CHARACTERIZATION OF AN EXPORTED PROTEASE FROM SHIGA TOXIN-PRODUCING ESCHERICHIA-COLI

The gene for a novel, high molecular weight protein secreted by Shiga toxin-producing Escherichia coil (STEC) has been cloned, sequenced and characterized with respect to its activity. This gene, designated pss A, is localized on the large plasmid that also harbours the STEC haemo lysin operon, Sequencing of a region comprising 10 630 nt revealed tha t the sequences flanking the pssA gene are composed of several remnant s of different insertion elements. The PssA protein is produced as a 1 42 kDa precursor molecule that, after N- and C-terminal processing, is released into the culture supernatant as a mature polypeptide of appr oximately 104 kDa. The primary sequence of PssA is highly related to a family of autonomously transported putative virulence factors from di fferent Gram-negative pathogens, which includes the Tsh protein of an avian-pathogenic E. coil strain, the SepA protein from Shigella flexne ri and the EspC protein from enteropathogenic E. coil. A common motif present in all four proteins is reminiscent of the catalytic centre of certain serine proteases. PssA (protease Secreted by STEC) indeed sho ws serine protease activity in a casein-based assay and is moreover cy totoxic for Vero cells. This activity of PssA and probably of other pr oteins of the Tsh family may be of functional importance during infect ion of the mucosal cell layer by the bacterial pathogen.