胃幽門螺旋桿菌26695菌株之ADP-L-glycero-D-manno-heptose-6-epimerase由HP0859基因所表現之特性研究

Abstract

Helicobacter pylori is a spiral-shaped gram-negative bacteria and is recognized as a human pathogen that infects 50% human population worldwide. Several human stomach diseases, such as gastric ulcer, gastroduodenal ulcer, gastric adenocarcinoma and mucosa-associated lymphatic tissue (MALT) lymphoma, appeared to be related with H. pylori infection and colonization. The lipopolysaccharide (LPS) of H. pylori is thought to be one of the most important factors in pathogenesis. This structure enables H. pylori to escape from host immune response. LPS is present in the outer membrane and composed of O-chain, core oligosaccharide and lipid A. This study focused on the HP0859 gene. According to the bioinformatic data, the HP0859 product is predicted to catalyze the interconversion of ADP-D-glycero-D-mannoheptose and ADP-L-glycero-D-mannoheptose. ADP-L-glycero-D- mannoheptose is the major component of inner core LPS. The conserved domains of HP0859 protein also suggested that this enzyme is a NAD(P) + or FAD binding protein. To further characterize the properties of this protein, we cloned HP0859 gene and overexpressed its product. The molecular weight of recombinant HP0859 protein is 39.6 kDa and the native molecular weight is 257.7 kDa, indicated that HP0859 protein is a hexamer. The cofactor of HP0859 protein is NADP+. We also constructed the HP0859 knockout mutant. The LPS produced by knockout mutant showed a truncated LPS structure. The knockout mutant also exhibited reduced growth rate, ineffective adhesion, weak motility and more susceptible to detergent and antibiotic.