Interaction of the GraRS Two-Component System with the VraFG ABC Transporter To Support Vancomycin-Intermediate Resistance in Staphylococcus aureus

Abstract

Current treatment for serious infections caused by methicillin-resistant Staphylococcus aureus relies heavily upon the glycopeptide antibiotic vancomycin. Unfortunately, this practice has led to an intermediate resistance phenotype that is particularly difficult to treat in invasive staphylococcal diseases, such as septicemia and its metastatic complications, including endocarditis. Although the vancomycin-intermediate resistance phenotype has been linked to abnormal cell wall structures and autolytic rates, the corresponding genetic changes have not been fully elucidated. Previously, whole-genome array studies listed numerous genes that are overexpressed in vancomycin-intermediate sensitive strains, including graRS (SACOL0716 to -0717), encoding a two-component regulatory system (TCRS), as well as the adjacent vraFG (SACOL0718 to -0720), encoding an ATP-binding cassette (ABC) transporter; but the exact contribution of these genes to increased vancomycin resistance has not been defined. In this study, we showed that isogenic strains with mutations in genes encoding the GraRS TCRS and the VraFG ABC transporter are hypersensitive to vancomycin as well as polymyxin B. Moreover, GraRS regulates the expression of the adjacent VraFG pump, reminiscent of gram-positive bacteriocin-immunity regulons. Mutations of graRS and vraFG also led to increased autolytic rates and a more negative net surface charge, which may explain, in part, to their increased sensitivity to cationic antimicrobial peptides. Taken together, these data reveal an important genetic mediator to the vancomycin-intermediate S. aureus phenotype and may hold clues to the selective pressures on staphylococci upon exposure to selective cationic peptide antibiotics used in clinical practice.

Reduced Vancomycin Susceptibility inStaphylococcus aureus, Including Vancomycin-Intermediate and Heterogeneous Vancomycin-Intermediate Strains: Resistance Mechanisms, Laboratory Detection, and Clinical Implications

“…The locus has also been called aps because of its more general antimicrobial-peptide-sensing capacity (180). The overexpression of GraR or GraS results in a slight increase in the MIC of vancomycin (57), and a knockout mutation results in hypersensitivity (122,204). However, in two different instances, point mutations have resulted in increased resistance to vancomycin, presumably by modifying the activity of the proteins (60,123).…”

“…Mutations, inactivation, and altered expression of graRS have been linked to changes in vancomycin susceptibility in S. aureus (57,60,111,123,204,225). GraRS (Aps) is known to control the dltABCD operon, which controls the alanylation of wall teichoic acids in response to antimicrobial challenge, indicating that the structure of teichoic acids can change in response to challenges (111,180,181).…”

Compound-gene interaction mapping reveals distinct roles forStaphylococcus aureusteichoic acids

“…Although our screen and follow up validation used tunicamycin concentrations that are two orders of magnitude below those that inhibit MraY, we wanted to confirm key results using a fully genetic approach that did not rely on a small molecule (10). This step seemed necessary because some of the knockouts, including ΔdltA, ΔgraR, and ΔvraFG, are known to have increased susceptibility to cationic antimicrobial peptides (CAMPs) and certain other antibiotics with cell envelope targets (7,8,25). Increased susceptibility is proposed to occur because the cell surface lacks the D-alanine modifications that normally repel positively charged toxins at the envelope interface (26).…”

Characterization of Two Component Systems of Lactobacillus casei BL23 and their involvement in stress response

“…cytochrome c (Cyt c), calculated pI 10.0 Meehl et al, 2007;Matsuo et al, 2011)). Subsequently the bacteria are removed by centrifugation and the amount of these substances remaining in the supernatants is determined fluorometrically (GFP) or photometrically (Cyt c), for example.…”

“…However, in the Bce-like systems it has been shown that the BceS-like HKs are not the sensors of the stimulus by itself, but instead depend on the ATP driven transport of the substrate by the cognate ABC transporter for signal transduction (Bernard et al, 2007;Meehl et al, 2007;.…”