Candida parapsilosis Protects Premature Intestinal Epithelial Cells from Invasion and Damage by Candida albicans

Abstract

Candida is a leading cause of late-onset sepsis in premature infants and is thought to invade the host via immature or damaged epithelial barriers. We previously showed that the hyphal form of Candida albicans invades and causes damage to premature intestinal epithelial cells (pIECs), whereas the non-hyphal Candida parapsilosis, also a fungal pathogen of neonates, has less invasion and damage abilities. In this study, we investigated the potential for C. parapsilosis to modulate pathogenic interactions of C. albicans with the premature intestine. While a mixed infection with two fungal pathogens may be expected to result in additive or synergistic damage to pIECs, we instead found that C. parapsilosis was able to protect pIECs from invasion and damage by C. albicans. C. albicans-induced pIEC damage was reduced to a similar extent by multiple different C. parapsilosis strains, but strains differed in their ability to inhibit C. albicans invasion of pIECs, with the inhibitory activity correlating with their adhesiveness for C. albicans and epithelial cells. C. parapsilosis cell-free culture fractions were also able to significantly reduce C. albicans adhesion and damage to pIECs. Furthermore, coadministration of C. parapsilosis cell-free fractions with C. albicans was associated with decreased infection and mortality in zebrafish. These results indicate that C. parapsilosis is able to reduce invasion, damage, and virulence functions of C. albicans. Additionally, the results with cellular and cell-free fractions of yeast cultures suggest that inhibition of pathogenic interactions between C. albicans and host cells by C. parapsilosis occurs via secreted molecules as well as by physical contact with the C. parapsilosis cell surface. We propose that non-invasive commensals can be used to inhibit virulence features of pathogens and deserve further study as a non-pharmacological strategy to protect the fragile epithelial barriers of premature infants.

Keywords: Candida albicans; Candida parapsilosis; fungal pathogenesis; intestinal epithelium; premature infant; zebrafish model system.

Figure 1

Candida parapsilosis reduces premature intestinal epithelial cell damage (A) and invasion (B) by C. albicans strains. (A) Cell damage (lactate dehydrogenase amount) is plotted as a percentage of that caused by Candida albicans (Ca) strains (SC5314 and A022b) alone (“normalized cytotoxicity”). Cp, C. parapsilosis strain 4175. (B) C. albicans hyphal invasion in the presence of C. parapsilosis (Cp 4175) is plotted as a percentage of that observed for C. albicans (Ca SC5314) alone. Data shown are the average of three individual experiments. For both panels (A,B), an asterisk (*) indicates a statistically significant difference with p ≤ 0.05. Error bars represent SEM.

Figure 2

Candida parapsilosis cell-free culture fraction reduces Candida albicans-induced premature intestinal epithelial cell damage. Cell damage (lactate dehydrogenase amount) is plotted as a percentage of that caused by C. albicans (Ca SC5314) alone (“normalized cytotoxicity”). Data shown are the average of three individual experiments. Letters are used to indicate statistical significance, where letters differ, p < 0.05. Error bars represent SEM.

Figure 3

Adhesiveness of Candida parapsilosis strains correlates with ability to reduce Candida albicans invasion, but not damage, of pIECs. (A) Cell damage (lactate dehydrogenase amount) is plotted as a percentage of that caused by C. albicans (Ca SC5314) alone (“normalized cytotoxicity”). Data shown are the average of three individual experiments. Letters are used to indicate statistical significance, where letters differ, p < 0.05. Error bars represent SEM. (B) C. albicans hyphal invasion in the presence of C. parapsilosis (Cp) strains is plotted as a percentage of that observed for C. albicans (Ca SC5314) alone. Data shown are the average of three individual experiments. An asterisk (*) indicates a statistically significant difference with p ≤ 0.05. Error bars represent SEM.

Figure 4

Candida albicans and Candida parapsilosis interactions on premature intestinal epithelial cells (pIECs). Representative photomicrographs of C. albicans (Ca GFP) coincubated with C. parapsilosis (Cp) strains on pIECs. A C. albicans strain expressing GFP was used to differentiate between C. albicans (red and green fluorescence) and C. parapsilosis (red fluorescence, no green fluorescence, see asterisks in GFP panels) yeast cells. Alexa 568 conjugated to phalloidin was used to stain fungal cells with red fluoroscence, as previously described (14). A penetrating C. albicans hypha lacks red fluorescent signal (Cp LOW, Red/DIC Merge panel, arrow). Non-penetrating Candida cells exhibit red fluorescent signal. Cp 4175 and Cp HIGH cells make contacts with C. albicans hyphae (top and center merge panels, asterisks), while Cp LOW cells do not (bottom merge panel, asterisk). Scale bar, 10 μm.

Figure 5

Candida parapsilosis cell-free culture fraction protects zebrafish from infection by Candida albicans. (A) Schematic of infection model. The larval zebrafish swimbladder offers a transparent vertebrate mucosal infection model that is amenable to non-invasive imaging of both the host and the pathogen. (B–D) Zebrafish at 4 days post-fertilization with inflated swimbladders were infected in their swimbladders by glass needle injection with C. albicans yeast cells (C.a.) suspended in control (H4) media or in supernatants from C. parapsilosis (C.p. Supt) or C. albicans (C.a. Supt.) cultures. (B) Relative survival of fish infected with C. albicans with or without Candida supernatants. All C. albicans-infected fish cohorts are significantly different from their respective controls. C. parapsilosis supernatants significantly reduce the mortality of a C. albicans infection (denoted by γ). Matching Greek letters label individual comparisons: α, p < 0.01; β, p < 0.0001; γ, p < 0.05. Survival data are pooled from two independent experiments, n = 20 per group. All pair-wise comparisons were made with the Mantel–Cox test. (C,D) Fish from the experiment in (B) were viewed by fluorescence microscopy at 24 h post-infection and scored for two indicators of infection, swimbladder deflation [(C) **p < 0.01] and breaching of epithelial barrier (D). Examples of these phenotypes are shown in Figure S1 in Supplementary Material. Data were pooled from two independent experiments and analyzed by Fisher’s exact test with Bonferroni correction (Control media, n = 22, C.a. supernatant, n = 21, C.p. supernatant, n = 22).

Figure 6

Diagram summarizing the Candida–premature intestinal epithelial cell (pIEC) interactions described in this study. Infection models: (A) Candida albicans alone, (B) C. albicans along with Candida parapsilosis cell-free culture fraction, (C) mix of C. albicans and C. parapsilosis cultures. For all panels: C. albicans yeast and hyphal cells, light blue; C. parapsilosis cells, purple; C. parapsilosis cell-free culture factors, purple dots; pIECs, gray; damaged pIECs, black explosion outlines.