Genome-wide mutagenesis identifies factors involved inEnterococcus faecalisvaginal adherence and persistence

Abstract

21Enterococcus faecalis is a Gram-positive commensal bacterium native to the gastrointestinal tract and an 22 opportunistic pathogen of increasing clinical concern. E. faecalis also colonizes the female reproductive 23 tract and reports suggest vaginal colonization increases following antibiotic treatment or in patients with 24 aerobic vaginitis. Currently, little is known about specific factors that promote E. faecalis vaginal 25 colonization and subsequent infection. We developed a mouse model of E. faecalis vaginal carriage and 26 demonstrate that both vancomycin resistant and sensitive strains colonize the murine vaginal tract. 27Following vaginal colonization, we observed E. faecalis in vaginal, cervical and uterine tissue. A mutant 28 lacking endocarditis-and biofilm-associated pili (Ebp) exhibited a decreased ability to associate with 29 human vaginal and cervical cells in vitro as well as ascend to the cervix and uterus in vivo. To further 30 identify novel factors that promote vaginal colonization, we screened a low-complexity transposon (Tn) 31 mutant library to identify genes important for E. faecalis persistence in the vaginal tract. This screen 32 revealed 383 mutants that were underrepresented during vaginal colonization at all time points compared 33 to growth in culture medium. We confirmed that mutants deficient in ethanolamine catabolism and the 34 type VII secretion system were attenuated in persisting during vaginal colonization. These results reveal 35 the complex nature of vaginal colonization and suggest that multiple factors contribute to E. 36 faecalis persistence in the reproductive tract. 37 38 IMPORTANCE 39Despite increasing prevalence and association of E. faecalis with aerobic vaginitis, essentially nothing is 40 known about the bacterial factors that influence E. faecalis vaginal colonization. We have established an 41 animal model of vaginal colonization that supports colonization of multiple E. faecalis strains. 42Additionally, we determined that pili, ethanolamine utilization, and type VII secretion system genes 43 3 contribute to vaginal persistence and ascension to reproductive tract tissues. Identification of factors 44 important for vaginal colonization and persistence provides potential targets for the development of 45 therapeutics. This study is the first to identify key determinants that promote vaginal colonization by E. 46 faecalis, which may represent an important reservoir for antibiotic resistant enterococci. 47 48 INTRODUCTION 49Enterococcus faecalis is an opportunistic pathogen that resides in the human gastrointestinal and 50 urogenital tracts [1, 2]. While E. faecalis colonization is normally asymptomatic, certain populations are 51 at risk for severe disease including urinary tract infections (UTIs), wound infections, pelvic inflammatory 52 disease (PID), infective endocarditis, and adverse birth effects during pregnancy [3, 4]. Enterococcal 53 infections are often associated with the production of biofilms, assemblages of microbes enclosed in an 54 extracellula...

Comparative biofilm assays usingEnterococcus faecalisOG1RF identify new determinants of biofilm formation

“…It is made The copyright holder for this preprint this version posted February 24, 2021. ; https://doi.org/10.1101/2021.02. 24…”

“…SmarT TnSeq library #2 is a subset of library #1 and contains 1,948 Tn insertions in intergenic regions or uncharacterized or poorly characterized genes (22,23). These Tn libraries have been used to identify OG1RF genes important for cholic acid resistance, biofilm formation and biofilm-associated antibiotic resistance in microtiter plates, response to phage infection, vaginal colonization, and augmentation of E. coli growth (9,(22)(23)(24)(25)(26)(27).…”

Optimized replication of arrayed bacterial mutant libraries increase access to biological resources

“…The second SmarT library consists of 1,946 Tn insertions in poorly characterized genes and intergenic regions and was designed to facilitate genetic screens targeting uncharacterized regions of the genome (13). Both libraries are also available in pooled formats and have been used extensively to identify E. faecalis genes required for biofilm formation, metabolism, response to antibiotics, phage infection, vaginal colonization in a mouse model, and polymicrobial interactions involving E. faecalis (14)(15)(16)(17)(18)(19)(20)(21).…”

Bacterial type VII secretion: An important player in host‐microbe and microbe‐microbe interactions

“…The bacteria‐ and host‐targeting capabilities of the T7SSb have been linked to a role in infectious disease. For example, the bacteria‐targeting T7SSb of E. faecalis was recently shown to be required for the colonization and persistence of this pathogen in a murine vaginal tract infection model (Alhajjar et al., 2020). Other studies have indicated a more direct role in pathogenesis with the T7SSb of S. aureus being implicated in direct targeting of host cells (Burts et al., 2005; Korea et al., 2014).…”