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 abilities of OmpT and CroP to cleave LL-37 and
CRAMP. EHEC OmpT degraded LL-37 and
CRAMP at similar rates. In contrast, C. rodentium CroP cleaved
CRAMP more rapidly than LL-37. The different cleavage rates of LL-37 and
CRAMP were independent of the bacterial background and substrate sequence specificity, as 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 rate of cleavage 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, and 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 specificities 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.