An ultrastructural and a cytochemical study of candidal invasion of reconstituted human oral epithelium.
ABSTRACT Opportunistic yeast, Candida albicans causes superficial and systemic mycoses in compromised patients. Adhesion to host tissues, morphogenesis and extracellular phospholipases (PL) are thought to contribute to its virulence. The nature of numerous host-parasite interactions at the invasive phase of oral candidiasis is not fully understood. Hence in this study, we explore the ultrastructural features of oral candidiasis using a tissue culture model based on reconstituted human oral epithelium (RHOE).
Reconstituted human oral epithelium (Skinethic Laboratory, Nice, France) was inoculated with C. albicans SC5314 and incubated up to 48 h. The infected tissue was harvested at 12, 24 and 48 h and examined using light, scanning (SEM) and transmission electron microscopy (TEM). Localized activity of PLs of C. albicans during tissue invasion was also examined using a cytochemical method.
Over a period of 48 h C. albicans invaded the RHOE, and histological examination revealed characteristic hallmarks of pathological tissue invasion. Hyphal penetration into the superficial epithelium, particularly at cell junctions, together with features of cellular internalization of yeasts was noted. Phospholipase activity was visible at the tips of hyphae and initial sites of bud formation. Further, SEM studies revealed cavitations on the surface epithelial cells particularly pronounced at the sites of hyphal invasion. Hyphal invasion was seen both at cell surfaces and intercellular cell junctions of the epithelium, the latter resembling thigmotropic behaviour.
Our findings confirm that multiple cellular interactions such as internalization, thigmotropism and extracellular PLs contribute to invasive candidiasis. The RHOE model, described here, appears to be a satisfactory model for the investigation of ultrastructural and histochemical features of invasive candidiasis in humans.
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ABSTRACT: Candida albicans is a low level commensal organism in normal human populations with the continuous potential to expand and cause a spectrum of clinical conditions. Using ex vivo human organ cultures and populations of primary human cells, we have developed several related experimental systems to examine early-stage interactions between C. albicans and mucosal surfaces. Experiments have been conducted both with exogenously added C. albicans and with overtly normal human mucosal surfaces supporting pre-existing infections with natural isolates of Candida. Under different culture conditions, we have demonstrated the formation of C. albicans colonies on human target cells and filament formation, equivalent to tissue invasion. These organ culture systems provide a valuable new resource to examine the molecular and cellular basis for Candida colonization of human mucosal surfaces.PLoS ONE 02/2008; 3(4):e2067. · 4.09 Impact Factor
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ABSTRACT: Cryptococcus neoformans is a common life-threatening human fungal pathogen. The size of cryptococcal cells is typically 5 to 10 microm. Cell enlargement was observed in vivo, producing cells up to 100 microm. These morphological changes in cell size affected pathogenicity via reducing phagocytosis by host mononuclear cells, increasing resistance to oxidative and nitrosative stress, and correlated with reduced penetration of the central nervous system. Cell enlargement was stimulated by coinfection with strains of opposite mating type, and ste3aDelta pheromone receptor mutant strains had reduced cell enlargement. Finally, analysis of DNA content in this novel cell type revealed that these enlarged cells were polyploid, uninucleate, and produced daughter cells in vivo. These results describe a novel mechanism by which C. neoformans evades host phagocytosis to allow survival of a subset of the population at early stages of infection. Thus, morphological changes play unique and specialized roles during infection.PLoS Pathogens 01/2010; 6(6):e1000953. · 9.13 Impact Factor
Article: Farnesol-induced apoptosis in Candida albicans is mediated by Cdr1-p extrusion and depletion of intracellular glutathione.[show abstract] [hide abstract]
ABSTRACT: Farnesol is a key derivative in the sterol biosynthesis pathway in eukaryotic cells previously identified as a quorum sensing molecule in the human fungal pathogen Candida albicans. Recently, we demonstrated that above threshold concentrations, farnesol is capable of triggering apoptosis in C. albicans. However, the exact mechanism of farnesol cytotoxicity is not fully elucidated. Lipophilic compounds such as farnesol are known to conjugate with glutathione, an antioxidant crucial for cellular detoxification against damaging compounds. Glutathione conjugates act as substrates for ATP-dependent ABC transporters and are extruded from the cell. To that end, this current study was undertaken to validate the hypothesis that farnesol conjugation with intracellular glutathione coupled with Cdr1p-mediated extrusion of glutathione conjugates, results in total glutathione depletion, oxidative stress and ultimately fungal cell death. The combined findings demonstrated a significant decrease in intracellular glutathione levels concomitant with up-regulation of CDR1 and decreased cell viability. However, addition of exogenous reduced glutathione maintained intracellular glutathione levels and enhanced viability. In contrast, farnesol toxicity was decreased in a mutant lacking CDR1, whereas it was increased in a CDR1-overexpressing strain. Further, gene expression studies demonstrated significant up-regulation of the SOD genes, primary enzymes responsible for defense against oxidative stress, with no changes in expression in CDR1. This is the first study describing the involvement of Cdr1p-mediated glutathione efflux as a mechanism preceding the farnesol-induced apoptotic process in C. albicans. Understanding of the mechanisms underlying farnesol-cytotoxicity in C. albicans may lead to the development of this redox-cycling agent as an alternative antifungal agent.PLoS ONE 01/2011; 6(12):e28830. · 4.09 Impact Factor