Abstract
BackgroundLipoproteins 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.MethodsWe used proteomics to confirm lipoprotein presence and relative abundance; fractionation, immunoblotting and confocal and electron immuno-microscopy for localisation and ELISA for serological analysis.ResultsProteomic 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.ConclusionsTwo 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.
Highlights
Lipoproteins are the major agonists of Wolbachia-dependent inflammatory pathogenesis in filariasis and a validated target for drug discovery
Previous studies have determined that the pro-inflammatory capacity of Brugia malayi and Onchocerca volvulus is dependent on the presence of Wolbachia and the molecular ligands responsible have been characterised as lipoproteins [7]
Analysis of the Wolbachia B. malayi genome revealed the absence of the gene for the apolipoprotein Nacyltransferase (Lnt), which converts di- to tri- acylated bacterial lipoprotein in gram-negative bacteria, suggesting lipoproteins in Wolbachia are di-acylated accounting for their recognition by Toll-like receptors 2 and 6 (TLR2/6) [7]
Summary
Lipoproteins are the major agonists of Wolbachia-dependent inflammatory pathogenesis in filariasis and a validated target for drug discovery. Loss of Wolbachia induces extensive apoptosis of germline and somatic cells, presumably due to the lack of provision of an essential nutrient or metabolite required to prevent apoptosis of these cells and tissues during periods of high metabolic demand [3] This mutualistic association has been exploited as a target for antibiotic therapy, which can cure patients infected with Wuchereria bancrofti and Onchocerca volvulus, providing an alternative treatment and control strategy [1,4,5]. In addition to their essential role in the biology of filarial nematodes, Wolbachia are a major driver of the inflammatory pathogenesis of filarial disease [6]. Analysis of the Wolbachia B. malayi (wBm) genome revealed the absence of the gene for the apolipoprotein Nacyltransferase (Lnt), which converts di- to tri- acylated bacterial lipoprotein in gram-negative bacteria, suggesting lipoproteins in Wolbachia are di-acylated accounting for their recognition by TLR2/6 [7]
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