Functional characterization of PMT2, encoding a protein-O-mannosyltransferase, in the human pathogen Cryptococcus neoformans.

Diazobenzoic acid B (DBB), also known as diazonium blue B or fast blue B, can be used to distinguish basidiomycetous yeasts from ascomycetes. This chemical has long been used for the taxonomic study of yeast species at the phylum level, but the mechanism underlying the DBB staining remains unknown. To identify molecular targets of DBB staining, we isolated Agrobacterium tumefaciens-mediated insertional mutants of Cryptococcus neoformans, a basidiomycetous pathogenic yeast, which were negative to DBB staining. In one of these mutants, we found that the PMT2 gene, encoding a protein-O-mannosyltransferase, was interrupted by a T-DNA insertion. A complete gene knockout of the PMT2 gene revealed that the gene was responsible for DBB staining in C. neoformans, suggesting that one of the targets of Pmt2-mediated glycosylation is responsible for interacting with DBB. We also determined that Cryptococcus gattii, a close relative of C. neoformans, was not stained by DBB when the PMT2 gene was deleted. Our finding suggests that the protein-O-mannosylation by the PMT2 gene product is required for DBB staining in Cryptococcus species in general. We also showed that glycosylation in Cryptococcus by Pmt2 plays important roles in controlling cell size, resistance to high temperature and osmolarity, capsule formation, sexual reproduction, and virulence.

Candida albicans Msi3p, a homolog of the Saccharomyces cerevisiae Sse1p of the Hsp70 family, is involved in cell growth and fluconazole tolerance.

We investigated the cellular function of Msi3p, belonging to the heat shock protein 70 family, in Candida albicans. The mutant strain tetMSI3 was generated, in which MSI3 was controlled by a tetracycline-repressive promoter, because there is evidence to suggest that MSI3 is an essential gene. We controlled the MSI3 expression level by doxycycline (DOX) and compared its phenotype with that of a control strain with the tetracycline-repressive promoter and a wild-type copy MSI3. The results indicated that MSI3 was essential for cell growth. In addition, all the tetMSI3-infected mice survived after DOX administration. Drug susceptibility tests indicated that repression of MSI3 expression resulted in hypersensitivity to fluconazole and conferred fungicidal activity to fluconazole. The expression levels of MSI3 and calcineurin-dependent genes were upregulated in response to fluconazole in the control strain. In tetMSI3, the upregulation of MSI3 was lost, and the expression level of the calcineurin-dependent genes was no longer elevated in response to fluconazole and was not affected by DOX, indicating that the upregulation of MSI3 expression was required for the induction of the calcineurin-dependent gene expression. These data suggest that Msi3p confers fluconazole tolerance by partially influencing the calcineurin signaling pathway and also other tolerance mechanisms.

Intestinal resident yeast Candida glabrata requires Cyb2p-mediated lactate assimilation to adapt in mouse intestine.

The intestinal resident Candida glabrata opportunistically infects humans. However few genetic factors for adaptation in the intestine are identified in this fungus. Here we describe the C. glabrata CYB2 gene encoding lactate dehydrogenase as an adaptation factor for survival in the intestine. CYB2 was identified as a virulence factor by a silkworm infection study. To determine the function of CYB2, we analysed in vitro phenotypes of the mutant Δcyb2. The Δcyb2 mutant grew well in glucose medium under aerobic and anaerobic conditions, was not supersensitive to nitric oxide which has fungicidal-effect in phagocytes, and had normal levels of general virulence factors protease, lipase and adherence activities. A previous report suggested that Cyb2p is responsible for lactate assimilation. Additionally, it was speculated that lactate assimilation was required for Candida virulence because Candida must synthesize glucose via gluconeogenesis under glucose-limited conditions such as in the host. Indeed, the Δcyb2 mutant could not grow on lactate medium in which lactate is the sole carbon source in the absence of glucose, indicating that Cyb2p plays a role in lactate assimilation. We hypothesized that Cyb2p-mediated lactate assimilation is necessary for proliferation in the intestinal tract, as the intestine is rich in lactate produced by bacteria flora, but not glucose. The Δcyb2 mutant showed 100-fold decreased adaptation and few cells of Saccharomyces cerevisiae can adapt in mouse ceca. Interestingly, C. glabrata could assimilate lactate under hypoxic conditions, dependent on CYB2, but not yeast S. cerevisiae. Because accessible oxygen is limited in the intestine, the ability for lactate assimilation in hypoxic conditions may provide an advantage for a pathogenic yeast. From those results, we conclude that Cyb2p-mediated lactate assimilation is an intestinal adaptation factor of C. glabrata.

