Bicarbonate enhances expression of the endocarditis and biofilm associated pilus locus, ebpR-ebpABC, in Enterococcus faecalis

Abstract

BackgroundWe previously identified ebpR, encoding a potential member of the AtxA/Mga transcriptional regulator family, and showed that it is important for transcriptional activation of the Enterococcus faecalis endocarditis and biofilm associated pilus operon, ebpABC. Although ebpR is not absolutely essential for ebpABC expression (100-fold reduction), its deletion led to phenotypes similar to those of an ebpABC mutant such as absence of pili at the cell surface and, consequently, reduced biofilm formation. A non-piliated ebpABC mutant has been shown to be attenuated in a rat model of endocarditis and in a murine urinary tract infection model, indicating an important participation of the ebpR-ebpABC locus in virulence. However, there is no report relating to the environmental conditions that affect expression of the ebpR-ebpABC locus.ResultsIn this study, we examined the effect of CO2/HCO3-, pH, and the Fsr system on the ebpR-ebpABC locus expression. The presence of 5% CO2/0.1 M HCO3- increased ebpR-ebpABC expression, while the Fsr system was confirmed to be a weak repressor of this locus. The mechanism by which the Fsr system repressed the ebpR-ebpABC locus expression appears independent of the effects of CO2- bicarbonate. Furthermore, by using an ebpA::lacZ fusion as a reporter, we showed that addition of 0.1 M sodium bicarbonate to TSBG (buffered at pH 7.5), but not the presence of 5% CO2, induced ebpA expression in TSBG broth. In addition, using microarray analysis, we found 73 genes affected by the presence of sodium bicarbonate (abs(fold) > 2, P < 0.05), the majority of which belong to the PTS system and ABC transporter families. Finally, pilus production correlated with ebpA mRNA levels under the conditions tested.ConclusionsThis study reports that the ebp locus expression is enhanced by the presence of bicarbonate with a consequential increase in the number of cells producing pili. Although the molecular basis of the bicarbonate effect remains unclear, the pathway is independent of the Fsr system. In conclusion, E. faecalis joins the growing family of pathogens that regulates virulence gene expression in response to bicarbonate and/or CO2.

Conservation of Ebp-Type Pilus Genes among Enterococci and Demonstration of Their Role in Adherence of Enterococcus faecalis to Human Platelets

“…Their genetic organization is highly conserved in each species, but the E. casseliflavus isolates lack an ebpB homolog. Similar to E. faecalis, E. faecium and E. gallinarum also have ebpR (encoding a positive regulator of the ebp operon in E. faecalis [5,6]) in the same position (Fig. 1).…”

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

“…pTEX5585 is a pTCV-lac-derived vector containing the ebpA promoter, the 5Ј-UTR, and the first 41 nucleotides (nt) of the coding region upstream of a LacZ coding region (6). As shown in Fig.…”

“…It has been demonstrated that deletion of ebpR leads to reduced levels of the ebpABC mRNA, as evaluated by quantitative real-time PCR (qRT-PCR), and of pili, as evaluated by Western blotting (5). Many environmental factors affect pilus production, including culture medium (tryptic soy broth [TSB] versus brain heart infusion [BHI]), serum, and bicarbonate (6). In a recent study, Bourgogne et al demonstrated that the addition of bicarbonate to culture media enhanced the expression of the ebpR and ebpABC locus (6).…”

“…Many environmental factors affect pilus production, including culture medium (tryptic soy broth [TSB] versus brain heart infusion [BHI]), serum, and bicarbonate (6). In a recent study, Bourgogne et al demonstrated that the addition of bicarbonate to culture media enhanced the expression of the ebpR and ebpABC locus (6). In general, however, the genetic regulation mechanism of pilin gene expression remains poorly understood.…”

“…The cell lysates were centrifuged at 13,600 rpm for 1 min to remove cell debris before being used in a ␤-galactosidase assay. The ␤-galactosidase assay was performed according to the protocol of Bourgogne et al (6), with some modifications. Dilutions of cell lysate were incubated with o-nitrophenyl-␤-D-galactoside (ONPG) until a yellow color developed.…”