[Virulence of Candida dubliniensis in comparison with Candida albicans using an experimental model of mouse oral candiddiasis].

Certain species of Candida are known as opportunistic fungal pathogens and Candida albicans has especially been isolated oral candidiasis patients at high frequency as a result of its strong pathogenicity. Recently C. dubliniensis is isolated mainly from immunocompromised patients, but is also detected from healthy persons. C. dubliniensis has similar cell morphology and molecular biological properties to C. albicans. Thus, in order to clarify the pathogenicity of C. dubliniensis, the activities of two extracellular enzymes, phospholipase (PL) and proteinase (PT), were measured, and pathological features were compared using mice. PL activity was examined in the improved Price's PL activity assay. In brief, the white precipitation zone was detected by spraying NaCl on egg yold plates without NaCl after colonies had grown. PL activity was no detected in any of the 31 C. dubliniensis strains tested. On the other hand, PT acitivty of C. dubliniensis was almost equivalent to that of C. albicans. Although we attempted to make an experimental model of mouse oral candidiasis using C. dubliniensis in yeast form as an inoculum following the conventional method, oral candidiasis did not develop in any mice. Thrush was successfully developed after inoculation with mycelial form cells, and there was no significant difference in histopathological findings of the thrush in comparison with C. albicans. These results strongly suggest that the two enzymes, PT and PL, do not play a crusial role in the establishment of mouse oral experimental candidiasis by C. dubliniensis.

Diversity of laccase among Cryptococcus neoformans serotypes.

The pathogenic yeast C. neoformans is classified into three varieties with five serotypes; var. grubii (serotype A), var. neoformans (serotype D), var. gattii (serotypes B and C), and serotype AD. Melanin is a virulence factor in the species, and its biosynthesis is catalyzed by laccase, encoded by the LAC1 gene. In order to estimate the natural variability of the LAC1 gene among Cryptococcus serotypes, the laccase protein sequence from 55 strains was determined and the phylogenetic relationships between cryptococcal and related fungal laccases revealed. The deduced laccase proteins consisted of 624 amino acid residues in serotypes A, D and AD, and 613 to 615 residues in serotypes B and C. Intra-serotype amino acid variation was marginal within serotypes A and D, and none was found within serotypes AD and C. Maximum amino acid replacement occurred in two serotype B strains. The similarity in the deduced sequence ranged from 80 to 96% between serotypes. The sequence in the copper-binding regions was strongly conserved in the five serotypes. The laccases of the five serotypes were grouped together in the same clade of the phylogenetic tree reconstructed from different fungal laccases, suggesting a monophyletic clade.

[Cell cycle control and CDC28/Cdc2 homologue and related gene cloning of Cryptococcus neoformans].

In Cryptococcus neoformans the DNA content of cells having tiny buds varied rather widely, depending on growth phases and strains used. Typically, buds of C. neoformans emerged soon after initiation of DNA synthesis in the early exponential phase. However, bud emergence was delayed to G2 during transition to the stationary phase, and in the early stationary phase budding scarcely occurred, although roughly half of the cells completed DNA synthesis. The timing of budding in C. neoformans was shifted to later cell cycle points with progression of the growth phase of the culture. Similarly, a deficit in oxygen was demonstrated to delay the timing of budding, prolong the G2 phase and cause accumulation of cells after DNA synthesis, but before commitment to budding. The C. neoformans homologue of the main cell cycle control gene CDC28/Cdc2 was isolated using degenerate RT-PCR. The full-length coding region was then amplified using primers to target the regions around the start and stop codons. The gene was called CnCdk1 and was found to have high homologies to S. cerevisiae CDC28 and S. pombe cdc2. To determine its function, its ability to rescue S. cerevisiae cdc28-temperature sensitive mutants was tested. S. cerevisiae cdc28-4 and cdc28-1N strains transformed with the pYES2-CnCdk1 construct exhibited growth at the restrictive temperature. Results of the sequence analysis and the ability of CnCdk1 to complement the S. cerevisiae cdc28-ts mutations support its assumed role as the CDC28/cdc2 homologue in C. neoformans.

