title: Increased Outer Membrane Vesicle Formation in a Helicobacter pylori tolB Mutant.
abstract: BACKGROUND
  Multiple 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.
  
  
  METHODS
  H. pylori ΔtolB, Δpal and ΔtolBpal 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.
  
  
  RESULTS
  H. pylori ΔtolB and Δpal 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 ΔtolB and Δpal bacteria harbored many of the major outer membrane and virulence proteins observed in wild-type (WT) OMVs. Interestingly, ΔtolB, Δpal and ΔtolBpal OMVs induced significantly higher levels of IL-8 production by host cells, compared with WT OMVs.
  
  
  CONCLUSIONS
  This 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.
authors: Centre for Innate Immunity and Infectious Diseases, MIMR-PHI Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, (3168), Victoria, Australia.
authors: Lorinda
authors: L
authors: Turner
authors: Centre for Innate Immunity and Infectious Diseases, MIMR-PHI Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, (3168), Victoria, Australia.
authors: Judyta
authors: J
authors: Praszkier
authors: Centre for Innate Immunity and Infectious Diseases, MIMR-PHI Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, (3168), Victoria, Australia.
authors: Melanie L
authors: ML
authors: Hutton
authors: Monash Biomedical Proteomics Facility, Monash University, Wellington Road, Clayton, (3800), Victoria, Australia.
authors: David
authors: D
authors: Steer
authors: Monash Micro Imaging, Monash University, Clayton, Victoria, Australia.
authors: Georg
authors: G
authors: Ramm
authors: Centre for Innate Immunity and Infectious Diseases, MIMR-PHI Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, (3168), Victoria, Australia.
authors: Maria
authors: M
authors: Kaparakis-Liaskos
authors: Centre for Innate Immunity and Infectious Diseases, MIMR-PHI Institute of Medical Research, Monash University, 27-31 Wright Street, Clayton, (3168), Victoria, Australia.
authors: Richard L
authors: RL
authors: Ferrero
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D002462:Cell Membrane / Q000502:physiology; D058501:Enzyme-Linked Immunospot Assay; D016481:Helicobacter Infections / Q000382:microbiology; D016480:Helicobacter pylori / Q000235:genetics / Q000472:pathogenicity; D016209:Interleukin-8 / Q000378:metabolism*; D013058:Mass Spectrometry; D008855:Microscopy, Electron, Scanning; D033903:Periplasmic Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; C508606:CXCL8 protein, human; D016209:Interleukin-8; D033903:Periplasmic Proteins
keywords: Helicobacter pylori; outer membrane protein; outer membrane vesicles