Abstract
VanA-type resistance to glycopeptide antibiotics in clinical enterococci is regulated by the VanSARA two-component signal transduction system. The nature of the molecular ligand that is recognised by the VanSA sensory component has not hitherto been identified. Here we employ purified, intact and active VanSA membrane protein (henceforth referred to as VanS) in analytical ultracentrifugation experiments to study VanS oligomeric state and conformation in the absence and presence of vancomycin. A combination of sedimentation velocity and sedimentation equilibrium in the analytical ultracentrifuge (SEDFIT, SEDFIT-MSTAR and MULTISIG analysis) showed that VanS in the absence of the ligand is almost entirely monomeric (molar mass M = 45.7 kDa) in dilute aqueous solution with a trace amount of high molar mass material (M ~ 200 kDa). The sedimentation coefficient s suggests the monomer adopts an extended conformation in aqueous solution with an equivalent aspect ratio of ~(12 ± 2). In the presence of vancomycin over a 33% increase in the sedimentation coefficient is observed with the appearance of additional higher s components, demonstrating an interaction, an observation consistent with our circular dichroism measurements. The two possible causes of this increase in s – either a ligand induced dimerization and/or compaction of the monomer are considered.
Highlights
Vancomycin is a tricyclic glycopeptide used to treat penicillin-resistant infections caused by Gram-positive bacteria including bloodstream infections and infective endocarditis of enterococcal origin and meningitis caused by methicillin-resistant Staphylococcus aureus[1,2]
High levels of vancomycin and teicoplanin in enterococci occurs as a result of acquisition of genes that encode enzymes that (1) synthesise an alternative target peptidoglycan precursor D-Ala-D-Lac which lacks one of the hydrogen bonding sites involved in glycopeptide binding, thereby constituting a low-affinity precursor that exhibits an approximately 1000-fold reduced affinity for the antibiotic; and (2) eliminate pre-existing high-affinity D-Ala-D-Ala precursors thereby removing the glycopeptide-binding target[20,21]
Studies of distantly-related VanS sensors in streptomycetes and enterococci (VanB-type) have provided evidence that the antibiotic itself or the antibiotic bound to the D-Ala-D-Ala substrate serve as the inducing effectors whilst in VanA-type resistances it has been speculated that the inducer might be a peptidoglycan precursor[26,27,28]
Summary
Vancomycin is a tricyclic glycopeptide used to treat penicillin-resistant infections caused by Gram-positive bacteria including bloodstream infections and infective endocarditis of enterococcal origin and meningitis caused by methicillin-resistant Staphylococcus aureus[1,2]. High levels of vancomycin and teicoplanin (the VanA phenotype) in enterococci occurs as a result of acquisition of genes that encode enzymes that (1) synthesise an alternative target peptidoglycan precursor D-Ala-D-Lac which lacks one of the hydrogen bonding sites involved in glycopeptide binding, thereby constituting a low-affinity precursor that exhibits an approximately 1000-fold reduced affinity for the antibiotic; and (2) eliminate pre-existing high-affinity D-Ala-D-Ala precursors thereby removing the glycopeptide-binding target[20,21]. The genes encoding these enzymes are regulated by the VanSR two-component signal transduction system[22,23]. The solution conformation of the VanS was analysed using the ELLIPS suite of conformation algorithms[34] and the effect of the addition of vancomycin was explored
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