Nutritional Control of Antibiotic Resistance via an Interface between the Phosphotransferase System and a Two-Component Signaling System

Abstract

Enterococci are ubiquitous inhabitants of the gastrointestinal (GI) tract. However, antibiotic-resistant enterococci are also major causes of hospital-acquired infections. Enterococci are intrinsically resistant to cephalosporins, enabling growth to abnormally high densities in the GI tract in patients during cephalosporin therapy, thereby promoting dissemination to other sites where they cause infection. Despite its importance, many questions about the underlying basis for cephalosporin resistance remain. A specific two-component signaling system, composed of the CroS sensor kinase and its cognate response regulator (CroR), is required for cephalosporin resistance in Enterococcus faecalis, but little is known about the factors that control this signaling system to modulate resistance. To explore the signaling network in which CroR participates to influence cephalosporin resistance, we employed a protein fragment complementation assay to detect protein-protein interactions in E. faecalis cells, revealing a previously unknown association of CroR with the HPr protein of the phosphotransferase system (PTS) responsible for carbohydrate uptake and catabolite control of gene expression. Genetic and physiological analyses indicate that association with HPr restricts the ability of CroR to promote cephalosporin resistance and gene expression in a nutrient-dependent manner. Mutational analysis suggests that the interface used by HPr to associate with CroR is distinct from the interface used to associate with other cellular partners. Our results define a physical and functional connection between a critical nutrient-responsive signaling system (the PTS) and a two-component signaling system that drives antibiotic resistance in E. faecalis, and they suggest a general strategy by which bacteria can integrate their nutritional status with diverse environmental stimuli. Enterococci are ubiquitous Gram-positive bacteria usually found as commensals of the gastrointestinal (GI) tract in humans, animals, and insects. In the competitive environment of the GI tract, the ability to utilize a diverse repertoire of nutrients for growth may represent an important selective advantage. Enterococci can utilize a wide variety of carbohydrates for growth (1-3), a trait that is reflected in the abundance of carbohydrate uptake and utilization pathways identified in the Enterococcus faecalis genome (4). The enterococcal genome is especially replete with genes encoding components of the phosphoenol pyruvate (PEP)-dependent phosphotransferase system (PTS) that is widely used by bacteria both to transport carbohydrates into the cell and to control gene expression in response to nutrient availability (see references 5 and 6 for thorough reviews). PTSs mediate transport and phosphorylation of substrate carbohydrates through the coupled action of carbohydrate-specific transporters (called "EIIs") and the general PTS components EI and HPr. EI and HPr participate in sequential phosphotransfer reactions in which phosphoryl groups derived from PEP are s...

The Bacterial Phosphoenolpyruvate:Carbohydrate Phosphotransferase System: Regulation by Protein Phosphorylation and Phosphorylation-Dependent Protein-Protein Interactions

“…This result suggests that phosphorylation of HPr by either PEP and EI or ATP and HprK/P prevents the interaction with CroR. In agreement with these results, cephalosporin resistance of E. faecalis was also influenced by the utilization of carbon sources (155). While growth on the non-PTS carbon sources pyruvate and citrate, which leads to high levels of PϳHis-HPr, caused enhanced cephalosporin resistance, growth on the PTS substrates glucose and N-acetylglucosamine, which leads to low levels of PϳHis-HPr, had no such effect.…”

“…A highly interesting interaction of HPr from enterococci with the response regulator of the CroRS two-component system was recently detected by carrying out a protein fragment complementation assay (155). CroR is required for intrinsic cephalosporin resistance in E. faecalis.…”

Requirement of the CroRS Two-Component System for Resistance to Cell Wall-Targeting Antimicrobials in Enterococcus faecium

“…Ectopic expression of croRS Efm was accomplished using the cCF10-inducible promoter in the enterococcal expression vector pBK2 (34). E. faecium pbp5 alleles were expressed from pJH123, a modified version of the enterococcal expression vector pJRG9 containing the constitutive P23s promoter (35). pJH123 was generated to have a versatile multiple cloning site for which N-terminal or C-terminal hemagglutinin (HA) epitope fusions could be generated if desired.…”

Glucose-Specific Enzyme IIA of the Phosphoenolpyruvate:Carbohydrate Phosphotransferase System Modulates Chitin Signaling Pathways in Vibrio cholerae

“…Several auxiliary factors are known that modulate the activities of HKs and RRs through protein-protein interactions. Although very little is known about connections between the PTS and TCSs, a functional interaction of Hpr from enterococci with the RR of the CroRS TCS has been recently reported (80). In addition, the EIIA Ntr protein, which is a component of a nitrogen-related PTS, paralogous to the canonical PTS, has been identified as another auxiliary protein that can modulate the activities of the HKs KdpD and PhoR in E. coli (81,82).…”