Abstract
Helicobacter pylori, a Gram-negative, spiral-shaped, microaerophilic bacterium that can inhabit various areas of human stomach and duodenum, has been considered as a risk for inducing human gastric cancer. Lipopolysaccharide (LPS) is thought to be associated with H. pylori adhesion and has the capability of modulating the immune response of infected hosts. This bacterium continuously sheds outer membrane vesicles (OMVs) during different phases of growth and these vesicles containing LPS have been suggested to have potential roles in bacterial survival and pathogenesis. Previously, our laboratory has demonstrated that the hp0859 gene encodes an ADP-L-glycero-D-manno-heptose-6-epimerase, which is involved in the synthesis of ADP-L-D-heptose for the assembly of LPS inner core in H. pylori. In this study, we first demonstrated that the HP0859 knockout mutant (WTΔ0859) not only exhibited a truncated LPS structure but also decreased its OMV production level. Surprisingly, two major H. pylori toxins, cytotoxin associated gene A (CagA) and vacuolating cytotoxin A (VacA), were both present in OMVs derived from H. pylori 26695 wild type strain but significantly lesser amounts were present in OMVs derived from WTΔ0859. To further investigate the effects of the LPS structure on OMV formation and CagA/VacA functions, WTΔVacA, WTΔCagA, and WTΔVacA/ΔCagA double knockout mutants were also constructed. The OMVs isolated from these mutants were co-cultured with AGS cells for infection studies. The AGS cells showed a typical hummingbird phenotype and cellular vacuolation after co-culturing with OMVs derived from H. pylori 26695 wild type strain. In contrast, the AGS cells significant lost the hummingbird phenotype when infected with OMVs isolated from the WTΔCagA, and the vacuolation phenomenon significant reduced when co-culturing with OMVs from WTΔVacA. When the AGS cells were treated with OMVs from WTΔ0859, they neither showed the hummingbird phenotype nor cellular vacuolation morphological change. We also conducted total reactive oxygen species (tROS), mitochondrial reactive oxygen species (mROS) and mitochondrial membrane potential (MMP) assays on AGS cells co-culturing with OMVs derived from these mutants, and found that there may exist a functional antagonism between the OMV-associated CagA and the OMV-associated VacA to control the entry of these two toxins into the host cell along with affecting the mitochondrial functions of the infected gastric epithelial cells.