The CAP64 gene is known to be involved in capsule formation in the basidiomycete yeast Cryptococcus neoformans . A null mutant of CAP64 , Δ cap64 , 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 Δ cap64 strain than in the wild-type strain, and the transcriptional levels of SAV1 and VPH1 were also higher in the Δ cap64 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.
A basidiomycetous anamorphic yeast-like fungus, isolated from new bamboo shoots collected in Japan, was assigned to Meira argovae by comparison of conidial morphology, physiological characteristics, rDNA sequences, and DNA–DNA relatedness with the ex-type strains of Meira species. This is the first record of the finding of M. argovae from other than mite cadavers and in regions other than Israel. Phylogenetic analysis based on the D1–D2 domain demonstrated that Meira species and teleomorphic Dicellomyces species, which include a bamboo leaf parasite, D. gloeosporus, formed sister clades.
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.
Secondary metabolite production and sporulation are tightly co-regulated in the Aspergilli and Fusaria. Here we discuss two conserved pathways, including a G-protein/cAMP/Protein kinase A cascade and an oxylipin-mediated signalling process, that genetically link sporulation and secondary metabolism in these genera. The G-protein alpha subunit FadA negatively regulates aflatoxin and cyclopiazonic acid production in Aspergillus, and positively regulates penicillin production in A. nidulans and tricothecene production in Fusarium sporotrichiodes. Inactivation of FadA eliminates or decreases asexual spore production in both genera. The G-protein cascade is conserved throughout eukaryotes, and regulation of sporulation and secondary metabolism by this signal transduction pathway appears to be conserved within filamentous fungi. On the other hand, the oxylipin-mediated signalling pathway appears to be restricted to filamentous fungi. We have identified novel genes encoding putative dioxygenases likely to be responsible for secreted oxylipins which act as sporulation factors. Deletion of these genes affects asexual sporulation and secondary metabolite production in A. nidulans and F. sporotrichiodes.
Calcineurin is a serine/threonine protein phosphatase that consists of catalytic (calcineurin A) and regulatory (calcineurin B) subunits. The conserved protein plays important roles in various biological processes. Drug combination of fluconazole and the calcineurin inhibitor (FK506) showed synergistic effects against dermatophytes. In the current study, we identified the calcineurin A homologous gene (TmcanA) in the dermatophyte Arthroderma vanbreuseghemii (anamorph: Trichophyton mentagrophytes). Knockdown mutants were produced from A. vanbreuseghemii, resulting in a defection in growth properties in accordance with dose of the suppressing reagent. The TmcanA gene restored the ability of calcineurin A-deficient Cryptococcus neoformans strain to grow at elevated temperatures. Repression of TmcanA at 37°C resulted in severely stunted growth, suggesting that this protein plays a role in tolerance to elevated temperatures. In addition, TMCANA showed an interaction with high osmolarity glycerol (HOG) signalling pathway by governing the secretion of a secondary metabolite. Moreover, expression of the hydrophobin A gene (TmHF) decreased significantly under the TmcanA-repressive condition, suggesting that TMCANA is involved in its regulation. In conclusion, calcineurin A is a multifunctional gene that is involved in the regulation of several biological processes and therefore is worth being considered as a drug target for treatment of dermatophytoses.
Although α-1,3-glucan is one of the major cell wall polysaccharides in filamentous fungi, the physiological roles of α-1,3-glucan remain unclear. The model fungus Aspergillus nidulans possesses two α-1,3-glucan synthase (AGS) genes, agsA and agsB. For functional analysis of these genes, we constructed several mutant strains in A. nidulans: agsA disruption, agsB disruption, and double-disruption strains. We also constructed several CagsB strains in which agsB expression was controlled by the inducible alcA promoter, with or without the agsA-disrupting mutation. The agsA disruption strains did not show markedly different phenotypes from those of the wild-type strain. The agsB disruption strains formed dispersed hyphal cells under liquid culture conditions, regardless of the agsA genetic background. Dispersed hyphal cells were also observed in liquid culture of the CagsB strains when agsB expression was repressed, whereas these strains grew normally in plate culture even under the agsB-repressed conditions. Fractionation of the cell wall based on the alkali solubility of its components, quantification of sugars, and 13C-NMR spectroscopic analysis revealed that α-1,3-glucan was the main component of the alkali-soluble fraction in the wild-type and agsA disruption strains, but almost no α-1,3-glucan was found in the alkali-soluble fraction derived from either the agsB disruption strain or the CagsB strain under the agsB-repressed conditions, regardless of the agsA genetic background. Taken together, our data demonstrate that the two AGS genes are dispensable in A. nidulans, but that AgsB is required for normal growth characteristics under liquid culture conditions and is the major AGS in this species.
