Understanding colistin resistance in Gram-negative bacteria

The antibiotic colistin is considered a "last resort" treatment for bacterial infections caused by multi-drug resistant Gram-negative bacteria. The efficacy of this antibiotic is now threatened by the emergence of the plasmid-borne mobilised colistin resistance (mcr) genes. MCR-1 is a zinc-dependent membrane-bound phosphoethanolamine transferase, that modifies the Lipid A in the bacterial outer membrane; conferring resistance against colistin. This project aims to better understand this recently discovered resistance mechanism, MCR-1, and its homologues, as well as develop a platform that could serve as a basis for the discovery and development of possible inhibitors. Structure and sequence-based analysis were performed to gather a better understanding of the position of MCR-1, and other phosphoethanolamine transferases, within the alkaline phosphatase protein superfamily to which these enzymes belong. MCR-1 was recombinantly produced and purified in detergent micelles and its integrity was analysed by Size-exclusion chromatography coupled to a multi-angle light scattering system (SEC-MALS), circular dichroism (CD) as well and mass spectrometry analysis. An activity assay was developed by employing using "catalytic domain only" constructs of MCR-1 its homologs MCR-2 in conjunction with the synthetic chromophoric substrate 4-nitrophenol-phosphoethanolamine (pNP-PEtN). Another phosphoethanolamine transferase, PmrC from Acinetobacter baumannii, was recombinantly produced and used, its activity was assessed, and crystallization attempts were made. Analysis of the sequence and structure revealed that MCR-1 has a partial second metal site resembling those of the multimeric members of the alkaline phosphatase superfamily. SEC-MALS, CD and melting temperature analysis showed that recombinantly produced MCR-1 is stable in detergent micelles. The developed activity assay efficiently monitors the cleavage of the phosphoethanolamine (PEtN) motif from the chromophoric pNP-PEtN and allowed the estimation of kinetic parameters. Finally, a "catalytic domain only" construct of PmrC was recombinantly produced, using the activity assay kinetic parameters were estimated and starting conditions for protein crystallization were found. The work presented here makes a significant contribution towards understanding phosphoethanolamine transferase-mediated colistin resistance in Gram-negative bacteria by producing and characterising relevant enzymes (MCR-1 and PmrC) and developing an activity assay that can serve as a platform for the identification and screening of inhibitors. Structural and sequence-based analysis highlighted key conserved residues involved in the catalytic action of this family of enzymes. Exploiting the findings from this project in the design of inhibitors for phosphoethanolamine transferases will help to preserve the efficacy of colistin against multi-drug resistant gram-negative bacterial infections.