, a member of the human gastrointestinal microbiota, is an opportunistic pathogen associated with hospital-acquired wound, bloodstream, and urinary tract infections. can subvert or evade immune-mediated clearance, although the mechanisms are poorly understood. In this study, we examined-mediated subversion of macrophage activation. We observed that actively prevents NF-κB signaling in mouse RAW264.7 macrophages in the presence of Toll-like receptor agonists and during polymicrobial infection with and coinfection in a mouse model of catheter-associated urinary tract infection (CAUTI) resulted in a suppressed macrophage transcriptional response in the bladder compared to that with infection alone. Finally, we demonstrated that coinoculation of with a commensal strain of into catheterized bladders significantly augmented CAUTI. Taken together, these results support the hypothesis that suppression of NF-κB-driven responses in macrophages promotes polymicrobial CAUTI pathogenesis, especially during coinfection with less virulent or commensal strains.

Enterococcus faecalis, a member of the human gastrointestinal microbiota, is an opportunistic pathogen associated with hospital-acquired wound, bloodstream, and urinary tract infections. E. faecalis can subvert or evade immune-mediated clearance, although the mechanisms are poorly understood. In this study, we examined E. faecalis-mediated subversion of macrophage activation. We observed that E. faecalis actively prevents NF-κB signaling in mouse RAW264.7 macrophages in the presence of Toll-like receptor agonists and during polymicrobial infection with Escherichia coli. E. faecalis and E. coli co-infection in a mouse model of catheter-associated urinary tract infection (CAUTI) resulted in a suppressed macrophage transcriptional response in the bladder compared to E. coli infection alone. Finally, we demonstrated that co-inoculation of E. faecalis with E. coli into catheterized bladders significantly augmented E. coli CAUTI. Taken together, these results support that E. faecalis suppression of NF-κB-driven responses in macrophages promotes polymicrobial CAUTI pathogenesis.Author SummarySynergistic polymicrobial infections can contribute to both disease severity and persistence. Enterococcus faecalis and Escherichia coli are frequently co-isolated from polymicrobial urinary tract infections. Immunomodulation by co-infecting microbes can result in a more permissive environment for pathogens to establish infection. Presently, we do not yet understand how these microbes overcome host immunity to establish polymicrobial infections. To address this, we investigated how the immunosuppressive function of E. faecalis can contribute to acute infection. We defined that E. faecalis is able to suppress macrophages in vitro, despite the presence of E. coli. We also demonstrated E. faecalis’ ability to augment E. coli titers in vivo to establish kidney infection. Our findings raise the prospect that E. faecalis can alter host immunity to increase susceptibility to other uropathogens.

Membrane vesicles (MVs) contribute to various biological processes in bacteria, including virulence factor delivery, antimicrobial resistance, host immune evasion, and cross-species communication. MVs are frequently released from the surface of both Gram-negative and Gram-positive bacteria during growth. In some Gram-positive bacteria, genes affecting MV biogenesis have been identified, but the mechanism of MV formation is unknown. In Enterococcus faecalis, a causative agent of life-threatening bacteraemia and endocarditis, neither mechanisms of MV formation nor their role in virulence has been examined. Since MVs of many bacterial species are implicated in host-pathogen interactions, biofilm formation, horizontal gene transfer, and virulence factor secretion in other species, we sought to identify, describe, and functionally characterize MVs from E. faecalis. Here we show that E. faecalis releases MVs that possess unique lipid and protein profiles, distinct from the intact cell membrane, and are enriched in lipoproteins. MVs of E. faecalis are specifically enriched in unsaturated lipids that might provide membrane flexibility to enable MV formation, providing the first insights into the mechanism of MV formation in this Gram-positive organism.