Cell Envelope Integrity and Capsule Characterization of Rhodotorula mucilaginosa Strains from Clinical and Environmental Sources.

Rhodotorula yeasts are pink, encapsulated basidiomycetes isolated from a variety of environments and clinical settings. They are increasingly linked with disease, particularly central venous catheter infections and meningitis, in immunocompromised patients. Eight clinical and eight environmental strains molecularly typed as Rhodotorula mucilaginosa were compared to six Cryptococcus neoformans strains for phenotypic variability. Growth on cell integrity-challenging media suggested that R. mucilaginosa cells possess differences in signaling pathways, cell wall composition, or assembly and that their membranes are more susceptible to perturbations than those of C. neoformans All 16 R. mucilaginosa strains produced urease, while none produced melanin with l-3,4-dihydroxyphenylalanine (l-DOPA) as a substrate. India ink staining reveals that clinical R. mucilaginosa capsules are larger than environmental capsules but that both are generally smaller than C. neoformans capsules. All R. mucilaginosa strains were resistant to fluconazole. Only two clinical strains were susceptible to voriconazole; all of the environmental strains were resistant. We generated an anticapsular antibody (Rh1) to R. mucilaginosa; Rh1 did not bind C. neoformans control strains, was specific to Rhodotorula species, and bound to all tested Rhodotorula strains. Binding assays performed with wheat germ agglutinin (WGA), concanavalin A (ConA), calcofluor white (CFW), and eosin Y dye (EY) cell surface probes suggested that chitin may be more accessible in R. mucilaginosa but that the total abundance of chitooligomers is less than in C. neoformans This report describes a novel reagent that can be used to identify Rhodotorula species and lays the foundation for future cell envelope composition analysis.IMPORTANCE Currently, there is very little known about the phenotypic variability within species of Rhodotorula strains and the role of their capsule. Cryptococcus neoformans has been considered the only encapsulated human fungal pathogen, but as more individuals come to live in states of immunocompromised health, they are more susceptible to fungal infections, including those by RhodotorulaR. mucilaginosa species are some of those most commonly associated with clinical infections. We wanted to know if clinical and environmental strains of R. mucilaginosa demonstrated disparate capsule phenotypes. With limited antifungal options available and clinical Rhodotorula spp. often resistant to common antifungal drugs such as fluconazole, caspofungin (1, 2), and voriconazole (2), a better understanding of the fungal biology could inform the design and use of future antifungal drugs. The generation of an antibody specific to Rhodotorula fungi could be a useful diagnostic tool, and this work presents the first mention of such in the literature.

The role of host gender in the pathogenesis of Cryptococcus neoformans infections.

Cryptococcus neoformans (Cn) is a pathogenic yeast and the cause of cryptococcal meningitis. Prevalence of disease between males and females is skewed, with males having an increased incidence of disease. Based on the reported gender susceptibility differences to Cn in the literature, we used clinical isolates from Botswanan HIV-infected patients to test the hypothesis that different gender environments exerted different selective pressures on Cn. When we examined this data set, we found that men had significantly higher risk of death despite having significantly higher CD4(+) T lymphocyte counts upon admittance to the hospital. These observations suggested that Cn strains are uniquely adapted to different host gender environments and that the male immune response may be less efficient in controlling Cn infection. To discriminate between these possibilities, we tested whether there were phenotypic differences between strains isolated from males and females and whether there was an interaction between Cn and the host immune response. Virulence phenotypes showed that Cn isolates from females had longer doubling times and released more capsular glucoronoxylomannan (GXM). The presence of testosterone but not 17-ß estradiol was associated with higher levels of GXM release for a laboratory strain and 28 clinical isolates. We also measured phagocytic efficiency, survival of Cn, and amount of killing of human macrophages by Cn after incubation with four isolates. While macrophages from females phagocytosed more Cn than macrophages from males, male macrophages had a higher fungal burden and showed increased killing by Cn. These data are consistent with the hypothesis that differential interaction between Cn and macrophages within different gender environments contribute to the increased prevalence of cryptococcosis in males. This could be related to differential expression of cryptococcal virulence genes and capsule metabolism, changes in Cn phagocytosis and increased death of Cn-infected macrophages.

An efficiently regulated promoter system for Cryptococcus neoformans utilizing the CTR4 promoter.

Cryptococcus neoformans is an opportunistic fungal pathogen responsible for serious meningitis. Although many useful molecular tools have been developed for its study, there are currently few inducible promoters available for general use. To address this need, we have constructed expression plasmids incorporating upstream elements of the C. neoformans copper transporter gene CTR4, and tested them in C. neoformans serotypes A and D. In response to copper deprivation, these plasmids mediate high-level expression of a reporter protein. This expression can be completely repressed using physiologically low concentrations of copper. Notably, this new family of copper-sensing promoters demonstrates excellent expression in serotype A, contrasting with other available promoters. These plasmids therefore offer efficient and regulated expression for both serotypes A and D, and should be valuable tools for the C. neoformans research community.

Biosynthesis of UDP-GlcA, a key metabolite for capsular polysaccharide synthesis in the pathogenic fungus Cryptococcus neoformans.

UDP-glucose dehydrogenase catalyses the conversion of UDP-glucose into UDP-GlcA, a critical precursor for glycan synthesis across evolution. We have cloned the gene encoding this important enzyme from the opportunistic pathogen Cryptococcus neoformans. In this fungus, UDP-GlcA is required for the synthesis of capsule polysaccharides, which in turn are essential for virulence. The gene was expressed in Escherichia coli and the 51.3-kDa recombinant protein from wild-type and five mutants was purified for analysis. The cryptococcal enzyme is strongly inhibited by UDP-xylose and NADH, has highest activity at pH 7.5 and demonstrates Km (app) values of 0.1 and 1.5 mM for NAD+ and UDP-glucose respectively. Its activity was significantly decreased by mutations in the putative sites of NAD+ and UDP-glucose binding. Unlike previously reported eukaryotic UDP-glucose dehydrogenases, which are hexamers, the cryptococcal enzyme is a dimer.