Growth defects resulting from inhibiting ERG20 and RAM2 in Candida glabrata.

Farnesyl pyrophosphate (FPP) is utilized for many cellular processes, including the production of dolichols, ubiquinone (CoQ), sterols, farnesylated heme A and prenylated proteins. This lipid synthesized by FPP synthetase (ERG20) becomes attached to target proteins by the prenyltransferases, CDC43/RAM2 and RAM1/RAM2 complexes after the formation of the C15 and C20 units, respectively. Defects in protein prenylation as a result of inhibiting these enzyme complexes lead to pleiotropic effects in all eukaryotes. In this study, using Candida glabrata conditional mutants, the importance of the ERG20 and RAM2 genes for growth using both in vivo and in vitro assays was assessed by placing the RAM2 and ERG20 genes under the control of a regulatable promoter. Repression of RAM2 gene expression revealed growth defects under both conditions. However, repression of ERG20 gene expression did not impair fungal growth in a mouse host, but did result in growth defects on laboratory media. Thus, FPP synthase is not required for survival in an infected mouse, but the RAM2-encoded prenyltransferase was critical for growth under both conditions. This study strongly suggests that inhibitors of prenyltransferase may be promising antifungals.

[A study for testing the antifungal susceptibility of yeast by the Japanese Society for Medical Mycology (JSMM) method. The proposal of the modified JSMM method 2009].

The Japanese Society for Medical Mycology (JSMM) method used for testing the antifungal susceptibility of yeast, the MIC end point for azole antifungal agents, is currently set at IC(80). It was recently shown, however that there is an inconsistency in the MIC value between the JSMM method and the CLSI M27-A2 (CLSI) method, in which the end- point was to read as IC(50). To resolve this discrepancy and reassess the JSMM method, the MIC for three azoles, fluconazole, itraconazole and voriconazole were compared to 5 strains of each of the following Candida species: C. albicans, C. glabrata, C. tropicalis, C. parapsilosis and C. krusei, for a total of 25 comparisons, using the JSMM method, a modified JSMM method, and the CLSI method. The results showed that when the MIC end- point criterion of the JSMM method was changed from IC(80) to IC(50) (the modified JSMM method) , the MIC value was consistent and compatible with the CLSI method. Finally, it should be emphasized that the JSMM method, using a spectrophotometer for MIC measurement, was superior in both stability and reproducibility, as compared to the CLSI method in which growth was assessed by visual observation.

Comparison of genotypes between environmental and clinical isolates of Cryptococcus neoformans var. grubii based on microsatellite patterns.

We applied multilocus microsatellite typing (MLMT) method to investigate the genetic relation between Cryptococcus neoformans var. grubii clinical and environmental isolates in São Paulo, Brazil. This MLMT method includes three functional gene sequences of C. neoformans var. grubii, which are dispersed on three chromosomes. In all, 89 strains (36 clinical and 53 environmental isolates) were analyzed. Of 36 clinical strains, 20 belonged to a major type of MLMT-13 (55.6%). They were mainly isolated from clinical specimens. About 52.8% of strains from the environment belong to a major type of MLMT-36, which are indigenous to environments and which were not isolated from clinical samples. Thus, we recognized two genotypes that distinguish majority of clinical and environmental strains. No differences were found in antifungal susceptibility and capsule size between major environmental and clinical MLMT types.

C-type lectin Mincle is an activating receptor for pathogenic fungus, Malassezia.

Mincle (also called as Clec4e and Clecsf9) is a C-type lectin receptor expressed in activated phagocytes. Recently, we have demonstrated that Mincle is an FcRgamma-associated activating receptor that senses damaged cells. To search an exogenous ligand(s), we screened pathogenic fungi using cell line expressing Mincle, FcRgamma, and NFAT-GFP reporter. We found that Mincle specifically recognizes the Malassezia species among 50 different fungal species tested. Malassezia is a pathogenic fungus that causes skin diseases, such as tinea versicolor and atopic dermatitis, and fatal sepsis. However, the specific receptor on host cells has not been identified. Mutation of the putative mannose-binding motif within C-type lectin domain of Mincle abrogated Malassezia recognition. Analyses of glycoconjugate microarray revealed that Mincle selectively binds to alpha-mannose but not mannan. Thus, Mincle may recognize specific geometry of alpha-mannosyl residues on Malassezia species and use this to distinguish them from other fungi. Malassezia activated macrophages to produce inflammatory cytokines/chemokines. To elucidate the physiological function of Mincle, Mincle-deficient mice were established. Malassezia-induced cytokine/chemokine production by macrophages from Mincle(-/-) mice was significantly impaired. In vivo inflammatory responses against Malassezia was also impaired in Mincle(-/-) mice. These results indicate that Mincle is the first specific receptor for Malassezia species to be reported and plays a crucial role in immune responses to this fungus.

