Enterococcus faecalis inhibits hyphal morphogenesis and virulence of Candida albicans

Abstract

The Gram-positive bacterium Enterococcus faecalis and the fungus Candida albicans are both found as commensals in many of the same niches of the human body, such as the oral cavity and gastrointestinal (GI) tract. However, both are opportunistic pathogens and have frequently been found to be coconstituents of polymicrobial infections. Despite these features in common, there has been little investigation into whether these microbes affect one another in a biologically significant manner. Using a Caenorhabditis elegans model of polymicrobial infection, we discovered that E. faecalis and C. albicans negatively impact each other's virulence. Much of the negative effect of E. faecalis on C. albicans was due to the inhibition of C. albicans hyphal morphogenesis, a developmental program crucial to C. albicans pathogenicity. We discovered that the inhibition was partially dependent on the Fsr quorum-sensing system, a major regulator of virulence in E. faecalis. Specifically, two proteases regulated by Fsr, GelE and SerE, were partially required. Further characterization of the inhibitory signal revealed that it is secreted into the supernatant, is heat resistant, and is between 3 and 10 kDa. The substance was also shown to inhibit C. albicans filamentation in the context of an in vitro biofilm. Finally, a screen of an E. faecalis transposon mutant library identified other genes required for suppression of C. albicans hyphal formation. Overall, we demonstrate a biologically relevant interaction between two clinically important microbes that could affect treatment strategies as well as impact our understanding of interkingdom signaling and sensing in the human-associated microbiome.

Fig 1

E. faecalis attenuates C. albicans killing of C. elegans. (A) The killing of C. elegans was significantly reduced when exposed to C. albicans (JRC52) and E. faecalis (SD234) on solid medium before being transferred to liquid medium compared with the killing when exposed to C. albicans alone (P < 0.0001). C. elegans worms feeding on E. coli (OP50) were used as a control. C. albicans > E. faecalis, exposure first to the fungus and subsequent exposure to the bacterium; E. faecalis > C. albicans, exposure first to the bacterium and subsequent exposure to the fungus. (B to D) Nomarski and fluorescent views of C. elegans fed on C. albicans, E. faecalis, or both on day 4. (B) C. albicans alone; (C) E. faecalis alone; (D) C. albicans and E. faecalis coculture. (E to G) Nomarski and fluorescent views of C. elegans fed on C. albicans, E. faecalis, or both on day 4. (E) C. albicans alone; (F) E. faecalis alone; (G) C. albicans and E. faecalis coculture. Following feeding on both microbes in the order indicated, the proportion of worms with either mixed or monomicrobial colonization was quantified on day 4 (H) or day 7 (I). Colonization was scored as described in Materials and Methods, and the percentage of worms in each category is indicated. n, number of worms observed.

Fig 2

TEM images of the C. elegans intestine when infected with C. albicans (A), E. faecalis (B), or both microbes (C). Ca, C. albicans (JRC52) cells; Ef, E. faecalis (SD234) cells; white boxes in the left panels, a region of the apical border of the intestinal cells which is magnified in the respective insets. Bars, 2 μm (right), 1 μm (left), and 0.2 μm (insets). All images are from longitudinal sections of C. elegans, except for the right of panel B, which is cross-sectional.

Fig 3

Protection of C. elegans from C. albicans killing and filamentation is dependent on the E. faecalis inoculum size. (A) C. elegans worms were exposed to C. albicans (SC5314) on solid medium and then placed in liquid medium containing E. faecalis (OG1RF) at the indicated inoculum size. Survival was scored daily, and differences in survival from the control with C. albicans alone were significant at P < 0.001 for 10² and 10⁴ CFU and P < 0.0001 for all others. No number indicates exposure to E. faecalis on solid medium following C. albicans exposure, as was done for Fig. 1A. (B) Inhibition of C. albicans filaments on day 7 depended on the size of the E. faecalis inoculum.

Fig 4

fsrB, gelE, and sprE are required for full protection from C. albicans killing and filamentation. (A) C. elegans worms were exposed to C. albicans (SC5314) on solid medium and then exposed to E. faecalis OG1RF or fsrB, gelE sprE, gelE, and sprE mutants in liquid medium and scored for survival daily. The mutants did not protect against C. albicans infection as well as wild-type OG1RF (P < 0.0001 for all mutants compared to OG1RF). (B) The mutants were scored for their ability to inhibit C. albicans filamentation on day 7. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant.

Fig 5

Culture supernatants of E. faecalis inhibit filamentation of host-associated C. albicans. (A) Inhibition of C. albicans (SC5314) filamentation in infected worms with stationary supernatants from OG1RF or fsrB, gelE sprE, gelE, and sprE mutants on day 7. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; NS, not significant. (B) Inhibition of C. albicans filamentation in infected worms with early-, middle-, and late-log-phase supernatants from OG1RF on day 4; (C) inhibition of C. albicans filamentation with various percentages of the indicated E. faecalis supernatants on day 7; (D) inhibition of filamentation with supernatant extracts from 0 to 10 kDa and a boiled extract on day 7; (E) inhibition of C. albicans filamentation in worms infected with OG1RF grown overnight in minimal medium with 100 mM glucose; (F) inhibition of C. albicans filamentation in worms infected with E. faecalis V583 on day 7.

Fig 6

E. faecalis does not inhibit C. albicans morphogenesis in vitro. (A) Overnight cultures of C. albicans SC5314 were diluted into RPMI or YPD–10% serum with or without E. faecalis OG1RF and incubated at 37°C for 2 h. Cultures were examined microscopically and scored for morphology (n > 200 cells in two experiments for each condition). (B) Overnight cultures of SC5314 were diluted into YPD–10% serum diluted with either fresh BHI medium or sterilized supernatants from saturated OG1RF cultures grown in BHI. Fungal morphology was assessed as described for panel A. (C) Representative images from the experiment quantified in panel A. (D) Representative images from the experiment quantified in panel B.

Fig 7

E. faecalis inhibits C. albicans morphogenesis in a biofilm. Overnight cultures of C. albicans (strain SC5314) were diluted into buffered Spider medium with 20% serum with or without E. faecalis and incubated at 37°C for 24 h. (A) Representative image of a C. albicans biofilm stained with calcofluor white (blue peripheral staining); (B) representative image of a biofilm comprised of calcofluor white-stained C. albicans (blue) and GFP-expressing E. faecalis (green; strain SD234); (C) representative DIC image of C. albicans forming a biofilm; (D) representative DIC image of C. albicans forming a biofilm in the presence of E. faecalis OG1RF supernatant; (E) percentage of C. albicans cells displaying a yeast, hyphal, or pseudohyphal morphology within a biofilm with and without E. faecalis supernatant. At least 10 fields and more than 500 cells were counted in three separate experiments. ***, a significant difference (P < 0.0001) between the E. faecalis supernatant addition compared to the control.