Candida albicans-epithelial interactions: dissecting the roles of active penetration, induced endocytosis and host factors on the infection process

Abstract

Candida albicans frequently causes superficial infections by invading and damaging epithelial cells, but may also cause systemic infections by penetrating through epithelial barriers. C. albicans is a remarkable pathogen because it can invade epithelial cells via two distinct mechanisms: induced endocytosis, analogous to facultative intracellular enteropathogenic bacteria, and active penetration, similar to plant pathogenic fungi. Here we investigated the contributions of the two invasion routes of C. albicans to epithelial invasion. Using selective cellular inhibition approaches and differential fluorescence microscopy, we demonstrate that induced endocytosis contributes considerably to the early time points of invasion, while active penetration represents the dominant epithelial invasion route. Although induced endocytosis depends mainly on Als3-E-cadherin interactions, we observed E-cadherin independent induced endocytosis. Finally, we provide evidence of a protective role for serum factors in oral infection: human serum strongly inhibited C. albicans adhesion to, invasion and damage of oral epithelial cells.

Figure 1. Transmission electron microscopy of C. albicans epithelial invasion.

Induced endocytosis of thimerosal killed hyphae (A), invasion of cytochalasin D treated epithelial cells by viable hyphae (B), invasion of untreated epithelial cells by viable hyphae (C), invasion of inactivated epithelial cells by viable hyphae (D).

Figure 2. Contributions of fungal invasins, induced endocytosis and active penetration to C. albicans oral epithelial invasion.

(A and B) Untreated or thimerosal-inactivated C. albicans cells were co-incubated with either untreated or cytochalasin D treated TR-146 oral epithelial cells for 2 h (A) and 3 h (B). After fixation, the samples were differentially stained and analyzed by fluorescence microscopy. The experiment was performed at least two times in duplicates. */**, indicates significant difference compared to the corresponding wild type control (Wt) (p<0.05/p<0.01, respectively). (C) Micrographs of invading hyphae. C. albicans appears blue (calcofluor white), with extracellular section of the hypha stained red (Concanavalin A); epithelial cells are stained yellow (Dil). Upper panel: a living hypha penetrating through several epithelial cells. Lower panel: a killed hypha, fully phagocytosed by an epithelial cell. Only viable hyphae are able to undergo inter-epithelial invasion. Numbers indicate the number of invaded epithelial cells and arrows mark internalized cells. Scale bar = 10 µm.

Figure 3. Differential

invasion of TR-146 or HeLa epithelial cells by C. albicans . (A) Oral TR-146 or HeLa epithelial cells were co-incubated with 10⁵ C. albicans cells (alive or thimerosal killed – Wt+Thim) for either 1, 2 or 3 h. (B) HeLa epithelial cells were co-incubated with 10⁵ indicated C. albicans strains (alive or thimerosal killed) for 3 h. Following fixation, samples were differentially stained and analyzed by fluorescence microscopy. The experiment was performed at least two times in duplicates. */**, indicates significant difference between cell lines (A) or between mutant and wild type (B) (p<0.05/p<0.01, respectively).

Figure 4. Adhesion, invasion and damage of oral epithelial cells by C. albicans in the presence of serum.

(A) Adhesion of viable C. albicans cells to oral epithelial cells in cell culture medium supplemented with 10% human [hSerum] or fetal bovine serum [FBS]; (+) heat-treated; (−) untreated; ctr, C. albicans without serum. (B) Induced endocytosis of thimerosal-inactivated C. albicans hyphae. Either oral epithelial cells were supplemented with untreated and heat-treated 10% hSerum or FBS [EC] or C. albicans hyphae [Ca] were pre-incubated with untreated or heat-treated 40% serum; ctr killed hyphae without serum. (C) Cell damage of epithelial monolayers caused by viable C. albicans cells after 24 h in cell culture medium supplemented with untreated or heat-treated 10% hSerum or FBS compared to C. albicans infection with the addition of 1% serum (ctr). **, significant difference compared to control adhesion, invasion or damage (p≤0.01).