Treatment of onychomycosis caused by dermatophytes--an opinion proposed by Committee for Standardization of the Japanese Society for Medical Mycology 2007.

After the rapid progress in therapeutic pharmaceutics against onychomycosis caused by dermatophytes in the 1990s, an optimal therapeutic strategy for individual patients with the onychomycosis has become possible for clinical dermatologists. In this review, we discuss on clinical problems concerning this disease and propose recommendable treatments for each patient with topical and/or systemic use of antifungal agents. Finally, with consideration of already published therapeutic guidelines, we stress the necessity of "order-made" therapy for each patient with his/her medical status and wishes taking into account.

Resposta imune humoral na paracoccidioidomicose experimental em camundongos ddY / Humoral immune response in experimental ddY mice paracoccidioidomycosis

A paracoccidioidomicose (PCM) é uma micose sistêmica, restrita à América Latina, com maior incidência noBrasil. O camundongo ddY tem sido empregado como modelo murino de PCM e, no entanto, não há informaçõesa respeito da resposta imune desse animal frente à infecção. O presente estudo tem como objetivo avaliar aresposta imune humoral específica para o principal antígeno, gp43, do fungo Paracoccidioides brasiliensis,em camundongos ddY infectados com a cepa virulenta Pb 18. Foram realizadas análises da antigenemia ehistopatológico em vários órgãos e em diferentes tempos pós-infecção. Os resultados obtidos demonstraramaumento nos níveis de IgG anti-gp43 nos dias 14, 17, 21, 24, 28 e 56 pós-infecção e aumento no nível de gp43solúvel aos 28 dias pós-infecção. As células fúngicas foram detectadas em todos os órgãos analisados(cérebro, coração, pulmão, fígado, baço e rim) e em todos os períodos. As lesões granulomatosas tornaramsepredominantes 14 dias pós-infecção. Os resultados evidenciaram que o camundongo ddY produz respostaimune humoral frente ao principal antígeno de P. brasiliensis, apresentando-se elevado até 56 dias pósinfecção.A redução do nível de gp43 solúvel na fase crônica, supostamente devido ao início do controle dainfecção, requer estudos complementares adicionais.

Paracoccidioidomycosis (PCM) is a systemic mycosis, restrict to Latin America, with higher incidence inBrazil. ddY mice have been used as experimental PCM model, although there is no data regarding immuneresponse. The aim of the present study was evaluated specific humoral response against the main specificantigen of the fungal Paracoccidioides brasiliensis, the gp43, in ddY mice infected with virulent Pb 18.Antigenemia analysis and histophatological exam in several organs were performed in different time post-infection The results showed increased levels of anti-gp43 IgG on days 14, 17, 21, 24, 28 and 56 post-infectionand increased levels of soluble gp-43 on day 28 post-infection. The fungal cells were detected in all organsanalyzed (brain, heart, lung, liver, spleen and kidney) in all investigated periods. The granulomatous lesionsbecame predominant 14 days after infection. The results evidence that ddY mice produce humoral immuneresponse to main P. brasiliensis antigen, with high levels until 56 days after infection. Further studies areneeded to show that reduction of soluble gp43 in chronic phase correlates with infection control.

Development of a highly efficient gene targeting system induced by transient repression of YKU80 expression in Candida glabrata.

In the pathogenic yeast Candida glabrata, gene targeting to generate knockouts and "knockins" is a potentially powerful method for the analysis of gene function. Its importance increased after the C. glabrata genome sequence project, but progress in the field is hampered by inefficient mechanisms for gene targeting. With the use of 40-bp homologous flanking DNA, no gene targeting was identified. To address this issue, YKU80 was disrupted, leading to an increase in targeting efficiency of 5.1% using 40-bp flanking homologous DNA. To harness the beneficial effects of YKU80 inactivation on gene targeting frequency without incurring any negative effects, such as synthetic sickness or lethality, we developed a new system whereby the expression of YKU80 was restored following a transient knockdown of expression during transformation. Strains used for this new system carried a SAT1 flipper in the YKU80 promoter region, which was used to repress expression during transformation but was spontaneously excised from the locus after the transformation. By using this strain, DNA damage induced by methyl methane sulfonate, H(2)O(2), UV irradiation, and hydroxyurea before and during gene targeting was evaluated and the mutation rate of URA3 was determined. No significant effects of the SAT1 flipper on these processes have been identified. After the SAT1 flipper is excised, a 34-bp FLP recombination target sequence is left in the promoter region. However, the levels of mRNA transcription were restored and no difference in the survival ratio in vivo compared to that with the YKU80 wild-type strain was identified.

