The Extracellular Domain of Two-component System Sensor Kinase VanS from Streptomyces coelicolor Binds Vancomycin at a Newly Identified Binding Site

Abstract

The glycopeptide antibiotic vancomycin has been widely used to treat infections of Gram-positive bacteria including Clostridium difficile and methicillin-resistant Staphylococcus aureus. However, since its introduction, high level vancomycin resistance has emerged. The genes responsible require the action of the two-component regulatory system VanSR to induce expression of resistance genes. The mechanism of detection of vancomycin by this two-component system has yet to be elucidated. Diverging evidence in the literature supports activation models in which the VanS protein binds either vancomycin, or Lipid II, to induce resistance. Here we investigated the interaction between vancomycin and VanS from Streptomyces coelicolor (VanS Sc), a model Actinomycete. We demonstrate a direct interaction between vancomycin and purified VanS Sc , and traced these interactions to the extracellular region of the protein, which we reveal adopts a predominantly α-helical conformation. the VanS Scbinding epitope within vancomycin was mapped to the N-terminus of the peptide chain, distinct from the binding site for Lipid II. In targeting a separate site on vancomycin, the effective VanS ligand concentration includes both free and lipid-bound molecules, facilitating VanS activation. This is the first molecular description of the VanS binding site within vancomycin, and could direct engineering of future therapeutics. Vancomycin is a glycopeptide antibiotic that exerts its antimicrobial effect on a variety of Gram-positive bacterial species, and has been seen as a last line of defence in the treatment of complicated clinical infections including Clostridium difficile and methicillin-resistant Staphylococcus aureus (MRSA). Despite the significant toxicity of vancomycin in patients, there is still strong clinical reliance on this drug as a last line of defence against such infections 1. In some cases, MRSA infections are incalcitrant and vancomycin-resistant Enterococcal (VRE) strains are resistant to a wide panel of antibiotics. As a result, VRE and MRSA strains are now on worldwide health authorities' high priority lists, and new treatments are desperately needed 1-3. Three recently approved semi-synthetic glycopeptide derivatives, oritavancin, dalbavancin and telavancin, are being treated as reserve antibiotics to be used sparingly to preserve their efficacy 4. The mode of action of vancomycin involves binding to the d-alanyl-d-alanine-terminating pentapeptide of the cell wall precursor Lipid II, preventing its utilisation in biosynthesis of bacterial peptidoglycan cell wall 5. Vancomycin is one of a number of antibiotics that target Lipid II rather than the enzymes that use Lipid II as a substrate 6. Inducible high-level resistance to vancomycin is commonly found in soil bacteria including the Actinomycetes 7 where it has been genome-encoded for millennia 8. Recently resistance genes have spread intergenerically into S. aureus, resulting in the first strains of vancomycin-and methicillin-resistant S. aureus (VRSA) causing widespread...

Antibiotic Production and Antibiotic Resistance: The Two Sides of AbrB1/B2, a Two-Component System of Streptomyces coelicolor

“…Our working hypothesis for future work is that the role of AbrB1/B2 could be to detect cell wall damage (similar to VraR/S and LiaR/S) and regulate systems that contribute to the maintain of cell wall integrity such as the van cluster whose TCS (VanR/S) directly detects vancomycin molecules ( Koteva et al, 2010 ; Novotna et al, 2015 ; Lockey et al, 2020 ) and promotes cell wall remodeling. Thus, a preliminary experiment testing the susceptibility to penicillin G agrees with the working hypothesis, being the Δ abrB mutant more sensitive to this ß-lactam ( Supplementary Figure 6 ).…”

Membrane Sensor Histidine Kinases: Insights from Structural, Ligand and Inhibitor Studies of Full-Length Proteins and Signalling Domains for Antibiotic Discovery

“…Use of pTTQ18His as expression plasmid has resulted in a high degree of success for producing intact SHKs compared with members of other membrane protein families, including successful expression of 15 of the 16 genome complement of membrane SHKs of Enterococcus faecalis [ 34 ], VanS (A-type) of E. faecium [ 75 , 89 ], BlpH, and ComD2 pheromone-sensing SHKs of Streptococcus pneumoniae (together with several other membrane proteins associated with antimicrobial resistance) [ 90 ] and the PrrB (or RegB) redox-sensing SHK protein of the Gram-negative bacterium Rhodobacter sphaeroides [ 87 , 91 ]. Other membrane overexpression plasmids include pT7-7 [ 92 ] based on T7 polymerase [ 93 ], pProEX available commercially [ 94 ] and pETCH [ 95 ] derived from SUMO fusion vectors [ 96 ]. Many studies have opted to express SHK genes as synthetic codon-optimised genes, though high success rates were obtained with plasmid pTTQ18His using the native gene sequences from both Gram-positive and Gram-negative species.…”

“…Studies of other more distantly-related VanS sensors such as VanS B and VanS SC also revealed direct interactions with vancomycin [ 94 , 138 ], or a signalling complex involving vancomycin [ 139 ]. As mentioned earlier, Koteva et al (2010) reported a k d value of 56 µM for the in vivo binding of a vancomycin photoaffinity probe with actinomycete VanS SC [ 138 ], a value comparable to the weak binding strengths observed for VanS A [ 134 ], whilst Lockey et al (2020) demonstrated direct interactions between full-length VanS SC and vancomycin in vitro, and identified regions of the extracellular sensory domain as the interacting site [ 94 ].…”

“…Studies of other more distantly-related VanS sensors such as VanS B and VanS SC also revealed direct interactions with vancomycin [ 94 , 138 ], or a signalling complex involving vancomycin [ 139 ]. As mentioned earlier, Koteva et al (2010) reported a k d value of 56 µM for the in vivo binding of a vancomycin photoaffinity probe with actinomycete VanS SC [ 138 ], a value comparable to the weak binding strengths observed for VanS A [ 134 ], whilst Lockey et al (2020) demonstrated direct interactions between full-length VanS SC and vancomycin in vitro, and identified regions of the extracellular sensory domain as the interacting site [ 94 ]. Inspection of the primary sequence of the sensory domain of VanS SC reveals no significant similarity with VanS A [ 138 , 140 ]; the alignment shown below ( Figure 6 ) is a CLUSTAL-O alignment of the predicted extracellular sensory domains of VanS A and VanS SC (as described in [ 94 ]), which share only 8.8% identity.…”

Covid-19: Aspectos da origem, fisiopatologia, imunologia e tratamento - uma revisão narrativa

“…Segundo a OMS, existiam 70 vacinas em desenvolvimento. E, qualquer candidato a vacina teria que direcionar os recursos presentes nos dois tipos de vírus: S e L identificados por pesquisadores de Pequim em pacientes infectados, para ser eficaz (LOCKEY C, et al, 2020). Esforços e ensaios clínicos de fase 1 para desenvolvimento de novas vacinas, estavam em andamento no Reino Unido, Estados Unidos, Alemanha, França e China.…”