Growth advantage and enhanced toxicity of Escherichia coli adherent to tissue culture cells due to restricted diffusion of products secreted by the cells.

Zafriri, D; Oron, Yoram; Eisenstein, B I; Ofek, Itzhak

doi:10.1172/jci112939

Cited by 86 publications

(41 citation statements)

References 15 publications

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“…Adhesion of bacteria to the target epithelium is felt to be an essential prerequisite for toxin delivery by ETEC (39) and, indeed, earlier in vitro studies demonstrated that both flagellar motility and the intimate interaction of ETEC with host cells were strict requirements for LT-mediated activation of cAMP (10). The present experiments demonstrate that EtpA, thought to mediate adhesion by acting as a molecular bridge between ETEC flagella and host cells (30), likewise plays an important role in the delivery of heatlabile toxin.…”

Section: Discussionmentioning

confidence: 99%

“…The involvement of EtpA in toxin delivery by ETEC suggests that generating antibodies against this adhesin could complement existing approaches, including the direct neutralization of toxin. Interestingly, there is scant existing data regarding whether anti-LT antibodies can neutralize toxin as delivered by the bacteria, and earlier published reports had suggested that ETEC can deliver toxin in a way that circumvents neutralization (23,39). Therefore, we conducted additional in vitro experiments to examine the utility of both antiEtpA and anti-LT antibodies in mitigating toxin delivery to target intestinal epithelial cells.…”

Section: Etpa Is Required For Optimal Delivery Of Lt To Epithelial Cementioning

confidence: 99%

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Cooperative Role of Antibodies against Heat-Labile Toxin and the EtpA Adhesin in Preventing Toxin Delivery and Intestinal Colonization by Enterotoxigenic Escherichia coli

Roy

Hamilton

Fleckenstein

2012

Clin. Vaccine Immunol.

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dEnterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease in developing countries, where it is responsible for hundreds of thousands of deaths each year. Vaccine development for ETEC has been hindered by the heterogeneity of known molecular targets and the lack of broad-based sustained protection afforded by existing vaccine strategies. In an effort to explore the potential role of novel antigens in ETEC vaccines, we examined the ability of antibodies directed against the ETEC heat-labile toxin (LT) and the recently described EtpA adhesin to prevent intestinal colonization in vivo and toxin delivery to epithelial cells in vitro. We demonstrate that EtpA is required for the optimal delivery of LT and that antibodies against this adhesin play at least an additive role in preventing delivery of LT to target intestinal cells when combined with antibodies against either the A or B subunits of the toxin. Moreover, vaccination with a combination of LT and EtpA significantly impaired intestinal colonization. Together, these results suggest that the incorporation of recently identified molecules such as EtpA could be used to enhance current approaches to ETEC vaccine development. Enterotoxigenic Escherichia coli (ETEC) strains comprise a genetically and phenotypically diverse group of organisms that share the ability to produce and effectively deliver heat-labile (LT) and/or heat-stable (ST) toxins to effector sites in the small intestine (14). Effective delivery of these toxins results in increases of intracellular concentrations of cyclic nucleotides, cyclic AMP (cAMP) and cGMP, respectively. Both toxins stimulate cellular kinases responsible for the phosphorylation of the cystic fibrosis transmembrane regulatory channel (CFTR) in the cell membrane (7,17). Activation of the CFTR in turn leads to efflux of chloride into intestinal lumen with commensurate salt and water losses responsible for the watery diarrhea associated with these pathogens.ETEC strains are a leading cause of diarrheal illness in developing countries, where they are responsible for hundreds of thousands of deaths, largely among young children. Although ETEC strains were discovered more than 40 years ago, the development of a broadly protective vaccine has been hampered by a number of factors (5, 35), including (i) the lack of complete sustained protection afforded by anti-LT immunity, (ii) the poor inherent immunogenicity of ST molecules, typically short peptides, and (iii) the antigenic heterogeneity of plasmid-encoded fimbrial colonization factors (20), one of the principle targets of ETEC vaccines to date.Despite these challenges, the development of immunity following either a naturally occurring (4, 34) or an experimental (22) infection with ETEC has suggested that the development of an ETEC vaccine is technically feasible. In addition, recent molecular (30, 31) and immunoproteomic (26) studies have demonstrated that the pathogenesis of these organisms is considerably more complex than previously appreciated, and conseque...

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Section: Discussionmentioning

confidence: 99%

Section: Etpa Is Required For Optimal Delivery Of Lt To Epithelial Cementioning

confidence: 99%

Cooperative Role of Antibodies against Heat-Labile Toxin and the EtpA Adhesin in Preventing Toxin Delivery and Intestinal Colonization by Enterotoxigenic Escherichia coli

Roy

Hamilton

Fleckenstein

2012

Clin. Vaccine Immunol.

