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

Roy, Koushik; Hamilton, D. R.; Allen, Kenneth P.; Randolph, Mildred P.; Fleckenstein, James M.

doi:10.1128/iai.01304-07

Cited by 63 publications

(60 citation statements)

References 41 publications

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“…5d). These results are similar to those previously demonstrated for EtpA (28,29), another antigen which is associated at least in part with vesicles (27). Together, these data suggest that OMV contain at least two protective antigens and that these structures potentially offer an effective strategy for immunization against ETEC infections.…”

Section: Resultssupporting

confidence: 79%

Outer Membrane Vesicles Induce Immune Responses to Virulence Proteins and Protect against Colonization by Enterotoxigenic Escherichia coli

Roy

Hamilton

Munson

et al. 2011

Clin Vaccine Immunol

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Enterotoxigenic Escherichia coli (ETEC) strains are a heterogeneous group of pathogens that produce heat-labile (LT) and/or heat-stable (ST) enterotoxins. Collectively, these pathogens are responsible for hundreds of thousands of deaths annually in developing countries, particularly in children under the age of 5 years. The heterogeneity of previously investigated molecular targets and the lack of complete sustained protection afforded by antitoxin immunity have impeded progress to date toward a broadly protective vaccine. Many pathogens, including ETEC, have the capacity to form outer membrane vesicles (OMV), which often contain one or more virulence proteins. Prompted by recent studies that identified several immunogenic virulence proteins in outer membrane vesicles of ETEC, we sought to examine the immunogenicity and protective efficacy of these structures in a murine model of infection. Here we demonstrate that immunization with OMV impairs ETEC colonization of the small intestine and stimulates antibodies that recognize the heat-labile toxin and two additional putative virulence proteins, the EtpA adhesin and CexE. Similar to earlier studies with EtpA, vaccination with LT alone also inhibited intestinal colonization. Together, these findings suggest that OMV could be exploited to deliver protective antigens relevant to development of ETEC vaccines.Enterotoxigenic Escherichia coli (ETEC) strains are a diverse group of diarrheal pathogens that share the ability to colonize the small intestine, where they produce heat-labile (LT) and/or heat-stable (ST) enterotoxins. These organisms are a leading cause of diarrhea in developing countries, where they are responsible for an estimated 300,000 to 500,000 deaths per year, mostly in young children (33).Unfortunately, there is no broadly protective vaccine available to prevent these infections (5). Although plasmid-encoded colonization factors (CFs) have been a major focus of vaccine development efforts for ETEC to date, the underlying plasticity of E. coli genomes (25) and the antigenic heterogeneity of CFs (5) have impeded vaccine strategies based solely on these antigens. Additional approaches that incorporate highly conserved molecular targets are therefore needed to focus future endeavors toward the design of effective vaccines.Recent immunoproteomic studies (27) identified a number of known and putative ETEC virulence factors associated with outer membrane vesicles (OMV), small spherical "blebs" released from the surfaces of E. coli and other . Given the established association of LT with OMV (16) and the potential utility of vesicle-based vaccines (4, 14, 31), we elected to examine the immunogenicity and protective efficacy of ETEC-derived OMV in an animal model of ETEC infection. MATERIALS AND METHODS Bacterial strains and plasmids.A complete list of bacterial strains and recombinant expression plasmids used in these experiments is included in Table 1.Preparation of outer membrane vesicles. Vesicles were prepared from culture supernatants of either strain H10...

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Section: Resultssupporting

confidence: 79%

Outer Membrane Vesicles Induce Immune Responses to Virulence Proteins and Protect against Colonization by Enterotoxigenic Escherichia coli

Roy

Hamilton

Munson

et al. 2011

Clin Vaccine Immunol

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“…However, the relatively large number of ETEC isolates without identifiable CFs found in this and other studies suggests that additional CFs may still be undetected, although new and interesting putative CFs have been proposed recently (9).…”

Section: Four Multiplex Pcr Assays For Detection Of 19 Enterotoxigenimentioning

confidence: 70%

Development of Multiplex PCR Assays for Detection of Enterotoxigenic Escherichia coli Colonization Factors and Toxins

