SILAC-based comparative analysis of pathogenic Escherichia coli secretomes.

Comparative studies of pathogenic bacteria and their non-pathogenic counterparts has led to the discovery of important virulence factors thereby generating insight into mechanisms of pathogenesis. Protein-based antigens for vaccine development are primarily selected among unique virulence-related factors produced by the pathogen of interest. However, recent work indicates that proteins that are not unique to the pathogen but instead selectively expressed compared to its non-pathogenic counterpart could also be vaccine candidates or targets for drug development. Modern methods in quantitative proteome analysis have the potential to discover both classes of proteins and hence form an important tool for discovering therapeutic targets. Adherent-invasive Escherichia coli (AIEC) and Enterotoxigenic E. coli (ETEC) are pathogenic variants of E. coli which cause intestinal disease in humans. AIEC is associated with Crohn's disease (CD), a chronic inflammatory condition of the gastrointestinal tract whereas ETEC is the major cause of human diarrhea which affects hundreds of millions annually. In spite of the disease burden associated with these pathogens, effective vaccines conferring long-term protection are still needed. In order to identify proteins with therapeutic potential, we have used mass spectrometry-based Stable Isotope Labeling with Amino acids in Cell culture (SILAC) quantitative proteomics method which allows us to compare the proteomes of pathogenic strains to commensal E. coli. In this study, we grew the pathogenic strains ETEC H10407, AIEC LF82 and the non-pathogenic reference strain E. coli K-12 MG1655 in parallel and used SILAC to compare protein levels in OMVs and culture supernatant. We have identified well-known virulence factors from both AIEC and ETEC, thus validating our experimental approach. In addition we find proteins that are not unique to the pathogenic strains but expressed at levels different from the commensal strain, including the colonization factor YghJ and the surface adhesin antigen 43, which is involved in pathogenesis of other Gram-negative bacteria. The described method provides a framework for further understanding E. coli pathogenesis but can also be applied to interrogate relative protein expression levels of other pathogens that have non-pathogenic counterparts thereby facilitating the discovery of new vaccine targets.

Glycosylation of the Escherichia coli TibA self-associating autotransporter influences the conformation and the functionality of the protein.

The self-associating autotransporters (SAATs) are multifunctional secreted proteins of Escherichia coli, comprising the AIDA-I, TibA and Ag43 proteins. One of their characteristics is that they can be glycosylated. Glycosylation of AIDA-I and Ag43 have been investigated, but not that of TibA. It is still not clear whether glycosylation of the SAATs affect their structure or their functionality. Therefore, we have looked at the effects of glycosylation on the TibA adhesin/invasin. TibA is glycosylated by TibC, a specific glycosyltransferase, and the two genes are encoded in an operon. In this study, we have found that the glycosylation of TibA is not limited to the extracellular functional domain, as previously observed with AIDA-I and Ag43. We have determined that unglycosylated TibA is not able to promote the adhesion of bacteria on cultured epithelial cell, even though it is still able to promote invasion, biofilm formation and autoaggregation of bacteria. We have purified the glycosylated and unglycosylated forms of TibA, and determined that TibA is less stable when not glycosylated. We finally observed that glycosylation affects the oligomerisation of TibA and that unglycosylated TibA is locked in a conformation that is not suited for adhesion. Our results suggest that the effect of glycosylation on the functionality of TibA is indirect.

Purification, crystallization and preliminary X-ray crystallographic analysis of the transport unit of the monomeric autotransporter AIDA-I from Escherichia coli.

The adhesin involved in diffuse adherence (AIDA-I) from Escherichia coli belongs to the group of autotransporters, specifically the type Va secretion system (T5aSS). All autotransporter systems contain a C-terminal ß-domain, which forms a barrel-like structure in the outer membrane with a hydrophilic pore allowing passenger translocation across the outer membrane. The passenger domain harbours the biological activity in the extracellular space and functions, for example, as an adhesin, an enzyme and a toxin. The exact transport mechanism of passenger translocation across the outer membrane is not clear at present. Thus, structure determination of the transport unit of AIDA-I could provide new insights into the transport mechanism. Here, the purification, crystallization and preliminary X-ray crystallographic studies of the transport unit of AIDA-I are reported.

