Permeability Barrier, Pathogenicity And Antibiotic Resis

Diabetes, Digestive, Kidney Diseases

Investigators

Linked publications & trials

Abstract

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 which makes these bateria more refractory to antibiotic therapy than their gram-positive counterparts (save mycobacteria). Research findings in our laboratory provided the initial genetic and biochemical evidence for two lipopolysaccharide (LPS) inner core biosynthetic genes (i.e., rfaC and rfaD) and their products. Subsequently, we produced diffraction-quality crystals of the rfaD gene product, ADP-L-glycero-D-mannoheptose 6-epimerase, allowing us to determine the complete structure of this enzyme at 2-Angstrom resolution. ADP-L-glycero-D-mannoheptose 6-epimerase (epimerase) is required for the synthesis of L-glycero-D-mannoheptose (heptose) in several genera of gram-negative bacteria. Our findings are relevant to the management of infection, build 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 pathway in bacteria presents a unique target for the design of novel antimicrobial agents. In a recent publication we reported biochemical, analytical and structural studies of epimerase that unequivocally demonstrated that epimerase binds NADP preferentially and tightly relative to NAD. However, inactive apo-ADP-hep 6-epimerase (epimerase minus NADP) can be reconstituted with NAD with 50 percent enzymatic activity restored. The apparent Kd values for NADP and NAD are 26 micromolar and 45 micromolar, respectively. UV CD spectra showed that apo-ADP-hep 6-epimerase reconstituted with NADP+ had more secondary structure than apo-ADP-hep 6-epimerase reconstituted with NAD+. The loss of secondary structure with the removal of a cofactor is not a common trait of enzymes that utilizes NADP or NAD as a cofactor. A structural comparison of ADP-hep 6-epimerase with UDP-galactose 4-epimerase, which utilizes an NAD+ cofactor, identified the regions of ADP-hep 6-epimerase, which defines its specificity for NADP+. 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. Based on these findings, we have constructed several chemically induced or site-directed epimerase mutant proteins (i.e., Y140C, Y140Q, Y140F, Y140H, Y140E, K144I, K144R, S116A, G6S and G6A). Our recent mutagenesis studies confirmed the importance of the Tyr140XXXLys144 sequence motif and Serine 116 to the catalytic activity of epimerase. In summary our results suggest 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 that Gly6 is required for NADP binding and that the secondary structure of G6S mutants of ADP- hep 6-pimerase is lost. 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), an in vivo expression technology to identify genes induced during infection. We have developed a non-invasive, sensitive and species-specific method to detect H. pylori infection of mice by PCR analysis of fecal pellets extracts. Our findings demonstrate that the H. pylori presence in mice can be detected reliably over time, thus allowing determination of the persistence/transience of the infection. Furthermore, our findings that the sensitivity in feces was 10 to 100 cells suggesing that it may be directly applicable to detection of H. pylori in feces of other live experimental animals and humans. 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. 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. Structural studies are planned for ADP-D-glycero-D-mannoheptose synthetase. In summary, both the E. coli and H. pylori studies should advance our understanding of gram-negative bacterial infection, pathogenesis and offers ways to manage them.