M. Alexander Schmidt

Molecular mechanisms underlying the probiotic effects of Escherichia coli Nissle 1917 involve ZO-2 and PKCzeta redistribution resulting in tight junction and epithelial barrier repair.

The probiotic Escherichia coli strain Nissle 1917 (EcN) has been used for decades in human medicine in Central Europe for the treatment and prevention of intestinal disorders and diseases. However, the molecular mechanisms underlying its beneficial effects are only partially understood. To identify molecular responses induced by EcN that might contribute to its probiotic properties polarized T84 cells were investigated employing DNA microarrays, quantitative RT‐PCR, Western blotting, immunofluorescence and specific protein kinase C (PKC) inhibitors. Polarized T84 epithelial cell monolayers were used as a model to monitor barrier disruption by infection with the enteropathogenic E. coli (EPEC) strain E2348/69. Co‐incubation of EPEC with EcN or addition of EcN following EPEC infection abolished barrier disruption and, moreover, restored barrier integrity as monitored by transepithelial resistance. DNA‐microarray analysis of T84 cells incubated with EcN identified 300+ genes exhibiting altered expression. EcN altered the expression, distribution of zonula occludens‐2 (ZO‐2) protein and of distinct PKC isotypes. ZO‐2 expression was enhanced in parallel to its redistribution towards the cell boundaries. This study provides evidence that EcN induces an overriding signalling effect leading to restoration of a disrupted epithelial barrier. This is transmitted via silencing of PKCζ and the redistribution of ZO‐2. We suggest that these properties contribute to the reported efficacy in the treatment of inflammatory bowel diseases and in part rationalize the probiotic nature of EcN.

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).

Characterization of translocation pores inserted into plasma membranes by type III-secreted Esp proteins of enteropathogenic Escherichia coli.

Many mucosal pathogens use type III secretion systems for the injection of effector proteins into target cells. The type III‐secreted proteins EspB and EspD of enteropathogenic Escherichia coli (EPEC) are inserted into the target cell membrane. Together with EspA, these proteins are supposed to constitute a molecular syringe, channelling other effector proteins into the host cell. In this model, EspB and EspD would represent the tip of the needle forming a pore into target cell membranes. Although contact‐dependent and Esp‐mediated haemolytic activity by EPEC has already been described, the formation of a putative pore resulting in haemolysis has not been demonstrated so far. Here, we show that (i) diffusely adhering (DA)‐EPEC strains exhibit a type III‐dependent haemolytic activity too; (ii) this activity resides in the secreted proteins and, for DA‐EPEC strains, in contrast to EPEC strains, does not require bacterial contact; and (iii) pores are introduced into the target cell membrane. Osmoprotection revealed a minimal pore size of 3–5 nm. The pores induced by type III‐secreted proteins of DA‐EPEC were characterized by electron microscopy techniques. Analysis by atomic force microscopy demonstrated the pores to be composed of six to eight subunits with a lateral extension of 55–65 nm and to be raised 15–20 nm above the membrane plane. We could also demonstrate an association of EspB and EspD with erythrocyte membranes and an interaction of both proteins with each other in vitro. These results, together with the homologies of EspB and EspD to proposed functional domains of other pore‐forming proteins (Yop/Ipa), strongly support the idea that both proteins are directly involved in pore formation, which might represent the type III secretion system translocon.

