Inga Benz

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.

Polar Localization of the Autotransporter Family of Large Bacterial Virulence Proteins

Autotransporters are an extensive family of large secreted virulence-associated proteins of gram-negative bacteria. Secretion of such large proteins poses unique challenges to bacteria. We demonstrate that autotransporters from a wide variety of rod-shaped pathogens, including IcsA and SepA of Shigella flexneri, AIDA-I of diffusely adherent Escherichia coli, and BrkA of Bordetella pertussis, are localized to the bacterial pole. The restriction of autotransporters to the pole is dependent on the presence of a complete lipopolysaccharide (LPS), consistent with known effects of LPS composition on membrane fluidity. Newly synthesized and secreted BrkA is polar even in the presence of truncated LPS, and all autotransporters examined are polar in the cytoplasm prior to secretion. Together, these findings are consistent with autotransporter secretion occurring at the poles of rod-shaped gram-negative organisms. Moreover, NalP, an autotransporter of spherically shaped Neisseria meningitidis contains the molecular information to localize to the pole of Escherichia coli. In N. meningitidis, NalP is secreted at distinct sites around the cell. These data are consistent with a model in which the secretion of large autotransporters occurs via specific conserved pathways located at the poles of rod-shaped bacteria, with profound implications for the underlying physiology of the bacterial cell and the nature of bacterial pathogen-host interactions.

Functional substitution of the TibC protein of enterotoxigenic Escherichia coli strains for the autotransporter adhesin heptosyltransferase of the AIDA system

ABSTRACT The plasmid-encoded AIDA (adhesin involved in diffuse adherence) autotransporter protein derived from diffuse-adhering clinical Escherichia coli isolate 2787 and the TibA (enterotoxigenic invasion locus B) protein encoded by the chromosomal tib locus of enterotoxigenic E. coli (ETEC) strain H10407 are posttranslationally modified by carbohydrate substituents. Analysis of the AIDA-I adhesin showed that the modification involved heptose residues. AIDA-I is modified by the heptosyltransferase activity of the product of the aah gene, which is located directly upstream of adhesin-encoding gene aidA. The carbohydrate modification of the TibA adhesin/invasin is mediated by the TibC protein but has not been elucidated. Based on the sequence similarities between TibC and AAH (autotransporter adhesin heptosyltransferase) and between the TibA and the AIDA proteins we hypothesized that the AIDA system and the Tib system encoded by the tib locus are structurally and functionally related. Here we show that (i) TibC proteins derived from different ETEC strains appear to be highly conserved, (ii) recombinant TibC proteins can substitute for the AAH heptosyltransferase in introducing the heptosyl modification to AIDA-I, (iii) this modification is functional in restoring the adhesive function of AIDA-I, (iv) a single amino acid substitution at position 358 completely abolishes this activity, and (v) antibodies directed at the functionally active AIDA-I recognize a protein resembling modified TibA in ETEC strains. In summary, we conclude that, like AAH, TibC represents an example of a novel class of heptosyltransferases specifically transferring heptose residues onto multiple sites of a protein backbone. A potential consensus sequence for the modification site is suggested.

Comparative Analysis of the Locus of Enterocyte Effacement and Its Flanking Regions

ABSTRACT The attaching-and-effacing (A/E) phenotype mediated by factors derived from the locus of enterocyte effacement (LEE) is a hallmark of clinically important intestinal pathotypes of Escherichia coli, including enteropathogenic (EPEC), atypical EPEC (ATEC), and enterohemorrhagic E. coli strains. Epidemiological studies indicate that the frequency of diarrhea outbreaks caused by ATEC is increasing. Hence, it is of major importance to further characterize putative factors contributing to the pathogenicity of these strains and to gain additional insight into the plasticity and evolutionary aspects of this emerging pathotype. Here, we analyzed the two clinical ATEC isolates B6 (O26:K60) and 9812 (O128:H2) and compared the genetic organizations, flanking regions, and chromosomal insertion loci of their LEE with those of the LEE of other A/E pathogens. Our analysis shows that the core LEE is largely conserved—particularly among genes coding for the type 3 secretion system—whereas genes encoding effector proteins display a higher variability. Chromosomal insertion loci appear to be restricted to selC, pheU, and pheV. In contrast, striking differences were found between the 5′- and 3′-associated flanking regions reflecting the different histories of the various strains and also possibly indicating different lines in evolution.

