Cryptococcus neoformans requires the TVF1 gene for thermotolerance and virulence.

Cryptococcus neoformans is the primary causative agent of cryptococcosis. Since C. neoformans thrives in environments and its optimal growth temperature is 25-30°C, it needs to adapt to heat stress in order to cause infection in mammalian hosts. In this study, we aimed to investigate the role of an uncharacterized gene, CNAG_03308. Although the CNAG_03308 deletion strain grew as well as the parent strain KN99, it produced yeast cells with abnormal morphology at 37°C and failed to propagate at 39°C. Furthermore, the deletion strain exhibited slower growth at 37°C in the presence of congo red, which is a cell wall stressor. When cultured at 39°C, the deletion strain showed strong staining with fluorescent probes for cell wall chitin and chitosan, including FITC-labeled wheat germ agglutinin, Eosin Y, and calcofluor white. The transmission electron microscopy of the deletion strain revealed a thickened inner layer of the cell wall containing chitin and chitosan under heat stress. This cell-surface altered deletion strain induced dendritic cells to secrete more interleukin (IL)-6 and IL-23 than the control strains under heat stress. In a murine infection study, C57BL/6 mice infected with the deletion strain exhibited lower mortality and lower fungal burden in the lungs and brain compared to those infected with the control strains. Based on these findings, we concluded that CNAG_03308 gene is necessary for C. neoformans to adapt to heat stress both in vitro and in the host environment. Therefore, we designated the CNAG_03308 gene as TVF1, which stands for thermotolerance and virulence-related factor 1.

Cryptococcus neoformans is a fungal pathogen causing cryptococcosis, which requires thermotolerance to proliferate in the host environment. In the present study, we identified a novel gene, TVF1 (CNAG_03308), required for thermotolerance and virulence by reverse genetics approach.

Biodiversity of Basidiomycetous Yeasts Associated with Cladonia rei Lichen in Japan, with a Description of Microsporomyces cladoniophilus sp. nov.

For more than a century, lichens have been used as an example of dual-partner symbiosis. Recently, this has been challenged by the discovery of various basidiomycetous yeasts that coexist in multiple lichen species, among which Cladonia lichens from Europe and the United States were discovered to be highly specifically associated with the basidiomycetous yeast of the family Microsporomycetaceae. To verify this highly specific relationship, we investigated the diversity of basidiomycetous yeasts associated with Cladonia rei, a widely distributed lichen in Japan, by applying two approaches: yeast isolation from the lichen thalli and meta-barcoding analysis. We obtained 42 cultures of Cystobasidiomycetous yeast which were grouped into six lineages within the family Microsporomycetaceae. Unexpectedly, although the cystobasidiomycetes-specific primer was used, not only the cystobasidiomycetous yeasts but species from other classes were also detected via the meta-barcoding dataset; in particular, pucciniomycetous yeasts were found at a high frequency in some samples. Further, Halobasidium xiangyangense, which was detected in every sample with high abundance, is highly likely a generalist epiphytic fungus that has the ability to associate with C. rei. In the pucciniomycetous group, most of the detected species belong to the scale insect-associated yeast Septobasidium genus. In conclusion, even though Microsporomyces species are not the only yeast group associated with Cladonia lichen, our study demonstrated that the thalli of Cladonia rei lichen could be a suitable habit for them.

Promising whole-cell vaccines against cryptococcosis.

Cryptococcosis is a mycosis caused by Cryptococcus neoformans and C. gattii species complexes. Although this infection is potentially lethal, no prophylactic vaccine is yet commercially available, and the immune memory that enables prevention is still under investigation. These pathogens have a capsule layer for immune evasion and a sophisticated mechanism to advance the infection, and it is expected that these characteristics will make it difficult to develop prophylactic vaccines and to decipher the protective immunity. The current vaccine studies are focused on subunit, mRNA, DNA, and viral vector vaccines, with whole-cell vaccines also proving successful against cryptococcal infections. Cryptococcal whole-cell vaccines have been composed of highly immunostimulating strains with low-pathogenicity that are modified by genetic recombination technology. Examples include the whole-cell vaccines H99γ, sgl1∆, fbp1∆, znf2oe , cda1/2/3∆, cap59∆, and cap60∆. Some of these whole-cell vaccines were found to be highly effective in prolonging life and suppressing the fungal burden after an infection challenge in mice, and to be cross-reactive to C. neoformans, C. gattii, and other fungal pathogens. Furthermore, for some vaccines, the protective effect can be retained even in an immunocompromised host depleted of CD4+ T cells. These findings have provided new insights into protective immunity that should aid in vaccine development. In this review, we highlight the upsides and downsides of whole-cell vaccines against cryptococcosis.

