Increased Outer Membrane Vesicle Formation in a Helicobacter pylori tolB Mutant

BackgroundMultiple studies have established the importance of the tol-pal gene cluster in bacterial cell membrane integrity and outer membrane vesicle (OMV) formation in Escherichia coli. In contrast, the functions of Tol-Pal proteins in pathogenic organisms, including those of the Epsilonproteobacteria, remain poorly if at all defined. The aim of this study was to characterize the roles of two key components of the Tol-Pal system, TolB and Pal, in OMV formation in the pathogenic bacterium, Helicobacter pylori. MethodsH.pylori tolB, pal and tolBpal mutants, as well as complemented strains, were generated and assessed for changes in morphology and OMV production by scanning electron microscopy and enzyme-linked immunoassay (ELISA), respectively. The protein content and pro-inflammatory properties of OMVs were determined by mass spectroscopy and interleukin-8 (IL-8) ELISA on culture supernatants from OMV-stimulated cells, respectively. ResultsH.pylori tolB and pal bacteria exhibited aberrant cell morphology and/or flagella biosynthesis. Importantly, the disruption of H.pylori tolB but not pal resulted in a significant increase in OMV production. The OMVs from H.pylori tolB and pal bacteria harbored many of the major outer membrane and virulence proteins observed in wild-type (WT) OMVs. Interestingly, tolB, pal and tolBpal OMVs induced significantly higher levels of IL-8 production by host cells, compared with WT OMVs. ConclusionsThis work demonstrates that TolB and Pal are important for membrane integrity in H.pylori. Moreover, it shows how H.pylori tolB-pal genes may be manipulated to develop hypervesiculating strains for vaccine purposes.