to environmental stresses (Praekelt & Meacock, 1990, Siderius et al., 1997). Unexpectedly, however, the HSP12 and HSP122 genes were found to be highly expressed genes in the WT strain under unstressed, glucose-rich condition (FIG. 11A). In a good agreement with the microarray data, HSP12 and HSP122 expression was significantly downregulated in the cAMP mutants, including gpa1Δ, cac1Δ, and pka1Δ pka2Δ mutants (FIG. 11A). In the aca1Δ and ras1Δ mutants, expression levels of the HSP12 and HSP122 genes were only slightly affected (FIG. 11A). These data not only confirmed our microarray data, but also indicated that HSP12 and HSP122 were positively regulated by the cAMP-signaling pathway.
Interestingly our previous array analysis showed that HSP12 and HSP122 may also be under control of the HOG pathway. HSP12 and HSP122 expression levels were considerably low in the hog1Δ and ssk1Δ, but not in the skn7Δ mutant (FIG. 11B). To confirm this, we performed Northern blot analysis and found that expression levels of HSP12 and HSP122 were very high in the WT and skn7Δ mutants, but was undetectable in the hog1Δ and ssk1Δ (FIG. 11B). All these data strongly indicated that the HSP12 and HSP122 gene was co-regulated by the cAMP and HOG signaling pathways.
As discussed in the above, hypersensitivity of the cAMP mutants to the polyene drug appeared to be partly contributed by decreased expression of the heat shock protein Hsp12 and Hsp122. In S. cerevisiae, Hsp12 plays a role in stabilizing the plasma membrane as a cell wall plasticizer and water replacement molecules (Sales et al., 2000, Shamrock & Lindsey, 2008) and therefore is involved in maintaining cell wall integrity under the stressful conditions in S. cerevisiae (Shamrock et al., 2009). Therefore, the hsp2Δ mutant is unable to grow in the presence of a cell wall destabilizer, Congo red (Motshwene et al., 2004). Therefore, perturbation of the cAMP-signaling pathway reduces basal expression levels of Hsp12, which subsequently weakened cell wall integrity and membrane plasticity of C. neoformans. Similarly, hypersensitivity of the HOG pathway mutants to the polyene drug in part results from decreased expression of HSP12. However, since the cac1Δ mutant is much more sensitive to AmpB than the hsp12Δ mutant, other factors, except ergosterol biosynthesis, may affect resistance to the polyene drug. Supporting this, the hog1Δ cac1Δ or hog1Δ pka1Δ double mutant exhibited even higher polyene drug sensitivity than each single mutant, which indicated that the two pathways play an independent role in the polyene drug susceptibility. Notably, the double mutation of the HOG1 and CAC1 genes renders C. neoformans cells to be hypersensitive to most of azole drugs, including fluconazole, ketoconazole, and itraconazole, with unknown reasons.
In any case, modulation of each Ras-, cAMP/PKA-, and HOG-signaling pathway (or combination of them) may provide a novel antifungal therapeutic approach in combination with polyene and azole drugs. Simultaneous inhibition of the cAMP and HOG pathways when treated with polyene drugs such as amphotericin B could be one of the most powerful combination therapy for treatment of cryptococcosis.

Claims

1. A method of treating fungal infection, comprising:

injecting an effective amount of an inhibitor against at least one protein selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of Cryptococcus neoformans into a subject.

2. The method of treating fungal infection of claim 1, wherein the inhibitor against at least one protein selected from the group consisting of Ssk1, Ena1 and Nha1.

3. The method of treating fungal infection of claim 1, wherein an ergosterol-binding antifungal agent or azole-based antifungal agent is sequentially or simultaneously injected with the inhibitor.

4. The method of treating fungal infection of claim 3, wherein the ergosterol-binding antifungal agent is a polyene-based antifungal agent.

5. The method of treating fungal infection of claim 4, wherein the polyene-based antifungal agent is at least one selected from the group consisting of amphotericin B, natamycin, rimocidin, filipin, nystatin and candicin.

6. The method of treating fungal infection of claim 5, wherein the polyene-based antifungal agent is amphotericin B.

7. The method of treating fungal infection of claim 3, wherein the azole-based antifungal agent is at least one selected from the group consisting of ketoconazole, fluconazole, itraconazole and voriconazole.

8. A method of treating fungal infection comprising:

Injecting an effective amount of an inhibitor against at least one gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of Cryptococcus neoformans into a subject.

9. The method of treating fungal infection of claim 8, wherein the inhibitor against at least one gene selected from the group consisting of SSK1, ENA1 and NHA1.

10. The method of treating fungal infection of claim 8, wherein an ergosterol-binding antifungal agent or azole-based antifungal agent is sequentially or continuously injected with the inhibitor.

11. The method of treating fungal infection of claim 10, wherein the ergosterol-binding antifungal agent is a polyene-based antifungal agent.