In-vitro antifungal activities of sulfa drugs against clinical isolates of Aspergillus and Cryptococcus species.

In vitro susceptibilities of ten clinical isolates, including five strains of Cryptococcus neoformans var. grubii and five strains of Aspergillus fumigatus, were determined against nine sulfa drugs using a microdilution method. Among the five tested media, minimum inhibitory concentration (MIC) values were observed only in YNB medium: no detectable level MIC value of less than 125 microg/ml was observed in the four remaining media against Cryptococcus species. Of the nine sulfa drugs, of which sulfaphenazole showed the highest antifungal activity, the MIC values for A. fumigatus and C. neoformans var. grubii were, respectively, 64 microg/ml and 4-8 microg/ml, suggesting high susceptibility of C. neoformans to sulfa drugs.

[Essential genes as potential targets of antifungal agents in pathogenic yeast Candida].

An important point in the development of an antimicrobial agent is whether its target molecules are essential for growth of the microorganism. From this viewpoint, we focused attention on essential genes as potential targets of antifungal agents in the pathogenic yeast Candida. Here we introduce recent attempts for screening, identification, and characterization of essential genes from a haploid yeast Candida glabrata, using temperature-sensitive mutants. Our experimental results suggesting the essentiality of C. albicans PHO85, the homologue of which is known as a negative regulator of the PHO system and as a non-essential gene in Saccharomyces cerevisiae are also described.

Mutation in IRO1 tightly linked with URA3 gene reduces virulence of Candida albicans.

Gene deletion in the pathogenic fungus Candida albicans has relied heavily on a URA3 cassette and a recipient delta ura3 strain CAI4. The IRO1 gene adjacent to URA3 was inadvertently deleted during construction of CAI4. We report here that a mutation in IRO1 reduces virulence of C. albicans.

Transvalencin A, a thiazolidine zinc complex antibiotic produced by a clinical isolate of Nocardia transvalensis. I. Taxonomy, fermentation, isolation and biological activities.

A new thiazolidine-type antibiotic with zinc in its structure, designated transvalencin A, was isolated from Nocardia sp. IFM 10065, a clinical isolate from a patient with actinomycotic mycetoma. The strain was identified as Nocardia transvalensis based on its morphological, phenotypic and phylogenetic characteristics. Transvalencin A showed antimicrobial activity against fungi such as Trichophyton mentagrophytes and Cryptococcus neoformans. The antibiotic is also active against Gram-positive bacteria such as Micrococcus luteus. We observed higher activity for fungi in an acidic medium than in a neutral medium.

Transvalencin A, a thiazolidine zinc complex antibiotic produced by a clinical isolate of Nocardia transvalensis. II. Structure elucidation.

A novel antifungal antibiotic, transvalencin A, is produced by Nocardia transvalensis IFM 10065 isolated from a patient with actinomycotic mycetoma in Japan. The antibiotic structure was elucidated using NMR, mass spectrometric investigations, and X-ray crystallographic analysis. Transvalencin A is a 1:1 complex of a zinc and an organic acid with a phenolic substituent. Transvalencin A is comprised of o-substituted p-chlorophenol, tetrasubstituted oxazoline, disubstituted thiazolyl-N-methylthiazolidine and monosubstituted N-methylthiazolidine.

Histoplasma capsulatum variety duboisii isolated in Japan from an HIV-infected Ugandan patient.

A strain of Histoplasma capsulatum var. duboisii (deposited as IFM 50954 in Chiba University) was isolated from the cerebrospinal fluid of a female Ugandan patient infected with HIV. The isolate had in vitro urease activity on Christensen's urea agar slants, although the common belief is that H. capsulatum var. duboisii is urease negative, and is, considered one of the characteristic markers that distinguishes the three varieties of H. capsulatum. Forty H. capsulatum var. capsulatum, five H. capsulatum var. duboisii, and five H. capsulatum var. farciminosum isolates were evaluated for urease activity on Christensen's urea agar slants and for other qualitative and quantitative urease assays of activity. All 50 isolates of H. capsulatum used in this study were positive for urease activity, suggesting that urease activity may be universal characteristic of H. capsulatum. We also compared the urease activity and pathogenicity of seven H. capsulatum isolates that convert into yeast-form cells. Although isolate IFM 50954 showed moderate virulence in mice and moderate urease activity among tested H. capsulatum isolates, there was no correlation between level of urease activity and pathogenicity. In addition, scanning electron microscopy revealed that some microconidia of isolate IFM 50954 formed "double-cell" configurations that were attached to each other by narrow bases.