Tacrolimus inhibits stress responses and hyphal formation via the calcineurin signaling pathway in Trichosporon asahii.

The pathogenic fungus Trichosporon asahii causes fatal deep-seated mycosis in immunocompromised patients. Calcineurin, which is widely conserved in eukaryotes, regulates cell growth and various stress responses in fungi. Tacrolimus (FK506), a calcineurin inhibitor, induces sensitivity to compounds that cause stress on the cell membrane and cell wall integrity. In this study, we demonstrated that FK506 affects stress responses and hyphal formation in T. asahii. In silico structural analysis revealed that amino acid residues in the binding site of the calcineurin-FKBP12 complex that interact with FK506 are conserved in T. asahii. The growth of T. asahii was delayed by FK506 in the presence of SDS or Congo red but not in the presence of calcium chloride. FK506 also inhibited hyphal formation in T. asahii. A mutant deficient of the cnb gene, which encodes the regulatory subunit B of calcineurin, exhibited stress sensitivities on exposure to SDS and Congo red and reduced the hyphal forming ability of T. asahii. In the cnb-deficient mutant, FK506 did not increase the stress sensitivity or reduce hyphal forming ability. These results suggest that FK506 affects stress responses and hyphal formation in T. asahii via the calcineurin signaling pathway.

Cryptococcus neoformans MET5 Gene is not Essential for Virulence in the Silkworm Infection Model.

The involvement of the MET5 gene in virulence of Cryptococcus neoformans was examined using the silkworm Bombyx mori infection model. In the virulence assay, the met5Δ mutant showed virulence not significantly different from the wild-type strain, suggesting that the MET5 gene is not essential for full virulence of C. neoformans. The effect of silkworm hemolymph on the survival of the met5Δ mutant was also tested. The C. neoformans met5Δ strain incubated in the silkworm hemolymph for five days remained viable, suggesting that silkworm hemolymph supports survival of the met5Δ strain.

Draft Genome Sequence of Purpureocillium takamizusanense, a Potential Bioinsecticide.

To investigate the biocontrol capability of the entomopathogenic fungus Purpureocillium takamizusanense, the genome of the wild-type strain isolated from synnemata on Meimuna opalifera, was sequenced using a combination of HiSeq and Nanopore technologies, and annotated using evidence from RNA sequences and protein sequences from its sister species Purpureocillium lilacinum.

Hyperosmotic medium partially restores the growth defect and the impaired production of sterigmatocystin of an Aspergillus nidulans ΔpmtC mutant in a HogA-independent manner.

The protein O-mannosyltransferase catalyzes O-mannosylation in the endoplasmic reticulum by transferring mannose to the seryl or threonyl residues of substrate proteins. We previously reported a deletion mutant of O-mannosyltransferase C (ΔpmtC) in Aspergillus nidulans with impaired vegetative growth and sterigmatocystin (ST) production. In this study, we investigated whether osmotic conditions contribute to the developmental processes and ST biosynthesis of the ΔpmtC deletion mutant. We found that hyphal growth and ST production partially improved in the presence of NaCl, KCl or sorbitol as osmotic stabilizers. Conidiation of the ΔpmtC deletion mutant was not restored under osmotic stress conditions when the hogA gene was deleted. The hogA gene encodes a protein required for the cellular response to osmotic pressure. However, the yield of ST and the vegetative growth of the ΔhogA ΔpmtC double deletant was restored by high osmolarity in a HogA-independent manner.

A capsule-associated gene of Cryptococcus neoformans, CAP64, is involved in pH homeostasis.