Production of a subunit vaccine candidate against porcine post-weaning diarrhea in high-biomass transplastomic tobacco.

Post-weaning diarrhea (PWD) in piglets is a major problem in piggeries worldwide and results in severe economic losses. Infection with Enterotoxigenic Escherichia coli (ETEC) is the key culprit for the PWD disease. F4 fimbriae of ETEC are highly stable proteinaceous polymers, mainly composed of the major structural subunit FaeG, with a capacity to evoke mucosal immune responses, thus demonstrating a potential to act as an oral vaccine against ETEC-induced porcine PWD. In this study we used a transplastomic approach in tobacco to produce a recombinant variant of the FaeG protein, rFaeG(ntd/dsc), engineered for expression as a stable monomer by N-terminal deletion and donor strand-complementation (ntd/dsc). The generated transplastomic tobacco plants accumulated up to 2.0 g rFaeG(ntd/dsc) per 1 kg fresh leaf tissue (more than 1% of dry leaf tissue) and showed normal phenotype indistinguishable from wild type untransformed plants. We determined that chloroplast-produced rFaeG(ntd/dsc) protein retained the key properties of an oral vaccine, i.e. binding to porcine intestinal F4 receptors (F4R), and inhibition of the F4-possessing (F4+) ETEC attachment to F4R. Additionally, the plant biomass matrix was shown to delay degradation of the chloroplast-produced rFaeG(ntd/dsc) in gastrointestinal conditions, demonstrating a potential to function as a shelter-vehicle for vaccine delivery. These results suggest that transplastomic plants expressing the rFaeG(ntd/dsc) protein could be used for production and, possibly, delivery of an oral vaccine against porcine F4+ ETEC infections. Our findings therefore present a feasible approach for developing an oral vaccination strategy against porcine PWD.

Adhesin-encoding genes from shiga toxin-producing Escherichia coli are more prevalent in atypical than in typical enteropathogenic E. coli.

Four of six adhesin-encoding genes (lpfA, paa, iha, and toxB) from Shiga toxin-producing Escherichia coli strains were detected in typical and atypical enteropathogenic E. coli (EPEC) strains of various serotypes. Although the most prevalent gene was lpfA in both groups, paa was the only potential diarrhea-associated gene in atypical EPEC.

Conformation change in a self-recognizing autotransporter modulates bacterial cell-cell interaction.

Bacteria mostly live as multicellular communities, although they are unicellular organisms, yet the mechanisms that tie individual bacteria together are often poorly understood. The adhesin involved in diffuse adherence (AIDA-I) is an adhesin of diarrheagenic Escherichia coli strains. AIDA-I also mediates bacterial auto-aggregation and biofilm formation and thus could be important for the organization of communities of pathogens. Using purified protein and whole bacteria, we provide direct evidence that AIDA-I promotes auto-aggregation by interacting with itself. Using various biophysical and biochemical techniques, we observed a conformational change in the protein during AIDA-AIDA interactions, strengthening the notion that this is a highly specific interaction. The self-association of AIDA-I is of high affinity but can be modulated by sodium chloride. We observe that a bile salt, sodium deoxycholate, also prevents AIDA-I oligomerization and bacterial auto-aggregation. Thus, we propose that AIDA-I, and most likely other similar autotransporters such as antigen 43 (Ag43) and TibA, organize bacterial communities of pathogens through a self-recognition mechanism that is sensitive to the environment. This could permit bacteria to switch between multicellular and unicellular lifestyles to complete their infection.

The prevalence of virulence genes of E. coli strains isolated from children with urinary tract infection.

To evaluate the prevalence of virulence genes in E. coli strains isolated from urine samples of children with urinary tract infection(UTI) and their correlation with clinical data, we isolated E. coli strains from urine samples of children with UTI during the period of August 2005 - August 2006 and studied them for the presence of the virulence genes by PCR. A total of 96 E. coli strains were isolated. The prevalence of genes, pyelonephritis associated pili (pap genes), S-family adhesions (sfa gene), hemolysin (hly gene), and cytotoxic nercotizing factor type 1 (cnf-1-1 gene) among the isolated strains was 27.1%, 14.6%, 13.5% and 22.9 %, respectively. Pyelonephritis was more prevalent in the cases with positive virulence genes. The results showed significant correlation between age of the patient and the presence of the genes (P< 0.05). Cnf-1 gene was significantly more common in samples of patients with abnormal finding on the ultrasound of kidneys (P= 0.049). Our study demonstrated higher prevalence of pyelonephritis in the presence of E. coli virulence genes. Detection of the genes in urine samples may help in the management of UTI.

