The polysaccharide capsule in E. coli is a major pathogenic factor. Loss of encapsulation results in complete attenuation in an otherwise lethal systemic murine infection model. We have previously identified a small molecule inhibitor of capsule biogenesis (designated DU011) and identified its target as MprA, a transcriptional repressor of multi-drug efflux pumps. Unlike other proposed MprA ligands such as salicylate and 2,4-dinitrophenol (DNP), DU011 does not alter E. coli antibiotic resistance and has significantly enhanced inhibition of capsule expression. We hypothesized that the potency and unique action of DU011 are due to novel interactions with the MprA binding pocket and the conformation assumed by MprA upon binding DU011 relative to other ligands. Experiments were performed to define the critical residues in the binding pocket to optimize inhibitor design.
To identify amino acid residues in MprA that involved in DU011 binding and action, mprA was randomly mutated in DNA repair deficient XL-Red cells, and mutants were screened for DU011 resistance by using a capsule dependent phage assay. Mutations were identified through Sanger sequencing. Based on a threaded model of MprA, the putative mprA ligand binding site was predicted and subjected to site-directed mutagenesis. Function of the mutated proteins was measured using a thermal shift assay and DNase protection assay. Hydrogen-Deuterium Exchange mass spectrometry (HDX-MS) was performed to understand the dynamics of MprA-DU011 interactions.
Mutant MprA clones were identified that conferred relative resistance to DU011. Mutations mapped to the predicted binding pocket as modeled in silico. Biophysical studies demonstrated altered binding of DU011 in purified mutant MprA proteins. HDX-MS identified 4 regions of MprA undergoing conformational shifts following DU011 binding. These regions were similar to those identified during random and site-directed mutagenesis further validating our results.
Mutational and HDX-MS analyses provided complementary identification of the DU011 binding pocket in the transcriptional virulence regulator, MprA. These data will inform future rational design of MprA-family inhibitors as a class of novel anti-virulence, anti-infective small molecules.
P. Seed, None