Understanding The Roles Of Transcriptional Regulators For The Development Of Natural And Novel Inhibitors Of Listeria Monocytogenes.
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Listeria monocytogenes is pathogenic intracellular foodborne bacterium that causes listeriosis, a rare, but serious disease in humans. Despite the use of antibiotics, the mortality rate remains at 20-30%. The ability of L. monocytogenes to survive transmission through food systems and to cause disease is attributed in part to the regulatory networks that control environmental stress adaptation and virulence functions. Therefore, a comprehensive understanding of the factors that are important to virulence, stress response and antimicrobial resistance will help us better develop novel inhibitors for therapeutics. With the information garnered from select regulators, it is possible to identify new drug targets and new drugs for treatment alternatives. The purpose of this research in L. monocytogenes is to (i) determine the contributions of select transcriptional regulators to virulence functions, (ii) assess the contributions of two regulators to antimicrobial peptides response, and (iii) identify novel small molecule inhibitors of the regulator sigma-B. In summary, we found that of central transcriptional regulators, sigma-B, PrfA, HrcA, CtsR, sigma-L, sigma-H, and sigma-C, sigma-B contributes to invasion, PrfA contributes to cell-to-cell growth and CtsR, in addition to PrfA and sigma-B, contributes to virulence in a guinea pig model of listeriosis. We determined that sigma-B and sigma-L are important to controlling expression of genes needed for resistance to the select antimicrobial peptides SdpC and Nisin, thus indicating that sigma-B has a role in virulence and stress survival as well as antimicrobial resistance. Therefore, we focused on sigma-B as a promising novel drug target for the treatment of listeriosis. From a library of 57,000 small molecules, we identified a novel compound, sigmastatin, which inhibits the activity of sigma-B and its regulon, inhibits Bacillus subtilis sigma-B and severely impedes L. monocytogenes enterocyte invasion. With a solid understanding of the contributions and roles of various regulators in L. monocytogenes, novel inhibitors can be used to target those regulators, like sigma-B, which are associated with survival, pathogenesis, and resistance. These novel agents can be used to treat listeriosis, extrapolated for use against other similar clinically relevant diseases, or used to gain insight into physiology of pathogenic bacteria and related gene regulation.
dissertation or thesis