Hog1-mediated stress tolerance in the pathogenic fungus Trichosporon asahii.

Trichosporon asahii is an opportunistic pathogenic fungus that causes severe and sometimes fatal infections in immunocompromised patients. Hog1, a mitogen-activated protein kinase, regulates the stress resistance of some pathogenic fungi, however its role in T. asahii has not been investigated. Here, we demonstrated that the hog1 gene-deficient T. asahii mutant is sensitive to high temperature, cell membrane stress, oxidative stress, and antifungal drugs. Growth of the hog1 gene-deficient T. asahii mutant was delayed at 40 °C. The hog1 gene-deficient T. asahii mutant also exhibited sensitivity to sodium dodecyl sulfate, hydrogen peroxide, menadione, methyl methanesulfonate, UV exposure, and antifungal drugs such as amphotericin B under a glucose-rich condition. Under a glucose-restricted condition, the hog1 gene-deficient mutant exhibited sensitivity to NaCl and KCl. The virulence of the hog1 gene-deficient mutant against silkworms was attenuated. Moreover, the viability of the hog1 gene-deficient mutant decreased in the silkworm hemolymph. These phenotypes were restored by re-introducing the hog1 gene into the gene-deficient mutant. Our findings suggest that Hog1 plays a critical role in regulating cellular stress responses in T. asahii.

A critical role of calcineurin in stress responses, hyphal formation, and virulence of the pathogenic fungus Trichosporon asahii.

Trichosporon asahii is a conditional pathogenic fungus that causes severe and sometimes fatal infections in immunocompromised patients. While calcineurin, an essential component of a calcium-dependent signaling pathway, is known to regulate stress resistance and virulence of some pathogenic fungi, its role in T. asahii has not been investigated. Here, we demonstrated that calcineurin gene-deficient T. asahii mutants are sensitive to high temperature as well as cell-membrane and cell-wall stress, and exhibit decreased hyphal formation and virulence against silkworms. Growth of T. asahii mutants deficient in genes encoding subunits of calcineurin, cna1 and cnb1, was delayed at 40 °C. The cna1 and cnb1 gene-deficient mutants also showed sensitivity to sodium dodecyl sulfate, Congo red, dithiothreitol, and tunicamycin. On the other hand, these mutants exhibited no sensitivity to caffeine, sorbitol, monensin, CaCl2, LiCl, NaCl, amphotericin B, fluconazole, or voriconazole. The ratio of hyphal formation in the cna1 and cnb1 gene-deficient mutants was decreased. Moreover, the virulence of the cna1 and cnb1 gene-deficient mutants against silkworms was attenuated. These phenotypes were restored by re-introducing each respective gene into the gene-deficient mutants. Our findings suggest that calcineurin has a role in regulating the cellular stress response and virulence of T. asahii.

A joint PCR-based gene-targeting method using electroporation in the pathogenic fungus Trichosporon asahii.

Trichosporon asahii is a pathogenic fungus that causes deep-seated fungal infections in immunocompromised patients. Established methods for generating gene-deficient T. asahii mutants exist, but the frequency of obtaining transformants by electroporation remains low. In the present study, we optimized the conditions for gene transfer by electroporation using a ku70 gene-deficient mutant with high recombination efficiency. Introducing a DNA fragment by electroporation into T. asahii cells on Sabouraud dextrose agar to generate a cnb1 gene-deficient mutant and incubating for 1 day led to the growth of approximately 100 transformants. When the incubation period was extended to 2 days or 5 days, however, only 2 or no transformants, respectively, were grown. The highest number of transformants was grown by electroporation when a square wave at 1.8 kV (9 kV/cm) was applied for 5 ms. In addition, the number of transformants increased with an increase in the length of the homologous region, and transformants did not grow when the homologous region was less than 500 base pairs. A DNA fragment was produced for deletion of the cnb1 gene by joint PCR, and the cnb1 gene-deficient mutant was obtained by introducing the DNA fragment by electroporation. These results indicate that DNA fragments produced by joint PCR can be used to generate gene-deficient mutants of T. asahii through gene transfer by electroporation.

Development of an efficient gene-targeting system for elucidating infection mechanisms of the fungal pathogen Trichosporon asahii.

Trichosporon asahii is a pathogenic fungus that causes severe, deep-seated fungal infections in neutropenic patients. Elucidating the infection mechanisms of T. asahii based on genetic studies requires a specific gene-targeting system. Here, we established an efficient gene-targeting system in a highly pathogenic T. asahii strain identified using the silkworm infection model. By comparing the pathogenicity of T. asahii clinical isolates in a silkworm infection model, T. asahii MPU129 was identified as a highly pathogenic strain. Using an Agrobacterium tumefaciens-mediated gene transfer system, we obtained a T. asahii MPU129 mutant lacking the ku70 gene, which encodes the Ku70 protein involved in the non-homologous end-joining repair of DNA double-strand breaks. The ku70 gene-deficient mutant showed higher gene-targeting efficiency than the wild-type strain for constructing a mutant lacking the cnb1 gene, which encodes the beta-subunit of calcineurin. The cnb1 gene-deficient mutant showed reduced pathogenicity against silkworms compared with the parental strain. These results suggest that an efficient gene-targeting system in a highly pathogenic T. asahii strain is a useful tool for elucidating the molecular mechanisms of T. asahii infection.

Induction of signal transduction pathways related to the pathogenicity of Cryptococcus neoformans in the host environment.

Cryptococcus neoformans, a human pathogenic fungus, infects immunocompromised humans and causes serious diseases such as cerebral meningitis. C. neoformans controls the expression of virulence factors in response to the host environment via various signal transduction pathways. Understanding the molecular mechanisms involved in C. neoformans infection will contribute to the development of methods to prevent and treat C. neoformans-related diseases. C. neoformans produces virulence factors, such as a polysaccharide capsule and melanin, to escape host immunity. Several proteins of C. neoformans are reported to regulate production of the virulence factors. In this review, on the basis of studies using gene-deficient mutants of C. neoformans and animal infection models, we outline the signal transduction pathways involved in the regulation of virulence factors.