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...

Infectious diseases caused by bacterial pathogens are a worldwide burden. Serious bacterial infection-related complications, such as sepsis, affect over a million people every year with mortality rates ranging from 30% to 50%. Crucial clinical microbiology laboratory responsibilities associated with patient management and treatment include isolating and identifying the causative bacterium and performing antibiotic susceptibility tests (ASTs), which are labor-intensive, complex, imprecise, and slow (taking days, depending on the growth rate of the pathogen). Considering the life-threatening condition of a septic patient and the increasing prevalence of antibiotic-resistant bacteria in hospitals, rapid and automated diagnostic tools are needed. This review summarizes the existing commercial AST methods and discusses some of the promising emerging AST tools that will empower humans to win the evolutionary war between microbial genes and human wits.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a diarrheal pathogen that causes attaching and effacing (A/E) lesions on intestinal epithelial cells. Strains of the O157 serogroup carry the large virulence plasmid pO157, which encodes the etp type II secretion system that secretes the genetically linked zinc metalloprotease StcE. The Ler regulator controls expression of many genes involved in A/E lesion formation, as well as StcE, suggesting StcE may be important at a similar time during colonization. Our laboratory has previously demonstrated that StcE cleaves C1-esterase inhibitor, a regulator of multiple inflammation pathways. Here we report two new substrates for StcE, mucin 7 and glycoprotein 340, and that purified StcE reduces the viscosity of human saliva. We tested the hypothesis that StcE contributes to intimate adherence of EHEC to host cells by cleavage of glycoproteins from the cell surface. The fluorescent actin stain (FAS) test was used to observe the intimate adherence represented by fluorescently stained bacteria colocalized with regions of bundled actin formed on HEp-2 cells. An E. coli O157:H7 strain with a stcE gene deletion was not affected in its ability to generally adhere to HEp-2 cells, but it did score threefold lower on the FAS test than wild-type or complemented strains. Addition of exogenous recombinant StcE increased intimate adherence of the mutant to wild-type levels. Thus, StcE may help block host clearance of E. coli O157:H7 by destruction of some classes of glycoproteins, and it contributes to intimate adherence of E. coli O157:H7 to the HEp-2 cell surface.

We used real-time polymerase chain reaction (PCR) targeting the cdc2 gene and direct fluorescent microscopy examination (DFME) to evaluate the prevalence of Pneumocystis jirovecii among immunocompetent patients without clinical pulmonary infection and immunosuppressed patients evaluated for opportunistic pulmonary infections. Among 102 bronchoalveolar lavage samples collected from immunocompetent patients without infection, none tested positive for P. jirovecii by either DFME or real-time PCR despite the presence of other comorbidities. Among patients with suspected pulmonary infection and tested with either assay, real-time PCR produced a higher number of positive results compared to DFME and increased P. jirovecii detection by 7% when added to DFME-negative samples. Real-time PCR may have increased sensitivity for P. jirovecii detection over DFME and decrease the risk of sample contamination compared to conventional and nested PCR. The use of single-copy gene targets (e.g., cdc2) may lower the rate of "colonization" detection and confer a high predictive value for Pneumocystis pneumonia.