The CAP64 gene is known to be involved in capsule formation in the basidiomycete yeast Cryptococcus neoformans. A null mutant of CAP64, Δcap64, lacks a capsule around the cell wall and its acidic organelles are not stained with quinacrine. In order to clarify whether the Cap64 protein indeed maintains vacuole or vesicle acidification, so that the vesicle containing the capsule polysaccharide or DBB substrate are transported to the cell membrane side, the relationship between CAP64 and intracellular transport genes and between CAP64 and enzyme-secretion activity were analysed. Laccase activity was higher in the Δcap64 strain than in the wild-type strain, and the transcriptional levels of SAV1 and VPH1 were also higher in the Δcap64 strain than in the wild-type strain. The intracellular localization of the Cap64 protein was analysed by overexpressing an mCherry-tagged Cap64 and observing its fluorescence. The Cap64 protein was accumulated within cells in a patch-like manner. The quinacrine-stained cells were observed to analyse the acidified cell compartments; quinacrine was found to be accumulated in a patch-like manner, with the patches overlapping the fluorescence of CAP64-mCherry fusion protein. Quinacrine was thus accumulated in a patch-like fashion in the cells, and the mCherry-tagged Cap64 protein position was consistent with the position of quinacrine accumulation in cells. These results suggest that CAP64 might be involved in intracellular acidification and vesicle secretion via exocytosis.

Cryptococcus gattii evades CD11b-mediated fungal recognition by coating itself with capsular polysaccharides.

Cryptococcus gattii is a capsular pathogenic fungus causing life-threatening cryptococcosis. Although the capsular polysaccharides (CPs) of C. gattii are considered as virulence factors, the physiological significance of CP biosynthesis and of CPs themselves is not fully understood, with many conflicting data reported. First, we demonstrated that CAP gene deletant of C. gattii completely lacked capsule layer and its virulence, and that the strain was susceptible to host-related factors including oxidizing, hypoxic, and hypotrophic conditions in vitro. Extracellular CPs recovered from culture supernatant bound specifically to C. gattii acapsular strains, not to other fungi and immune cells, and rendered them the immune escape effects. In fact, dendritic cells (DCs) did not efficiently uptake the CP-treated acapsular strains, which possessed no visible capsule layer, and a decreased amount of phosphorylated proteins and cytokine levels after the stimulation. DCs recognized C. gattii acapuslar cells via an immune receptor CD11b- and Syk-related pathway; however, CD11b did not bind to CP-treated acapsular cells. These results suggested that CPs support immune evasion by coating antigens on C. gattii and blocking the interaction between CD11b and C. gattii cells. Here, we describe the importance of CPs in pathogenicity and immune evasion mechanisms of C. gattii.

Identification and characterization of a sulfite reductase gene and new insights regarding the sulfur-containing amino acid metabolism in the basidiomycetous yeast Cryptococcus neoformans.

The amino acid biosynthetic pathway of invasive pathogenic fungi has been studied as a potential antifungal drug target. Studies of the disruption of genes involved in amino acid biosynthesis have demonstrated the importance of this pathway in the virulence of Cryptococcus neoformans. Here, we identified the MET5 (CNL05500) and MET10 (CNG03990) genes in this pathway, both encoding sulfite reductase, which catalyzes the reduction of sulfite to sulfide. The MET14 (CNE03880) gene was also identified, which is responsible for the conversion of sulfate to sulfite. The use of cysteine as a sulfur source led to the production of methionine via hydrogen sulfide synthesis mediated by CYS4 (CNA06170), CYS3 (CNN01730), and MST1 (CND03690). MST1 exhibited high homology with the TUM1 gene of Saccharomyces cerevisiae, which has functional similarity with the 3-mercaptopyruvate sulfurtransferase (3-MST) gene in humans. Although the hypothesis that hydrogen sulfide is produced from cysteine via CYS4, CYS3, and MST1 warrants further study, the new insight into the metabolic pathway of sulfur-containing amino acids in C. neoformans provided here indicates the usefulness of this system in the development of screening tools for antifungal drug agents.

Vaccines and Protective Immune Memory against Cryptococcosis.

Cryptococcosis is a potentially lethal disease caused by fungal pathogens including Cryptococcus neoformans and Cryptococcus gattii species complex. These fungal pathogens live in the environment and are associated with certain tree species and bird droppings. This infectious disease is not contagious, and healthy individuals may contract cryptococcal infections by inhaling the airborne pathogens from the environment. Although cleaning a contaminated environment is a feasible approach to control environmental fungal pathogens, prophylactic immunization is also considered a promising method to regulate cryptococcal infections. We review the history of the development of cryptococcal vaccines, vaccine components, and the various forms of immune memory induced by cryptococcal vaccines.

Cryptococcus gattii alters immunostimulatory potential in response to the environment.

