title: Functional characterization of Helicobacter pylori 26695 sedoheptulose 7-phosphate isomerase encoded by hp0857 and its association with lipopolysaccharide biosynthesis and adhesion.
abstract: Helicobacter pylori is a notorious human pathogen and the appearance of antibiotic resistance of this bacterium has posed a serious threat to human health. Lipopolysaccharide (LPS) is a key virulence factor and plays important roles in pathogenesis of H. pylori infection. Sedoheptulose 7-phosphate isomerase (GmhA), as an enzyme participating in the first step of heptose biosynthesis, is indispensable for the formation of inner core oligosaccharide of LPS. In this study, we cloned one putative gmhA ortholog, hp0857, from H. pylori 26695 and overexpressed it in Eschericha coli. Based on the results of molecular weight determination, the recombinant HP0857 is likely a homodimer. Analysis of enzymatic kinetic properties of this protein confirmed that hp0857 is indeed encoded a phosphoheptose isomerase which can utilize sedoheptulose 7-phosphate as the substrate in the ADP-L-glycero-D-manno-heptose (ADP- L,D-Hep) biosynthesis pathway. We also generated an HP0857 knockout mutant and explored its phenotypic changes. This mutant exhibited a decreased growth rate and displayed a "deep rough" type of LPS structure. In addition, it also had a slight decrease in its motility and was more susceptible to hydrophobic antibiotic novobiocin and detergents Triton X-100 and SDS. Furthermore, the adhesive capacity of the HP0857 knockout mutant to AGS cells was reduced significantly, and most of the infected cells didn't show a classic hummingbird phenotype. However, complementation of the HP0857 knockout mutation restored most of these phenotypic changes. In conclusion, we demonstrated that HP0857 protein is essential for inner core biosynthesis of H. pylori LPS and is a potential target for developing new antimicrobial agents against H. pylori infection.
authors.affiliation: Institute of Molecular Medicine & Department of Life Science, College of Life Sciences, National Tsing Hua University, No. 101, Sec. 2, Kuang-Fu Road, Hsinchu 30013, Taiwan.
authors.forename: Chung-Kai
authors.initials: CK
authors.lastname: Yu
authors.affiliation: Institute of Molecular Medicine & Department of Life Science, College of Life Sciences, National Tsing Hua University, No. 101, Sec. 2, Kuang-Fu Road, Hsinchu 30013, Taiwan.
authors.forename: Chun-Jen
authors.initials: CJ
authors.lastname: Wang
authors.affiliation: Institute of Molecular Medicine & Department of Life Science, College of Life Sciences, National Tsing Hua University, No. 101, Sec. 2, Kuang-Fu Road, Hsinchu 30013, Taiwan.
authors.forename: Yongyu
authors.initials: Y
authors.lastname: Chew
authors.affiliation: Department of Gastroenterology, MacKay Memorial Hospital, No. 690, Sec. 2, Kuang-Fu Road, Hsinchu 30071, Taiwan.
authors.forename: Po-Chuan
authors.initials: PC
authors.lastname: Wang
authors.affiliation: Institute of Bioinformatics and Structure Biology, College of Life Sciences, National Tsing Hua University, No. 101, Sec. 2, Kuang-Fu Road, Hsinchu 30013, Taiwan.
authors.forename: Hsien-Sheng
authors.initials: HS
authors.lastname: Yin
authors.affiliation: Institute of Molecular Medicine & Department of Life Science, College of Life Sciences, National Tsing Hua University, No. 101, Sec. 2, Kuang-Fu Road, Hsinchu 30013, Taiwan. Electronic address: mckao@life.nthu.edu.tw.
authors.forename: Mou-Chieh
authors.initials: MC
authors.lastname: Kao
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000378:metabolism*; D002448:Cell Adhesion / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000378:metabolism*; D016480:Helicobacter pylori / Q000145:classification / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000096:biosynthesis*; D019998:Racemases and Epimerases / Q000378:metabolism*; D013045:Species Specificity; D013403:Sugar Phosphates / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D008070:Lipopolysaccharides; D013403:Sugar Phosphates; C442865:lipopolysaccharide, Helicobacter pylori; C020495:sedoheptulose 7-phosphate; D019998:Racemases and Epimerases; C445301:GmhA protein, E coli; D019747:Aldose-Ketose Isomerases; C023917:phosphoheptose isomerase
keywords: Adhesion; Helicobacter pylori; Lipopolysaccharide; Sedoheptulose 7-phosphate isomerase