The major pilin subunit of the AAF/II fimbriae from enteroaggregative Escherichia coli mediates binding to extracellular matrix proteins.

Enteroaggregative Escherichia coli (EAEC) adherence to human intestinal tissue is mediated by aggregative adherence fimbriae (AAF); however, the receptors involved in EAEC adherence remain uncharacterized. Adhesion to extracellular matrix proteins is commonly observed among enteric pathogens, so we addressed the hypothesis that EAEC may bind to extracellular matrix proteins commonly found in the intestine. We found that EAEC prototype strain 042 adhered more abundantly to surfaces that were precoated with the extracellular matrix proteins fibronectin, laminin, and type IV collagen. Differences in fibronectin binding of almost 2 orders of magnitude were observed between EAEC 042 and a mutant in the AAF/II major pilin gene, aafA. Purified AafA, refolded as a donor strand complementation construct, bound fibronectin in a dose-dependent manner. Addition of fibronectin to the apical surfaces of polarized T84 cell monolayers augmented EAEC 042 adherence, and this effect required expression of aafA. Finally, increased bacterial adherence was observed when apical secretion of fibronectin was induced by adenosine in polarized T84 cells. Binding to fibronectin may contribute to colonization of the gastrointestinal tract by EAEC.

Binding of Dr adhesins of Escherichia coli to carcinoembryonic antigen triggers receptor dissociation.

Carcinoembryonic antigen (CEA)-related cell adhesion molecules (CEACAMs) are host receptors for the Dr family of adhesins of Escherichia coli. To define the mechanism for binding of Dr adhesins to CEACAM receptors, we carried out structural studies on the N-terminal domain of CEA and its complex with the Dr adhesin. The crystal structure of CEA reveals a dimer similar to other dimers formed by receptors with IgV-like domains. The structure of the CEA/Dr adhesin complex is proposed based on NMR spectroscopy and mutagenesis data in combination with biochemical characterization. The Dr adhesin/CEA interface overlaps appreciably with the region responsible for CEA dimerization. Binding kinetics, mutational analysis and spectroscopic examination of CEA dimers suggest that Dr adhesins can dissociate CEA dimers prior to the binding of monomeric forms. Our conclusions include a plausible mechanism for how E. coli, and perhaps other bacterial and viral pathogens, exploit CEACAMs. The present structure of the complex provides a powerful tool for the design of novel inhibitory strategies to treat E. coli infections.

Functional organization of the autotransporter adhesin involved in diffuse adherence.

The Escherichia coli adhesin involved in diffuse adherence (AIDA-I) is a multifunctional autotransporter protein that mediates bacterial aggregation and biofilm formation, as well as adhesion and invasion of cultured epithelial cells. To elucidate the structure-function relationships of AIDA-I, we performed transposon-based linker scanning mutagenesis and constructed mutants with site-directed deletions. Twenty-nine different mutants with insertions that did not affect protein expression were obtained. Eleven mutants were deficient for one or two but not all of the functions associated with the expression of AIDA-I. Functional characterization of the transposon mutants and of an additional deletion mutant suggested that the N-terminal third of mature AIDA-I is involved in binding of this protein to cultured epithelial cells. The purified product of the putative domain could bind to cultured epithelial cells, confirming the importance of this region in adhesion. We also identified several different mutants in which invasion and adhesion were changed to different extents and two mutants in which autoaggregation and biofilm formation were also affected differently. These results suggest that although conceptually linked, adhesion and invasion, as well as autoaggregation and biofilm formation, are phenomena that may rely on distinct mechanisms when they are mediated by AIDA-I. This study sheds new light on the workings of a protein belonging to an emerging family of strikingly versatile virulence factors.

Quaternary structure of a SPATE autotransporter protein.

