Chemical or Genetic Alteration of Proton Motive Force Results in Loss of Virulence of Burkholderia glumae, the Cause of Rice Bacterial Panicle Blight

Rice is an important source of food for more than half of the world’s population. Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae. B. glumae synthesizes toxoflavin, an essential virulence factor that is required for symptoms of the disease. The products of the tox operons, ToxABCDE and ToxFGHI, are responsible for the synthesis and the proton motive force (PMF)-dependent secretion of toxoflavin, respectively. The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Our previous work has demonstrated that absence of certain DedA family members results in pleiotropic effects, impacting multiple pathways that are energized by PMF. We have demonstrated that a member of the DedA family from Burkholderia thailandensis, named DbcA, is required for the extreme polymyxin resistance observed in this organism. B. glumae encodes a homolog of DbcA with 73% amino acid identity to Burkholderia thailandensis DbcA. Here, we created and characterized a B. glumae DdbcA strain. In addition to polymyxin sensitivity, the B. glumae DdbcA strain is compromised for virulence in several BPB infection models and secretes only low amounts of toxoflavin (;15% of wild-type levels). Changes in membrane potential in the B. glumae DdbcA strain were reproduced in the wild-type strain by the addition of subinhibitory concentrations of sodium bicarbonate, previously demonstrated to cause disruption of PMF. Sodium bicarbonate inhibited B. glumae virulence in rice, suggesting a possible non-toxic chemical intervention for bacterial panicle blight. IMPORTANCE Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae. The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Here, we constructed a B. glumae mutant with a deletion in a DedA family member named dbcA and report a loss of virulence in models of BPB. Physiological analysis of the mutant shows that the proton motive force is disrupted, leading to reduction of secretion of the essential virulence factor toxoflavin. The mutant phenotypes are reproduced in the virulent wild-type strain without an effect on growth using sodium bicarbonate, a nontoxic buffer that has been reported to disrupt the PMF. The results presented here suggest that bicarbonate may be an effective antivirulence agent capable of controlling BPB without imposing an undue burden on the environment

A DedA Family Membrane Protein Is Required for Burkholderia thailandensis Colistin Resistance

© Copyright © 2019 Panta, Kumar, Stafford, Billiot, Douglass, Herrera, Trent and Doerrler. 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 \u3e100-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

A Klebsiella pneumoniae DedA family membrane protein is required for colistin resistance and for virulence in wax moth larvae

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