Cryptococcus gattii is a capsular fungal pathogen, which causes life-threatening cryptococcosis in immunocompetent individuals. This emerging pathogen is less likely to be recognized by innate immunity compared to traditional Cryptococcus neoformans strains. Previous studies indicate that C-type lectin receptors (CLRs), including dectin-1 and dectin-2, play a role in recognizing cryptococcal cells; however, it remains to be elucidated whether the receptors physically associate with C. gattii yeast cell surfaces. Based on the previous findings, we hypothesized that culture conditions influence the expression or exposure of CLR ligands on C. gattii. Therefore, in the present study, we first investigated the culture conditions that induce exposure of CLR ligands on C. gattii yeast cells via the binding assay using recombinant fusion proteins of mouse CLR and IgG Fc, Fc dectin-1 and Fc dectin-2. Common fungal culture media, such as yeast extract-peptone-dextrose (YPD) broth, Sabouraud broth, and potato dextrose agar, did not induce the exposure of dectin-1 ligands, including ß-1,3-glucan, on both capsular and acapsular C. gattii strains, in contrast to Fc dectin-1 and Fc dectin-2 bound to C. gattii cells growing in the conventional synthetic dextrose (SD) medium [may also be referred to as a yeast nitrogen base with glucose medium]. The medium also induced the exposure of dectin-1 ligands on C. neoformans, whereas all tested media induced dectin-1 and dectin-2 ligands in a control fungus Candida albicans. Notably, C. gattii did not expose dectin-1 ligands in SD medium supplemented with yeast extract or neutral buffer. In addition, compared to YPD medium-induced C. gattii, SD medium-induced C. gattii more efficiently induced the phosphorylation of Syk, Akt, and Erk1/2 in murine dendritic cells (DCs). Afterwards, the cells were considerably engulfed by DCs and remarkably induced DCs to secrete the inflammatory cytokines. Overall, the findings suggest that C. gattii alters its immunostimulatory potential in response to the environment.

Neutrophil-mediated antifungal activity against highly virulent Cryptococcus gattii strain R265.

Vaccine-induced immune responses, including neutrophil, macrophage, and T-cell responses, ameliorate cryptococcosis caused by Cryptococcus gattii. However, whether neutrophils can exert fungicidal activity against C. gattii remains to be elucidated. Therefore, in this study, we investigated the neutrophil-mediated fungicidal effect against C. gattii R265 in vitro and compared it to the related fungal pathogen, Cryptococcus neoformans standard strain H99. We found that neutrophils recognized, phagocytosed, and killed C. gattii R265 in the presence of fresh mouse serum. This antifungal effect required phagocytosis and serine protease activity but not nicotinamide adenine dinucleotide phosphate oxidase activity. We also demonstrated that C. gattii R265 was more resistant to oxidative and nitrosative stress than C. neoformans H99. Together, these findings indicate that neutrophils can exert fungicidal activity against highly virulent C. gattii, at least under in vitro conditions.

A dendritic cell-based systemic vaccine induces long-lived lung-resident memory Th17 cells and ameliorates pulmonary mycosis.

Tissue-resident memory T cells (TRMs) are a novel nonvascular memory T cell subset. Although CD8+ TRMs are well-characterized, CD4+ TRMs-especially lung-resident memory Th17 cells-are still being defined. In this study, we characterized lung-resident memory Th17 cells (lung TRM17) and their role in protection against the highly virulent fungus Cryptococcus gattii. We found that intravenously transferred DCs preferentially migrated to lungs and attracted recipient DCs and led to the induction of long-lived Th17 cells expressing characteristic markers. This population could be clearly discriminated from circulating T cells by intravascular staining and was not depleted by the immunosuppressive agent FTY720. The C. gattii antigen re-stimulation assay revealed that vaccine-induced lung Th17 cells produced IL-17A but not IFNγ. The DC vaccine significantly increased IL-17A production and suppressed fungal burden in the lungs and improved the survival of mice infected with C. gattii. This protective effect was significantly reduced in the IL-17A knockout (KO) mice, but not in the FTY720-treated mice. The protective effect also coincided with the activation of neutrophils and multinucleated giant cells, and these inflammatory responses were suppressed in the vaccinated IL-17A KO mice. Overall, these data demonstrated that the systemic DC vaccine induced lung TRM17, which played a substantial role in anti-fungal immunity.

Mouse LIMR3/CD300f is a negative regulator of the antimicrobial activity of neutrophils.

