Colistin is a “last resort” antibiotic for treatment of infections caused by some multidrug resistant Gram-negative bacterial pathogens. Resistance to colistin varies between bacterial species. Some Gram-negative bacteria such as Burkholderia spp. are intrinsically resistant to very high levels of colistin with minimal inhibitory concentrations (MIC) often above 0.5 mg/ml. We have previously shown DedA family proteins YqjA and YghB are conserved membrane transporters required for alkaline tolerance and resistance to several classes of dyes and antibiotics in Escherichia coli. Here, we show that a DedA family protein in Burkholderia thailandensis (DbcA; DedA of Burkholderia required for colistin resistance) is a membrane transporter required for resistance to colistin. Mutation of dbcA results in >100-fold greater sensitivity to colistin. Colistin resistance is often conferred via covalent modification of lipopolysaccharide (LPS) lipid A. Mass spectrometry of lipid A of ΔdbcA showed a sharp reduction of aminoarabinose in lipid A compared to wild type. Complementation of colistin sensitivity of B. thailandensis ΔdbcA was observed by expression of dbcA, E. coli yghB or E. coli yqjA. Many proton-dependent transporters possess charged amino acids in transmembrane domains that take part in the transport mechanism and are essential for function. Site directed mutagenesis of conserved and predicted membrane embedded charged amino acids suggest that DbcA functions as a proton-dependent transporter. Direct measurement of membrane potential shows that B. thailandensis ΔdbcA is partially depolarized suggesting that loss of protonmotive force can lead to alterations in LPS structure and severe colistin sensitivity in this species.

Enterobacteria, including Escherichia coli, bloom to high levels in the gut during inflammation and strongly contribute to the pathology of inflammatory bowel diseases. To survive in the inflamed gut, E. coli must tolerate high levels of antimicrobial compounds produced by the immune system, including toxic metals like copper and reactive chlorine oxidants such as hypochlorous acid (HOCl). Here, we show that extracellular copper is a potent detoxifier of HOCl and that the widely conserved bacterial HOCl resistance enzyme RclA, which catalyzes the reduction of copper(II) to copper(I), specifically protects E. coli against damage caused by the combination of HOCl and intracellular copper. E. coli lacking RclA was highly sensitive to HOCl when grown in the presence of copper and was defective in colonizing an animal host. Our results indicate that there is unexpected complexity in the interactions between antimicrobial toxins produced by innate immune cells and that bacterial copper status is a key determinant of HOCl resistance and suggest an important and previously unsuspected role for copper redox reactions during inflammation. IMPORTANCE During infection and inflammation, the innate immune system uses antimicrobial compounds to control bacterial populations. These include toxic metals, like copper, and reactive oxidants, including hypochlorous acid (HOCl). We have now found that RclA, a copper(II) reductase strongly induced by HOCl in proinflammatory Escherichia coli and found in many bacteria inhabiting epithelial surfaces, is required for bacteria to resist killing by the combination of intracellular copper and HOCl and plays an important role in colonization of an animal host. This finding indicates that copper redox chemistry plays a critical and previously underappreciated role in bacterial interactions with the innate immune system.

Ineffectiveness of carbapenems against multidrug resistant pathogens led to the increased use of colistin (polymyxin E) as a last resort antibiotic. A gene belonging to the DedA family encoding conserved membrane proteins was previously identified by screening a transposon library of K. pneumoniae ST258 for sensitivity to colistin. We have renamed this gene dkcA (dedA of Klebsiella required for colistin resistance). DedA family proteins are likely membrane transporters required for viability of Escherichia coli and Burkholderia spp. at alkaline pH and for resistance to colistin in a number of bacterial species. Colistin resistance is often conferred via modification of the lipid A component of bacterial lipopolysaccharide with aminoarabinose (Ara4N) and/or phosphoethanolamine. Mass spectrometry analysis of lipid A of the ∆dkcA mutant shows a near absence of Ara4N in the lipid A, suggesting a requirement for DkcA for lipid A modification with Ara4N. Mutation of K. pneumoniae dkcA resulted in a reduction of the colistin minimal inhibitory concentration to approximately what is found with a ΔarnT strain. We also identify a requirement of DkcA for colistin resistance that is independent of lipid A modification, instead requiring maintenance of optimal membrane potential. K. pneumoniae ΔdkcA displays reduced virulence in Galleria mellonella suggesting colistin sensitivity can cause loss of virulence.

Neutrophils generate hypochlorous acid (HOCl) and related reactive chlorine species as part of their defence against invading microorganisms. In isolation, bacteria respond to reactive chlorine species by upregulating responses that provide defence against oxidative challenge. Key questions are whether these responses are induced when bacteria are phagocytosed by neutrophils, and whether this provides them with a survival advantage. We investigated RclR, a transcriptional activator of the rclABC operon in Escherichia coli that has been shown to be specifically activated by reactive chlorine species. We first measured induction by individual reactive chlorine species, and showed that HOCl itself activates the response, as do chloramines (products of HOCl reacting with amines) provided they are cell permeable. Strong RclR activation was seen in E. coli following phagocytosis by neutrophils, beginning within 5 min and persisting for 40 min. RclR activation was suppressed by inhibitors of NOX2 and myeloperoxidase, providing strong evidence that it was due to HOCl production in the phagosome. RclR activation demonstrates that HOCl, or a derived chloramine, enters phagocytosed bacteria in sufficient amount to induce this response. Although RclR was induced in wild-type bacteria following phagocytosis, we detected no greater sensitivity to neutrophil killing of mutants lacking genes in the rclABC operon.

Despite advances in treatment options for people with CF, complications of bacterial infections remain the greatest driver of morbidity and mortality in this patient population. These infections often involve more than one bacterial pathogen, and our understanding of how interspecies interactions impact disease progression is lacking.