Research in my laboratory focuses on the understanding of the mechanisms of bacterial pathogenesis, antibiotic resistance of gram-negative bacteria, and the development of novel antimicrobial agents and vaccines targeted to ulcer disease and other bacterial infections. Gram-negative bacteria (pathogens and nonpathogens) have a unique structure called the outer membrane that makes these bacteria more refractory to antibiotic therapy than their gram-positive counterparts (save mycobacteria). Research findings in my laboratory provided the genetic, biochemical, and structural evidence for the role of ADP-L-glycero-D-mannoheptose 6-epimerase (epimerase) in the synthesis of the lipopolysaccharide (LPS) precursor L-glycero-D-mannoheptose (heptose) in several genera of gram-negative bacteria. These findings are relevant to the management of infection, taking advantage of the observations that gram-negative bacteria with defective heptose biosynthesis have altered growth rate, virulence, increased susceptibility to antibiotics and that heptose is not found in mammalian host cells. Therefore, the heptose biosynthetic enzymic steps in bacteria are unique targets for the design of novel antimicrobial agents. Our structural studies of epimerase (done in collaboration with Drs. Steve E. Ealick and Ashley M. Deacon) indicated a high structural similarity to the short-chain dehydrogenases/reductases superfamily and the presence of the conserved catalytic motif TyrXXXLys. ? In two studies, in collaboration, with Dr. Martin Tanner?s group at the University of British Columbia, we reported the elucidation of the catalytic mechanism of ADP-L-glycero-D-mannoheptose 6-epimerase (epimerase) catalyzed reactions. These studies demonstrated that epimerase (1) catalyzes a sterochemical inversion at an unactivated stereocenter, (2) employs a transient oxidation strategy involving a tightly bound cofactor, NADP+, (3) utilizes non-stereospecific oxidation/reduction directly at C-6"""""""", and (4) utilizes a two-site mechanism for the reaction. The new knowledge of the structural characteristics and the catalytic mechanism of epimerase provide rationale for designing specific inhibitors of LPS synthesis. Efforts to screen for specific inhibitors are planned as the next phase.? We are currently preparing a manuscript describing our mutagenic and kinetic studies to confirm a role for Ser116, Tyr140, Lys144 and Lys178 residues in ADP-L-glycero-D-mannoheptose 6-epimerase catalysis. ? We currently focus our investigation of the outer membrane of the gram-negative strain, Helicobacter pylori. H. pylori chronically infect a large portion (50 to 90%) of the world?s population and is the causative agent for gastritis, ulcer disease and some gastric cancers. A biochemical approach is used to identify and characterize novel targets for the development of novel antibiotics and/or protective vaccines directed against H. pylori. After cloning the LPS core biosynthetic genes (rfaD and rfaE) of H. pylori, study their protein products. Studies characterizing the rfaE gene product, ADP-D-glycero-D-mannoheptose synthetase (the native enzyme has a molecular mass of 312 kDa and a subunit molecular weight of 52,000), from Helicobacter pylori are near completion. We also observed that a H. pylori rfaE mutant is defective in its ability to infect and colonize the stomachs of mice when compared to wild-type H. pylori. The result suggests that H. pylori strains with severely truncated (LPS) structures are more susceptible to host defenses. Additional studies have been initiated to define the role of wild-type LPS structures in H. pylori infection and colonization of infected mice.? ? To date, the mechanism of H. pylori pathogenesis is not completely understood. We are using mouse and cell line models to study of H. pylori infection, pathogenic mechanism(s) and detection methods. To study host requirements and immune responses necessary for establishing successful infection and subsequent pathogenesis, we are utilizing a mouse model in which we infect mutant C57BL/6 mice. The mutant C57BL/6 mice carry genetic alterations that may or my not influence the virulence of H. pylori. For this study several C57BL/6 mutant mouse strains were selected: an interleukin 10-deficient (IL-10), p47phox, MyD88-/-, TLR2-/-, TLR4-/- knockout strains and TLR2-/-/TLR4-/- double knockout animals. ? Using these knockout mice, we are evaluating the role of endogenous IL-10 on the regulation of the immune response to H. pylori infection. The p47phox knockout mice allow us to examine the in vivo role of NADP oxidase-mediated inflammatory responses to H. pylori infection and pathology. In a recently concluded 9 month study, involving IL-10 and p47phox knockout mice, we monitored histological changes by pathology scoring, cytokine expression and the expression of CD8, CD25 and foxp3 via immunochemistry of stomach tissues associated with H. pylori infection. At present these data are being quantified and analyzed. ? Additionally we have initiated studies (approximately 9 month studies) to compare immune responses to H. pylori infection involving a common adaptor molecule, myeloid differentiation protein MyD88 and selected Toll-like receptors in MyD88-/-, TLR2-/-, and TLR4-/- knockout and TLR2-/-/TLR4-/- double knockout animals. Our goal is to assess the relative contribution of MyD88 and Toll-like receptors TLR2 and TLR4 in the host response to H. pylori infection.? We are also studying pathogenic mechanism(s) of H. pylori using mouse macrophage cell lines RAW 264.7 and J774.1A as well as primary dendritic or macrophage cells from the bone marrow of several knockout mice. Our preliminary fluorescence and confocal microscopic studies demonstrate that H. pylori can infect macrophage cells and can survive intracellularly for 24 hours. In collaboration with Dr. Ding Jin, NCI, we are also testing whether ∆spoT and rfaE mutants of H. pylori have the ability to survive intracellularly in macrophage as compared to their wild-type counterparts.? ? ? Finally, we are employing a genetic method known as in vivo expression technology (IVET) to search for microbial genes important in pathogenicity. IVET is a practical strategy for identifying a subset of genes induced preferentially during infection of an animal host. We use a variant of IVET involving random DNA fragments of H. pylori fused to a tandem-reporter system of chloramphenicol acetyltransferase (cat) and beta-galactosidase (lacZ). We constructed a unique promoter-screening vector for use in H. pylori strains and a H. pylori genomic bank. This system allows the use of a mouse as a selective medium to identify genes that H. pylori specifically expresses when infecting host tissues. A subset of the identified in vivo induced genes (ivi genes) may play a role in H. pylori infection and pathogenesis. We have identified several putative ivi genes that we are presently studying. Novel, H. pylori ivi genes will be mutated and mutant bacterial strains will be tested for defects in colonization and virulence. Putative, ivi genes will be subjected to microarray analysis for confirmation.? In summary, ongoing H. pylori studies in our lab should advance our understanding of gram-negative bacterial infection, pathogenesis, and host immune response. Collectively, this information is invaluable for infection control and management protocols.
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