Leukocyte mono-immunoglobulin-like receptor (LMIR)/CD300 proteins comprise a family of immunoglobulin-like receptors that are widely expressed on the immune cell surface in humans and mice. In general, LMIR3/CD300f suppresses the inflammatory response, but it can occasionally promote it. However, the precise roles of LMIR3 in the function of neutrophils remain to be elucidated. In the present study, we investigated LMIR3 expression in mature and immature neutrophils, and evaluated the effects of LMIR3 deficiency in mouse neutrophils. Our results indicated that bone marrow (BM) neutrophils expressed LMIR3 on their cell surface during cell maturation and that surface LMIR3 expression increased in response to Pseudomonas aeruginosa infection in a TLR4/MyD88-dependent manner. LMIR3-knockout (KO) neutrophils displayed significantly increased hypochlorous acid production, and elastase release, as well as significantly augmented cytotoxic activity against P. aeruginosa and Candida albicans; meanwhile, inhibitors of elastase and myeloperoxidase offset this enhanced antimicrobial activity. Furthermore, LMIR3-KO mice were significantly more resistant to Pseudomonas peritonitis and systemic candidiasis, although this may not be entirely due to the enhanced activity of neutrophils. These results demonstrate that LMIR3/CD300f deficiency augments the antimicrobial activity of mouse neutrophils.

Protein O-mannosyltransferases are required for sterigmatocystin production and developmental processes in Aspergillus nidulans.

Aspergillus nidulans produces sterigmatocystin (ST), a precursor of a carcinogenic secondary metabolite aflatoxin (AF), during its developmental process. ST biosynthesis has been shown to be affected by various regulatory factors. In this study, we investigated the involvement of O-mannosyltransferases (PmtA, PmtB, PmtC), in ST production and morphological development. Deletion of pmtA (ΔpmtA), pmtB (ΔpmtB) or pmtC (ΔpmtC) caused no spore production and a significant decline of vegetative growth. A tremendous decline of ST level was observed in all Δpmt mutants at the third day after inoculation. By extending the growth period, ST production of ΔpmtA and ΔpmtB increased to the wild-type level 7 days after inoculation. On the other hand, ST was not detected from 7- or 14-day cultures in ΔpmtC. Expression levels of aflR gene, an essential regulator of the ST biosynthesis pathway, were also down-regulated in the Δpmt strains. By introducing the aflR overexpression cassette, ST production in the ΔpmtA and ΔpmtB significantly increased to levels comparable to the wild type. However, the presence of the aflR overexpression cassette could not improve ST production in the ΔpmtC mutant. These data suggest that the PMT family is a new endogenous factor that is required for ST biosynthesis in A. nidulans. These findings provide better understanding of the regulatory mechanisms of AF/ST biosynthesis, which can ultimately contribute to our ability to control aflatoxin contamination.

Novel biosynthetic pathway for sulfur amino acids in Cryptococcus neoformans.

We elucidated a unique feature of sulfur metabolism in Cryptococcus neoformans. C. neoformans produces cysteine solely by the O-acetylserine pathway that consists of serine-O-acetyl transferase and cysteine synthase. We designated the gene encoding the former enzyme CYS2 (locus tag CNE02740) and the latter enzyme CYS1 (locus tag CNL05880). The cys1Δmutant strain was found to be avirulent in a murine infection model. Methionine practically does not support growth of the cys1Δ strain, and cysteine does not serve as a methionine source, indicating that the transsulfuration pathway does not contribute to sulfur amino acid synthesis in C. neoformans. Among the genes encoding enzymes catalyzing the reactions from homoserine to methionine, the gene corresponding to the Saccharomyces cerevisiae MET17 encoding O-acetylhomoserine sulfhydrylase (Met17p) had remained to be identified in C. neoformans. By genetic analysis of Met- mutants obtained by Agrobacterium tumefaciens-mediated mutagenesis, we concluded that Cnc01220, most similar to Str2p (36% identity), cystathionine-γ-synthase, in the Saccharomyces genome, is the C. neoformans version of O-acetylhomoserine sulfhydrylase. We designated CNC01220 as MET17. The C. neoformans met3Δ mutant defective in the first step of the sulfate assimilation pathway, sulfate adenylyltransferase, barely uses methionine as a sulfur source, whereas it uses cysteine efficiently. The poor utilization of methionine by the met3Δ mutant is most probably due to the absence of the transsulfuration pathway, causing an incapability of C. neoformans to produce cysteine and hydrogen sulfide from methionine. When cysteine is used as a sulfur source, methionine is likely produced de novo by using hydrogen sulfide derived from cysteine via an unidentified pathway. Altogether, the unique features of sulfur amino acid metabolism in C. neoformans will make this fungus a valuable experimental system to develop anti-fungal agents and to investigate physiology of hydrogen sulfide.