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“…In vitro, fimbriae may be positively selected for by growth in static broth, whereas fimbriate cells grow more slowly than their afimbriate counterparts in aerated broth or on agar plates (7,8,14,17,21,28,34). In vivo, fimbriae may promote bacterial colonization, which in turn may be beneficial for bacterial growth (33,44), or they may promote killing of the bacteria (32) or clearance of the bacteria from the host (26). The two mechanisms of phase variation may cooperate to avoid the inappropriate expression of fimbriae that could result in dire consequences for the cell.…”

Section: Discussionmentioning

confidence: 99%

Inversion-independent phase variation of type 1 fimbriae in Escherichia coli

et al. 1993

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The roles of fimB and fimE in the phase-variable expression of type 1 fimbriae in Escherichia coli were examined. A method was developed to study the effects offimB and fimE on both recombination of the fim invertible element and fimbrial expression. The method used an allelic exchange procedure consisting of two steps. The first step, construction of intermediate strains, deletedfimB andfimE. This step locked the invertible element in either the on or the off orientation. The second step of the exchange procedure introduced either wild-type or mutant alleles offimB and/orfimE into the chromosome of the intermediate strains. Analysis of the resulting strains supported the current, plasmid-based model of recombination. Unexpectedly, strains in which the invertible element was locked in the on orientation (either by mutation of bothfimB andfimE or, in a control strain, by mutation of the left inverted repeat sequence of the invertible element) continued to exhibit phase-variable expression of type 1 fimbriae. A strain in which fimA was transcribed from the tac promoter continued to exhibit phase-variable fimbrial expression, suggesting that inversion-independent phase variation cannot be explained by variable transcription initiation offimA.The expression of type 1 fimbriae in Escherichia coli exhibits on-off phase variation. This phase variation correlates with the orientation of a short, invertible DNA sequence located immediately upstream of finiA, the gene encoding the major fimbrial subunit (1, 15, 18). The promoter for fimA is believed to reside within this invertible element and to direct the transcription offimA when the element is in one orientation ("on") but not when the element is in the alternate orientation ("off'). Furthermore, the orientation of thefim invertible element correlates with the Fim phenotype (8, 18).Two genes, fimB and fimE, map immediately adjacent to the fim invertible element and are believed to encode sitespecific recombinases (8,24,29,35,36,38). The current model of the roles of fimB andfimE in inversion suggests that fimB promotes recombination of thefim invertible element in both directions, whereas fimE promotes recombination primarily from on to off (29). However, this model is based on a recombinant plasmid-based assay with which fimbrial expression cannot be examined (29).In the present study, the roles of fimB and fimE in the phase-variable expression of type 1 fimbriae were examined by use of a method that preserved the native location, organization, stoichiometry, and topology of the recombination elements. Thus, in contrast to the plasmid-based assay (29), in this assay both recombination and fimbrial expression could be examined. Analysis of the recombination results supported the current, plasmid-based model of the roles offimB andfimE. Additionally, examination of fimbrial production indicated that the on orientation of the fim invertible element was necessary but not sufficient for fimbrial expression; strains in which the invertible element was * Corresponding autho...

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“…Effective toxin delivery is thought to occur upon colonization of the small intestine and likely requires intimate association of the organism with target epithelial cells (15,45) of the intestinal mucosa.…”

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confidence: 99%

The EtpA Exoprotein of Enterotoxigenic Escherichia coli Promotes Intestinal Colonization and Is a Protective Antigen in an Experimental Model of Murine Infection

Roy

Hamilton²,

Allen³

et al. 2008

Infect Immun

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The enterotoxigenic Escherichia coli (ETEC) strains are major causes of morbidity and mortality due to diarrheal illness in developing countries. At present, there is no broadly protective vaccine for this diverse group of pathogens. The EtpA protein, identified in ETEC H10407 in a recent search for candidate immunogens, is a large glycosylated exoprotein secreted via two-partner secretion (TPS). Similar to structurally related molecules, EtpA functions in vitro as an adhesin. The studies reported here use a recently developed murine model of ETEC intestinal colonization to examine the immunogenicity and protective efficacy of EtpA. We report that mice repeatedly exposed to ETEC are protected from subsequent colonization and that they mount immune responses to both EtpA and its presumed two-partner secretion transporter (EtpB) during the course of experimental infection. Furthermore, isogenic etpA deletion mutants were impaired in the colonization of mice, and intranasal immunization of mice with recombinant EtpA conferred protection against ETEC H10407 in this model. Together, these data suggest that EtpA is required for optimal colonization of the intestine, findings paralleling those of previous in vitro studies demonstrating its role in adherence. EtpA and other TPS proteins may be viable targets for ETEC vaccine development.

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Growth advantage and enhanced toxicity of Escherichia coli adherent to tissue culture cells due to restricted diffusion of products secreted by the cells.

Cited by 86 publications

References 15 publications

Cooperative Role of Antibodies against Heat-Labile Toxin and the EtpA Adhesin in Preventing Toxin Delivery and Intestinal Colonization by Enterotoxigenic Escherichia coli

Cooperative Role of Antibodies against Heat-Labile Toxin and the EtpA Adhesin in Preventing Toxin Delivery and Intestinal Colonization by Enterotoxigenic Escherichia coli

Inversion-independent phase variation of type 1 fimbriae in Escherichia coli

The EtpA Exoprotein of Enterotoxigenic Escherichia coli Promotes Intestinal Colonization and Is a Protective Antigen in an Experimental Model of Murine Infection

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