Effects of a HP0859 (rfaD) knockout mutation on lipopolysaccharide structure of Helicobacter pylori 26695 and the bacterial adhesion on AGS cells.
Lipopolysaccharide (LPS) is considered as an important virulence factor of Helicobacter pylori, and contributes to infection persistence and disease severity. ADP-L-glycero-D-manno-heptose-6-epimerase is an enzyme essential for LPS synthesis and understanding of its biochemistry is critical for drug development. We cloned one putative ortholog of Escherichia colirfaD, HP0859, from H. pylori 26695. Determination of the native molecular weight of the recombinant HP0859 protein suggests that the protein is likely a hexamer. NADP+, instead of NAD+, was proved to be the physiological cofactor for HP0859 protein. Circular dichroism spectrum analysis demonstrated that the secondary structure of this protein is significantly altered when the cofactor is removed. We also constructed an HP0859 knockout mutant and examined its phenotypic properties. The HP0859 knockout mutant exhibited a severe truncation of LPS, a decreased growth rate, and a higher susceptibility to novobiocin. Disruption of HP0859 also reduced the adhesive capacity of H. pylori to AGS cells, and the infected cells failed to display the classic hummingbird phenotype. Complementation of the HP0859 knockout mutation restored these phenotypes completely. In conclusion, we demonstrate that HP0859 codes for a protein essential for the LPS inner core biosynthesis in H. pylori and an intact LPS structure contributes to the adherence ability of this bacterium.
Institute of Molecular Medicine and Department of Life Science, National Tsing Hua University, 101, Sec. 2, Kuang-Fu Rd., Hsinchu 30013, Taiwan, ROC.
Po-Chun
PC
Chang
Chun-Jen
CJ
Wang
Chung-Kai
CK
You
Mou-Chieh
MC
Kao
D001422:Bacterial Adhesion / Q000235:genetics; D002238:Carbohydrate Epimerases / Q000235:genetics*; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D055786:Gene Knockout Techniques; D016480:Helicobacter pylori / Q000235:genetics / Q000472:pathogenicity*; D006801:Humans; D008070:Lipopolysaccharides / Q000737:chemistry* / Q000235:genetics*; D009154:Mutation
D008070:Lipopolysaccharides; C442865:lipopolysaccharide, Helicobacter pylori; C037213:ADP-L-glycero-D-mannoheptose-6-epimerase; D002238:Carbohydrate Epimerases