Abstract
Helicobacter pylori is a gram-negative spiral-shaped microaerophilic bacterium which infects 50% human population and is associated with chronic gastritis, peptic ulcer, gastroduodenal ulcer, gastric adenocarcinoma and mucosa-associated lymphatic tissue (MALT) lymphoma. Because of the appearance of antibiotic resistance, it is essential to develop new antibiotics against this notorious bacterium. Lipopolysaccharide (LPS) plays significant roles in pathogenesis of H. pylori infection. This phosphorylated lipoglycan maintains the structural integrity of bacterial outer membrane and provides a protective barrier against the entry of toxic hydrophobic compounds like bile salts, detergents and lipophilic antibiotics into the bacterial cell. It is composed of lipid A, core oligosaccharide and O-chain polysaccharide. In this study, we identified that the H. pylori open reading frame (ORF) hp0857 codes for a phosphoheptose isomerase which catalyzes the isomerization of D-Sedoheptulose 7-phosphate into D-glycero-D-manno-heptose 7-phosphate and is involved in inner core biosynthesis of lipopolysaccharide. We cloned hp0857 and overexpressed it in Eschericha coli. The recombinant protein was purified to homogeneity with the subunit molecular weight of 23.2 kDa. The gel filtration and analytical ultracentrifugation studies indicated that HP0857 protein has a native molecular mass of 40.5 ± 2.2 kDa and 49.0 ± 0.1 kDa, respectively, suggesting that HP0857 protein is likely a dimer under the present experimental conditions. The result of circular dichroism spectrum analysis indicated that HP0857 contains 67.8 ± 6.8% α-helix and 2.7 ± 0.8% β-strand. The results of the enzymatic activity assay indicated that the purified protein could convert the D-Sedoheptulose 7-phosphate into D-glycero-D-manno-heptose 7-phosphate. Moreover, a HP0857 knockout mutant was constructed and its phenotypic properties was characterized. The HP0857 knockout mutant showed a truncated LPS structure, a reduced growth rate, a weak motility and more susceptible to detergent Triton X-100 and antibiotic novobiocin. In addition, the AGS cells infected by the HP0857 knockout mutant were unable to display a classic hummingbird phenotype. Complementation of the HP0857 in the knockout mutant was found to restore most of these phenotypic changes. In conclusion, we demonstrated that HP0857 protein is essential for LPS inner core biosynthesis of H. pylori and is a potential target for developing new antimicrobial agents against H. pylori infection.