Large-Scale Screening of a Targeted Enterococcus faecalis Mutant Library Identifies Envelope Fitness Factors

Abstract

Spread of antibiotic resistance among bacteria responsible for nosocomial and community-acquired infections urges for novel therapeutic or prophylactic targets and for innovative pathogen-specific antibacterial compounds. Major challenges are posed by opportunistic pathogens belonging to the low GC% Gram-positive bacteria. Among those, Enterococcus faecalis is a leading cause of hospital-acquired infections associated with life-threatening issues and increased hospital costs. To better understand the molecular properties of enterococci that may be required for virulence, and that may explain the emergence of these bacteria in nosocomial infections, we performed the first large-scale functional analysis of E. faecalis V583, the first vancomycin-resistant isolate from a human bloodstream infection. E. faecalis V583 is within the high-risk clonal complex 2 group, which comprises mostly isolates derived from hospital infections worldwide. We conducted broad-range screenings of candidate genes likely involved in host adaptation (e.g., colonization and/or virulence). For this purpose, a library was constructed of targeted insertion mutations in 177 genes encoding putative surface or stress-response factors. Individual mutants were subsequently tested for their i) resistance to oxidative stress, ii) antibiotic resistance, iii) resistance to opsonophagocytosis, iv) adherence to the human colon carcinoma Caco-2 epithelial cells and v) virulence in a surrogate insect model. Our results identified a number of factors that are involved in the interaction between enterococci and their host environments. Their predicted functions highlight the importance of cell envelope glycopolymers in E. faecalis host adaptation. This study provides a valuable genetic database for understanding the steps leading E. faecalis to opportunistic virulence.

Involvement of Enterococcus faecalis Small RNAs in Stress Response and Virulence

“…E. coli DH5␣ (36) was used as the recipient for cloning. Mutants were constructed from the parental E. faecalis strain EryS, an erythromycin-sensitive strain derived from the vancomycin-resistant clinical isolate V583 (37). E. faecalis EryS and its derivates were grown without shaking at 37°C in M17 medium supplemented with 0.5% glucose (M17glu).…”

Enterococcal Rgg-Like Regulator ElrR Activates Expression of the elrA Operon

“…A 1.28-kb EaeI-EcoRI fragment from pVE14045 (see below) was cloned in pJIM2242, generating pVE14147, which was introduced into strain OG1RF⌬gelE (44) by electroporation. An integrant of pVE14147 was obtained as previously described (45), generating a strain expressing chromosomal ElrR-6ϫHis under the control of its own promoter. Another OG1RF strain expressing ElrR-6ϫHis under the control of the constitutively expressed E. faecalis P aphA3 promoter (46) on a replicating plasmid was used as a positive control.…”

Cold-shock RNA-binding protein CspR is also exposed to the surface of Enterococcus faecalis

“…The DcspR mutant was constructed from the parental E. faecalis strain EryS, an erythromycin-sensitive-cured derivative of the vancomycin-resistant clinical isolate V583 (Rigottier-Gois et al, 2011). E. faecalis EryS and its derivatives were grown without shaking at 37 uC in M17 medium supplemented with 0.5 % glucose (GM17).…”