EspPα 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 EspPα 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. EspPα cleaved the
serpins α1-protease inhibitor (α1-PI), α1-antichymotrypsin,
angiotensinogen, and α2-antiplasmin.
Serpin cleavage led to loss of inhibitory function as demonstrated for α1-PI while EspPα activity was not affected. Notably, EspPα showed pronounced specificity and cleaved procoagulatory
serpins such as α2-antiplasmin while the anticoagulatory
antithrombin III was not affected. Together with recently published research, this underlines the interference of EspPα with hemostasis or inflammatory responses during
infection, while the observed interaction of EspI with
serpins is likely to be not physiologically relevant. EspPα-mediated
serpin cleavage occurred always in flexible loops, indicating that this structural motif might be required for substrate recognition.