Abstract
Helicobacter pylori is a microaerophilic Gram-negative, spiral-shaped and flagellated bacterium that colonizes more than half of the world’s population and frequently causes chronic infection. Gram-negative bacteria have been reported to release outer membrane vesicles (OMVs) from the outer membrane during their growth. The functions of OMVs include the delivery of virulence factors, modulation of the host’s immune system and stress response. LPS is considered as a key virulence factor of H. pylori and may contribute to sorting of proteins into OMVs. It is composed of lipid A, core oligosaccharide and O-antigen. Previously, our laboratory had constructed various H. pylori LPS truncated mutants, including those having defects in the inner-core, outer-core, O-antigen, heptose biosynthetic pathway, and the cag type Ⅳ secretion system. In this study, we examined the different LPS profiles of these mutants on OMVs by sliver staining. The amounts of OMVs produced were found to be related to the length (or the structure) of LPS. Using dynamic light scattering analysis, we found that the average size of OMVs derived from the heptose biosynthetic pathway knockout mutants were larger than others. In addition, we observed the presence of key virulence factors CagA, VacA and various adhesins on OMVs by immunoblotting. The results also suggested that the major route of releasing CagA into OMVs is not through the type Ⅳ secretion system because the disruption of this system by knocking out cagL, an important component of the type Ⅳ secretion system, did not affect the presence of CagA in OMVs. In addition, CagA and the tested adhesins were significantly reduced in OMV samples collected from the heptose biosynthetic pathway knockout mutants. Furthermore, the change of LPS structure also altered the glycosylation status of the adhesins on OMVs. Moreover, the amount of CagA and VacA present in the whole cell extract of AGS cells were significantly decreased after treating with OMVs derived from the heptose biosynthetic pathway knockout mutants while compared to those treated with OMVs from the wild-type strain. In conclusion, we reported that LPS structure plays an important role not only in the formation of OMVs, but also in the sorting of proteins into OMVs.