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ABSTRACTBiofilm-associated polymicrobial infections tend to be challenging to treat. Candida albicansand Staphylococcus aureusare leading pathogens due to their ability to form biofilms on medical devices. However, the therapeutic implications of their interactions in a host is largely unexplored. In this study, we used a mouse subcutaneous catheter model for in vivo-grown polymicrobial biofilms to validate our in vitro findings on C. albicans-mediated enhanced S. aureus tolerance to vancomycin in vivo. Comparative assessment of S. aureusrecovery from catheters with single- or mixed-species infection demonstrated failure of vancomycin against S. aureusin mice with co-infected catheters. To provide some mechanistic insights, RNA-seq analysis was performed on catheter biofilms to delineate transcriptional modulations during polymicrobial infections. C. albicansinduced the activation of the S. aureusbiofilm formation network viadown-regulation of the lrg operon, repressor of autolysis, and up-regulation of the ica operon and production of polysaccharide intercellular adhesin (PIA), indicating an increase in eDNA production, and extracellular polysaccharide matrix, respectively. Interestingly, virulence factors important for disseminated infections, and superantigen-like proteins were down-regulated during mixed-species infection, whereas capsular polysaccharide genes were up-regulated, signifying a strategy favoring survival, persistence and host immune evasion. In vitrofollow-up experiments using DNA enzymatic digestion, lrg operon mutant strains, and confocal scanning microscopy confirmed the role of C. albicans-mediated enhanced eDNA production in mixed-biofilms on S. aureustolerance to vancomycin. Combined, these findings provide mechanistic insights into the therapeutic implications of interspecies interactions, underscoring the need for novel strategies to overcome limitations of current therapies.

ABSTRACTMicrobial species utilize secreted-signaling molecules to coordinate their behavior. Our previous investigations demonstrated a key role for the Candida albicans-secreted quorum-sensing molecule farnesol in modulating Staphylococcus aureusresponse to antimicrobials in mixed biofilms. In this study, we aimed to provide mechanistic insights into the impact of farnesol on S. aureuswithin the context of inter-species interactions. To mimic biofilm dynamics, farnesol-sensitized S. aureuscells were generated viasequential farnesol exposure. The sensitized phenotype exhibited dramatic loss of the typical pigment, which we identified as staphyloxanthin, an important virulence factor synthesized by the Crt operon in S. aureus. Additionally, farnesol exposure exerted oxidative-stress as indicated by transcriptional analysis demonstrating alterations in redox-sensors and major virulence regulators. Paradoxically, the activated stress-response conferred S. aureuswith enhanced tolerance to H2O2and phagocytic killing. Since expression of enzymes in the staphyloxanthin biosynthesis pathway was not impacted by farnesol, we generated a theoretical-binding model which indicated that farnesol may block staphyloxanthin biosynthesis viacompetitive-binding to the CrtM enzyme crucial for staphyloxanthin synthesis, due to high structural similarity to the CrtM substrate. Finally, mixed growth with C. albicanswas found to similarly induce S. aureusdepigmentation, but not during growth with a farnesol-deficient C. albicansstrain. Collectively, the findings demonstrate that a fungal molecule acts as a redox-cycler eliciting a bacterial stress response viaactivation of the thiol-based redox system under the control of global regulators. Therefore, farnesol-induced transcriptional modulations of key regulatory networks in S. aureusmay modulate the pathogenesis of C. albicans-S. aureusco-infections.