title: Wolbachia lipoproteins: abundance, localisation and serology of Wolbachia peptidoglycan associated lipoprotein and the Type IV Secretion System component, VirB6 from Brugia malayi and Aedes albopictus.
abstract: BACKGROUND
  Lipoproteins are the major agonists of Wolbachia-dependent inflammatory pathogenesis in filariasis and a validated target for drug discovery. Here we characterise the abundance, localisation and serology of the Wolbachia lipoproteins: Wolbachia peptidoglycan associated lipoprotein and the Type IV Secretion System component, VirB6.
  
  
  METHODS
  We used proteomics to confirm lipoprotein presence and relative abundance; fractionation, immunoblotting and confocal and electron immuno-microscopy for localisation and ELISA for serological analysis.
  
  
  RESULTS
  Proteomic analysis of Brugia malayi adult female protein extracts confirmed the presence of two lipoproteins, previously predicted through bioinformatics: Wolbachia peptidoglycan associated lipoprotein (wBmPAL) and the Type IV Secretion System component, VirB6 (wBmVirB6). wBmPAL was among the most abundant Wolbachia proteins present in an extract of adult female worms with wBmVirB6 only detected at a much lower abundance. This differential abundance was reflected in the immunogold-labelling, which showed wBmPAL localised at numerous sites within the bacterial membranes, whereas wBmVirB6 was present as a single cluster on each bacterial cell and also located within the bacterial membranes. Immunoblotting of fractionated extracts confirmed the localisation of wBmPAL to membranes and its absence from cytosolic fractions of C6/36 mosquito cells infected with wAlbB. In whole worm mounts, antibody labelling of both lipoproteins were associated with Wolbachia. Serological analysis showed that both proteins were immunogenic and raised antibody responses in the majority of individuals infected with Wuchereria bancrofti.
  
  
  CONCLUSIONS
  Two Wolbachia lipoproteins, wBmPAL and wBmVirB6, are present in extracts of Brugia malayi with wBmPAL among the most abundant of Wolbachia proteins. Both lipoproteins localised to bacterial membranes with wBmVirB6 present as a single cluster suggesting a single Type IV Secretory System on each Wolbachia cell.
authors.affiliation: Department of Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK. voronind@gmail.com.
authors.forename: Denis
authors.initials: D
authors.lastname: Voronin
authors.affiliation: Department of Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK. ana.filipaguedes@gmail.com.
authors.forename: Ana F
authors.initials: AF
authors.lastname: Guimarães
authors.affiliation: Department of Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK. Gemma.Molyneux@lstmed.ac.uk.
authors.forename: Gemma R
authors.initials: GR
authors.lastname: Molyneux
authors.affiliation: Department of Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK. Kelly.Johnston@lstmed.ac.uk.
authors.forename: Kelly L
authors.initials: KL
authors.lastname: Johnston
authors.affiliation: Department of Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK. Louise.Ford@lstmed.ac.uk.
authors.forename: Louise
authors.initials: L
authors.lastname: Ford
authors.affiliation: Department of Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK. Mark.Taylor@lstmed.ac.uk.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Taylor
mesh_terms: D000330:Aedes / Q000737:chemistry*; D000818:Animals; D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D058947:Bacterial Secretion Systems / Q000502:physiology*; D017178:Brugia malayi / Q000737:chemistry*; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D007074:Immunoglobulin G; D008074:Lipoproteins / Q000737:chemistry / Q000378:metabolism*; D010457:Peptidoglycan / Q000737:chemistry / Q000378:metabolism*; D020577:Wolbachia / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000907:Antibodies, Bacterial; D001425:Bacterial Outer Membrane Proteins; D058947:Bacterial Secretion Systems; D007074:Immunoglobulin G; D008074:Lipoproteins; D010457:Peptidoglycan