Arrangement of the Translocator of the Autotransporter Adhesin Involved in Diffuse Adherence on the Bacterial Surface

ABSTRACT Autotransporters of gram-negative bacteria are single-peptide secretion systems that consist of a functional N-terminal α-domain (“passenger”) fused to a C-terminal β-domain (“translocator”). How passenger proteins are translocated through the outer membrane has not been resolved, and at present essentially three different models are discussed. In the widely accepted “hairpin model” the passenger proteins are translocated through a channel formed by the β-barrel of the translocator that is integrated in the outer membrane. This model has been challenged by a recent proposal for a general autotransporter model suggesting that there is a hexameric translocation pore that is generated by the oligomerization of six β-domains. A third model suggests that conserved Omp85 participates in autotransporter integration and passenger protein translocation. To examine these models, in this study we investigated the presence of putative oligomeric structures of the translocator of the autotransporter adhesin involved in diffuse adherence (AIDA) in vivo by cross-linking techniques. Furthermore, the capacity of isolated AIDA fusion proteins to form oligomers was studied in vitro by several complementary analytical techniques, such as analytical gel filtration, electron microscopy, immunogold labeling, and cross-linking of recombinant autotransporter proteins in which different passenger proteins were fused to the AIDA translocator. Our results show that the AIDA translocator is mostly present as a monomer. Only a fraction of the AIDA autotransporter was found to form dimers on the bacterial surface and in solution. Higher-order structures, such as hexamers, were not detected either in vivo or in vitro and can therefore be excluded as functional moieties for the AIDA autotransporter.

Diffuse Adherence of Enteropathogenic Escherichia coli Strains — Processing of AIDA-I

The adherence of pathogenic Escherichia coli to the mucosa of the small intestine is an important step in the development of diarrhoea. To study the molecular basis of the diffuse adherence (DA) pattern of E. coli strains expressing the classical serotypes of enteropathogenic E. coli (EPEC), strain 2787 (O126:H27) was investigated. By expression cloning, a plasmid-derived 6.0 kb DNA fragment was identified which conferred the DA phenotype on recipient K-12 strains. This fragment encoded the 100 kDa adhesin involved in diffuse adherence (AIDA-I) which by mild heat shock treatment was isolated from the surface of the wild-type and recombinant DA-positive strains. Analysis of the entire DNA fragment revealed two open reading frames coding for proteins of 45 kDa and 132 kDa, respectively. The 132 kDa protein has been identified as the AIDA-I precursor protein which after cleavage of the signal sequence undergoes additional C-terminal processing for maturation to AIDA-I. Though the function of the cytoplasmic 45 kDa protein is not known, preliminary evidence indicates that authentic expression of the protein is a prerequisite for the correct processing of the 132 kDa precursor to AIDA-I. The AIDA-I precursor exhibits significant homology to the virG (icsA) protein of Shigella flexneri which apparently plays a major role in the events leading to the intercellular spread of invasive Shigella organisms.

Characterization of Escherichia coli Strains Isolated from Patients with Diarrhea in São Paulo, Brazil: Identification of Intermediate Virulence Factor Profiles by Multiplex PCR

ABSTRACT Intestinal pathogenic Escherichia coli is a major causative agent of severe diarrhea. In this study the prevalences of different pathotypes among 702 E. coli isolates from Brazilian patients with diarrhea were determined by multiplex PCR. Interestingly, most strains were enteroaggregative E. coli (EAEC) strains, followed by atypical EPEC (ATEC) strains. Classical enteropathogenic E. coli (EPEC) strains were not detected.

Characterization of Escherichia coli Strains Isolated from Diarrhea Patients in São Paulo: Identification of Intermediate Virulence Factor Profiles by Multiplex PCR

ABSTRACT Intestinal pathogenic Escherichia coli is a major causative agent of severe diarrhea. In this study the prevalences of different pathotypes among 702 E. coli isolates from Brazilian patients with diarrhea were determined by multiplex PCR. Interestingly, most strains were enteroaggregative E. coli (EAEC) strains, followed by atypical EPEC (ATEC) strains. Classical enteropathogenic E. coli (EPEC) strains were not detected.