Interaction of Candida albicans with an intestinal pathogen, Salmonella enterica serovar Typhimurium

Abstract

Candida albicans is an opportunistic human fungal pathogen that normally resides in the gastrointestinal tract and on the skin as a commensal but can cause life-threatening invasive disease. Salmonella enterica serovar Typhimurium is a gram-negative bacterial pathogen that causes a significant amount of gastrointestinal infection in humans. Both of these organisms are also pathogenic to the nematode Caenorhabditis elegans, causing a persistent gut infection leading to worm death. In the present study, we used a previously developed C. elegans polymicrobial infection model to assess the interactions between S. Typhimurium and C. albicans. We observed that when C. elegans is infected with C. albicans and serovar Typhimurium, C. albicans filamentation is inhibited. The inhibition of C. albicans filamentation by S. Typhimurium in C. elegans appeared to be mediated by a secretary molecule, since filter-sterilized bacterial supernatant was able to inhibit C. albicans filamentation. In vitro coculture assays under planktonic conditions showed that S. Typhimurium reduces the viability of C. albicans, with greater effects seen at 37 degrees C than at 30 degrees C. Interestingly, S. Typhimurium reduces the viability of both yeast and filamentous forms of C. albicans, but the killing appeared more rapid for the filamentous cells. The antagonistic interaction was also observed in a C. albicans biofilm environment. This study describes the interaction between two diverse human pathogens that reside within the gastrointestinal tract and shows that the prokaryote, S. Typhimurium, reduces the viability of the eukaryote, C. albicans. Identifying the molecular mechanisms of this interaction may provide important insights into microbial pathogenesis.

FIG. 1.

S. Typhimurium (ST) inhibits C. albicans (CA) filamentation in the C. elegans coinfection model. (A) The degree of inhibition of C. albicans filamentation was dependent on the initial inoculum of S. Typhimurium in the liquid medium of the assay. (B) S. Typhimurium supernatant taken from stationary-phase growth (16 h of growth) (SUP stat.) caused a significant inhibition of C. albicans filamentation in the C. elegans model, whereas that taken from exponential-phase growth (4 h of growth) (SUP exp.) did not. All images were taken at 24 h. Column bars represent the means, and error bars represent the standard deviations. Confocal microscopy showed the marked phenotypic differences between C. elegans infection with C. albicans alone (C) and infection with C. albicans and S. Typhimurium (D). (E) By using a green fluorescent protein-linked S. Typhimurium strain (SL1344-GFP), we observed not only bacteria and fungal cells within the gut of the worm (white arrow) but also bacteria associating with sparse filaments that had protruded through the worm cuticle (black arrow). Asterisks denote comparison of percentage filamentation with that for C. albicans strain DAY185 alone: ***, P ≤ 0.001; **, P ≤ 0.01; *, P ≤ 0.05 (two-tailed t test). Scale bar: 50 μm (C), 38 μm (D), or 18.6 μm (E).

FIG. 2.

S. Typhimurium (ST) reduces the viability of C. albicans (CA) in an in vitro environment. (A) The viability of the C. albicans DAY185 strain in the presence of S. Typhimurium is significantly lower at 37°C than at 30°C. The C. albicans suv3 mutant, which is in the yeast form at 30°C, was more resistant to killing by S. Typhimurium than the constitutively filamentous C. albicans tup1 mutant strain at 30°C (B). Error bars represent the standard deviations. In panel A, asterisks denote comparison of log CFU/ml of C. albicans DAY185 at 30°C and 37°C. In panel B, asterisks denote comparison of log CFU/ml of the C. albicans suv3 strain to the C. albicans tup1 strain at 30°C: ***, P ≤ 0.001; **, P ≤ 0.01 (two-tailed t test).

FIG. 3.

Confocal laser microscopy of in vitro cultures after staining with the Live/Dead staining system, whereby dead cells stain red and live cells stain green. C. albicans strain DAY185 cultured alone (A and B) or in the presence of S. Typhimurium (C and D), showing a nonviable filamentous cell when cocultured with S. Typhimurium (D) despite an absence of pronounced cell-cell association. (E and F) The constitutively filamentous C. albicans tup1 mutant cultured alone (E and F) or in the presence of S. Typhimurium (G and H), showing a similar finding of reduced viability (red stain) when cocultured with S. Typhimurium (H). Images were taken after 24 h of incubation in LB broth at 37°C. Scale bar: 17.65 μm (A and B), 10 μm (C and D), 20 μm (E and F), or 11.7 μm (G and H).

FIG. 4.

S. Typhimurium supernatant (SUP) isolated at 37°C inhibits C. albicans strain DAY185 (CA) in a growth-dependent fashion (A). Inhibition of C. albicans DAY185 (CA) and suv3 mutant growth was not observed until they were exposed to supernatant taken from S. Typhimurium grown to late stationary phase (16 h, optical density at 600 nm [OD₆₀₀] of 1.7) (A and B, respectively). tup1 mutant growth was inhibited in S. Typhimurium supernatant (SUP) isolated from early stationary phase (12 h, OD₆₀₀ of 1) (C). When C. albicans strains DAY185 (CA) and suv3 were grown in S. Typhimurium supernatant taken from a 24-h growth (OD₆₀₀ of 1.9), the density of C. albicans was more than 7 log CFU/ml lower than that with growth in fresh LB medium (A and B). In the same supernatant, there was almost no growth for the tup1 strain (C). All determinations of C. albicans CFU/ml were performed after 24 h of growth at 37°C. Column bars represent the means and error bars represent the standard deviations. Asterisks denote comparison of log CFU/ml with that of C. albicans in LB medium: ***, P ≤ 0.001; **, P ≤ 0.01 (two-tailed t test).

FIG. 5.

S. Typhimurium (ST) inhibits the development of C. albicans (CA) biofilm on silicone pads. (A) The degree of inhibition of C. albicans (CA) biofilm formation was dependent on the density of S. Typhimurium (ST) inoculated into the biofilm environment. (B) S. Typhimurium supernatant (ST SUP) isolated at 37°C, when taken from a bacterial growth with an optical density (OD) at 600 nm of at least 1.2, led to a significant reduction in C. albicans biofilm mass compared to its development in fresh spider medium. (C and D) Fluorescent images of mature C. albicans biofilms (48 h of growth) when grown in the absence (C) or presence (D) of S. Typhimurium. with use of the Live/Dead staining system, nonviable (red stain) filaments are observed with incubation in the presence of S. Typhimurium. Column bars represent the means, and error bars represent the standard deviations. Asterisks denote comparison of C. albicans biofilm mass (mg) to C. albicans alone (A) or to C. albicans grown in spider medium (B): ***, P ≤ 0.001; **, P ≤ 0.01 (two-tailed t test). Scale bar: 42.86 μm (C) or 26.72 μm (D).