The researchers used X-ray crystallography to determine the structure of ToxT. The process involves taking DNA from V. cholerae and using non-pathogenic E. coli bacteria to produce large amounts of the target protein, in this case, ToxT. Once protein has been purified, it is concentrated and crystallized. Then the crystal, which is an ordered array of protein molecules, is subjected to a powerful X-ray beam. The pattern of diffracted X-rays is collected on a detector and then analyzed using mathematical algorithms, eventually revealing the atomic structure of the protein.
Doctors have known that bile, found in the intestine, inhibits the expression of the virulence genes in V. cholerae, but until now, the mechanism behind this was not completely understood. This study provides a direct link between the environment of the gut and the regulation of virulence genes, and it also identifies the regulatory molecule.
"Finding a fatty acid in the structure was quite a surprise," says F. Jon Kull, associate professor of chemistry at Dartmouth and senior author on the paper."The exciting thing about this finding is that we might be able to use a small, natural molecule to treat and/or prevent cholera. We will also use the structure of the fatty acid as a framework to try and design a small molecule inhibitor of ToxT."
"The results of the study are exciting from the points of view of both the mechanistic aspect of the complex regulation of V. cholerae virulence gene expression and the potential medical impact as we now move forward to apply this new knowledge to influence this mechanism to control infection in humans," said co-author Ronald Taylor, a researcher at the Dartmouth Medical School.
Citation: Lowden et al., 'Structure of Vibrio cholerae ToxT reveals a mechanism for fatty acid regulation of virulence genes', PNAS, February 2010; doi: 10.1073/pnas.0915021107
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