StcE, a metalloprotease secreted by Escherichia coli O157:H7, specifically cleaves C1 esterase inhibitor

Abstract

SummaryEscherichia coli O157:H7 causes diarrhoea, haemorrhagic colitis, and the haemolytic uraemic syndrome. We have identified a protein of previously unknown function encoded on the pO157 virulence plasmid of E. coli O157:H7, which is the first described protease that specifically cleaves C1 esterase inhibitor (C1-INH), a member of the serine protease inhibitor family. The protein, named StcE for secreted protease of C1 esterase inhibitor from EHEC (formerly Tagn) Factor XIa, and kallikrein of the coagulation and the inflammation systems respectively. Additionally, C1-INH competes with Factor B for binding to C3b to inhibit the activation of the alternative complement pathway (Jiang et al., 2001). Circulating C1-INH has a M r of 105 kDa, with post-translational glycosylations accounting for nearly half of its mass. Most glycosylations occur in the N-terminal 100 amino acids that are unique to this serpin. Desialylation does not affect the in vitro inhibitory activity of C1-INH, but reduces its circulating half-life in rabbits from >24 h to 3-5 min (Minta, 1981). C1-INH interacts with its target proteases to form large SDS-insoluble complexes that are subsequently removed from circulation (reviewed in Caliezi et al., 2000). Also, data suggest that surfaceassociated C1-INH protects cells from proinflammatory events at their surface (Schmaier et al., 1989;Schmaier et al., 1993;Caliezi et al., 2000).In this article, we describe the identification and characterization of a zinc metalloprotease produced by E. coli O157:H7 that cleaves C1-INH. This protein, termed StcE, is encoded by a gene on pO157, secreted via the etp type II secretion pathway, is positively regulated by Ler, and is highly specific for its substrate. Results Lysates of E. coli carrying pO157 aggregate cultured human T cell linesTo find novel E. coli O157:H7 virulence factors, we looked for cytopathic effects in Jurkat cells, a human T cell lymphoma line, treated with lysates of STEC strains EDL933 and EDL933cu, E. coli K-12 strain C600, C600 carrying pO157 (WAM2035), enteropathogenic E. coli strain E2348/69, and the mouse pathogen Citrobacter rodentium strain DBS100. Lysates of wild-type and transformed E. coli containing pO157 aggregated Jurkat cells (Fig. 1A); lysates of E. coli without pO157 (Fig. 1B) or other bacteria able to induce the A/E phenotype, such as E2348/69 and DBS100, did not. Lysates of EDL933, but not EDL933cu, aggregated MOLT-4 cells ( Fig. 1C and D) but not HL-60, U937, or Raji cells (data not shown), suggesting T cell-lineage specificity for this effect. Identification and cloning of stcETo localize the gene(s) on pO157 responsible for this unusual phenotype, we subjected pO157 to mutagenesis using a minitransposon. The location of the transposon insertion of one mutant whose lysate was unable to aggregate Jurkat cells (WAM2553) was determined to be position 23 772 of pO157 (position based on GenBank accession no. #AF074613). The ORF into which the transposon inserted was designated L7031 (tagA) (Burland et al., 1998), and is...

Proteomic Analysis of the Vibrio cholerae Type II Secretome Reveals New Proteins, Including Three Related Serine Proteases

“…This protein is homologous to TagA, a ToxR-regulated lipoprotein of V. cholerae (40% identity), and to the metalloprotease StcE of E. coli O157:H7 (34% identity). The observation that StcE is secreted to the extracellular environment by the E. coli T2S apparatus, Etp, provides further support for the suggestion that the TagArelated protein is a true T2S substrate in V. cholerae (67). Our iTRAQ approach also revealed a new putative trypsin-like protease encoded by the ORF VCA0803, in addition to the previously identified ORF VC1649, and hypothetical proteins encoded by ORFs VCA0738 and VC2298.…”

Differences in adherence and virulence gene expression between two outbreak strains of enterohaemorrhagic Escherichia coli O157 : H7

“…In this study, two effectors of type II secretion previously suggested to contribute to adherence, StcE (Grys et al, 2005) and AdfO (Ho et al, 2008), were upregulated in TW14359. The StcE metalloprotease, the expression of which is positively influenced by Ler (Lathem et al, 2002), is hypothesized to promote adherence by cleaving proteins in the glycocalyx and mucin layers atop the intestinal epithelium, facilitating close contact with the intestinal mucosa (Grys et al, 2005). The contribution of AdfO to adherence is not clear, as an adfO mutation has been shown to markedly decrease epithelial cell adherence in vitro, but does not attenuate colonization following oral challenge of infant rabbits (Ho et al, 2008).…”

Analysis of the expression, regulation and export of NleA–E in Escherichia coli O157 : H7

“…Using tandem MS, we observed many of the established proteins that have been shown to be secreted by EHEC O157. These included the Esp family of translocon proteins (EspA, B, D and F) (Abe et al, 1998;Viswanathan et al, 2004), the translocated intimin receptor, Tir (DeVinney et al, 1999), the mitochondrion-associated protein, Map (Kenny & Jepson, 2000), secreted proteases EspP (Brunder et al, 1997) and StcE (Lathem et al, 2002) (both encoded on pO157), and enterohaemolysin (Schmidt et al, 1995) (also on pO157). Using this wild-type strain, only NleA of the Nle A subpopulation of bacteria expressed nleA : : gfp, and only these bacteria were analysed for fluorescence output.…”