Functional characterization of BcrR:a one-component transmembrane signal transduction system for bacitracin resistance

Bacitracin is a cell wall targeting antimicrobial with clinical and agricultural applications. With the growing mismatch between antimicrobial resistance and development, it is essential we understand the molecular mechanisms of resistance in order to prioritize and generate new effective antimicrobials. BcrR is a unique membrane-bound one-component system that regulates high-level bacitracin resistance in Enterococcus faecalis. In the presence of bacitracin, BcrR activates transcription of the bcrABD operon conferring resistance through a putative ATP-binding cassette (ABC) transporter (BcrAB). BcrR has three putative functional domains, an N-terminal helix-turn-helix DNA-binding domain, an intermediate oligomerization domain and a C-terminal transmembrane domain. However, the molecular mechanisms of signal transduction remain unknown. Random mutagenesis of bcrR was performed to generate loss- and gain-of-function mutants using transcriptional reporters fused to the target promoter PbcrA. Fifteen unique mutants were isolated across all three proposed functional domains, comprising 14 loss-of-function and one gain-of-function mutant. The gain-of-function variant (G64D) mapped to the putative dimerization domain of BcrR, and functional analyses indicated that the G64D mutant constitutively expresses the PbcrA-luxABCDE reporter. DNA-binding and membrane insertion were not affected in the five mutants chosen for further characterization. Homology modelling revealed putative roles for two key residues (R11 and S33) in BcrR activation. Here we present a new model of BcrR activation and signal transduction, providing valuable insight into the functional characterization of membrane-bound one-component systems and how they can coordinate critical bacterial responses, such as antimicrobial resistance.</p

Functional characterization of BcrR:a one-component transmembrane signal transduction system for bacitracin resistance

Bacitracin is a cell wall targeting antimicrobial with clinical and agricultural applications. With the growing mismatch between antimicrobial resistance and development, it is essential we understand the molecular mechanisms of resistance in order to prioritize and generate new effective antimicrobials. BcrR is a unique membrane-bound one-component system that regulates high-level bacitracin resistance in Enterococcus faecalis. In the presence of bacitracin, BcrR activates transcription of the bcrABD operon conferring resistance through a putative ATP-binding cassette (ABC) transporter (BcrAB). BcrR has three putative functional domains, an N-terminal helix-turn-helix DNA-binding domain, an intermediate oligomerization domain and a C-terminal transmembrane domain. However, the molecular mechanisms of signal transduction remain unknown. Random mutagenesis of bcrR was performed to generate loss- and gain-of-function mutants using transcriptional reporters fused to the target promoter PbcrA. Fifteen unique mutants were isolated across all three proposed functional domains, comprising 14 loss-of-function and one gain-of-function mutant. The gain-of-function variant (G64D) mapped to the putative dimerization domain of BcrR, and functional analyses indicated that the G64D mutant constitutively expresses the PbcrA-luxABCDE reporter. DNA-binding and membrane insertion were not affected in the five mutants chosen for further characterization. Homology modelling revealed putative roles for two key residues (R11 and S33) in BcrR activation. Here we present a new model of BcrR activation and signal transduction, providing valuable insight into the functional characterization of membrane-bound one-component systems and how they can coordinate critical bacterial responses, such as antimicrobial resistance.</p

Investigating resistance in Enterococcus faecalis against the new antibiotic teixobactin

The rapid rise and spread of multi-drug resistant (MDR) pathogens has become a daunting reality for many countries world-wide driving the need for new antimicrobials with novel targets. Enterococci, a natural inhabitant of the gut microbiota, are now recognised as the second most common cause of hospital-acquired infection due to their physiological hardiness and ability to acquire antimicrobial resistance determinants from their surrounding environment. The discovery of antimicrobials that are bactericidal against enterococci is paramount in the treatment of endocarditis and other severe infections caused by this group of microorganisms. Teixobactin is a promising new class of antibiotic isolated from a soil bacterium using iChip technology. Teixobactin is active against a range of MDR Gram-positive pathogens including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE), targeting the sugar-phosphate moieties of cell wall precursors Lipid II and Lipid III. No resistance to teixobactin has been reported thus far. A major concern in the development of new antibiotics is the possibility of pre-existing resistance determinants conferring cross-resistance to these new antibiotics. Bacitracin, like teixobactin is a peptide antibiotic which targets the undecaprenyl pyrophosphate (UPP) carrier associated with cell wall precursors such as Lipid II and Lipid III. The high-level bacitracin resistance cassette BcrRABD is well established in New Zealand enterococcal isolates due to the extensive use of bacitracin as a growth promoter in broiler chickens. We investigated the possibility of a cross-resistance mechanism between the high-level bacitracin-resistance cassette BcrRABD and teixobactin. Antibiotic susceptibility assays and time-dependent kill kinetics showed that bacitracin-resistant and sensitive strains of Enterococcus faecalis share identical teixobactin sensitivities, indicating a lack of cross-resistance between BcrRABD and teixobactin. Predicting how bacteria will generate resistance to teixobactin is essential for further clinical development. To address this goal we set out to isolate teixobactin-resistant and hyper-susceptible mutants using transposon and spontaneous mutagenesis techniques. We were successful in isolating transposon mutants displaying changes in teixobactin susceptibility. However, when sequenced, the transposon insertions mapped to a variety of plasmid encoded genes, with 50% of the mutants containing an insertion in a DDE transposase and/or an adjacent recombinase. It remains unclear how these insertions play a role in teixobactin resistance and hyper-susceptibility. Through serial passaging, we successfully isolated three spontaneous mutants with increased resistance and tolerance to not only teixobactin, but other cell wall-acting antimicrobials (daptomycin, ampicillin and penicillin G). Whole genome sequencing revealed multiple mutations were required for teixobactin tolerance, with an initial mutation acquired in liaF, a key regulator of cell envelope biosynthesis and turnover. We hypothesise this mutation (insLiaF177) results in the constitutive up-regulation of a putative cell wall recycling pathway (as seen in two of our mutants using qPCR), in order to overcome teixobactin-mediated peptidoglycan biosynthesis inhibition. In addition, subsequent mutations were acquired in genes such as tagO, catalysing Lipid III synthesis, and mvaE, involved in the production of UPP, ultimately reducing the availability of the teixobactin target Lipid III. In two of these mutants (viz. MKB3 and MKB5), the growth rate of the cells in the absence of teixobactin was identical to the isogenic wild-type parent demonstrating that these mutations did not have an effect on cell fitness. We predict this broader cellular response encompassing mutations affecting multiple pathways, is what allows these mutants to be tolerant to not only teixobactin, but other cell wall acting antibiotics

