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.

Wolbachia endosymbionts induce neutrophil extracellular trap formation in human onchocerciasis

“…All investigated onchocercomata from placebo (P) matching doxycycline +/− ivermectin-treated patients contained worms positive for Wolbachia, as assessed using immunohistochemistry (IHC) with a purified anti- w PAL IgG 11 32 , whilst all nodules derived from doxycycline (D) or doxycycline + ivermectin (DI) treated patients were Wolbachia -negative ( Fig. 1A,B ).…”

“…The diacylated N-terminus of Wolbachia lipoprotein, Wo LP, has been defined as the predominant Wolbachia Pathogen-Associated Molecular Pattern, interacting with the host immune system via the TLR2/6-MyD88 signalling pathway 11 . Two native Wolbachia surface-associated lipoproteins have been characterised in both filarial and insect Wolbachia 11 32 . Filarial w PAL is abundantly expressed in female O. volvulus tissues (Fig.…”

Identification of putative effectors of the Type IV secretion system from the Wolbachia endosymbiont of Brugia malayi

“…w Bm0152, conserved across Wolbachia isolates of diverse hosts, is a predicted peptidoglycan associated lipoprotein (PAL) that was previously identified as a component of the wBm secretome [ 80 ]. PAL proteins are thought to be intimately involved in regulating Gram-negative cell envelope integrity via interactions with both periplasmic peptidoglycan and the outer membrane [ 81 ].…”

AmiD Is a Novel Peptidoglycan Amidase in Wolbachia Endosymbionts of Drosophila melanogaster

“…Thus, AmiD wol is probably transported to the periplasm by the Sec pathway and cleaved between residues 30 and 31. The only Wolbachia lipoproteins identified from w Bm and insect Wolbachia pipientis strain A. albopictus B ( w AlbB) to date are the peptidoglycan associated lipoprotein (wPAL) and the type IV secretion system component VirB6 (Voronin et al, 2014 ). Both are probably diacylated due to the absence of the gene encoding the N-acetyltransferase Ltn.…”