SummaryShiga toxin-producing Escherichia coli (STEC) induce so-called attaching and effacing lesions that enable the tight adherence of these pathogens to the gut epithelium. All of the genes necessary for this process are present in the locus of enterocyte effacement, which encodes a type III secretion system, the secreted Esp proteins and the surface protein intimin. In this study we sequenced the espA gene of STEC, generated and characterized a corresponding deletion mutant and raised EspA-specific monoclonal antibodies to analyse the functional role of this protein during infection. EspA was detected in often filament-like structures decorating all bacteria that had attached to HeLa cells. These appendages were especially prominent on bacteria that had not yet induced the formation of actin pedestals, indicating that they mediate the initial contact of STEC to their target cells. Consistently, a deletion of the espA gene completely abolished the capacity of such STEC mutants to bind to HeLa cells and to induce actin rearrangements. Surface appendages similar to those described in this study are also formed by Pseudomonas syringae and may represent a structural element common to many bacterial pathogens that deliver proteins into their target cells via a type III secretion system.

SummaryShiga toxin-producing Escherichia coli (STEC), enteropathogenic E. coli (EPEC) and some strains of Hafnia alvei are capable of inducing attaching and effacing (A/E) lesions, characterized by tight apposition of the bacteria to the eukaryotic membrane and formation of actin-based pedestals. In this study, we report on the identification of EspE, a novel secreted 80 kDa protein of A/E bacteria. During infection, EspE is delivered into the cytoplasm of the infected host cell, where it is detected as a higher-molecular-weight form of 90 kDa. We present evidence that translocated EspE becomes tyrosine phosphor ylated and that this modified form of EspE may be identical to Hp90, the putative receptor of EPEC intimin. Bacteria of the classic enterohaemorrhagic E. coli (EHEC) serotype O157:H7 fail to induce a tyrosine phosphor ylation of EspE and differ in this respect from other A/E bacteria. Translocated EspE, whether tyrosine phosphor ylated or not, becomes incorporated into the bacteria-induced cytoskeletal structures, where it normally colocalizes with filamentous actin. EPEC are also able to induce 'pseudopods', elongated pedestals that have recently been implicated in a novel kind of actin-based motility. EspE is enriched at the tip of these structures, suggesting its involvement in the process of actin dynamics, which is triggered during the attaching and effacing process.

SummaryThe gene for a novel, high molecular weight protein secreted by Shiga toxin-producing Escherichia coli (STEC) has been cloned, sequenced and characterized with respect to its activity. This gene, designated pssA, is localized on the large plasmid that also harbours the STEC haemolysin operon. Sequencing of a region comprising 10 630 nt revealed that the sequences flanking the pssA gene are composed of several remnants of different insertion elements. The PssA protein is produced as a 142 kDa precursor molecule that, after N-and C-terminal processing, is released into the culture supernatant as a mature polypeptide of approximately 104 kDa. The primary sequence of PssA is highly related to a family of autonomously transported putative virulence factors from different Gram-negative pathogens, which includes the Tsh protein of an avian-pathogenic E. coli strain, the SepA protein from Shigella flexneri and the EspC protein from enteropathogenic E. coli. A common motif present in all four proteins is reminiscent of the catalytic centre of certain serine proteases. PssA (protease secreted by STEC) indeed shows serine protease activity in a casein-based assay and is moreover cytotoxic for Vero cells. This activity of PssA and probably of other proteins of the Tsh family may be of functional importance during infection of the mucosal cell layer by the bacterial pathogen.

Infections due to Shiga toxin-producing Escherichia coli (STEC) are responsible for severe diarrheal disease in humans and livestock, and these bacteria have recently emerged as a leading cause of renal failure in children. In this study, we have examined medium-and temperature-dependent production of secreted proteins from a STEC O26 serotype strain. Growth of bacteria in Luria broth led to the detection of secreted polypeptides of 104, 55, 54, and 37 kDa (p104, p55, p54, and p37, respectively). When grown in serum-free tissue culture medium, only p104, p37 and two additional polypeptides of 25 and 22 kDa (p25 and p22) were present in supernatant fluids. Production of these polypeptides was growth temperature dependent and induced in cultures grown at 37؇C. N-terminal amino acid sequencing revealed that p104 was homologous to the secreted p110 of enteropathogenic Escherichia coli (EPEC), and both proteins belong to a family of secreted proteins in pathogenic bacteria of which the immunoglobulin A protease of Neisseria gonorrhoeae is the prototype. The N-terminal amino acid sequences of p55 and p54 were unique to the STEC strain, while p37 and p25 were found to be highly homologous to the similarly sized EspA and EspB proteins, previously detected in culture supernatants of EPEC. Molecular cloning and sequencing of STEC espB alleles from two different serotypes showed that the encoded polypeptides were about 80% homologous. A monoclonal antibody raised against STEC EspB also cross-reacted with its EPEC analog and allowed us to demonstrate medium-and temperature-dependent production of this important virulence factor in STEC and EPEC strains of differing serotypes.

Enterohemorrhagic Escherichia coli (EHEC) exhibits a pattern of localized adherence to host cells, with the formation of microcolonies, and induces a specific histopathological phenotype collectively known as the attaching and effacing lesion. The genes encoding the products responsible for this phenotype are located on a 35-kb pathogenicity island designated the locus of enterocyte effacement, which is also shared by enteropathogenic E. coli. We have identified an open reading frame (ORF) which is located upstream of the espA, espB, andespD genes on the complementary strand and which exhibits high homology to the genes spiB fromSalmonella, yscD from Yersinia, andpscD from Pseudomonas. Localization studies showed that the encoded product is present in the cytoplasmic and inner membrane fractions of EHEC. The construction and characterization of a recombinant clone containing an in-frame deletion of this ORF demonstrated that the encoded product is a putative member of a type III system required for protein secretion. Disruption of this ORF, designated pas (protein associated with secretion), abolished the secretion of Esp proteins. The mutant adhered only poorly and lost its capacities to trigger attaching and effacing activity and to invade HeLa cells. These results demonstrate that Pas is a virulence-associated factor that plays an essential role in EHEC pathogenesis.

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