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 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.

FIG 1

Cleavage of LL-37 and CRAMP by EHEC OmpT. LL-37 or CRAMP was incubated with the indicated EHEC strain or PBS alone (Ctl.) for the indicated time. Resulting peptide cleavage products were resolved by 10 to 20% Tris-Tricine SDS-PAGE and visualized with Coomassie blue staining.

FIG 2

Cleavage of LL-37 and CRAMP by C. rodentium CroP. LL-37 or CRAMP was incubated with the indicated C. rodentium strain or PBS alone (Ctl.) for the indicated time. Resulting peptide cleavage products were resolved by 10 to 20% Tris-Tricine SDS-PAGE and visualized with Coomassie blue staining.

FIG 3

Cleavage of LL-37 and CRAMP by purified CroP. LL-37 or CRAMP was incubated with CroP and LPS or LPS alone (Ctl.) for the indicated time. Resulting peptide cleavage products were resolved by 10 to 20% Tris-Tricine SDS-PAGE and visualized with Coomassie blue staining.

FIG 4

Analysis of LL-37 and CRAMP cleavage products by purified CroP. CroP-dependent LL-37 and CRAMP cleavage products were detected by liquid chromatography and analyzed by MS/MS. Shown is a schematic of the LL-37 and CRAMP amino acid sequences. Dibasic motifs are highlighted in red, major cleavage products are indicated by solid arrows, and a minor cleavage product is indicated by a dashed arrow.

FIG 5

Effect of buffer on peptide conformation and CroP activity. (A and B) Circular dichroism of LL-37 (A) and CRAMP (B) was measured in PBS (black), 10 mM phosphate buffer (red), 1 mM phosphate buffer (blue), and distilled water (dH₂O) (green) in the far-UV spectrum from 200 to 260 nm. (C and D) LL-37 (C) and CRAMP (D) were incubated for 15 min with or without CroP in the indicated buffers at pH 7.4. Resulting peptide cleavage products were resolved by 10 to 20% Tris-Tricine SDS-PAGE and visualized with Coomassie blue staining.

FIG 6

Effect of AMP α-helicity on CroP activity. (A and B) Circular dichroism of LL-37 (A) and CRAMP (B) was measured in PBS with 4% TFE (red), with 8% TFE (blue), and without TFE (black) within the far-UV spectrum from 200 to 260 nm. (C and D) LL-37 (C) and CRAMP (D) were incubated with or without CroP for 5 min in the presence of the indicated amount of TFE. Resulting peptide cleavage products were resolved by 10 to 20% Tris-Tricine SDS-PAGE and visualized with Coomassie blue staining.