12. The method of treating fungal infection of claim 11, wherein the polyene-based antifungal agent is at least one selected from the group consisting of amphotericin B, natamycin, rimocidin, filipin, nystatin, and candicin.

13. The method of treating fungal infection of claim 12, wherein the polyene-based antifungal agent is amphotericin B.

14. The method of treating fungal infection of claim 10, wherein the azole-based antifungal agent is at least one selected from the group consisting of ketoconazole, fluconazole, itraconazole and voriconazole.

15. An antifungal combined formulation, comprising:

an inhibitor against at least one protein selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of Cryptococcus neoformans; and

an ergosterol-binding antifungal agent or azole-based antifungal agent.

16. A method of screening an antifungal agent comprising:

contacting at least one protein selected from the group consisting of Ssk1, Tco2, Ssk2, Pbs2, Hog1, Ena1 and Nha1 of Cryptococcus neoformans with a candidate material;

and

determining whether the candidate material inhibits or stimulates an activity of the protein.

17. The method of claim 16, wherein the antifungal agent is an inhibitor against an Ssk1, Ena1 or Nha1 protein.

18. A method of screening an antifungal agent comprising:

contacting at least one gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of Cryptococcus neoformans with a candidate material;

and

determining whether the candidate material inhibits or stimulates an activity of the gene.

19. The method of claim 18, wherein the antifungal agent is an inhibitor against an SSK1, ENA1 or NHA1 gene.

20. A method of treating fungal infection, comprising:

injecting an effective amount of an inhibitor against at least one protein selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of Cryptococcus neoformans into a subject.

21. The method of treating fungal infection of claim 20, wherein the inhibitor is an inhibitor against a Cac1 or Pka1 protein.

22. The method of treating fungal infection of claim 20, wherein a polyene- or azole-based antifungal agent is sequentially or simultaneously injected with the inhibitor.

23. The method of treating fungal infection of claim 22, wherein the polyene-based antifungal agent is at least one selected from the group consisting of amphotericin B, natamycin, rimocidin, filipin, nystatin, and candicin.

24. The method of treating fungal infection of claim 23, wherein the polyene-based antifungal agent is amphotericin B.

25. The method of treating fungal infection of claim 22, wherein the azole-based antifungal agent is at least one selected from the group consisting of ketoconazole, fluconazole, itraconazole and voriconazole.

26. The method of treating fungal infection of claim 20, wherein an inhibitor against at least one protein selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of Cryptococcus neoformans is sequentially or simultaneously injected with the inhibitor.

27. A method of treating fungal infection, comprising:

injecting an effective amount of an inhibitor against at least one gene selected from the group consisting of RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 and HSP122 of Cryptococcus neoformans into a subject.

28. The method of treating fungal infection of claim 27, wherein the inhibitor is an against CAC1 or PKA1 gene.

29. The method of treating fungal infection of claim 27, wherein a polyene- or azole-based antifungal agent is sequentially or simultaneously injected with the inhibitor.

30. The method of treating fungal infection of claim 29, wherein the polyene-based antifungal agent is at least one selected from the group consisting of amphotericin B, natamycin, rimocidin, filipin, nystatin, and candicin.

31. The method of treating fungal infection of claim 30, wherein the polyene-based antifungal agent is amphotericin B.

32. The method of treating fungal infection of claim 29, wherein the azole-based antifungal agent is at least one selected from the group consisting of ketoconazole, fluconazole, itraconazole and voriconazole.

33. The method of treating fungal infection of claim 27, wherein an inhibitor against at least one gene selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of Cryptococcus neoformans is sequentially or simultaneously injected with the inhibitor.

34. An antifungal combined formulation, comprising:

an inhibitor against at least one protein selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of Cryptococcus neoformans; and

at least one antifungal agent selected from the group consisting of a polyene-based antifungal agent, an azole-based antifungal agent, and an inhibitor against at least one protein or gene coding for the same selected from the group consisting of SSK1, TCO2, SSK2, PBS2, HOG1, ENA1 and NHA1 of Cryptococcus neoformans.

35. A method of screening an antifungal agent comprising:

contacting at least one protein selected from the group consisting of Ras1, Ras2, Cdc24, Gpa1, Cac1, Aca1, Pka1, Hsp12 and Hsp122 of Cryptococcus neoformans with a candidate material; and

36. The method of claim 35, wherein the antifungal agent is an inhibitor against a Cac1 or Pka1 protein.

37. A method of screening an antifungal agent comprising:

contacting at least one gene selected from the group consisting of RAS1, RAS2, CDC24, GPA1, CAC1, ACA1, PKA1, HSP12 and HSP122 of Cryptococcus neoformans with a candidate material; and

determining whether the candidate material inhibits or stimulates the activity of the gene.

38. The composition method of claim 37, wherein the antifungal agent is an inhibitor against a CAC1 or PKA1 gene.