Candida albicans, an opportunistic fungal pathogen, and Staphylococcus aureus, a ubiquitous pathogenic bacterium, are among the most prevalent causes of nosocomial infections and cause severe morbidity and mortality. Moreover, they are frequently coisolated from central venous catheters and deep-seated infections, including intra-abdominal sepsis. Relatively little is known about the complex interactions and signaling events that occur between microbes and even less so how microbial “cross-talk” shapes human health and disease. Using a murine model of polymicrobial intra-abdominal infection (IAI), we have previously shown that coinfection with C. albicans and S. aureus leads to synergistic lethality whereas monomicrobial infection is nonlethal. Therefore, we aimed to identify staphylococcal virulence determinants that drive lethal synergism in polymicrobial IAI. Using the toxigenic S. aureus strain JE2, we observed that co-infection with C. albicans led to a striking 80-100% mortality rate within 20 h p.i while monomicrobial infections were non-lethal. Use of a GFP-P3 promoter S. aureus reporter strain revealed enhanced activation of the staphylococcal agr quorum sensing system during in vitro polymicrobial versus monomicrobial growth. Analyses by qPCR, Western blot, and toxin functional assays confirmed enhanced agr-associated gene transcription and increases in secreted α- and δ-toxins. C. albicans-mediated elevated toxin production and hemolytic activity was determined to be agrA-dependent and genetic knockout and complementation of hla identified ⍺-toxin as the key staphylococcal virulence factor driving lethal synergism. Analysis of mono- and polymicrobial infection 8 h p.i. demonstrated equivalent bacterial burden in the peritoneal cavity, but significantly elevated levels of α-toxin (3-fold) and the eicosanoid PGE2 (4-fold) during co-infection. Importantly, prophylactic passive vaccination using the monoclonal anti-⍺-toxin antibody MEDI4893* led to significantly improved survival rates as compared to treatment with isotype control antibody. Collectively, these results define α-toxin as an essential virulence determinant during C. albicans-S. aureus IAI and describe a novel mechanism by which a human pathogenic fungus can augment the virulence of a highly pathogenic bacterium in vivo. We next sought to unravel the mechanism by which C. albicans drives enhanced staphylococcal ⍺-toxin production. Using a combination of functional and genetic approaches, we determined that an intact agr quorum sensing regulon is necessary for enhanced ⍺-toxin production during coculture and that a secreted candidal factor likely is not implicated in elevating agr activation. As the agr system is pH sensitive, we observed that C. albicans raises the pH during polymicrobial growth and that this correlates with increased agr activity and ⍺-toxin production. By using a C. albicans mutant deficient in alkalinization (stp2Δ/Δ), we confirmed that modulation of the extracellular pH by C. albicans can drive agr expression and toxin production. Additionally, the use of various Candida species (C. glabrata, C. dubliniensis, C. tropicalis, C. parapsilosis, and C. krusei) demonstrated that those capable of raising the extracellular pH correlated with elevated agr activity and ⍺-toxin production during coculture. Overall, we demonstrated that alkalinization of the extracellular pH by the Candida species leads to sustained activation of the staphylococcal agr system. Finally, we correlated ⍺-toxin production with significant increases in biomarkers of liver and kidney damage during coinfection and determined that functional toxin was required for morbidity and mortality. We next sought to determine the candidal effector(s) mediating this enhanced virulence by employing an unbiased screening approach. C. albicans transcription factor mutants were evaluated for their ability to induce S. aureus agr activation in polymicrobial culture. Incredibly, we identified several mutants that displayed defects in augmenting S. aureus agr activity in vitro. Two of the mutants failed to completely synergize with S. aureus in vivo and further analysis revealed the necessity of the uncharacterized C. albicans transcription factor, ZCF13, in driving enhanced toxin production both in vitro and in vivo. Collectively, we identified a novel effector by which C. albicans augments S. aureus virulence and identified a potential mechanism of fungal-bacterial lethal synergism.
Acidic extracellular pH of tumors induces octamer-binding transcription factor 4 expression in murine fibroblasts in vitro and in vivo