AIDA‐I, the adhesin involved in diffuse adherence of the diarrhoeagenic Escherichia coli strain 2787 (O126:H27), is synthesized via a precursor molecule

The adherence mechanisms of enteropathogenic Escherichia coli (EPEC) to epithelial cells are still not understood. To study the molecular basis of the diffuse adherence (DA) phenotype exhibited by diarrhoeagenic E. coli expressing classical EPEC serotypes we investigated strain 2787 (O126:H27) isolated from a case of infantile diarrhoea. A 6.0 kb plasmid‐derrved DNA fragment mediates the DA phenotype and encodes the 100 kDa adhesin protein AIDA‐I (adhesin involved in diffuse adherence). Sequencing of the entire fragment revealed two open reading frames which encoded proteins of 45 kDa and 132 kDa, respectively. The 132 kDa protein has been identified as an AIDA‐I precursor protein. After cleavage of the signal sequence further processing at the C‐terminus of the 132 kDa precursor leads to the mature ∼100 kDa AIDA‐I. While the exact function of the cytopiasmic 45 kDa protein is not known, preliminary evidence indicates that it is necessary for the correct maturation of AIDA‐I. The AIDA‐l precursor exhibits significant homology with the virG(icsA) protein of Shigella flexneri which seems to be involved in the intercellular spread of invasive Shigella organisms.

Never say never again: protein glycosylation in pathogenic bacteria.

In recent years, accumulating evidence for glycosylated bacterial proteins has overthrown an almost dogmatic belief that prokaryotes are not able to synthesize glycoproteins. Now it is widely accepted that eubacteria express glycoproteins. Although, at present, detailed information about glycosylation and structure–function relationships is available for only few eubacterial proteins, the variety of different components and structures observed already indicates that the variations in bacterial glycoproteins seem to exceed the rather limited display found in eukaryotes. Numerous virulence factors of bacterial pathogens have been found to be covalently modified with carbohydrate residues, thereby identifying these factors as true glycoproteins. In several bacterial species, gene clusters suggested to represent a general pro‐tein glycosylation system have been identified. In other cases, genes encoding highly specific glycosyltransferases have been found to be directly linked with virulence genes. These findings raise interesting questions concerning a potential role of glycosylation in pathogenesis. In this review, we will therefore focus on protein glycosylation in Gram‐negative bacterial pathogens.

Glycosylation with heptose residues mediated by the aah gene product is essential for adherence of the AIDA‐I adhesin

The diffuse adherence of Escherichia coli strain 2787 (O126:H27) is mediated by the autotransporter adhesin AIDA‐I (adhesin‐involved‐in‐diffuse‐adherence) encoded by the plasmid‐borne aidA gene. AIDA‐I exhibits an aberrant mobility in denaturing gel electrophoresis. Deletion of the open reading frame (ORF) A immediately upstream of aidA restores the predicted mobility of AIDA‐I, but the adhesin is no longer functional. This indicates that the mature AIDA‐I adhesin is post‐translationally modified and the modification is essential for adherence function. Labelling with digoxigenin hydrazide shows AIDA‐I to be glycosylated. Using carbohydrate composition analysis, AIDA‐I contains exclusively heptose residues (ratio heptose:AIDA‐I ≈19:1). The deduced amino acid sequence of the cytoplasmic open reading frame (ORF) A gene product shows homologies to heptosyltransferases. In addition, the modification was completely abolished in an ADP–glycero‐manno‐heptopyranose mutant. Our results provide direct evidence for glycosylation of the AIDA‐I adhesin by heptoses with the ORF A gene product as a specific (mono)heptosyltransferase generating the functional mature AIDA‐I adhesin. Consequently, the ORF A gene has been denoted ‘aah’ (autotransporter‐adhesin‐heptosyltransferase). Glycosylation by heptoses represents a novel protein modification in eubacteria.

Insertion of EspD into epithelial target cell membranes by infecting enteropathogenic Escherichia coli.

