Antimicrobial Peptide Conformation as a Structural Determinant of Omptin Protease Specificity

Abstract

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 ␣-helical and CRAMP was unstructured under our experimental conditions. By altering the ␣-helicity of LL-37 and CRAMP, we found that decreasing LL-37 ␣-helicity increased its rate of cleavage by CroP. Conversely, increasing CRAMP ␣-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. IMPORTANCEOmptins 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. Bacterial proteases are key virulence factors involved in hostpathogen interactions. Omptins are outer membrane (OM) proteases commonly found in Gram-negative pathogens. They play important roles at the host-pathogen interface by processing or degrading a variety of host and bacterial proteins (1-10). Many omptins are found among pathogens of the Enterobacteriaceae family (11). The prototypical omptins are Escherichia coli OmpT and Yersinia pestis Pla, which inactivate antimicrobial peptides (AMPs) and activate plasminogen into plasmin, respectively (4, 6). Omptins are ␤-barrel proteases embedded ...

Identification and characterization of OmpT‐like proteases in uropathogenic Escherichia coli clinical isolates

“…ArlC cleaved mCRAMP, C18G, and Magainin II by the first time point tested (2 min C18G, 15 min mCRAMP, and Magainin II), but only a small amount of LL‐37 cleavage was observed after 60 min. Substrate properties, such as size and secondary structure, are known to influence omptin activity (Brannon, Thomassin, Gruenheid, & Le Moual, ; Hritonenko & Stathopoulos, ). Peptide secondary structure also influences omptin activity (Brannon, Thomassin, et al, ); therefore, we used circular dichroism spectroscopy to determine the secondary structure of these AMPs (Figure c).…”

“…Substrate properties, such as size and secondary structure, are known to influence omptin activity (Brannon, Thomassin, Gruenheid, & Le Moual, ; Hritonenko & Stathopoulos, ). Peptide secondary structure also influences omptin activity (Brannon, Thomassin, et al, ); therefore, we used circular dichroism spectroscopy to determine the secondary structure of these AMPs (Figure c). Under our experimental conditions, only LL‐37 is α‐helical, while mCRAMP, C18G, and Magainin II are unstructured (Figure c).…”

“…Omptins belonging to the OmpT‐like subfamily are known to have subtle differences in substrate specificity (Brannon, Thomassin, et al, ; Hwang et al, ; McCarter et al, ). Studies using peptide libraries to compare OmpP and OmpT activity showed both omptins preferentially cleave substrates between two consecutive basic residues, but that OmpP appears to have a slight preference for Lys in the P and P’ sites (Hwang et al, ).…”

“…Studies using peptide libraries to compare OmpP and OmpT activity showed both omptins preferentially cleave substrates between two consecutive basic residues, but that OmpP appears to have a slight preference for Lys in the P and P’ sites (Hwang et al, ). In addition to subtle differences in amino acid motif preference, peptide size and secondary structure also impact substrate specificity (Brannon, Thomassin, et al, ; Haiko et al, ; Hritonenko & Stathopoulos, ). For example, AMP α‐helicity was shown to be a determining factor for proteolytic activity of the OmpT‐like omptin, CroP, from Citrobacter rodentium (Brannon, Thomassin, et al, ).…”

Targeting cancer-specific glycans by cyclic peptide lectinomimics

“…10B). Peptide conformation and thus accessibility to proteases present in human serum may explain the observed differences in serum stability of peptides 1–3 (Brannon et al 2015; Cline and Waters 2009). …”