Regulation of Central Carbon Metabolism in Enterococcus faecalis

Abstract

Enterococcus faecalis has emerged as a multidrug-resistant (MDR) nosocomial pathogen that causes life-threatening healthcare-associated infections at various host anatomic sites. Enterococcal colonization of these biological niches is, in part, dependent on its adaptive response to host conditions. Some important strategies to adapt to varying host environments include biofilm formation, immune evasion, and the utilization of secondary nutrients in the absence of preferred carbon sources, which are often host limited. The regulation of these factors is governed by environmental or metabolic cues that influence alterations of a bacterium’s transcriptome, often orchestrated by alternative sigma factors or other transcriptional regulators. Investigating the role of various transcriptional regulators and identifying the genes and cellular processes that these factors regulate may lead to new treatments against multidrug resistant enterococcal infections. In this study, we show that the alternative sigma factor 54 (σ54; RpoN) and its cognate enhancer binding protein MptR are essential for mannose utilization and principally responsible for glucose uptake by directly regulating the expression of the Mpt phosphotransferase system in Enterococcus faecalis. We performed a microarray transcriptional analysis of the parental strain V583 and an isogenic rpoN mutant grown in a chemically defined medium supplemented with glucose as the sole carbon source to gain further insight into how the disruption of RpoN-mediated glucose utilization influences global transcriptional changes. Transcripts of approximately 340 genes were differentially affected in the rpoN mutant. Predicted functions of these differentially affected genes were primarily related to nutrient acquisition. Predicted catabolite responsive element (cre) sites were identified in 42.74% of the differentially upregulated genes in the rpoN mutant, consistent with loss of repression by the transcriptional regulator Carbon Catabolite Protein A (CcpA). Three gene clusters (ef0114, ef0362-61, and ef2863) that encode the respective enterococcal endoglycosyl hydrolases were highly upregulated in the rpoN mutant with identified putative cre sites within their promoter regions. These glycosyl hydrolases possess putative signal peptide sequences and signal peptide cleavage sites, suggesting that these glycosidases are secreted into the surrounding environment and may contribute to the survival of this bacterium in nutrient limited environments. We confirmed that CcpA negatively regulates the expression of ef0114, ef0362-61, and ef2863 in a CCR-dependent manner. Further characterization of the enzymatic function of EF0362-61, EF0114, and EF2863 revealed that they target the β1,4-linked N-acetylglucosamine glycosidic bond present in chitinous substrates, complex-type glycoproteins, and high-mannose type glycoproteins, respectively. The amino sugar, N-acetylglucosamine (GlcNAc) is among the most abundant natural sugars and provides bacteria with a source of carbon and nitrogen when metabolized. Here, we propose a model of the metabolic pathway by which E. faecalis utilizes poly-β-1,4-linked N-acetylglucosamine (i.e. chitinous substrates) as a valuable carbon and nitrogen source.