Influence of the Alternative Sigma Factor RpoN on Global Gene Expression and Carbon Catabolism in Enterococcus faecalis V583

Abstract

The alternative sigma factor σ54 has been shown to regulate the expression of a wide array of virulence-associated genes, as well as central metabolism, in bacterial pathogens. In Gram-positive organisms, the σ54 is commonly associated with carbon metabolism. In this study, we show that the Enterococcus faecalis alternative sigma factor σ54 (RpoN) and its cognate enhancer binding protein MptR are essential for mannose utilization and are primary contributors to glucose uptake through the Mpt phosphotransferase system. To gain further insight into how RpoN contributes to global transcriptional changes, we performed microarray transcriptional analysis of strain V583 and an isogenic rpoN mutant grown in a chemically defined medium with glucose as the sole carbon source. Transcripts of 340 genes were differentially affected in the rpoN mutant; the predicted functions of these genes mainly related to nutrient acquisition. These differentially expressed genes included those with predicted catabolite-responsive element (cre) sites, consistent with loss of repression by the major carbon catabolite repressor CcpA. To determine if the inability to efficiently metabolize glucose/mannose affected infection outcome, we utilized two distinct infection models. We found that the rpoN mutant is significantly attenuated in both rabbit endocarditis and murine catheter-associated urinary tract infection (CAUTI). Here, we examined a ccpA mutant in the CAUTI model and showed that the absence of carbon catabolite control also significantly attenuates bacterial tissue burden in this model. Our data highlight the contribution of central carbon metabolism to growth of E. faecalis at various sites of infection. IMPORTANCE Hospital-acquired infections account for 2 billion dollars annually in increased health care expenses and cause more than 100,000 deaths in the United States alone. Enterococci are the second leading cause of hospital-acquired infections. They form biofilms at surgical sites and are often associated with infections of the urinary tract following catheterization. Nutrient uptake and growth are key factors that influence their ability to cause disease. Our research identified a large set of genes that illuminate nutrient uptake pathways in enterococci. Perturbation of the metabolic circuit reduces virulence in a rabbit endocarditis model, as well as in catheter-associated urinary tract infection in mice. Targeting metabolic pathways that are important in infection may lead to new treatments against multidrug-resistant enterococcal infections.

The NagY regulator: A member of the BglG/SacY antiterminator family conserved in Enterococcus faecalis and involved in virulence

“…In this context, HylA could be used as a backup to favorize E. faecalis survival and the competition with other microorganisms in gastro-intestinal microbiota. Contrary to nagY-nagE operon, it was shown that hylA gene expression is under the control of the RpoN sigma factor and CCR, suggesting a multifactorial regulation of this gene (Keffeler et al, 2021a). Thus, the nagY, nagE, and hylA genes could be involved in the adaptation of Enterococcaceae through the use of different carbohydrate sources.…”

“…Thus, the NagE PTS transporter allows the NagY antiterminator to sense carbohydrate source in the environment (Tortosa et al, 2001). NAG is then phosphorylated by NagE during its import into the cell (Keffeler et al, 2021b) and is directly used in glycolysis and metabolized. We showed that nagY-nagE is not 10.3389/fmicb.2022.1070116 Frontiers in Microbiology submitted to catabolic repression.…”

“…Thus, the nagY, nagE, and hylA genes could be involved in the adaptation of Enterococcaceae through the use of different carbohydrate sources. As hyaluronidases, i.e., endolytic glycoside hydrolases, HylA protein would be complementary to EfChi18A-EfCBM33A and EndoE to retrieve NAG from environment, described in a recent study by Keffeler et al (2021b). These enzymes allow E. faecalis to utilize poly-β1,4-linked N-acetylglucosamine, found in chitin, as carbon source.…”

“…The EF1515 (EF_RS07320 in the new nomenclature) protein presents 36% identity with SacY from B. subtilis, and 31% amino acid identity with BglG from E. coli (blastP alignment). This gene is followed by the ef1516 (EF_RS07325) gene (Figure 2), recently renamed nagE on V583 genome, encoding a NAG specific EIICBA PTS transporter (Keffeler et al, 2021b). Thus, the NagY and NagE proteins belong to multigene families.…”

“…Herein, we investigate the role of a BglG/SacY antiterminator homolog as a link between the metabolism and the opportunistic features of E. faecalis. An interesting aspect is that the β-glucosides metabolism was shown to be induced during infection (Muller 10.3389/fmicb.2022.1070116 Frontiers in Microbiology 03 frontiersin.org et al, 2015), so we studied the regulation of this metabolism in E. faecalis by the ef1515-ef1516 operon encoding a BglG/SacY-like antiterminator (NagY) and a NAG PTS transporter (NagE; Keffeler et al, 2021b). We analyzed the autoregulation mechanism of NagY, and its action on the expression of a hyaluronidase HylA, identified as a MSCRAMM (Microbial Surface Components Recognizing Adhesive Matrix Molecules).…”