Structural and functional characterization of cleavage and inactivation of human serine protease inhibitors by the bacterial SPATE protease EspP? from enterohemorrhagic E. coli.

ABSTRACT: EspP? and EspI are serine protease autotransporters found in enterohemorrhagic Escherichia coli. They both belong to the SPATE autotransporter family and are believed to contribute to pathogenicity via proteolytic cleavage and inactivation of different key host proteins during infection. Here, we describe the specific cleavage and functional inactivation of serine protease inhibitors (serpins) by EspP? and compare this activity with the related SPATE EspI. Serpins are structurally related proteins that regulate vital protease cascades, such as blood coagulation and inflammatory host response. For the rapid determination of serpin cleavage sites, we applied direct MALDI-TOF-MS or ESI-FTMS analysis of coincubations of serpins and SPATE proteases and confirmed observed cleavage positions using in-gel-digest of SDS-PAGE-separated degradation products. Activities of both serpin and SPATE protease were assessed in a newly developed photometrical assay using chromogenic peptide substrates. EspP? cleaved the serpins ?1-protease inhibitor (?1-PI), ?1-antichymotrypsin, angiotensinogen, and ?2-antiplasmin. Serpin cleavage led to loss of inhibitory function as demonstrated for ?1-PI while EspP? activity was not affected. Notably, EspP? showed pronounced specificity and cleaved procoagulatory serpins such as ?2-antiplasmin while the anticoagulatory antithrombin III was not affected. Together with recently published research, this underlines the interference of EspP? with hemostasis or inflammatory responses during infection, while the observed interaction of EspI with serpins is likely to be not physiologically relevant. EspP?-mediated serpin cleavage occurred always in flexible loops, indicating that this structural motif might be required for substrate recognition.

SUBMITTER: Weiss A 

PROVIDER: S-EPMC4210187 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature