Activity of CcpA-Regulated GH18 Family Glycosyl Hydrolases That Contributes to Nutrient Acquisition and Fitness in Enterococcus faecalis

Abstract

The ability of Enterococcus faecalis to colonize host anatomic sites is dependent on its adaptive response to host conditions. Three glycosyl hydrolase gene clusters, each belonging to the GH18 family ( ef0114 , ef0361 , and ef2863 ) in E. faecalis were previously found to be upregulated under glucose-limiting conditions. The GH18 catalytic domain is present in proteins that are classified as either chitinases or ENGases based on their β-1,4 endo-N-acetyl-beta-D-glucosaminidase activity, and ENGase activity is commonly associated with cleaving N-linked glycoproteins, an abundant glycan structure on host epithelial surfaces. Here we show that all three hydrolases are negatively regulated by the transcriptional regulator Carbon Catabolite Protein A (CcpA). Additionally, we demonstrate that a constitutively active CcpA variant represses the expression of CcpA-regulated genes irrespective of glucose availability. Previous studies showed that the GH18 catalytic domain of EndoE (EF0114) and EfEndo18A (EF2863) were capable of deglycosylating RNase B, a model high-mannose type glycoprotein. However, it remained uncertain which glycosidase is primarily responsible for deglycosylation of high-mannose type glycoproteins. In this study, we show by mutation analysis as well as a dose-dependent analysis of recombinant protein expression that EfEndo18A is primarily responsible for deglycosylating high-mannose glycoproteins and the glycans removed by EfEndo18A support growth under nutrient-limiting conditions, in vitro . In contrast, IgG is representative of a complex type glycoprotein, and we demonstrate that the GH18 domain of EndoE is primarily responsible for removal of this glycan decoration. Lastly, our data highlight the combined contribution of glycosidases to virulence of E. faecalis , in vivo. Importance Enterococcus faecalis has emerged as a multidrug-resistant (MDR) nosocomial pathogen that causes life-threatening healthcare-associated infections. Nutrient acquisition systems and immune evasions strategies are key factors that contribute to enterococcal colonization and influence the overall pathogenic potential of this organism. The regulation of these factors is governed by metabolic cues, specifically the availability of glucose as a preferred carbon source. Our research identifies CcpA as a major regulator of secondary nutrient acquisition and expands on the importance of GH18 family glycosyl hydrolases in E. faecalis . These hydrolases contribute to the direct targeting of host glycoproteins both for nutrient acquisition, as well as potentially evading both the innate and adaptive immune response. Disrupting the function of these microbial enzymes may lead to new treatments against multidrug resistant enterococcal infections.

Human gut microbes express functionally distinct endoglycosidases to metabolize the same N-glycan substrate

“…Thus, the catalytic activity of BT1285 and BT3987 processing HM glycoprotein substrates from the host and/or food sources could play a role in controlling some inflammatory processes in different conditions in the human gut environment, through the generation of SCFAs and/or direct through removing HM glycans on relevant human glycoproteins such as immunoglobulins, Fc γ Receptors and complement components, etc. Moreover, it has been reported that Ef Endo18A, one of the two GH18s encoded by Enterococcus faecalis 67 , 68 , is up-regulated in blood and urine 69 , 70 , where E. faecalis frequently causes infection. It was proposed that this ENGase can inactivate host glycoproteins to provide an advantage to the bacteria during infection.…”