[A Silkworm Infection Model to Evaluate Antifungal Drugs for Cryptococcosis].

The development of effective drugs against fungal diseases involves performing infection experiments in animals to evaluate candidate therapeutic compounds. Cryptococcus neoformans is a pathogenic fungus that causes deep mycosis, resulting in respiratory illness and meningitis. Here we describe a silkworm system established to evaluate the safety and efficacy of therapeutic drugs against infection by Cryptococcus neoformans and the advantages of this system over other animal models. The silkworm assay system has two major advantages: 1) silkworms are less expensive to rear and their use is less problematic than that of mammals in terms of animal welfare, and 2) in vivo screenings for identifying candidate drugs can be easily performed using a large number of silkworms. The pharmacokinetics of compounds are consistent between silkworms and mammals. Moreover, the ED50 values of antibiotics are concordant between mammalian and silkworm infection models. Furthermore, the body size of silkworms makes them easy to handle in experimental procedures compared with other invertebrate infectious experimental systems, and accurate amounts of pathogens and chemicals can be injected fairly easily. These advantages of silkworms as a host animal make them useful for screening candidate drugs for cryptococcosis.

Structome analysis of Escherichia coli cells by serial ultrathin sectioning reveals the precise cell profiles and the ribosome density.

Structome analysis, the quantitative three-dimensional structural analysis of whole cells at the electron microscopic level, of Exophiala dermatitidis (black yeast), Saccharomyces cerevisiae, Mycobacterium tuberculosis (MTB) and Myojin spiral bacteria (MSB) have already been reported. Here, the results of the structome analysis of Escherichia coli cells based on transmission electron microscope observation of serial ultrathin sections was reported, and compared with the data obtained from phase contrast microscopy and scanning electron microscopy. On average, the cells had 0.89 µm in diameter, 2.47 µm in length and 1.16 fl (µm3) in cell volume in the structome analysis. Furthermore, E. coli cells had 26 100 ribosomes per whole cell with density of 2840 per 0.1 fl cytoplasm. The total ribosome number per cell was 15 times larger than that of MTB and about one-eighth of those of the yeast cells above. On the other hand, the ribosome density of E. coli cells are more than 13 times, 4 times, 2.5-times and 1.5-times higher than MSB, MTB, E. dermatitidis and S. cerevisiae, respectively. Finally, our ribosome enumeration data were compared between the structome-analyzed species and the relationship between the ribosome density and the growth rate among these species was discussed.

Creation, characterization and utilization of Cryptococcus neoformans mutants sensitive to micafungin.

We constructed deletion mutants of Cryptococcus neoformans var neoformans (serotype D) genes encoding late ergosterol biosynthetic pathway enzymes and found that the mutations enhanced susceptibility to various drugs including micafungin, one of the echinocandins, to which wild-type Cryptococcus strains show no susceptibility. Furthermore, through isolation of a mutant resistant to micafungin from a micafungin-sensitive erg mutant and genetic analysis of it, we found that the responsible mutation occurred in the hotspot 2 of FKS1 encoding ß-1, 3-glucan synthase, indicating that micafungin inhibited the growth of the erg mutant via inhibiting Fks1 activity. Addition of ergosterol to the culture of the erg mutants recovered the resistance to micafungin, suggesting that the presence of ergosterol in membrane inhibits the accession of micafungin to its target. We found that a loss of one of genes encoding subunits of v-ATPase, VPH1, made Cryptococcus cells sensitive to micafungin. Our observation that the erg2 vph1 double mutant was more sensitive to micafungin than either single mutant suggests that these two genes act differently in becoming resistant to micafungin. The erg mutants allowed us to study the physiological significance of ß-1, 3-glucan synthesis in C. neoformans; the inhibition of ß-1, 3-glucan synthesis induced cell death and changes in cellular morphology. By observing the erg mutant cells recovering from the growth inhibition imposed by micafungin, we recognized ß-1, 3-glucan synthesis would suppress filamentous growth in C. neoformans.