Structure and Function Relationship of the Autotransport and Proteolytic Activity of EspP from Shiga Toxin-Producing Escherichia coli

BACKGROUND: The serine protease autotransporter EspP is a proposed virulence factor of Shiga toxin-producing Escherichia coli (STEC). We recently distinguished four EspP subtypes (EspPalpha, EspPbeta, EspPgamma, and EspPdelta), which display large differences in transport and proteolytic activities and differ widely concerning their distribution within the STEC population. The mechanisms underlying these functional variations in EspP subtypes are, however, unknown. METHODOLOGY/PRINCIPAL FINDINGS: The structural basis of proteolytic and autotransport activity was investigated using transposon-based linker scanning mutagenesis, site-directed mutagenesis and structure-function analysis derived from homology modelling of the EspP passenger domain. Transposon mutagenesis of the passenger domain inactivated autotransport when pentapeptide linker insertions occurred in regions essential for overall correct folding or in a loop protruding from the beta-helical core. Loss of proteolytic function was limited to mutations in Domain 1 in the N-terminal third of the EspP passenger. Site-directed mutagenesis demonstrated that His(127), Asp(156) and Ser(263) in Domain 1 form the catalytic triad of EspP. CONCLUSIONS/SIGNIFICANCE: Our data indicate that in EspP i) the correct formation of the tertiary structure of the passenger domain is essential for efficient autotransport, and ii) an elastase-like serine protease domain in the N-terminal Domain 1 is responsible for the proteolytic phenotype. Lack of stabilizing interactions of Domain 1 with the core structure of the passenger domain ablates proteolytic activity in subtypes EspPbeta and EspPdelta

Analysis of collection of hemolytic uremic syndrome-associated enterohemorrhagic Escherichia coli

Multilocus sequence typing of 169 non-O157 enterohemorrhagic Escherichia coli (EHEC) isolated from patients with hemolytic uremic syndrome (HUS) demonstrated 29 different sequence types (STs); 78.1% of these strains clustered in 5 STs. From all STs and serotypes identified, we established a reference panel of EHEC associated with HUS (HUSEC collection).</p

Translocation of outer membrane vesicles from enterohemorrhagic Escherichia coli O157 across the intestinal epithelial barrier

Outer membrane vesicles (OMVs) carrying virulence factors of enterohemorrhagic Escherichia coli (EHEC) are assumed to play a role in the pathogenesis of life-threatening hemolytic uremic syndrome (HUS). However, it is unknown if and how OMVs, which are produced in the intestinal lumen, cross the intestinal epithelial barrier (IEB) to reach the renal glomerular endothelium, the major target in HUS. We investigated the ability of EHEC O157 OMVs to translocate across the IEB using a model of polarized Caco-2 cells grown on Transwell inserts and characterized important aspects of this process. Using unlabeled or fluorescently labeled OMVs, tests of the intestinal barrier integrity, inhibitors of endocytosis, cell viability assay, and microscopic techniques, we demonstrated that EHEC O157 OMVs translocated across the IEB. OMV translocation involved both paracellular and transcellular pathways and was significantly increased under simulated inflammatory conditions. In addition, translocation was not dependent on OMV-associated virulence factors and did not affect viability of intestinal epithelial cells. Importantly, translocation of EHEC O157 OMVs was confirmed in human colonoids thereby supporting physiological relevance of OMVs in the pathogenesis of HUS

Shiga Toxin-Mediated Hemolytic Uremic Syndrome: Time to Change the Diagnostic Paradigm?

Hemolytic uremic syndrome (HUS) is caused by enterohemorrhagic Escherichia coli (EHEC) which possess genes encoding Shiga toxin (stx), the major virulence factor, and adhesin intimin (eae). However, the frequency of stx-negative/eae-positive E. coli in stools of HUS patients and the clinical significance of such strains are unknown.Between 1996 and 2006, we sought stx-negative/eae-positive E. coli in stools of HUS patients using colony blot hybridization with the eae probe and compared the isolates to EHEC causing HUS. stx-negative/eae-positive E. coli were isolated as the only pathogens from stools of 43 (5.5%) of 787 HUS patients; additional 440 (55.9%) patients excreted EHEC. The majority (90.7%) of the stx-negative/eae-positive isolates belonged to serotypes O26:H11/NM (nonmotile), O103:H2/NM, O145:H28/NM, and O157:H7/NM, which were also the most frequent serotypes identified among EHEC. The stx-negative isolates shared non-stx virulence and fitness genes with EHEC of the corresponding serotypes and clustered with them into the same clonal complexes in multilocus sequence typing, demonstrating their close relatedness to EHEC.At the time of microbiological analysis, approximately 5% of HUS patients shed no longer the causative EHEC, but do excrete stx-negative derivatives of EHEC that lost stx during infection. In such patients, the EHEC etiology of HUS is missed using current methods detecting solely stx or Shiga toxin; this can hamper epidemiological investigations and lead to inappropriate clinical management. While maintaining the paradigm that HUS is triggered by Shiga toxin, our data demonstrate the necessity of considering genetic changes of the pathogen during infection to adapt appropriately diagnostic strategies

