Deciphering Colonies of Phenotypic Switching-Derived Morphotypes of the Pathogenic Yeast Candida tropicalis.

BACKGROUND: Phenotypic switching generates fungal colonies with altered morphology and allows pathogens to adapt to changing environments. OBJECTIVE: This study investigated the structure and genetic factors of switched morphotypes colonies in Candida tropicalis. METHODS: Morphotypes of C. tropicalis comprised the clinical strain 49.07 that exhibited smooth colony phenotype and switched (crepe and rough) morphotypes that showed colonies with marked structural variations, including wrinkled surface, depressions areas, and irregular edges (structured morphology). The morphotypes were analyzed for the presence and distribution of the extracellular matrix (ECM) at the ultrastructural level-SEM. The composition of the ECM and the percentage of hyphae in colonies were evaluated. The expression of EFG1 (Enhanced filamentous growth protein 1), WOR1 (White-opaque regulator 1), and BCR1 (Biofilm and cell wall regulator 1) in the morphotypes was measured by RT-qPCR. RESULTS: Colonies of the switched variants exhibited distinct arrangements of ECM compared to the smooth phenotype (clinical strain). In addition, rough variant colonies showed higher amounts of total carbohydrates and proteins in ECM (p < 0.05). Switched (crepe and rough) colonies exhibited a higher percentage of hyphae throughout their development (p < 0.05). The mRNA expression levels of EFG1, WOR1, and BCR1 in the rough morphotype were significantly higher than they were in the smooth morphotype. In addition, there was a positive correlation between the expression of these genes and filamentation (hyphae formation) of the rough morphotype (r2 > 0.9472, p < 0.05). CONCLUSION: Structural variations in switched morphotypes colonies of C. tropicalis seem to be associated with increased hyphae growth and the amount and distribution of ECM. Switched colonies have distinct expressions of the EFG1, WOR1, and BCR1 master regulators genes.

Changes in Adhesion of Candida tropicalis Clinical Isolates Exhibiting Switch Phenotypes to Polystyrene and HeLa Cells.

BACKGROUND: Candida tropicalis is an important human pathogen that can undergo multiple forms of phenotypic switching. AIM: We aimed to evaluate the effect of phenotypic switching on the adhesion ability of C. tropicalis. METHODS: C. tropicalis morphotypes included parental phenotypes (clinical isolates) and switch phenotypes (crepe, revertant of crepe-CR, rough, revertant of rough-RR, irregular center and revertant of irregular center-ICR). Adhesion to polystyrene and HeLa cells was determined by crystal violet assay. The percentage of HeLa cells with adhered yeasts and the number of adhered yeasts per HeLa cell were determined by light microscopy. Filamentation among adhered cells was assessed by direct counting. RESULTS: On polystyrene, 60% of the switch strains showed difference (p < 0.05) on adhesion ability compared to their parental counterpart strains, and altered thickness of adhered cells layers. Filamentation was increased among adhered cells of the switched strains compared to parental strains. A positive correlation was observed between adhesion on polystyrene and filamentation for morphotypes of the system 49.07. The majority of the switched strains showed higher adhesion capability to HeLa cells in comparison to the adherence of the clinical strains. All revertant strains showed a higher number of yeast cells per HeLa cell compared to their variant counterparts (p < 0.05), with exception of the ICR. CONCLUSIONS: Our findings indicate that switching events in C. tropicalis affect adhesion and filamentation of adhered cells on polystyrene and HeLa cells. The rise of switch strains with increased adhesion ability may contribute to the success of infection associated with C. tropicalis.

Inhibitory effect of bacteriocins from enterococci on developing and preformed biofilms of Listeria monocytogenes, Listeria ivanovii and Listeria innocua.

The biofilm-forming ability of Listeria spp. is a concern to the food industry and health sectors. The aim of this study was to verify the inhibitory activity of bacteriocins produced by enterococci (Enterococcus faecium 20, 22 and 24 and Enterococcus faecalis 27) on developing biofilm and preformed biofilm of Listeria species. Bacteriocins were partially purified from cell free supernatant (CFS). L. monocytogenes 2032, L. innocua 2050 and L. ivanovii 2056 were selected to analyse the inhibitory effect of bacteriocins on biofilm biomass (crystal violet staining) and biofilm viability (XTT-reduction). The biomass of the developing and preformed biofilms of Listeria species were reduced (p < 0.05) in the presence of all bacteriocins tested. Overall, the reduction in biofilm biomass of developing biofilms was up to 87.4% for bacteriocin produced by E. faecium 22 (CFS22) against L. ivanovii and up to 87.1% for CFS22 against L. monocytogenes. These findings are in accordance with those observed in confocal microscopy analysis. Most of the CFS-containing bacteriocin (CFS22, CFS24, CFS27) were effective at decreasing the viability of biofilm cells from all Listeria species. The highest reduction in viability was observed for L. monocytogenes preformed biofilm cells (up to 98.7%), evidenced by fluorescence microscopy of propidium iodide-labelled cells. Scanning electron microscopy showed that cells of biofilm-treated bacteriocins displayed degenerative changes that may be indicative of cellular leakages. This study suggests that bacteriocins produced by enterococci have prospective applications to prevent biofilm formation and/or to reduce cell viability of formed biofilms of distinct Listeria species.