Antifungal agents from the roots of Cudrania cochinchinensis against Candida, Cryptococcus, and Aspergillus species.

Bioassay-guided fractionation resulted in the isolation of four antifungal agents from the roots of Cudrania cochinchinensis. Two of these were new compounds, cudraxanthone S [1, 1,3,5,6-tetrahydroxy-2-(1,1-dimethyl-2-propenyl)xanthone] and cudraflavanone B (2, 2',4',5,7-tetrahydroxy-6-prenylflavanone). The remaining two compounds were known compounds, toxyloxanthone C (3) and wighteone (4). Among these compounds, 1, 3, and 4 exhibited antifungal activities against Cryptococcus neoformans, Aspergillusfumigatus, and A. nidulans (MICs = 2-8 microg/mL). Compounds 1 and 3 also showed antifungal activity against Candida glabrata (MICs = 4-8 microg/mL).

In vitro antifungal activity of Micafungin (FK463) against dimorphic fungi: comparison of yeast-like and mycelial forms.

The characteristics of in vitro micafungin (FK463) antifungal activity against six species of dimorphic fungi were investigated in accordance with the NCCLS M27-A microdilution methods. MICs of micafungin, amphotericin B, itraconazole, and fluconazole for Histoplasma capsulatum var. capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis, Penicillium marneffei, and Sporothrix schenckii were determined both for the yeast-like form and mycelial form. Coccidioides immitis was tested only in its mycelial form. We have clearly demonstrated that the in vitro activity of micafungin depends considerably on the growth form of dimorphic fungi. Micafungin exhibited potent activity against the mycelial forms of H. capsulatum, B. dermatitidis, and C. immitis (MIC range, 0.0078 to 0.0625 micro g/ml), while it was very weakly active against their yeast-like forms (MIC range, 32 to >64 micro g/ml). Micafungin was also more active against the mycelial forms than the yeast-like forms of Paracoccidioides brasiliensis, Penicillium marneffei, and S. schenckii. The MICs of amphotericin B were 2 to 5 dilutions lower for the mycelial forms than for the yeast-like forms of B. dermatitidis and Paracoccidioides brasiliensis. There was no apparent difference in the activity of itraconazole between the two forms. The MICs of fluconazole for the yeast-like forms were generally lower than those for the mycelial forms, and considerably so for B. dermatitidis. These results suggest that the growth form employed in antifungal susceptibility testing of dimorphic fungi can considerably influence the interpretation of results. At present, it cannot be judged whether micafungin has clinical usefulness for dimorphic fungus infections, since for most fungi it remains uncertain which growth form correlates better with therapeutic outcome. However, the results of this study warrant further investigations of micafungin as a therapeutic agent for infections caused by dimorphic fungi.

Detection of the gp43 gene and (1-3)-beta-D-glucan of Paracoccidioides brasiliensis in the blood of experimentally infected mice.

Paracoccidioidomycosis (PCM) is a deep mycosis caused by the thermo-dependent dimorphic fungus Paracoccidioides brasiliensis and is prevalent in Latin American countries. Diagnosis of PCM is sometimes difficult outside the endemic countries, thus a rapid and conclusive method for diagnosis of PCM has been anticipated. We compared the sensitivities of a nested PCR method for detecting the gp43 gene and a commercial kit for detecting (1-3)-beta-D-glucan in the blood of experimentally infected mice. Blood samples were collected from mice at 0 (soon after inoculation), 6, 12, 24, 48, and 72 hours and 5, 7, 10, 14, 17, 21, 24, 28 and 56 days after the intravenous inoculation of 10(6) yeast cells of P. brasiliensis, and were separated into clots and plasma. The (1-3)-beta-D-glucan detection kit in the plasma showed positive reactions in some samples within 7 days and 28 and 56 days after infections. In contrast, the PCR method was more sensitive than the (1-3)-beta-D-glucan detection kit throughout the observation period. The clot samples yielded more sensitive PCR-results than did the plasma samples. Although 24 hours is required for the PCR detection, it was confirmed to provide an accurate diagnosis of PCM.