Shiga Toxin–Producing \u3ci\u3eEscherichia coli\u3c/i\u3e in Montana: Bacterial Genotypes and Clinical Profiles

The diseases and virulence genes associated with Shiga toxin–producing Escherichia coli (STEC) are characterized incompletely. We analyzed, by polymerase chain reaction, 82 STEC isolates collected prospectively in Montana and profiled associated illnesses by patient chart review. All E. coli O157:H7 contained stx2-group genes, as well as eae, iha, espA, and ehxA; 84% contained stx1. Non-O157:H7 STEC less frequently contained stx1( P = .046 ), stx2 (P \u3c .001), iha (P \u3c .001), eae, and espA (P = .039 for both), were isolated less often from patients treated in emergency departments (P = .022), and tended to be associated less frequently with bloody diarrhea (P = .061). There were no significant associations between stx genotype and bloody diarrhea, but isolates containing stx2c or stx2d-activatable were recovered more often from patients who underwent diagnostic or therapeutic procedures (P = .033). Non-O157:H7 STEC are more heterogeneous and cause bloody diarrhea less frequently than do E. coli O157:H7. Bloody diarrhea cannot be attributed simply to the stx genotype of the infecting organism

Phylogeny and disease association of Shiga toxin-producing Escherichia coli O91

The diversity and relatedness of 100 Shiga toxin–producing Escherichia coli O91 isolates from different patients were examined by multilocus sequence typing. We identified 10 specific sequence types (ST) and 4 distinct clonal groups. ST442 was significantly associated with hemolytic uremic syndrome

Prospective Genomic Characterization of the German Enterohemorrhagic Escherichia coli O104:H4 Outbreak by Rapid Next Generation Sequencing Technology

An ongoing outbreak of exceptionally virulent Shiga toxin (Stx)-producing Escherichia coli O104:H4 centered in Germany, has caused over 830 cases of hemolytic uremic syndrome (HUS) and 46 deaths since May 2011. Serotype O104:H4, which has not been detected in animals, has rarely been associated with HUS in the past. To prospectively elucidate the unique characteristics of this strain in the early stages of this outbreak, we applied whole genome sequencing on the Life Technologies Ion Torrent PGM™ sequencer and Optical Mapping to characterize one outbreak isolate (LB226692) and a historic O104:H4 HUS isolate from 2001 (01-09591). Reference guided draft assemblies of both strains were completed with the newly introduced PGM™ within 62 hours. The HUS-associated strains both carried genes typically found in two types of pathogenic E. coli, enteroaggregative E. coli (EAEC) and enterohemorrhagic E. coli (EHEC). Phylogenetic analyses of 1,144 core E. coli genes indicate that the HUS-causing O104:H4 strains and the previously published sequence of the EAEC strain 55989 show a close relationship but are only distantly related to common EHEC serotypes. Though closely related, the outbreak strain differs from the 2001 strain in plasmid content and fimbrial genes. We propose a model in which EAEC 55989 and EHEC O104:H4 strains evolved from a common EHEC O104:H4 progenitor, and suggest that by stepwise gain and loss of chromosomal and plasmid-encoded virulence factors, a highly pathogenic hybrid of EAEC and EHEC emerged as the current outbreak clone. In conclusion, rapid next-generation technologies facilitated prospective whole genome characterization in the early stages of an outbreak

Shiga Toxin-Producing E. coli: Methods and Protocols

This volume provides a comprehensive outline of the current methods used to detect, characterize, and investigate Shiga toxin-producing E. coli (STEC) and Shiga toxins. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Shiga Toxin-Producing E. coli: Methods and Protocols aims to be a valuable resource for clinicians, epidemiologists, and researchers interested in STEC pathogenesis