Enterococcus faecalis rnjB Is Required for Pilin Gene Expression and Biofilm Formation

Abstract

Pili in Gram-positive bacteria play a major role in the colonization of host tissue and in the development of biofilms. They are promising candidates for vaccines or drug targets since they are highly immunogenic and share common structural and functional features among various Gram-positive pathogens. Numerous publications have helped build a detailed understanding of pilus surface assembly, yet regulation of pilin gene expression has not been well defined. Utilizing a monoclonal antibody developed against the Enterococcus faecalis major pilus protein EbpC, we identified mutants from a transposon (Tn) insertion library which lack surface-exposed Ebp pili. In addition to insertions in the ebp regulon, an insertion in ef1184 (dapA) significantly reduced levels of EbpC. Analysis of in-frame dapA deletion mutants and mutants with the downstream gene rnjB deleted further demonstrated that rnjB was responsible for the deficiency of EbpC. Sequence analysis revealed that rnjB encodes a putative RNase J2. Subsequent quantitative real-time PCR (qRT-PCR) and Northern blotting demonstrated that the ebpABC mRNA transcript level was significantly decreased in the rnjB deletion mutant. In addition, using a reporter gene assay, we confirmed that rnjB affects the expression of the ebpABC operon. Functionally, the rnjB deletion mutant was attenuated in its ability to produce biofilm, similar to that of an ebpABC deletion mutant which lacks Ebp pili. Together, these results demonstrate the involvement of rnjB in E. faecalis pilin gene expression and provide insight into a novel mechanism of regulation of pilus production in Gram-positive pathogens.Enterococcus faecalis, a normal commensal of the human gastrointestinal tract, is also an opportunistic pathogen and a major cause of nosocomial infections. E. faecalis is one of many Gram-positive pathogens recently discovered to possess surface pili. These Gram-positive pili are distinct from Gramnegative pili in their structure and mechanism of assembly (35,42). Pilus expression has been closely associated with virulence in multiple human pathogens, including group A Streptococcus (33), group B Streptococcus (24), and Corynebacterium diphtheriae (43). In E. faecalis, the biofilm-associated pili (Ebp) are considered to be among its major virulence factors and play an important role in biofilm formation and the development of endocarditis (35). Mutations in ebp structural genes have been shown to significantly reduce E. faecalis biofilm formation in vitro (35) and to decrease the ability of E. faecalis to form vegetations in a rat endocarditis model (20). The Ebp pilus also plays a role in murine urinary tract infection (UTI), as was demonstrated using an ascending UTI model (40), which provides further evidence for the importance of pili in bacterial infection.Three genes encoding the E. faecalis pilus proteins (ebpA, -B, and -C) are located in the same operon (35). Ebp pili are formed by the cross-linking of all three pilus proteins (35). EbpR, encoded by the gene upstream of ebpABC,...

Library Screen Identifies Enterococcus faecalis CcpA, the Catabolite Control Protein A, as an Effector of Ace, a Collagen Adhesion Protein Linked to Virulence

“…Our library screen method once again (8,10) proved to be of great value in studying genetic determinants involved in bacterial surface protein expression, since it can detect changes that occur at any level from transcription to surface anchoring. Since Ace mRNA levels are not affected by deletion of ccpA, the link between these two factors would not have been discovered through traditional genetic or transcriptome analysis.…”

“…The whole-cell ELISA screen was performed as previously described (10), with some modifications. The Tn library of cells was cultured for 24 h before being diluted 1:10 into new wells containing BHI medium and subsequently cultured at 37°C for 1.5 h. Anti-Ace monoclonal antibody (MAb) 70 at 2 g/ml was used to label cells, followed by the addition of 100 l of a 1:3,000 dilution of goat anti-mouse IgG-horseradish peroxidase (HRP) (Jackson ImmunoResearch).…”

“…Growth at 46°C also significantly increases Ace expression compared to growth at 37°C; furthermore, reverse transcription-quantitative PCR (qRT-PCR) revealed an 18-fold increase in ace mRNA levels in cells grown in the presence of collagen IV compared to levels in the control (9). However, many of the genetic determinants that regulate Ace expression have yet to be evaluated.Recently, we developed a whole-cell enzyme-linked immunosorbent assay (ELISA)-based library screening method to identify genetic elements that are involved in the display of E. faecalis cell surface proteins (8,10). From a 536-membered E. faecalis OG1RF transposon (Tn) insertion mutant library, one mutant, an fsrB insertion mutant, was identified with significantly higher Ace expression levels at late growth phase.…”

“…Recently, we developed a whole-cell enzyme-linked immunosorbent assay (ELISA)-based library screening method to identify genetic elements that are involved in the display of E. faecalis cell surface proteins (8,10). From a 536-membered E. faecalis OG1RF transposon (Tn) insertion mutant library, one mutant, an fsrB insertion mutant, was identified with significantly higher Ace expression levels at late growth phase.…”

Enterococcus faecalis Ebp pili are important for cell-cell aggregation and intraspecies gene transfer

“…A pilusspecific class C sortase (Bps, for biofilm-and pilusassociated sortase), catalyses the assembly of the structural subunits into pili, before the housekeeping sortase A (SrtA) covalently binds the elongated pili to the cell wall via EbpB (Nallapareddy et al, 2006;Sillanpää et al, 2013). Studies on ebp regulation identified the upstream ebpR gene (Bourgogne et al, 2007) and the rnjB gene (Gao et al, 2010) as activators at the mRNA level, while the Fsr quorum-sensing system was described as a weak repressor (Bourgogne et al, 2006). In addition, a recent report by Montealegre et al (2015) demonstrated that E. faecalis uses the rare initiation codon ATT, found in all E. faecalis strains sequenced, as the start codon of the tip pilin EbpA and that this start codon results in reduced expression, at the translational level, relative to an engineered ATG, and negatively affects Ebp-associated functions (Montealegre et al, 2015).…”