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.

Figure 1. Venn diagram representation of the results of the five phenotypic tests performed on the mutants obtained in this study.

Only genes that are common to at least two phenotypes are listed. The increased adhesion phenotype was not included due to difficulties in constructing the Venn diagram with these data. Stars indicate mutants with increased adhesion phenotype. ^§ The 10 mutants combining phenotypes for H₂O₂ stress and virulence are not shown due to lack of space in the figure. They were inactivated in ef0073, ef0465, ef0814, ef1212, ef1420, ef1493, ef1741, ef1760, ef1798 or ef2442.

Figure 2. Schematic representation of E. faecalis V583 cell envelope, showing functions involved in pleiotropic stress protection.

The E. faecalis V583 cell envelope includes the peptidoglycan-anchored teichoic acid (WTA; burgundy), the membrane-anchored lipoteichoic acid (LTA; green), the rhamnopolysaccharide (EPA; blue), and a capsular polysaccharide (CPS; purple). Names of genes predicted to participate in glycopolymer synthesis are indicated with the cognate colour code. Membrane protein MprF-like is depicted in light orange.