Gastroenterology

Gastroenterology

Volume 130, Issue 6, May 2006, Pages 1688-1695
Gastroenterology

Basic–alimentary tract
A Recombinant Probiotic for Treatment and Prevention of Cholera

https://doi.org/10.1053/j.gastro.2006.02.005Get rights and content

Background & Aims: We have developed a therapeutic strategy based on molecular mimicry of host receptors for bacterial toxins on the surface of harmless gut bacteria. In the present study, this has been applied to the development of a recombinant probiotic for treatment and prevention of cholera, caused by Vibrio cholerae. Methods: We expressed glycosyltransferase genes from Neisseria gonorrhoeae and Campylobacter jejuni in a harmless Escherichia coli strain, resulting in production of a chimeric lipopolysaccharide terminating in a mimic of the ganglioside GM1. Results: The recombinant bacterium was capable of binding cholera toxin, a sine qua non of virulence, with high avidity; when tested with purified cholera toxin, it was capable of adsorbing >5% of its own weight of toxin in vitro. Administration of the GM1-expressing probiotic also protected infant mice against challenge with virulent V cholerae, even when treatment was delayed until after establishment of infection. When treatment commenced 1 hour after challenge, 12 of 12 mice given the probiotic survived, compared with only 1 of 12 for control mice (P < .00001). Conclusions: Toxin-binding probiotics such as that described here have considerable potential for prophylaxis and treatment of cholera in humans.

Section snippets

Bacterial Strains, Plasmids, and Oligonucleotides

The bacterial strains and plasmids used in this study are listed in Table 1, whereas oligonucleotides are listed in Table 2. All E coli strains were routinely grown in Luria–Bertani (LB) medium11 with or without 1.5% agar. When appropriate, ampicillin (Amp) or kanamycin (Kan) was added to growth media at a concentration of 50 μg/mL.

Manipulation and Analysis of DNA

Routine DNA manipulations (restriction digestion, agarose gel electrophoresis, ligation, transformation of E coli, and others) were carried out essentially as

Construction of Recombinant E coli Expressing a GM1 Mimic

E coli CWG308 was chosen as the host for expression of the GM1 receptor mimic because it has a waaO mutation, which results in truncation of its LPS, such that it comprises just the lipid A and inner oligosaccharide core components, terminating in glucose (Glc)6 (Figure 1). We have previously shown that several Neisseria glycosyltransferases responsible for assembly of outer core lipooligosaccharide are able to use this truncated LPS as an acceptor, and, when these transferases are expressed in

Discussion

Current treatment of cholera is centered on replacement of lost fluids, correction of metabolic acidosis and potassium deficiency, and replacement of continuing fluid losses. In mild cases, use of oral rehydration solutions (ORS) containing appropriate electrolytes and a carbohydrate such as glucose or rice is sufficient, but, in more severe cases, rapid intravenous rehydration is initially required, followed by ORS.4 ORS therapy needs to be maintained until the V cholerae has been eliminated

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    Supported by Australian Research Council (grant DP0208502) and the National Health and Medical Research Council of Australia (grant 250355).

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