et al. 2009

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Four multiplex PCR assays for detection of 19 enterotoxigenic Escherichia coli (ETEC) colonization factorsand an improved ETEC toxin multiplex PCR were developed and tested on Bangladeshi and Bolivian ETEC strain collections. The assays will be useful for surveillance of ETEC infections in diagnostic laboratories that have access to PCR.Infection with enterotoxigenic Escherichia coli (ETEC) is one of the main causes of childhood diarrhea in developing countries and in travelers to these areas (7). ETEC strains express one or both of two different enterotoxins, the heatstable toxins (STh and STp) and a heat-labile toxin (LT), and more than 23 different colonization factors (CFs) that are named CFA/I and CS1 to CS22 (2). The LT enterotoxin and the CF antigens are immunogenic, and one or both of these components are included in most vaccines that are being developed against ETEC diarrhea (14,15). To evaluate the putative impact of different vaccine compositions on ETEC diarrhea, it is important to characterize clinical ETEC isolates from different parts of the world with regard to toxin and CF profiles, since they have been observed to vary from one geographic region to another (4,7,8,10,12).In order to simplify and accelerate the detection of the different CFs and toxins by PCR, we have established a set of multiplex PCR assays for detection of the most-prevalent CFs and the toxins using previously established primers (11), as well as new primers when appropriate. The CF primers were assembled into four panels designed to amplify 19 of the most common CFs, and a new set of STh primers was included in the previously established toxin multiplex PCR (Table 1).To establish the multiplex assays for the CFs and toxins, 10-to 100-ng amounts of DNA from ETEC reference strains boiled in water (Table 1) were mixed with master mix as described previously (11), except that 400 nM deoxynucleoside triphosphates and 8 to 10 forward and reverse primers at a final concentration of 200 nM each were added to the mix. The PCRs were amplified by an initial denaturation at 94°C (1 min), followed by 35 cycles of amplification (94°C for 30 s, 52°C for 30 s, and 72°C for 1 min), and finally, 5 min at 72°C. The products were run on 3% agarose gels or 3% MetaPhor gels (MetaPhor agarose; Cambrex Bio Science Rockland, Inc.) for better resolution and visualized under UV light (Fig. 1). Only the correct CF and toxin PCR products were amplified, and by mixing several strains and performing multiplex PCR on the mixed DNA, we verified that the primers in each panel could detect the presence of several CFs in one reaction.To evaluate the new multiplex PCR methods for ETEC toxins and CFs, 106 clinical ETEC isolates were analyzed by multiplex PCR and the results were compared with results obtained by using the previously established ETEC toxin multiplex PCR and with the dot blot method using available monoclonal antibodies (11). We used two different sets of ETEC strains that were isolated in two geographically different developing countries: 65 Boli...

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“…Furthermore, there are reports suggesting that these proteins could be used as antigens to stimulate protective immunity against ETEC infection. Intranasal administration of purified EtpA conferred protection against subsequent ETEC H10407 infection in mice (25). Although the immunogenic potential of EatA has not been determined, proteins of the same family of autotransporters, such as Pic and Pet, are recognized by human sera obtained from infected patients (1).…”

Section: Discussionmentioning

confidence: 99%

Distribution of Classical and Nonclassical Virulence Genes in Enterotoxigenic Escherichia coli Isolates from Chilean Children and tRNA Gene Screening for Putative Insertion Sites for Genomic Islands

et al. 2011

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Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea. Three adhesins (Tia, TibA, EtpA), an iron acquisition system (Irp1, Irp2, and FyuA), a GTPase (LeoA), and an autotransporter (EatA) are ETEC virulence-related proteins that, in contrast to the classical virulence factors (enterotoxins and fimbrial colonization factors) have not heretofore been targets in characterizing isolates from epidemiological studies. Here, we determined the occurrence of these nonclassical virulence genes in 103 ETEC isolates from Chilean children with diarrhea and described their association with O serogroups and classical virulence determinants. Because tia, leoA, irp2, and fyuA are harbored by pathogenicity islands inserted into the selC and asnT tRNA genes (tDNAs), we analyzed the regions flanking these loci. Ten additional tDNAs were also screened to identify hot spots for genetic insertions. Associations between the most frequent serogroups and classical colonization factor (CF)-toxin profiles included O6/LT-STh/CS1-CS3-CS21 (i.e., O6 serogroup, heat-labile [LT] and human heat-stable [STh] enterotoxins, and CFs CS1, -3 and -21), O6/LT-STh/CS2-CS3-CS21, and O104-O127/STh/ CFAI-CS21. The eatA and etpA genes were detected in more than 70% of the collection, including diverse serogroups and virulence profiles. Sixteen percent of the ETEC strains were negative for classical and nonclassical adhesins, suggesting the presence of unknown determinants of adhesion. The leuX, thrW, and asnT tDNAs were disrupted in more than 65% of strains, suggesting they are hot spots for the insertion of mobile elements. Sequences similar to integrase genes were identified next to the thrW, asnT, pheV, and selC tDNAs. We propose that the eatA and etpA genes should be included in characterizations of ETEC isolates in future epidemiological studies to determine their prevalence in other geographical regions. Sequencing of tDNAassociated genetic insertions might identify new ETEC virulence determinants.Enterotoxigenic Escherichia coli (ETEC) causes nearly 400 million diarrhea episodes every year in children younger than 5 years of age and is responsible for approximately 50% of all traveler's diarrhea episodes (35). ETEC colonizes the smallbowel epithelium, producing heat-labile (LT) and/or heat-stable (ST) enterotoxins (7) that cause secretion of water and electrolytes. Toxin variants LT-I and STh are produced only by strains that infect humans. LT-II and STp are expressed by strains that infect mostly piglets, although they occasionally also infect humans (20).Other classical ETEC virulence determinants are the colonization factors (CFs), also referred to as coli surface antigens (20), adhesins that direct colonization of the small bowel epithelium. Currently, 22 different putative ETEC CFs have been identified, designated CS followed by a number depending on their order of discovery, with the exception of CFAI, which maintains its original denomination (10, 31). The most prevalent CFs associated with the occurrence of diarrhea worldwi...

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

Cited by 63 publications

References 41 publications

Outer Membrane Vesicles Induce Immune Responses to Virulence Proteins and Protect against Colonization by Enterotoxigenic Escherichia coli

Outer Membrane Vesicles Induce Immune Responses to Virulence Proteins and Protect against Colonization by Enterotoxigenic Escherichia coli

Development of Multiplex PCR Assays for Detection of Enterotoxigenic Escherichia coli Colonization Factors and Toxins

Distribution of Classical and Nonclassical Virulence Genes in Enterotoxigenic Escherichia coli Isolates from Chilean Children and tRNA Gene Screening for Putative Insertion Sites for Genomic Islands

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