Genome-Wide Mutagenesis Identifies Factors Involved in Enterococcus faecalis Vaginal Adherence and Persistence

Abstract

Enterococcus faecalis is a Gram-positive commensal bacterium native to the gastrointestinal tract and an opportunistic pathogen of increasing clinical concern. E. faecalis also colonizes the female reproductive tract and reports suggest vaginal colonization increases following antibiotic treatment or in patients with aerobic vaginitis. Currently, little is known about specific factors that promote E. faecalis vaginal colonization and subsequent infection. We modified an established mouse vaginal colonization model to explore E. faecalis vaginal carriage and demonstrate that both vancomycin resistant and sensitive strains colonize the murine vaginal tract. Following vaginal colonization, we observed E. faecalis in vaginal, cervical and uterine tissue. A mutant lacking endocarditis- and biofilm-associated pili (Ebp) exhibited a decreased ability to associate with human vaginal and cervical cells in vitro, but did not contribute to colonization in vivo. Thus, we screened a low-complexity transposon (Tn) mutant library to identify novel genes important for E. faecalis colonization and persistence in the vaginal tract. This screen revealed 383 mutants that were underrepresented during vaginal colonization at 1, 5 and 8 days post-inoculation compared to growth in culture medium. We confirmed that mutants deficient in ethanolamine catabolism or in the type VII secretion system were attenuated in persisting during vaginal colonization. These results reveal the complex nature of vaginal colonization and suggest that multiple factors contribute to E. faecalis persistence in the reproductive tract. IMPORTANCE Despite increasing prevalence and association of E. faecalis with aerobic vaginitis, essentially nothing is known about the bacterial factors that influence E. faecalis vaginal colonization. We have adapted an animal model of vaginal colonization that supports colonization of multiple E. faecalis strains. Additionally, we determined that ethanolamine utilization and type VII secretion system genes contribute to vaginal colonization and persistence. Identification of factors important for vaginal colonization and persistence provides potential targets for the development of therapeutics. This study is the first to identify key determinants that promote vaginal colonization by E. faecalis, which may represent an important reservoir for antibiotic resistant enterococci.

Phage infection and sub-lethal antibiotic exposure mediate Enterococcus faecalis type VII secretion system dependent inhibition of bystander bacteria

“…faecalis T7SS gene esxA are found throughout three of the four Enterococcus species groups [ 35 ], including Enterococcus faecium , suggesting a wide distribution of T7SS loci in enterococci ( S1 Fig ). In addition to EsxA, OG1RF encodes the core T7SS structural components EsaA, EssB, and EssC, which are predicted to localize to the membrane, and EsaB, a small predicted cytoplasmic protein ( Fig 1A ) [ 36 ]. OG1RF_11102 encodes an additional putative membrane protein, although it does not share sequence homology with staphylococcal or streptococcal EssA.…”

“…Indeed, E . faecalis T7SS mutants are defective in their ability to colonize the murine reproductive tract, which like the intestine is a polymicrobial environment [ 36 ].…”

Phage infection and sub-lethal antibiotic exposure mediateEnterococcus faecalistype VII secretion system dependent inhibition of bystander bacteria

“…In addition to EsxA, OG1RF encodes the core T7SS structural components EsaA, EssB, and EssC, which are predicted to localize to the membrane, and EsaB, a small predicted cytoplasmic protein (Fig. 1A) [36]. OG1RF_11102 encodes an additional putative membrane protein, although it does not share sequence homology with staphylococcal or streptococcal EssA.…”

“…Although exposure to a sub-inhibitory level of primary bile salt (a common molecule found in the intestine) did not elicit T7SS expression, it is possible that other stressors encountered in the intestinal tract, including lysozyme, antimicrobial proteins, and nutrient availability could influence T7SS activity in E. faecalis . Indeed, E. faecalis T7SS mutants are defective in their ability to colonize the murine reproductive tract, which like the intestine is a polymicrobial environment [36].…”

A type VII secretion system in Group B Streptococcus mediates cytotoxicity and virulence

“…CJB111 encodes a putative LXG toxin just downstream of the T7SS core machinery locus that has not yet been characterized. LXG toxin-encoding genes are prevalent in bacteria that comprise the human gut microbiota 30 and the T7SS of E. faecalis has been shown to be important for colonization of the murine vaginal tract 74 . Thus, GBS LXG toxins may promote interbacterial competition of GBS with normal flora in both the gastrointestinal and female reproductive tracts and would be of interest to study in the future.…”