Antimicrobial Peptide Conformation as a Structural Determinant of Omptin Protease Specificity

Bacterial proteases contribute to virulence by cleaving host or bacterial proteins to promote survival and dissemination. Omptins are a family of proteases embedded in the outer-membrane of Gram-negative bacteria that cleave various substrates, including host antimicrobial peptides, with a preference for cleaving at dibasic motifs. OmpT, the enterohemorrhagic Escherichia coli (EHEC) omptin, cleaves and inactivates the human cathelicidin LL-37. Similarly, the omptin CroP found in the murine pathogen Citrobacter rodentium , which is used as a surrogate model to study human-restricted EHEC, cleaves the murine cathelicidin-related antimicrobial peptide (CRAMP). Here, we compared the ability of OmpT and CroP to cleave LL-37 and CRAMP. EHEC OmpT degraded both LL-37 and CRAMP at similar rates. In contrast, C. rodentium CroP cleaved CRAMP more rapidly than LL-37. The different cleavage rate of LL-37 and CRAMP was independent of the bacterial background and substrate sequence specificity, as both OmpT and CroP have the same preference for cleaving at dibasic sites. Importantly, LL-37 was α-helical and CRAMP was unstructured under our experimental conditions. By altering the α-helicity of LL-37 and CRAMP, we found that decreasing LL-37 α-helicity increased its cleavage rate by CroP. Conversely, increasing CRAMP α-helicity decreased its cleavage rate. This structural basis for CroP substrate specificity highlights differences between the closely related omptins of C. rodentium and E. coli . In agreement with previous studies, this difference in CroP and OmpT substrate specificity suggests that omptins evolved in response to the substrates present in their host microenvironments. IMPORTANCE Omptins are recognized as key virulence factors for various Gram-negative pathogens. Their localization to the outer-membrane, their active site facing the extracellular environment, as well as their unique catalytic mechanism make them attractive targets for novel therapeutic strategies. Gaining insights into similarities and variations between the different omptin active sites and subsequent substrate specificities will be critical to develop inhibitors that can target multiple omptins. Here we describe subtle differences between the substrate specificity of two closely related omptins, CroP and OmpT. This is the first reported example of substrate conformation acting as a structural determinant for omptin activity between OmpT-like proteases.