Investigating resistance in Enterococcus faecalis against the new antibiotic teixobactin

The rapid rise and spread of multi-drug resistant (MDR) pathogens has become a daunting reality for many countries world-wide driving the need for new antimicrobials with novel targets. Enterococci, a natural inhabitant of the gut microbiota, are now recognised as the second most common cause of hospital-acquired infection due to their physiological hardiness and ability to acquire antimicrobial resistance determinants from their surrounding environment. The discovery of antimicrobials that are bactericidal against enterococci is paramount in the treatment of endocarditis and other severe infections caused by this group of microorganisms. Teixobactin is a promising new class of antibiotic isolated from a soil bacterium using iChip technology. Teixobactin is active against a range of MDR Gram-positive pathogens including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE), targeting the sugar-phosphate moieties of cell wall precursors Lipid II and Lipid III. No resistance to teixobactin has been reported thus far. A major concern in the development of new antibiotics is the possibility of pre-existing resistance determinants conferring cross-resistance to these new antibiotics. Bacitracin, like teixobactin is a peptide antibiotic which targets the undecaprenyl pyrophosphate (UPP) carrier associated with cell wall precursors such as Lipid II and Lipid III. The high-level bacitracin resistance cassette BcrRABD is well established in New Zealand enterococcal isolates due to the extensive use of bacitracin as a growth promoter in broiler chickens. We investigated the possibility of a cross-resistance mechanism between the high-level bacitracin-resistance cassette BcrRABD and teixobactin. Antibiotic susceptibility assays and time-dependent kill kinetics showed that bacitracin-resistant and sensitive strains of Enterococcus faecalis share identical teixobactin sensitivities, indicating a lack of cross-resistance between BcrRABD and teixobactin. Predicting how bacteria will generate resistance to teixobactin is essential for further clinical development. To address this goal we set out to isolate teixobactin-resistant and hyper-susceptible mutants using transposon and spontaneous mutagenesis techniques. We were successful in isolating transposon mutants displaying changes in teixobactin susceptibility. However, when sequenced, the transposon insertions mapped to a variety of plasmid encoded genes, with 50% of the mutants containing an insertion in a DDE transposase and/or an adjacent recombinase. It remains unclear how these insertions play a role in teixobactin resistance and hyper-susceptibility. Through serial passaging, we successfully isolated three spontaneous mutants with increased resistance and tolerance to not only teixobactin, but other cell wall-acting antimicrobials (daptomycin, ampicillin and penicillin G). Whole genome sequencing revealed multiple mutations were required for teixobactin tolerance, with an initial mutation acquired in liaF, a key regulator of cell envelope biosynthesis and turnover. We hypothesise this mutation (insLiaF177) results in the constitutive up-regulation of a putative cell wall recycling pathway (as seen in two of our mutants using qPCR), in order to overcome teixobactin-mediated peptidoglycan biosynthesis inhibition. In addition, subsequent mutations were acquired in genes such as tagO, catalysing Lipid III synthesis, and mvaE, involved in the production of UPP, ultimately reducing the availability of the teixobactin target Lipid III. In two of these mutants (viz. MKB3 and MKB5), the growth rate of the cells in the absence of teixobactin was identical to the isogenic wild-type parent demonstrating that these mutations did not have an effect on cell fitness. We predict this broader cellular response encompassing mutations affecting multiple pathways, is what allows these mutants to be tolerant to not only teixobactin, but other cell wall acting antibiotics