[Cell biology of respiration-deficient mutants of Candida albicans].

Respiration-deficient (petite) mutation is caused by hereditary impairment in mitochondrial functions. Yeasts have been grouped into "petite-positive" and "petite-negative" yeasts. Candida albicans has been regarded as a member of the petite-negative yeasts in which the respiration deficiency cannot be easily induced. We have succeeded in inducing the petite mutation in C. albicans by culturing in the presence of a chemical mutagen, acriflavine, at an elevated temperature. In the present review, we describe the cell biology of C. albicans petite mutants on the basis of experiments performed by our research group: namely, on respiratory activity and cytochrome composition, fine structures of cells and mitochondria, mitochondrial DNA structure, pathogenicity, oxidative stress sensitivity, generation of reactive oxygen species (ROS) and the roles of ROS in antifungal actions. We discuss also the usefulness of petite mutants in Candida research.

Comparisons of the laccase gene among serotypes and melanin-deficient variants of Cryptococcus neoformans.

Cryptococcus neoformans is a fungus causing life-threatening infections in immunocompromised hosts. Melanin production is a major virulence factor of this fungus and the initial steps of dihydroxyphenylalanine (DOPA)-melanin biosynthesis pathways are catalyzed by laccase. To understand phylogenetic relationships among serotypes of three varieties, partial sequences (about 600 bases) of the laccase gene (CNLAC1) were determined in a total of 64 strains, including 10 melanin-deficient variants. The phylogenetic tree constructed from the nucleotide sequence grouped the 64 strains into the clusters corresponding to the three varieties. The diversity of the fragment sequences was very minor among strains of each of var. grubii and var. neoformans. Strains in var. gattii, however, were subdivided into two groups, although differences between serotypes B and C were not large. The sequences of the melanin-deficient variants were almost completely homologous to those of the melanin-producing strains in the same serotype. Results of laccase assay and northern blot analysis suggested that the lower melanin production in the variants was associated with lower transcription of the laccase gene.

Differences in extracellular enzymatic activity between Candida dubliniensis and Candida albicans isolates.

Twenty-six Candida dubliniensis and 27 Candida albicans oral strains isolated from patients infected by the human immunodeficiency virus (HIV) were tested for germ tube production and 21 extracellular enzymatic activities. Assessment of the enzymatic profile was performed by using the API-ZYM commercial kit system (bioMerieux, France), which tests 19 different enzymes. Protease activity was expressed during the first days of incubation by 100% of the strains studied and resulted higher than phospholipase activity in the C. dubliniensis and C. albicans strains tested. The API-ZYM profile of the C. dubliniensis and C. albicans strains differs with respect to the number and percentage of the enzymes considered, as well as with the intensity of the substrate metabolized by the strains, in particular for the enzymes n 8 (cystine-arylamidase), n 12 (naphtol-AS-BI-phosphohydrolase) and n 16 (alpha-glucosidase). These enzymes may be useful to differentiate C. dubliniensis and C. albicans together with other phenotypic characteristics proposed in the literature. No relationship among protease, phospholipase and other extracellular enzymatic activities was observed in C. dubliniensis. The average percentage of strains filamentation after 4 h was between 32 and 42%.

Adherence of Candida albicans and Candida dubliniensis to buccal and vaginal cells.

Twenty-seven Candida albicans strains and 26 Candida dubliniensis strains, isolated from HIV patients, were tested for their adherence to buccal and vaginal epithelial cells. Both species showed important levels of adhesion to buccal and vaginal epithelial cells, although C. albicans showed the highest levels of adhesion. These results suggest that both Candida species are well adapted, in terms of adhesion capability, to the oral and vaginal environment.