Genome-wide analysis of <i>Enterococcus faecalis</i> genes that facilitate interspecies competition with <i>Lactobacillus crispatus</i>

Enterococci are opportunistic pathogens notorious for causing a variety of infections. While both <i>Enterococcus faecalis</i> and <i>Lactobacillus crispatus</i> are commensal residents of the vaginal tract, the molecular mechanisms that enable <i>E. faecalis</i> to take advantage of a vaginal biome with lower counts of lactobacilli to colonize the vaginal tract and induce aerobic vaginitis remain unknown. Here, we show that <i>L. crispatus</i> eradicates <i>E. faecalis</i> in a contact-independent manner. Using transposon sequencing to identify <i>E. faecalis</i> OG1RF transposon (Tn) mutants that are either under-represented or over-represented when co-cultured with <i>L. crispatus</i>, we found that Tn mutants with disruption in the <i>dltABCD</i> operon, that encodes the proteins responsible for the D-alanylation of teichoic acids, and <i>OG1RF_11697</i> encoding for an uncharacterized hypothetical protein are more susceptible to killing by <i>L. crispatus</i>. Inversely, Tn mutants with disruption in <i>ldh1,</i> which encodes for L-lactate dehydrogenase, are more resistant to <i>L. crispatu</i>s killing. Using the <i>Galleria mellonella</i> infection model, we show that co-injection of <i>L. crispatus</i> with <i>E. faecalis</i> OG1RF enhances larvae survival while this <i>L. crispatus</i>-mediated protection was lost in larvae co-infected with either <i>L. crispatus</i> and <i>E. faecalis</i>Δ<i>ldh1</i> or Δ<i>ldh1</i>Δ<i>ldh2</i> strains. Last, using RNA sequencing to identify <i>E. faecalis</i> genes that are differently expressed in the presence of <i>L. crispatus</i>, we found major changes in the expression of genes associated with glycerophospholipid metabolism, central metabolism, and general stress responses. The findings in this study provide insights into how <i>E. faecalis</i> mitigate assaults by <i>L. crispatus</i>.IMPORTANCE<i>Enterococcus faecalis</i> is an opportunistic pathogen notorious for causing a multitude of infections. As vaginal commensals, <i>E. faecalis</i> must interact with <i>Lactobacillus crispatus</i>, but how <i>E. faecalis</i> overcomes or mitigate assaults by <i>L. crispatus</i> killing remains unknown. We show that <i>L. crispatus</i> eradicates <i>E. faecalis</i> temporally in a contact-independent manner. Using high-throughput molecular approaches, we identified genetic determinants that enable <i>E. faecalis</i> to compete with <i>L. crispatus</i>. This study represents an important first step for the identification of adaptive genetic traits required for enterococci to tolerate assaults by lactobacilli.