Timothy Randall Hoover

Research in my lab focuses on three projects. The first of these projects examines the regulation of flagellar biogenesis in Helicobacter pylori, a significant human pathogen that is the causative agent of several gastric diseases including chronic gastritis, peptic ulcer disease and gastric cancer. Flagella are used for motility in H. pylori, which is needed for colonization of the gastric mucosa by the bacterium. Flagellar biosynthesis in H. pylori requires the coordinated regulation of over 40 genes through a transcriptional hierarchy that involves all three of the sigma factors found in this bacterium - sigma80, sigma54 (RpoN), and sigma28 (FliA). The sigma80-dependent flagellar genes encode components of the basal body and flagellar protein export apparatus, and are expressed early in the transcriptional hierarchy. The RpoN-dependent flagellar genes encode hook and filament proteins and are expressed later in the hierarchy. Transcription of these genes requires the activator FlgR and its cognate sensor kinase FlgS. The FliA-dependent genes encode components of the flagellar filament and are the last set of flagellar genes to be expressed. Expression of the FliA regulon is inhibited by the anti-sigma factor FlgM. Results from my lab, as well as work from other labs, suggest that transcription of the RpoN and FliA regulons is coupled to flagellar assembly through the activity of the flagellar protein export apparatus. Current goals of my lab include: (1) determine the molecular basis for how the flagellar protein export apparatus influences expression of the RpoN and FliA regulons; (2) identify a potential master regulator that initiates cascade for expression of the flagellar genes in H. pylori; and (3) determine the roles of genes of unknown function within the H. pylori RpoN regulon.

The second project in the lab examines the metabolism of acetone and other ketone bodies in H. pylori and the role catabolism of these compounds plays in host colonization. H. pylori possesses a cluster of genes that encode proteins that are predicted to convert acetone to acetyl-CoA. The first enzyme of the pathway is acetone carboxylase, which catalyzes the conversion of acetone to acetoacetate. Inactivation of the operon that encodes acetone carboxylase interferes with the ability of H. pylori to colonize the mouse stomach. Current goals are: (1) determine the reason disruption of acetone carboxylase affects colonization; (2) verify the proposed pathway for acetone utilization in H. pylori; (3) identify inhibitors of H. pylori acetone carboxylase and assess their potentials as therapeutic agents for treating H. pylori infections; (4) determine how disrupting other enzymes in the acetone metabolic pathway influence chronic infection by H. pylori; and (5) examine how ketogenic diets impact colonization and long-term survival of H. pylori.

The third project in the lab focuses on developing better methods for predicting RpoN-type promoters from genomic sequences and characterizing the roles of intragenic sequences the are remarkably similar to RpoN-type promoters. Recent studies from our lab suggest that RpoN-RNA polymerase holoenzyme binds these 'promoter-like' sequences in Salmonella typhimurium. Using DNA microarrays, we have sought to catalog the genes regulated by RpoN in S. typhimurium and identify the RpoN-dependent activators responsible for controlling expression of these genes. In addition, we have used chromatin immunoprecipitation (ChIP) assays in conjunction with DNA microarrays (ChIP-chip assays) to catalog the sites recognized by RpoN-holoenzyme or the various RpoN-dependent activators in the S. typhimurium genome. Taken together, the results of these studies will provide a comprehensive view of the S. typhimurium RpoN regulon.

Since acetone carboxylase is needed for efficient host colonization by H. pylori, compounds that inhibit the activity of this enzyme have potential therapeutic value for treatment of H. pylori infections. It is possible that genes encoding other enzymes in the acetone metabolism pathway are also essential for colonization, and therefore potential targets for new therapeutics.