The temperature-sensitive hemagglutinin (Tsh) is a representative of the growing subfamily of secreted bacterial virulence factors, known as serine protease autotransporters of the Enterobacteriaceae (SPATEs). Expressed by avian and human pathogenic strains of Escherichia coli Tsh acts as a serine protease and an adhesin to erythrocytes, hemoglobin, and extracellular matrix proteins. Mature Tsh is comprised of a 106-kDa secreted domain (Tshs) and a 33-kDa outer membrane beta-domain (Tshbeta). Based on the size of beta-domains and functional properties of their passenger domains, all SPATEs are considered to be conventional autotransporters. However, it is unsettled if the conventional autotransporters exist as monomers, oligomers, or multimers (e.g., hexamers). To determine the quaternary structure of Tsh in vitro, we purified Tshbeta from the outer membranes and showed that it is natively folded because it is heat modifiable and resistant to protease digestion. Blue-native polyacrylamide gel electrophoresis of Tshbeta indicated that Tshbeta exists as a monomer or a dimer. The cross-linking analysis demonstrated that purified Tshbeta exists as a monomer. The size-exclusion chromatography and cross-linking analyses of purified Tshs also showed that the passenger domain of Tsh is a monomer. Overall, our data indicated that Tsh is a monomeric protein in vitro and support the concept that the SPATE autotransporters exist as monomers rather than as multimers. Implications of our findings on the mechanism of autotransporter secretion across the outer membrane are discussed.

Characterization of adhesin variants in Indian isolates of enteroaggregative Escherichia coli.

Enteroaggregative Escherichia coli (EAEC) are causative agents of diarrhea, being characterized by aggregative adherence to cultured epithelial cells. In this study, phenotypic properties of EAEC were analyzed with respect to AA, hemagglutination, clump and biofilm formation, all of which are mediated by aggregative adherence fimbriae (AAF). The strains were also screened for AAF types, AAF adhesin variants and Dr adhesin by PCR. Of the three known AAF types, AAF/I and AAF/II adhesin variants were identified. An association between the AAF/adhesin genotypes and the subtypes/scores of phenotypic properties was sought and it was observed that strains harboring same adhesins displayed different subtypes/scores and vice versa.

Characterization and immuno-detection of AIDA-I adhesin isolated from porcine Escherichia coli.

A relatively high percentage of porcine Escherichia coli isolates from cases associated with neonatal and post-weaning diarrhea are positive for the gene encoding adhesin involved in diffuse adhesion I (AIDA-I). This gene and its corresponding protein were first identified and characterized in E. coli strain 2787 isolated from human infantile diarrhea. Little is known about the properties of the AIDA-I protein and its immuno-detection on surface of AIDA-I positive porcine E. coli isolates. In this study, we demonstrated that the AIDA-I adhesin isolated from porcine AIDA-I positive E. coli is an acidic protein consisting of five isoforms. It has a similar molecular weight (100 kDa) and relatively high amino acid homology (78-87%) with the AIDA-I adhesin expressed by human AIDA-I positive E. coli strain 2787. Based on limited comparison, it appears that there is a very high homology among AIDA-I proteins expressed by porcine AIDA-I positive E. coli isolates. Sensitivity of detection of surface AIDA-I adhesin of PCR-positive AIDA-I E. coli by immuno-dot-blot and coagglutination tests was 76 and 71%, respectively, whereas specificity was 89 and 84%, respectively. These tests are unlikely to be used for diagnostic detection of AIDA-I positive E. coli due to the relatively low sensitivity; however, they may be potentially useful for identification of false positive reactions generated by other diagnostic tests.

Barriers to folding of the transmembrane domain of the Escherichia coli autotransporter adhesin involved in diffuse adherence.

