Role of uropathogenic<i>Escherichia coli</i>OmpT in the resistance against human cathelicidin LL-37

Brannon, John R.; Thomassin, Jenny‐Lee; Desloges, Isabelle; Gruenheid, Samantha; Moual, Hervé Le

doi:10.1111/1574-6968.12185

Cited by 30 publications

(44 citation statements)

References 37 publications

(50 reference statements)

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“…3B). Previously, we reported that OmpT cleaves AMPs such as the human cathelicidin LL-37 (35,44). Similarly, C. rodentium CroP rapidly cleaves the murine cathelicidin CRAMP (Fig.…”

Section: Discussionmentioning

confidence: 77%

“…The bacterial pellets were then either used for Sarkosyl extraction to obtain OM fractions or resuspended in Laemmli sample buffer and boiled for 5 min to obtain whole-cell lysates. Protein samples were resolved by SDS-PAGE, and Western blotting with a rabbit polyclonal antibody raised against CroP was conducted as previously described (44). ImageJ software was used to measure the density of CroP bands on Western blot membranes.…”

Section: Methodsmentioning

confidence: 99%

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Inhibition of Outer Membrane Proteases of the Omptin Family by Aprotinin

et al. 2015

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Bacterial proteases are important virulence factors that inactivate host defense proteins and contribute to tissue destruction and bacterial dissemination. Outer membrane proteases of the omptin family, exemplified by Escherichia coli OmpT, are found in some Gram-negative bacteria. Omptins cleave a variety of substrates at the host-pathogen interface, including plasminogen and antimicrobial peptides. Multiple omptin substrates relevant to infection have been identified; nonetheless, an effective omptin inhibitor remains to be found. Here, we purified native CroP, the OmpT ortholog in the murine pathogen Citrobacter rodentium. Purified CroP was found to readily cleave both a synthetic fluorescence resonance energy transfer substrate and the murine cathelicidin-related antimicrobial peptide. In contrast, CroP was found to poorly activate plasminogen into active plasmin. Although classical protease inhibitors were ineffective against CroP activity, we found that the serine protease inhibitor aprotinin displays inhibitory potency in the micromolar range. Aprotinin was shown to act as a competitive inhibitor of CroP activity and to interfere with the cleavage of the murine cathelicidin-related antimicrobial peptide. Importantly, aprotinin was able to inhibit not only CroP but also Yersinia pestis Pla and, to a lesser extent, E. coli OmpT. We propose a structural model of the aprotinin-omptin complex in which Lys 15 of aprotinin forms salt bridges with conserved negatively charged residues of the omptin active site.

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“…3B). Previously, we reported that OmpT cleaves AMPs such as the human cathelicidin LL-37 (35,44). Similarly, C. rodentium CroP rapidly cleaves the murine cathelicidin CRAMP (Fig.…”

Section: Discussionmentioning

confidence: 77%

Section: Methodsmentioning

confidence: 99%

Inhibition of Outer Membrane Proteases of the Omptin Family by Aprotinin

et al. 2015

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“…AMP cleavage assays were carried out as previously described with a few modifications (4,5). Either the specified bacterial strain (ϳ3 ϫ 10 8 CFU/ml) or purified CroP (14 g/ml) was incubated with the indicated AMP (400 g/ml).…”

Section: Methodsmentioning

confidence: 99%

“…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)(2)(3)(4)(5)(6)(7)(8)(9)(10). Many omptins are found among pathogens of the Enterobacteriaceae family (11).…”

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confidence: 99%

“…An early study showed that OmpT readily degrades the AMP protamine (43). More recently, we showed that LL-37 is cleaved and inactivated to different extents by OmpT present in enterohemorrhagic (EHEC), enteropathogenic (EPEC), and uropathogenic E. coli (4,5,32). The enteric murine pathogen Citrobacter rodentium and the human pathogens EHEC and EPEC share numerous virulence factors and cause similar intestinal lesions (44).…”

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confidence: 99%

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Antimicrobial Peptide Conformation as a Structural Determinant of Omptin Protease Specificity

et al. 2015

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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 ...

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A Bottom‐Up Proteomic Approach to Identify Substrate Specificity of Outer‐Membrane Protease OmpT

et al. 2017

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Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity,guides development of inhibitors,and improves assays ensitivity.P eptide arrays and SAMDI mass spectrometry were used to identify at etrapeptide substrate exhibiting high activity for the bacterial outer-membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1')a nd nearest-neighbor positions (P2, P2')a nd their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity.S ubstituting FRRV into af ragment of LL37, an atural substrate of OmpT,l ed to ag reater than 400-fold improvement in OmpT catalytic efficiency,with ak cat /K m value of 6.1 10 6 Lmol À1 s À1 .Wild-type and mutant OmpT displayed significant differences in their substrate specificities,d emonstrating that even modest mutants mayn ot be suitable substitutes for the native enzyme.

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Role of uropathogenicEscherichia coliOmpT in the resistance against human cathelicidin LL-37

Cited by 30 publications

References 37 publications

Inhibition of Outer Membrane Proteases of the Omptin Family by Aprotinin

Inhibition of Outer Membrane Proteases of the Omptin Family by Aprotinin

Antimicrobial Peptide Conformation as a Structural Determinant of Omptin Protease Specificity

A Bottom‐Up Proteomic Approach to Identify Substrate Specificity of Outer‐Membrane Protease OmpT

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