| Summary: | 碩士 === 國立清華大學 === 分子醫學研究所 === 100 === Helicobacter pylori, a Gram-negative, microaerophilic spiral bacterium that colonizes the mucosa of human stomach, has been considered a risk factor for human gastric diseases. Lipopolysaccharwide (LPS), one of the virulence factors in H. pylori, is composed of lipid A, core oligosaccharide and O-antigen polysaccharide, and is thought to be toxic with potent immunomodulating and immunostimulating properties. In the current study, the HP0860 gene from H. pylori, which was predicted to encode the D-D-Heptose-1,7-bisphosphate phosphatase (GmhB) involved in the synthesis of ADP-L-D-heptose for the assembly of LPS inner core, was cloned and characterized. We reported HP0860 protein under native conditions is monomeric in solution, and confirmed HP0860 functions as a phosphatase by converting D-glycero-D-manno
-heptose-1,7-bisphosphate into D-glycero-D-manno-heptose-1-phosphate (kcat/Km = 2188 mM-1s-1). Subsequently, we constructed an HP0860 knockout mutant and examined its phenotypic properties. The HP0860 knockout mutant contained both mature and immature forms of LPS structure, suggesting another protein present in the HP0860 knockout mutant might be able to partially compensate for the loss of HP0860 activity. In addition, we also revealed that the HP0860 knockout mutant exhibited a decreased growth rate, less classic hummingbird phenotype showing by the infected AGS cells and a lower adherence on H. pylori-infected AGS cells, suggesting that H. pylori lacking HP0860 appeared to be less virulent. Furthermore, we provided the evidence that mutation of the genes involved in LPS biosynthesis altered the sorting of cargo proteins into outer membrane vesicles (OMVs). In conclusion, the findings in this report confirmed the involvement of HP0860 in the LPS inner core biosynthesis and added a new understanding for future developing novel antimicrobial agents against H. pylori infection.
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