The immunomodulatory roles of helicobacter pylori outer membrane vesicles
thesisposted on 23.02.2017, 01:08 by Turner, Lorinda
During normal growth, Gram-negative bacteria shed nano-sized outer membrane vesicles (OMVs), ranging from 50-250 nm in diameter. OMVs have been studied in many Gram-negative bacterial species, yet little is still known about the biology of OMV release, nor how these vesicles interact enter host cells or how they induce immune responses. To investigate OMV production by the gastric pathogen, Helicobacter pylori, we inactivated the H. pylori orthologues of Escherichia coli tolB and pal, encoding two of the critical components of the Tol-Pal system known to help maintain bacterial membrane integrity. We demonstrated that H. pylori mutant strains lacking an intact copy of tolB exhibited distinct outer membrane abnormalities, accompanied by defects in flagella formation. Importantly, H. pylori lacking functional TolB or Pal proteins exhibited extensive OMV production and release. The OMVs from these mutant strains induced high levels of pro-inflammatory cytokine (interleukin-8, IL-8) production. This work describes, for the first time, the importance of the TolB and Pal proteins in cell membrane integrity and OMV formation by H. pylori. Work performed previously in our laboratory reported that Gram-negative OMVs enter epithelial cells in vitro, resulting in the generation of pro-inflammatory and adaptive immune responses. However, to date the precise mechanism of entry has not been determined. Studies have reported that not all OMVs enter cells using the same endocytic pathways. Using a panel of chemical inhibitors of key endocytosis pathways, we found that heterogeneously sized populations of OMVs were internalised by epithelial cells via macropinocytosis, clathrin and caveolin dependant endocytosis. Interestingly, however, when we fractionated OMVs into either small (< 50 nm) or large (> 50 nm) sizes, we found that small OMVs entered host cells by each of the aforementioned pathways. In contrast, cell entry of large OMVs involved macropinocytosis and clathrin but not caveolin dependent endocytosis. Proteomic analyses of different sized OMVs revealed both shared and unique sets of proteins, indicating that particle size may also determine protein content. Following internalisation by polarised epithelial cells, we found that H. pylori OMVs induce the basolateral secretion of the pro-inflammatory chemokine, IL-8. Interestingly, we found that internalised OMVs up-regulated the expression of HLA Class I and II molecules in epithelial cells. Moreover, stimulation of polarised epithelial cells with H. pylori OMVs resulted in the production of exosomes secreted at their basolateral surface containing OMV proteins. Importantly, we demonstrated that exosomes containing OMV proteins induced proliferation of human T cells in an antigen dependent manner, indicating that exosomes could function to present OMV antigens to mucosal T cells. Collectively, our data suggest that proteins derived from internalised OMVs are packaged into secreted exosomes for presentation to immune cells. We speculate that through interactions with antigen presenting cells, these exosomes are capable of activating antigen specific T cells located beneath the epithelial cell layer, thereby providing a link between the generation of innate and adaptive immune responses to H. pylori OMVs at the mucosal epithelium.