Abstract
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.
Highlights
Colistin is a last resort antibiotic for treatment of infections caused by Gramnegative pathogenic bacteria (Paterson and Harris, 2016)
These results collectively suggest that the transport activity of DedA and tight control of proton motive force (PMF) is required for lipid A modification with Ara4N and colistin resistance of B. thailandensis
By screening several sequence-defined B. thailandensis transposon mutants obtained from the Manoil lab (Gallagher et al, 2013) in genes encoding DedA family members we discovered that several with confirmed insertions in the gene Bth_I1321 were highly sensitive to colistin and polymyxin B
Summary
Colistin (polymyxin E) is a last resort antibiotic for treatment of infections caused by Gramnegative pathogenic bacteria (Paterson and Harris, 2016). Discovered in 1947 (Ainsworth et al, 1947), polymyxins were rarely used internally due to nephrotoxicity (Koch-Weser et al, 1970) Their use has increased recently due to ineffectiveness of approved antibiotics against multidrug-resistant bacteria, including carbapenemase-producing Enterobacteriaceae such as Klebsiella pneumoniae (Queenan and Bush, 2007). The genus Burkholderia is a group of highly adaptable, Gram-negative bacteria that includes a number of animal and plant pathogens (Lipuma, 2010; Waters, 2012). While B. thailandensis is considered a suitable surrogate for B. pseudomallei and only rarely causes infections in humans, it is infectious in a number of mammalian tissue culture, murine, insect and plant models and possesses virulence factors and drug resistance mechanisms that are found in its more virulent relatives (Gallagher et al, 2013). The existence of an ordered transposon library makes B. thailandensis a valuable model organism to study Burkholderia virulence (Gallagher et al, 2013)
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