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Development and accuracy of quantitative real-time polymerase chain reaction assays for detection and quantification of enterotoxigenic Escherichia coli (ETEC) heat labile and heat stable toxin genes in travelers' diarrhea samples.

ABSTRACT: Enterotoxigenic Escherichia coli (ETEC), the leading bacterial pathogen of travelers' diarrhea, is routinely detected by an established DNA hybridization protocol that is neither sensitive nor quantitative. Quantitative real-time polymerase chain reaction (qPCR) assays that detect the ETEC toxin genes eltA, sta1, and sta2 in clinical stool samples were developed and tested using donor stool inoculated with known quantities of ETEC bacteria. The sensitivity of the qPCR assays is 89%, compared with 22% for the DNA hybridization assay, and the limits of detection are 10,000-fold lower than the DNA hybridization assays performed in parallel. Ninety-three clinical stool samples, previously characterized by DNA hybridization, were tested using the new ETEC qPCR assays. Discordant toxin profiles were observed for 22 samples, notably, four samples originally typed as ETEC negative were ETEC positive. The qPCR assays are unique in their sensitivity and ability to quantify the three toxin genes in clinical stool samples.

SUBMITTER: Youmans BP

PROVIDER: S-EPMC3886408 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Development and accuracy of quantitative real-time polymerase chain reaction assays for detection and quantification of enterotoxigenic Escherichia coli (ETEC) heat labile and heat stable toxin genes in travelers' diarrhea samples.

Youmans Bonnie P BP Ajami Nadim J NJ Jiang Zhi-Dong ZD Petrosino Joseph F JF DuPont Herbert L HL Highlander Sarah K SK

The American journal of tropical medicine and hygiene 20131104 1

Enterotoxigenic Escherichia coli (ETEC), the leading bacterial pathogen of travelers' diarrhea, is routinely detected by an established DNA hybridization protocol that is neither sensitive nor quantitative. Quantitative real-time polymerase chain reaction (qPCR) assays that detect the ETEC toxin genes eltA, sta1, and sta2 in clinical stool samples were developed and tested using donor stool inoculated with known quantities of ETEC bacteria. The sensitivity of the qPCR assays is 89%, compared wit ...[more]

PMID: 24189361

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Project description:Enterotoxigenic Escherichia coli (ETEC) strains producing heat-labile toxin (LT) and/or heat-stable toxin (STa) are a top cause of children's diarrhea and travelers' diarrhea. Holotoxin-structured GM1-binding LT is a strong immunogen and an effective adjuvant, and can serve a carrier or a platform for multivalent vaccine development. However, the significance of peptide domains or epitopes of LT particularly enzymatic LTA subunit in association with LT enterotoxicity and immunogenicity has not been characterized. In this study, we identified B-cell epitopes in silico from LTA subunit and examined epitopes for immunogenicity and association with LT enterotoxicity. Epitopes identified from LTA subunit were individually fused to a modified chicken ovalbumin carrier protein, and each epitope-ovalbumin fusion was used to immunize mice. Data showed all 11 LTA epitopes were immunogenic; epitope 7 (105SPHPYEQEVSA115) induced greater titers of anti-LT antibodies which neutralized LT enterotoxicity more effectively. To examine these epitopes for the significance in LT enterotoxicity, we constructed LT mutants by substituting each of 10 epitopes at the toxic A1 domain of LTA subunit with a foreign epitope and examined LT mutants for enterotoxicity and GM1-binding activity. Data showed that LT mutants exhibited no enterotoxicity but retained GM1-binding activity. The results from this study indicated that while not all immunodominant LTA epitopes were neutralizing, LT mutants with an individual epitope substituted lost enterotoxicity but retained GM1-binding activity. These results provided additional information to understand LT immunogenicity and enterotoxicity and suggested the potential application of LT platform for multivalent vaccines against ETEC diarrhea and other diseases.IMPORTANCE No vaccine is licensed for enterotoxigenic Escherichia coli (ETEC) strains, which remain a leading cause of diarrhea in children from developing countries and international travelers. GM1-binding heat-labile toxin (LT) which is a key virulence factor of ETEC diarrhea is a strong vaccine antigen and a self-adjuvant. LT can also serve a backbone or platform for MEFA (multiepitope fusion antigen), a newly developed structural vaccinology technology, to present heterogeneous epitopes (by replacing LT epitopes) and to mimic epitope antigenicity for development of broadly protective vaccines. Data from this study identified neutralizing LT epitopes and demonstrated that substitution of LT epitopes eliminated LT enterotoxicity without altering GM1-binding activity, suggesting LT is potentially a versatile MEFA platform to present heterogeneous epitopes for multivalent vaccines against ETEC and other pathogens.

Project description:Diarrheal illness contributes to malnutrition, stunted growth, impaired cognitive development, and high morbidity rates in children worldwide. Enterotoxigenic Escherichia coli (ETEC) is a major contributor to this diarrheal disease burden. ETEC cause disease in the small intestine by means of colonization factors and by production of a heat-labile enterotoxin (LT) and/or a small non-immunogenic heat-stable enterotoxin (ST). Overall, the majority of ETEC produce both ST and LT. LT induces secretion via an enzymatically active A-subunit (LT-A) and a pentameric, cell-binding B-subunit (LT-B). The importance of anti-LT antibodies has been demonstrated in multiple clinical and epidemiological studies, and a number of potential ETEC vaccine candidates have included LT-B as an important immunogen. However, there is limited information about the potential contribution of LT-A to development of protective immunity. In the current study, we evaluate the immune response against the A-subunit of LT as well as the A-subunit's potential as a protective antigen when administered alone or in combination with the B-subunit of LT. We evaluated human sera from individuals challenged with a prototypic wild-type ETEC strain as well as sera from individuals living in an ETEC endemic area for the presence of anti-LT, anti-LT-A and anti-LT-B antibodies. In both cases, a significant number of individuals intentionally or endemically infected with ETEC developed antibodies against both LT subunits. In addition, animals immunized with the recombinant proteins developed robust antibody responses that were able to neutralize the enterotoxic and cytotoxic effects of native LT by blocking binding and entry into cells (anti-LT-B) or the intracellular enzymatic activity of the toxin (anti-LT-A). Moreover, antibodies to both LT subunits acted synergistically to neutralize the holotoxin when combined. Taken together, these data support the inclusion of both LT-A and LT-B in prospective vaccines against ETEC.

Dataset Information

Development and accuracy of quantitative real-time polymerase chain reaction assays for detection and quantification of enterotoxigenic Escherichia coli (ETEC) heat labile and heat stable toxin genes in travelers' diarrhea samples.

Publications

Development and accuracy of quantitative real-time polymerase chain reaction assays for detection and quantification of enterotoxigenic Escherichia coli (ETEC) heat labile and heat stable toxin genes in travelers' diarrhea samples.

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