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, specifically as they relate 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 our laboratory provided the initial genetic and biochemical and recent structural evidence for the role of ADP-L-glycero-D-mannoheptose 6-epimerase (epimerase) in the synthesis of the lipopolysaccharide precursor L-glycero-D-mannoheptose (heptose) in several genera of gram-negative bacteria. Our findings are relevant to the management of infection, built on 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 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. We have completed mutagenic and kinetic studies to confirm a role for Ser116, Tyr140 and Lys144 residues in ADP-L-glycero-D-mannoheptose 6-epimerase catalysis. CD studies of several of these mutant epimerases indicated that significant structural alterations were not characteristic of the mutant enzymes relative to wild type epimerase. In summary our results suggested that the phenolic hydroxyl group of tyrosine, the epsilon-amino group of lysine and the hydroxyl group of serine are essential for the catalytic activity of ADP-hep 6-epimerase. In addition, we have demonstrated using epimerase mutants, G6S and G6A, that Gly6 is required for NADP binding and that the secondary structure of G6S mutants of ADP- hep 6-epimerase is lost. In collaboration, with Dr. Martin Tanner?s group of the University of British Columbia, we added to our understanding of the catalytic mechanism of ADP-L-glycero-D-mannoheptose 6-epimerase catalyzed reactions. Epimerase is known to require a tightly bound NADP+ cofactor for activity and presumably employs a mechanism involving transient oxidation of the substrate. Four mechanistic possibilities are considered that involve transient oxidation at either C-7"""""""", C-6"""""""" or C-4"""""""" of the heptose nucleotide. In our recent paper,the use of solvent isotope incorporation studies and alternate substrates provided strong evidence for a mechanism involving non-stereospecific oxidation/reduction directly at C-6"""""""". It was found that the epimerization proceeds without any detectable incorporation of solvent-derived deuterium or 18O-isotope into the product. This argues against mechanisms involving either proton transfers at carbon or dehydration/rehydration events. In addition, the deoxygenated analogs, 7""""""""-deoxy-ADP-L,D-Hep and 4""""""""-deoxy-ADP-L,D-Hep, were both found to serve as substrates for the enzyme, indicating that oxidation at either C-7"""""""" or C-4"""""""" is not required for catalysis. We have extended our investigation of the outer membrane to the gram-negative strain, Helicobacter pylori. H. pylori is the causative agent for gastritis, ulcer disease and some gastric cancers. The mechanism of H. pylori pathogenesis is not known at this time. We are using a mouse model for the study of H. pylori infection, detection methods, pathogenic mechanism(s) and, an in vivo expression technology system to identify a subset genes induced during infection. We have developed a non-invasive, sensittttive annd species-specific method to detect H. pylori infection of mice by PCR analysis of fecal pellets extracts. These findings were the subject of a recently filed patent application and a published manuscript indicating the importance our findings and suggesting that the non-invasive detection for H. pylori infection is applicable to detection of H. pylori in feces of other live experimental animals and humans. Another ongoing project has been designed to identify and characterize novel targets for the development of antibiotics and protective vaccines directed against H. pylori. Two LPS core biosynthetic genes (rfaD and rfaE) from H. pylori have been cloned and the gene products purified. This is the first purification and characterization of the rfaE gene product, ADP-D-glycero-D-mannoheptose synthetase, from any gram-negative bacterium. An assay for ADP-D-glycero-D-mannoheptose synthetase has been developed. The rfaE gene encodes a protein with two distinct domains. The two domains of the rfaE gene product have been cloned, expressed, and assayed for enzyme activity. Domain II of the synthetase has been shown to catalyze the formation of ADP-D-glycero-D-mannoheptose from heptose-1-phosphate and ATP. Gel filtration and SDS-PAGE analysis of the purified synthetase revealed that the native enzyme has a molecular mass of 312 kDa and a subunit molecular weight of 52,000. The km and Vmax for the substrate is 0.025 mM and 22.5 nmol/min/mg. The kcat for this enzyme is 0.020 s-1 Additional physico-chemical studies of the synthetase are ongoing. Maltose binding-rfaE fusions have been constructed to increase the yield of soluble protein and crystallization trials have been initiated for ADP-D-glycero-D-mannoheptose synthetase. To study host requirements for establishing successful infection and subsequent pathogenesis, we have infected mutant C57BL/6 mice with genetic alterations that may influence the virulence of H. pylori. For this study two C57BL/6 mutant mouse strains were selected: an interleukin 10-deficient (IL-10) knockout and p47phox knockout strain. 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 have allowed us to examine the in vivo role of NADP oxidase mediated inflammatory responses to Helicobacter pylori infection and pathology. We are also studying pathogenic mechanism(s)of H. pylori using mouse macrophage cell lines RAW 264.7 and J774.1A. Our preliminary fluorescence and confocal microscopic studies demonstrate that H. pylori can infect macrophage cells and can survive inter-cellularly for 24 hours. In summary, both the E. coli and H. pylori studies should advance our understanding of LPS core synthesis, gram-negative bacterial infection, pathogenesis and offers ways to manage them.
