Basic–alimentary tractA Recombinant Probiotic for Treatment and Prevention of Cholera
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).