Outer Membrane Vesicle Production by Helicobacter pylori Represents an Approach for the Delivery of Virulence Factors CagA, VacA and UreA into Human Gastric Adenocarcinoma (AGS) Cells.

Helicobacter pylori infection is the etiology of several gastric-related diseases including gastric cancer. Cytotoxin associated gene A (CagA), vacuolating cytotoxin A (VacA) and α-subunit of urease (UreA) are three major virulence factors of H. pylori, and each of them has a distinct entry pathway and pathogenic mechanism during bacterial infection. H. pylori can shed outer membrane vesicles (OMVs). Therefore, it would be interesting to explore the production kinetics of H. pylori OMVs and its connection with the entry of key virulence factors into host cells. Here, we isolated OMVs from H. pylori 26,695 strain and characterized their properties and interaction kinetics with human gastric adenocarcinoma (AGS) cells. We found that the generation of OMVs and the presence of CagA, VacA and UreA in OMVs were a lasting event throughout different phases of bacterial growth. H. pylori OMVs entered AGS cells mainly through macropinocytosis/phagocytosis. Furthermore, CagA, VacA and UreA could enter AGS cells via OMVs and the treatment with H. pylori OMVs would cause cell death. Comparison of H. pylori 26,695 and clinical strains suggested that the production and characteristics of OMVs are not only limited to laboratory strains commonly in use, but a general phenomenon to most H. pylori strains.

Functional characterization of Helicobacter pylori 26695 sedoheptulose 7-phosphate isomerase encoded by hp0857 and its association with lipopolysaccharide biosynthesis and adhesion.

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.

The Effects of HP0044 and HP1275 Knockout Mutations on the Structure and Function of Lipopolysaccharide in Helicobacter pylori Strain 26695.

Helicobacter pylori infection is associated with several gastric diseases, including gastritis, peptic ulcer, gastric adenocarcinoma and mucosa-associated lymphatic tissue (MALT) lymphoma. Due to the prevalence and severeness of H. pylori infection, a thorough understanding of this pathogen is necessary. Lipopolysaccharide, one of the major virulence factors of H. pylori, can exert immunomodulating and immunostimulating functions on the host. In this study, the HP0044 and HP1275 genes were under investigation. These two genes potentially encode GDP-D-mannose dehydratase (GMD) and phosphomannomutase (PMM)/phosphoglucomutase (PGM), respectively, and are involved in the biosynthesis of fucose. HP0044 and HP1275 knockout mutants were generated; both mutants displayed a truncated LPS, suggesting that the encoded enzymes are not only involved in fucose production but are also important for LPS construction. In addition, these two gene knockout mutants exhibited retarded growth, increased surface hydrophobicity and autoaggregation as well as being more sensitive to the detergent SDS and the antibiotic novobiocin. Furthermore, the LPS-defective mutants also had significantly reduced bacterial infection, adhesion and internalization in the in vitro cell line model. Moreover, disruptions of the HP0044 and HP1275 genes in H. pylori altered protein sorting into outer membrane vesicles. The critical roles of HP0044 and HP1275 in LPS biosynthesis, bacterial fitness and pathogenesis make them attractive candidates for drug inventions against H. pylori infection.