The Enterococcus faecium Enterococcal Biofilm Regulator, EbrB, Regulates the esp Operon and Is Implicated in Biofilm Formation and Intestinal Colonization

Abstract

Nowadays, Enterococcus faecium is one of the leading nosocomial pathogens worldwide. Strains causing clinical infections or hospital outbreaks are enriched in the enterococcal surface protein (Esp) encoding ICEEfm1 mobile genetic element. Previous studies showed that Esp is involved in biofilm formation, endocarditis and urinary tract infections. In this study, we characterized the role of the putative AraC type of regulator (locus tag EfmE1162_2351), which we renamed ebrB and which is, based on the currently available whole genome sequences, always located upstream of the esp gene, and studied its role in Esp surface exposure during growth. A markerless deletion mutant of ebrB resulted in reduced esp expression and complete abolishment of Esp surface exposure, while Esp cell-surface exposure was restored when this mutant was complemented with an intact copy of ebrB. This demonstrates a role for EbrB in esp expression. However, during growth, ebrB expression levels did not change over time, while an increase in esp expression at both RNA and protein level was observed during mid-log and late-log phase. These results indicate the existence of a secondary regulation system for esp, which might be an unknown quorum sensing system as the enhanced esp expression seems to be cell density dependent. Furthermore, we determined that esp is part of an operon of at least 3 genes putatively involved in biofilm formation. A semi-static biofilm model revealed reduced biofilm formation for the EbrB deficient mutant, while dynamics of biofilm formation using a flow cell system revealed delayed biofilm formation in the ebrB mutant. In a mouse intestinal colonization model the ebrB mutant was less able to colonize the gut compared to wild-type strain, especially in the small intestine. These data indicate that EbrB positively regulates the esp operon and is implicated in biofilm formation and intestinal colonization.

Growth condition‐dependent cell surface proteome analysis of Enterococcus faecium

“…The enterococcal surface protein Esp (EfmE1162_2353) was only identified in TSBg. This is in line with a recent study where it was shown that expression of Esp in BHI was very low compared to growth in TSBg . Esp is considered a virulence factor and can be specifically linked to nosocomial clonal lineages that are genetically distinct from indigenous commensal E. faecium strains .…”

Modulators of Enterococcus faecalis Cell Envelope Integrity and Antimicrobial Resistance Influence Stable Colonization of the Mammalian Gastrointestinal Tract

“…For example, EpaX, an enzyme involved in the synthesis of the surface polysaccharide Epa was found to be critical for GIT colonization by E. faecalis (4). Both the biofilm transcription regulator EbrB and PtsD, a subunit of an uncharacterized phosphotransferase system, were observed to promote colonization by E. faecium (5,6). The genes encoding EpaX, EbrB, and PtsD are carried on mobile genetic elements and/or are enriched in infection-causing clinical enterococcal strains relative to commensal strains and are therefore not part of the core enterococcal genome.…”

“…biofilm formation, sugar transport, and the synthesis of cell wall polysaccharides, to promote GIT colonization (4)(5)(6). The genes identified in these studies are located on mobile genetic elements or are enriched in clinical isolates, suggesting that they enhance GIT colonization under some circumstances but do not represent the essential determinants of GIT colonization in the core enterococcal genome that evolved over millions of years to enable enterococci to colonize the GIT.…”

Deletion Mutant Library for Investigation of Functional Outputs of Cyclic Diguanylate Metabolism in Pseudomonas aeruginosa PA14

“…Given the known role of c-di-GMP in biofilm formation, we tested the biofilm formation of each mutant under two growth conditions used routinely in our laboratory. The first, glucose supplemented with Casamino Acids (glucoseϩCAA), is the standard assay medium used in most of our previously reported studies (16,17), and glucose is a common growth substrate used by many groups studying biofilm formation (48)(49)(50)(51). The second substrate analyzed is arginine, one of the few carbon sources which P. aeruginosa can ferment and which we have shown previously boosts the levels of c-di-GMP by ϳ4-fold compared to growth on glucoseϩCAA (52).…”