Adhesin involved in diffuse adherence (AIDA) is an autotransporter protein that confers the diffuse adherence phenotype to certain diarrheagenic Escherichia coli strains. It consists of a 49 amino acid signal peptide, a 797 amino acid passenger domain, and a 440 amino acid beta-domain integrated in the outer membrane. The beta-domain consists of two parts: the beta(1)-domain, which is predicted to form two beta-strands on the bacterial cell surface, and the beta(2)-domain, which constitutes the transmembrane domain. We here present a detailed biophysical analysis of the AIDA beta-domain addressing its refolding properties and its different conformational states and their stability. We find that the beta(2)-domain in solution can fold only when the beta(1)-domain is present and only with 50% efficiency. However, 100% refolding of the beta(2)-domain, with or without the beta(1)-domain, can be achieved in the presence of a solid support. Folding can only take place above the cmc of the detergent used, but the refolded state is retained if diluted below the cmc, revealing a kinetic barrier to dissociation of the detergent molecules from the folded protein. Refolding attempts of the beta(2)-domain in the absence of a solid support result in the formation of an oligomeric misfolded state both in the absence and in the presence of detergent. Despite being misfolded, these states unfold cooperatively with a T(m) approximately 70 degrees C. The refolded protein in the nonionic detergent octylpolyoxyethylene (oPOE) can only be thermally unfolded in the presence of SDS. The linear relationship between SDS mole fraction and unfolding temperature, T(m), predicts a T(m) of 112.9 +/- 1.2 degrees C for the beta(2)-domain and 132.7 +/- 12.2 degrees C for the entire beta-domain in pure oPOE. Thus, the beta(1)-domain also stabilizes the beta(2)-domain. In conclusion, our data show that the in vitro refolding of the AIDA beta-domain is critically dependent on a solid support, suggesting that in vivo specific biological factors may assist in folding the protein correctly into the outer membrane to avoid the formation of stably misfolded conformations.

Functional analysis of the Tsh autotransporter from an avian pathogenic Escherichia coli strain.

The temperature-sensitive hemagglutinin (Tsh) is an autotransporter protein secreted by avian-pathogenic Escherichia coli strains that colonize the respiratory tract and lead to airsacculitis, pericarditis, and colisepticemia. It is synthesized as a 140-kDa precursor protein, whose processing results in a 106-kDa passenger domain (Tshs) and a 33-kDa beta-domain (Tsh(beta)). The presence of a conserved 7-amino-acid serine protease motif within Tshs classifies the protein in a subfamily of autotransporters, known as serine protease autotransporters of the Enterobacteriaceae. In this study, we report that purified Tshs is capable of adhering to red blood cells, hemoglobin, and the extracellular matrix proteins fibronectin and collagen IV. We also demonstrate that Tshs exerts proteolytic activity against casein, and we provide experimental evidence demonstrating that serine 259 is essential for the protease function. However, this residue is not required for adherence to substrates, and its replacement by an alanine does not abolish binding activity. In summary, our results demonstrate that Tsh is a bifunctional protein with both adhesive and proteolytic properties.

Recognition of the cellular beta1-chain integrin by the bacterial AfaD invasin is implicated in the internalization of afa-expressing pathogenic Escherichia coli strains.

The afa operons from Escherichia coli associated with extra-intestinal and intestinal infections have been characterized and the AfaD protein has been shown to be involved in the low internalization of laboratory strains expressing the afa-3 operon. The aim of this study was to determine the role of the AfaD invasin during the interaction of pathogenic E. coli with epithelial cells. We show that AfaD is implicated in the entry of a clinical isolate into both HeLa and undifferentiated Caco-2 cells. Once in the cytoplasm of these cells, the bacteria formed inclusions in which they were able to survive for at least 72 h. Internalization assays using polystyrene beads coated with His6-tagged purified AfaD (rAfaD) demonstrated that this invasin mediates entry into cells derived from various tissues (intestine and urothelium) that are targets for afa-positive strains. Consistent with the previous observation that an antibody blockade involving anti-alpha5beta1 integrin abolishes bacterial internalization, we show here that the entry of rAfaD-coated beads was dependent on the production and accessibility of beta1 integrins on the cells. The AfaD proteins belong to a family of invasins that are at least 45% identical. Despite their differences, the recombinant rAfaD-III and rAfaD-VIII proteins both bound to beta1 integrins. Our results suggest that beta1 integrin is a common receptor for AfaD invasins and that additional AfaD-type-specific receptors exist.

Occurrence of the temperature-sensitive hemagglutinin among avian Escherichia coli.

