Adherence of Candida albicans to epithelial cells: studies using fluorescently labelled yeasts and flow cytometry

Abstract

Candida albicans adherence to epithelial cells is the first step in the infectious process, but in Spite O f its importance, CUrTent methods for the quantitative measurement of adherence of C. albicans to epithelial cells in vitm have some serious limitations. They are based on filtration assays and either microscopic or radiometric analysis. The adherence reaction is usually carried out with a large excess of yeasts (100-fold) over epithelial cells in order to perform the microscopic analysis, which is slow# subjective and limited to 100-200 cells and thus lacks statistical power. The radiometric analysis fails to measure individual cells. A method for measuring yeast adherence that overcomes these problems has been developed. It is based on labelling the yeasts with the f luorogenic marker 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF) prior to the adherence reaction, and analysing 104 epithelial cells by flow cytometry, while nonbound yeasts are excluded by gating. Two subpopulations of buccal epithelial cells (BECs) which differ in their mean fluorescence intensities per cell (MFls) were observed: one with MFI which did not exceed nonspecific fluorescence, and the other with MFI as high or higher than the MFI of labelled yeasts. The two subpopulations represent yeast-free and yeast-binding epithelial cells, respectively, and the MFI increment of the BECs is a quantitative measure of the extent of yeast adherence. Control experiments confirming previously described basic features of adherence, such as enhanced adherence at increasing yeast excess, diminished adherence of trypsin-treated or heat-inactivated yeasts, and the differential adherence of various Candida species, supported the validity of the assay. The possibility of studying adherence reliably at low yeast:epithelial cell ratios, which better mimic adhesion as it occurs in wiwo, is an important advantage of the assay. New findings, using this method, included the observation that exfoliated BECs from diabetic patients exhibited the same capacity for C. albicans adherence as cells from healthy controls, and that epithelial cells from early human ontogenic stages had a significantly lower adherence level than those from later stages.

Oral Colonization By Candida Albicans

“…Palatal epithelial cells from acrylic-denture-wearers with non-insulin-dependent diabetes bound more fungal cells than did epithelial cells of non-diabetic individuals (Dorocka-Bobkowska et al, 1996). In another study, adherence to BECs from diabetic individuals was similar to adherence to BECs from normal individuals (Polacheck et al, 1995). Thus, adherence to BECs is affected by many host factors, and hormonal effects on adherence could be mediated by altering the expression of adhesins on C. albicans cells or ligands on host cells.…”

“…The binding capacity of BECs from newborn full-term infants for Candida is less than that of BECs from premature infants, school-age children, and adults, but increases over the infants' first few days (Davidson et al, 1984;Cox, 1986;Polacheck et al, 1995). Adherence was greater to BECs from children with oral infection or colonization than to BECs from uninfected controls but increased during a course of antibiotic therapy in previously uninfected children (Cox, 1983).…”

“…The site of adherence between the yeast cells and epithelial cells labeled intensely with wheat germ agglutinin, a lectin-recognizing chitin. In a porcine stratum corneum model, three isolates from oral infections adhered more than a commensal isolate (Law et al, 1997 (Ray et al, 1984 (Sandin et al, 1987b), the number of C. albicans cells that bind to individual BECs is variable (Sandin et al, 1987a), there are both binding and non-binding BECs (Gorman et al, 1986;Polacheck et al, 1995), and both viable and nonviable C. albicans cells bind to BECs, with the non-viable cells having a greater adherence (Gorman et al, 1986).…”

Toxicity Mechanisms of Amphotericin B and Its Neutralization by Conjugation with Arabinogalactan

“…We examined vacuolar pH with BCECF-AM (Fig. 2), a fluorescent pH indicator that accumulates in the yeast vacuole (1,13,24,40,59). The treatment of C. albicans with AMB resulted in a sharp pH increase from 6.20 to between 6.64 and 6.75 (depending on AMB concentration), followed by a decrease to pH 6.0 to 6.3.…”

A novel flow cytometric protocol for assessment of yeast cell adhesion

“…Although it proved to be beneficial over conventional methods, the potential of this tool is still underestimated. Recently, flow cytometric adherence assays have been used to measure the interaction between bacteria and fungi with eukaryotic cells (22)(23)(24).…”