Elucidating the mechanisms of transient polymyxin resistance in pathogenic E. coli.

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Defining Differences in how LPS modification is Regulated in Different E. coli pathotypes Project Summary Cells encounter a constant barrage of extracellular cues to which they respond using only the finite number of signal transduction pathways encoded within their genome. While we understand how individual signal transduction systems operate, little is known about how distinct signaling systems interact to integrate information and/or expand their signal responses. The overall goal of this project is to understand how signaling flexibility benefits the responses of different E. coli strains to cationic polypeptides. Although much attention is placed on the acquisition of antibiotic resistance markers by the Enterobacteriaceae, there is increasing evidence that bacteria can also mount transient resistance to antibiotics via the upregulation of chromosomally encoded markers. For example, the pmrC gene encoded by Salmonella spp and E. coli species is an orthologue of the mcr-1 gene that imparts resistance to colistin antibiotics. We have recently demonstrated that transient resistance to polymyxin B arises in strains of uropathogenic E. coli, following stimulation with ferric iron. We subsequently found that the transient polymyxin B resistance is brought about via the activation of the PmrB sensor kinase, a member of the PmrAB two-component system (TCS). Bacterial TCSs comprise a membrane- embedded histidine kinase that is the signal receptor, and a response regulator protein that directs the corresponding cellular changes. Although there are sequence-based determinants that dictate specificity among cognate TCS partners, we discovered strong interactions between the PmrAB and QseBC TCSs, in which the PmrB histidine kinase readily activates both its cognate partner PmrA and the non-cognate response regulator QseB in response to ferric iron, leading to a 16-fold increase in the MIC. I hypothesize that coordinated regulation of PmrA and QseB leads to upregulation of genes critical for lipid A modification that in turn protects bacteria from the insults of polymyxin B and other cationic polypeptides. I will test this hypothesis in three aims, in which I will: (1) Define the PmrA and QseB regulons in response to polymyxin B and define the mechanism by which PmrA and QseB activation leads to polymyxin B resistance. (2) Determine how the QseBC and PmrAB interactions have evolved to benefit bacterial fitness in different niches, and; (3) Ascertain how the amount of conservation present in the QseBC-PmrAB signaling cascade in E. coli strains from different phylogenetic clades and with different pathogenic strategies. Towards these goals, an inter-disciplinary approach will be followed, encompassing molecular biology, genome-wide analyses of transcription and robust murine models of infection. Combined these studies will provide mechanistic details into a mechanism that allows bacteria to survive one of the last resort antibiotics and will provide insights into the conservation of the QseBC- PmrAB circuitry in different E. coli pathotypes.