Diffusely adhering Escherichia coli (DAEC) strains have been implicated in epidemiological studies as a cause of diarrhoea in children. However, the molecular interactions of these pathogens with target cells have remained largely obscure. We found that some DAEC strains contain homologues of the locus of enterocyte effacement (LEE) pathogenicity island and secrete EspA, EspB and EspD proteins necessary for the formation of the attaching and effacing (A/E) lesions. To characterize the function of the EspD protein further, we cloned and sequenced the espD genes of two DA‐EPEC strains and compared their deduced amino‐acid sequences with known EspD sequences. A pattern of two conserved transmembrane regions and one conserved coiled‐coil region is predicted in EspD and also in the type III system secreted proteins YopB, PopB, IpaB and SipB of Yersinia, Pseudomonas, Shigella and Salmonella respectively. The EspD protein is inserted into a trypsin‐sensitive location in the HeLa cell membrane at sites of bacterial contact, but is not translocated into the cytoplasm. Secretion of EspD increases upon contact with host cells. We propose that the membrane‐located EspD protein is part of the translocation apparatus for Esp proteins into the target host cell performing functions similar to YopB in Yersinia.

Identification of Unconventional Intestinal Pathogenic Escherichia coli Isolates Expressing Intermediate Virulence Factor Profiles by Using a Novel Single-Step Multiplex PCR

ABSTRACT Intestinal pathogenic Escherichia coli represents a global health problem for mammals, including humans. At present, diarrheagenic E. coli bacteria are grouped into seven major pathotypes that differ in their virulence factor profiles, severity of clinical manifestations, and prognosis. In this study, we developed and evaluated a one-step multiplex PCR (MPCR) for the straightforward differential identification of intestinal pathotypes of E. coli. The specificity of this novel MPCR was validated by using a subset of reference strains and further confirmed by PCR-independent pheno- and genotypic characterization. Moreover, we tested 246 clinical E. coli isolates derived from diarrhea patients from several distinct geographic regions. Interestingly, besides strains belonging to the defined and well-described pathotypes, we identified five unconventional strains expressing intermediate virulence factor profiles. These strains have been further characterized and appear to represent intermediate strains carrying genes and expressing factors associated with enteropathogenic E. coli, Shiga toxin-producing E. coli, enterotoxigenic E. coli, and enteroaggregative E. coli alike. These strains represent further examples of the extraordinary plasticity of the E. coli genome. Moreover, this implies that the important identification of specific pathotypes has to be based on a broad matrix of indicator genes. In addition, the presence of intermediate strains needs to be accounted for.

Cytolethal Distending Toxin Gene Cluster in Enterohemorrhagic Escherichia coli O157:H− and O157:H7: Characterization and Evolutionary Considerations

ABSTRACT We identified a cytolethal distending toxin (cdt) gene cluster in 87, 6, and 0% of sorbitol-fermenting (SF) enterohemorrhagic Escherichiacoli (EHEC) O157:H−, EHEC O157:H7, and E. coli O55:H7/H− strains, respectively. The toxin was expressed by the wild-type EHEC O157 strains and by a cdt-containing cosmid from a library of SF EHEC O157:H− strain 493/89. The cdt flanks in strain 493/89 were homologous to bacteriophages P2 and lambda. Our data demonstrate that cdt, encoding a potential virulence factor, is present in the EHEC O157 complex and suggest that cdt may have been acquired by phage transduction.

LEEways: tales of EPEC, ATEC and EHEC

Intestinal pathogenic Escherichia coli are a major cause of worldwide morbidity and mortality. Currently seven intestinal pathovars are recognized causing a wide range of intestinal disorders that are sometimes associated with severe and even lethal complications. The arsenal of virulence factors is used to subvert cellular functions of the host thereby enhancing adaptation, virulence and pathogenicity. Virulence factor profiles are largely the result of the acquisition of mobile genetic elements such as prophages and pathogenicity islands. A group of highly adapted intestinal pathogenic E. coli that are characterized by the induction of ‘attaching‐and‐effacing (A/E) lesions’ have acquired a decisive pathogenicity island, the ‘locus of enterocyte effacement – LEE’ by horizontal gene transfer. This review focuses on recent advances in our understanding of A/E E. coli. It highlights novel functions of effector proteins, addresses the LEE flanking regions where additional genetic elements such as the LifA/Efa1 region have been identified, and points to implications for diagnostics and therapy due to the putative interconversion of A/E E. coli during infection.