Direct interaction between phosphotransferase LpxT and ArnT modulates polymyxin B resistance in Pseudomonas aeruginosa

초록

Pseudomonas aeruginosa is a common human opportunistic pathogen that causes a variety of acute and chronic infections. Polymyxins are considered last-resort options for treating serious infections caused by multidrug-resistant gram-negative bacteria. Previously, we evolved PAO1 by serial passage in the presence of sublethal concentrations of polymyxin B, resulting in the polymyxin B-resistant strain PAO1-D14. In this study, we investigate the molecular mechanisms of resistance to polymyxin B in PAO1-D14. We found that the pmrBL189Q missense mutation results in a fourfold increase in the minimum inhibitory concentration (MIC), while deletion of lpxT, the phosphotransferase-encoding gene, confers an additional eightfold increase in the MIC of the pmrBL189Q strain but not in the wild-type PAO1 strain. Using 1-N-phenylnaphthylamine and Dansyl-polymyxin B binding assays, we observed that the lpxT mutation decreased the amount of polymyxin B bound to the bacterial surface and reduced membrane permeability in the pmrBL189Q mutant. A co-purification assay revealed an interaction between L-Ara4N transferase ArnT and LpxT. The amino acid-substituted version of LpxTG52AK137A lost the interaction with ArnT and no longer restored the polymyxin B resistance of pmrBL189Q Delta lpxT. Additionally, deletion of arnT in pmrBL189Q Delta lpxT restored susceptibility to polymyxin B to the levels seen in the pmrBL189Q strain. Collectively, our findings reveal a novel role for lpxT and its molecular mechanism to defend against polymyxin B in P. aeruginosa.IMPORTANCETreatment of Pseudomonas aeruginosa infections is challenging due to its antibiotic resistance. Polymyxins are considered last-resort options for treating serious infections caused by multidrug-resistant P. aeruginosa. Understanding the molecular mechanisms of polymyxin resistance may provide clues for the development of new therapeutic strategies against P. aeruginosa. In this study, we demonstrated that the phosphatidic acid-phosphatase family (PAP2) protein LpxT interacts with L-Ara4N transferase ArnT and controls polymyxin B resistance in P. aeruginosa. Our findings reveal a novel role for lpxT and its molecular mechanism to defend against polymyxin B in P. aeruginosa.

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