Outer Membrane Protease Regulation and Function in Enterobacterales

Bacteria that make their living inside a host face the constant threat of clearance by host defenses. Such is the case for Escherichia coli, which often harmlessly colonize humans but are also capable of causing disease. Adherent-invasive E. coli (AIEC) are isolated from inflammatory bowel disease (IBD) patients at a higher rate than from healthy individuals. AIEC strains are genetically diverse suggesting that a phenotypic approach may be better suited for understanding their mechanisms of pathogenesis. In a collection of clinical E. coli isolates, we observed elevated host defense peptides (HDPs) in IBD-associated AIEC; this may be an adaptive phenotype converged upon by these strains on the basis of a shared environment. Ulcerative colitis-associated strains showed statistically greater resistance to LL-37-mediated killing. High-level resistance to LL-37 depended upon carriage of omptin protease genes, but this did not wholly explain the heterogeneity of omptin activity in these strains. This suggests that the molecular basis of the observed phenotype varies from strain-to-strain. We further investigated the mechanism of omptin protease regulation in E. coli BW25113, and found OmpT, OmpP and ArlC of E. coli and CroP of Citrobacter rodentium to be directly regulated by the PhoPQ system, a master regulator of virulence and antimicrobial peptide resistance. We identified conserved PhoP-binding sites in the promoters of these omptin genes and show that mutation of this site in the ompT promoter abrogates PhoP-dependent expression. We also show that despite the conserved PhoP-dependent regulation, each omptin has differential activity towards specific substrates, suggesting that each omptin may contribute to resistance to a particular repertoire of host immune peptides, depending on the particular environment in which each evolved. A screen to identify other potential regulators of omptin activity revealed EnvY as a repressor of omptin activity. Further characterization is consistent with EnvY functioning as a repressor of the PhoPQ system, allowing for the integration of temperature as a signal influencing expression of PhoP-regulated genes. The function of EnvY is broadly conserved in strains that possess the envY gene, but the gene itself is not present in all E. coli strains; this is another source of strain-to-strain variability allowing for fine-tuning of virulence phenotypes. Together, these results demonstrate diversity in virulence-associated phenotypes and their regulation in E. coli, potentially reflecting adaptations based on the specific lifestyles of each strain.