In this study, we determined the occurrence of the tsh gene among 305 Escherichia coli isolates from chickens by means of the polymerase chain reaction and agglutination of chicken erythrocytes; 200 of those isolates were obtained from chickens with colisepticemia, 52 isolates were from lesions of cellulitis, and 53 were from feces of normal chickens. The tsh gene was found in 79 (39.5%) isolates from colisepticemia, in 10 (19%) cellulitis-derived E. coli isolates, and in two (3.8%) fecal isolates. Among the tsh+ strains, 68 (86%) isolates from colisepticemia and nine (90%) from cellulitis agglutinated chicken erythrocytes in the presence of mannose, after growing the strains on colonization factor antigen agar plates at 26 C, which confirms a correlation between mannose-resistant hemagglutination and expression of hemagglutinin Tsh. These results show, for the first time, the presence of the gene tsh in cellulitis-derived E. coli isolates; the high frequency of this gene among avian pathogenic E. coli isolates in Brazil indicates that its putative role as a virulence factor should be studied more thoroughly.

Afa, a diffuse adherence fibrillar adhesin associated with enteropathogenic Escherichia coli.

O55 is one of the most frequent enteropathogenic Escherichia coli (EPEC) O serogroups implicated in infantile diarrhea in developing countries. Multilocus enzyme electrophoresis analysis showed that this serogroup includes two major electrophoretic types (ET), designated ET1 and ET5. ET1 corresponds to typical EPEC, whilst ET5 comprises strains with different combinations of virulence genes, including those for localized adherence (LA) and diffuse adherence (DA). Here we report that ET5 DA strains possess a DA adhesin, designated EPEC Afa. An 11.6-kb chromosomal region including the DA adhesin operon from one O55:H(-) ET5 EPEC strain was sequenced and found to encode a protein with 98% identity to AfaE-1, an adhesin associated with uropathogenic E. coli. Although described as an afimbrial adhesin, we show that both AfaE-1 and EPEC Afa possess fine fibrillar structures. This is the first characterization and demonstration of an Afa adhesin associated with EPEC.

The major structural subunits of Dr and F1845 fimbriae are adhesins.

Fimbrial adhesins mediate the attachment of pathogenic Escherichia coli to various host tissues leading to the development of disease. The Dr hemagglutinin and F1845 fimbriae belong to the Dr family of adhesins, which is associated with urinary tract infections and diarrheal disease. These adhesins bind to the Dr(a) blood-group antigen present on decay-accelerating factor (DAF). The Dr hemagglutinin is unique in this family since it also binds to type IV collagen and its binding is inhibited by the presence of chloramphenicol. We have purified the major structural subunits of Dr and F1845 fimbriae, DraE and DaaE, as fusions to maltose-binding protein and to oligohistidine tags and examined their binding to erythrocytes, Chinese hamster ovary cell transfectants expressing DAF, and a DAF fusion protein. The DraE and DaaE fusion proteins bind to the DAF receptor in a specific manner resembling the distinct phenotypes of the corresponding Dr and F1845 fimbriae. In contrast to binding studies with the DAF receptor, the DraE fusion proteins did not bind to type IV collagen.

Genotypic prevalence of the fimbrial adhesins (F4, F5, F6, F41 and F18) and toxins (LT, STa, STb and STx2e) in Escherichia coli isolated from postweaning pigs with diarrhoea or oedema disease in Korea.

A PCR was used to determine the genotypic prevalence of five fimbrial adhesins (F4, F5, F6, F41 and F18), two heat-stable enterotoxins (STa and STb), the heat-labile enterotoxin (LT), and the shiga toxin 2e (Stx2e) in 230 isolates of Escherichia coli from postweaning pigs with diarrhoea or oedema disease. Ninety-four (40.9 per cent) of the isolates carried genes for at least one of the fimbrial adhesins or toxins. Genes for the F18 fimbrial adhesin were detected in 18.3 per cent, and genes for F4, F6, F5 and F41 were detected in 10.0 per cent, 4.3 per cent, 1.7 per cent and 0.8 per cent of the isolates, respectively. Genes for STa, STb and LT were detected in 25.7 per cent, 15.2 per cent and 8.7 per cent of the isolates, respectively. Genes for Stx2e were detected in 36 (15.6 per cent) of the isolates, and among them 24 also contained the gene for F18ab and four also contained the gene for F18ac.