According to the World Health Organization, diarrhea caused by enteric infections affects some four billion individuals annually throughout the world, resulting in an estimated four million to six million deaths, many in children under five years of age. In the United States, diarrhea is the second most common infectious illness, and in some countries diarrheal diseases account for up to one third of all deaths. In many cases, enteric disease is caused by the ingestion of pathogenic bacteria via contaminated food or water. Once ingested by the host, environmental signals in the gut initiate a virulence gene cascade leading to the production of virulence factors. The expression of these virulence factors, many of which are toxins, effector molecules, and colonization factors, results in a wide variety of debilitating and often deadly diarrheal diseases including travelers'diarrhea, dysentery, and cholera. In cholera the primary virulence factors are the toxin- coregulated pilus (TCP), which is required for the organism to colonize the host, and cholera toxin (CT), which is responsible for the pathology of the disease. The expression of both virulence factors is regulated by ToxT, the paramount regulator of virulence gene expression in Vibrio cholerae. ToxT, a member of the AraC/XylS (A/X) superfamily of regulators, of which there are numerous members throughout the bacterial world, is inhibited by fatty acids present in bile. In the structure of ToxT, a palmitoleic acid bridges the regulatory and DNA binding domains of the transcription factor, locking it in an inactive conformation. Two positively charged lysine side chains, one from each domain, play a critical role in fatty acid binding and, thereby, in the mechanism of virulence gene regulation. The studies proposed in this application will test the hypothesis that many other enteric bacteria use a similar, if not identical, mechanism to regulate pathogenic activity. The approach will utilize secondary structure prediction and homology modeling to identify candidates from the A/X protein superfamily containing positively charged amino acid residues at positions homologous to those found in ToxT. The effect of fatty acids on virulence gene production in the various candidate organisms will be measured utilizing a lacZ fusion system and confirmed using electrophoretic mobility shift assays (EMSAs), an NMR-based ligand binding assay, and site directed mutagenesis. Preliminary analysis has indicated that a number of pathogenic bacteria, including several types of Escherichia coli, Salmonella typhi, Salmonella typhimurium, Shigella flexneri, and Yersinia enterocolitica, contain A/X regulators with homologous positively charged residues and ligand binding pockets. The studies proposed here will confirm if these bacteria use a common, fatty acid mediated mechanism to regulate pathogenic activity, thereby laying the groundwork for development of therapeutics that could be broadly applied to treat enteric bacterial infections causing diseases such as travelers'diarrhea, dysentery, and typhoid fever.

Public Health Relevance

Enteric infectious diarrheal diseases affect more than 4 billion individuals yearly throughout the world, resulting in an estimated 4 million to 6 million deaths, primarily among the elderly and children less than 5 years of age. This proposal investigates how pathogenic bacteria use fatty acids to regulate virulence activity. Understanding this process could lead to the development of therapeutics to treat or prevent enteric bacterial infections including travelers'diarrhea, salmonella, dysentery, and typhoid fever.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21AI092161-02
Application #
8339441
Study Section
Special Emphasis Panel (ZRG1-IDM-A (80))
Program Officer
Baqar, Shahida
Project Start
2011-09-30
Project End
2013-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
2
Fiscal Year
2012
Total Cost
$197,500
Indirect Cost
$72,500
Name
Dartmouth College
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
041027822
City
Hanover
State
NH
Country
United States
Zip Code
03755