Potential Role for a Carbohydrate Moiety in Anti-CandidaActivity of Human Oral Epithelial Cells

Abstract

Candida albicans is both a commensal and a pathogen at the oral mucosa. Although an intricate network of host defense mechanisms are expected for protection against oropharyngeal candidiasis, anti-Candida host defense mechanisms at the oral mucosa are poorly understood. Our laboratory recently showed that primary epithelial cells from human oral mucosa, as well as an oral epithelial cell line, inhibit the growth of blastoconidia and/or hyphal phases of several Candida species in vitro with a requirement for cell contact and with no demonstrable role for soluble factors. In the present study, we show that oral epithelial cell-mediated anti-Candida activity is resistant to gamma-irradiation and is not mediated by phagocytosis, nitric oxide, hydrogen peroxide, and superoxide oxidative inhibitory pathways or by nonoxidative components such as soluble defensin and calprotectin peptides. In contrast, epithelial cell-mediated anti-Candida activity was sensitive to heat, paraformaldehyde fixation, and detergents, but these treatments were accompanied by a significant loss in epithelial cell viability. Treatments that removed existing membrane protein or lipid moieties in the presence or absence of protein synthesis inhibitors had no effect on epithelial cell inhibitory activity. In contrast, the epithelial cell-mediated anti-Candida activity was abrogated after treatment of the epithelial cells with periodic acid, suggesting a role for carbohydrates. Adherence of C. albicans to oral epithelial cells was unaffected, indicating that the carbohydrate moiety is exclusively associated with the growth inhibition activity. Subsequent studies that evaluated specific membrane carbohydrate moieties, however, showed no role for sulfated polysaccharides, sialic acid residues, or glucose-and mannose-containing carbohydrates. These results suggest that oral epithelial cell-mediated anti-Candida activity occurs exclusively with viable epithelial cells through contact with C. albicans by an as-yet-undefined carbohydrate moiety.

Oral and vaginal epithelial cell anti‐Candida activity is acid labile and does not require live epithelial cells

“…Previous studies showed that the epithelial cell anti-Candida activity was partially resistant to fixation (14). This property was examined more closely to elucidate the viability (permeability) requirement for the antifungal activity.…”

“…Human oral epithelial cells were isolated as previously described (14,15). Briefly, 10-15 ml of unstimulated saliva from each participant was expectorated into a polypropylene centrifuge tube and centrifuged at 800 × g for 5 min.…”

“…[ 3 H]-glucose uptake-The growth inhibition assay was performed as previously described (13)(14)(15). Briefly, stationary-phase blastoconidia were added to individual wells of a 96-well microtiter plate (Costar, Cambridge, MA) at 1 × 10 5 cells/ml in a volume of 100 μl of PP broth supplemented with 10% FBS and 1% penicillin (100 U/ml) and streptomycin (100 μg/ml).…”

“…Quantitative plate count-Experiments involving periodic acid-treated epithelial cells require a quantitative plate count method to measure the growth inhibition of C. albicans due to high constitutive [ 3 H]-glucose uptake by periodic acid-treated epithelial cells (13)(14)(15). Briefly, effector and target cell cocultures were prepared as described above in the absence of [ 3 H]-glucose.…”

“…This property was examined more closely to elucidate the viability (permeability) requirement for the antifungal activity. For this, oral epithelial cells were fixed with 1% paraformaldehyde as previously described (14), and cell viability was determined by PI/FDA vital staining in addition to Trypan blue dye exclusion. As the images in Fig.…”