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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.

Voronin D, Guimarães AF, Molyneux GR, Johnston KL, Ford L, Taylor MJ - Parasit Vectors (2014)

Bottom Line: 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.In whole worm mounts, antibody labelling of both lipoproteins were associated with Wolbachia.Both lipoproteins localised to bacterial membranes with wBmVirB6 present as a single cluster suggesting a single Type IV Secretory System on each Wolbachia cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK. voronind@gmail.com.

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.

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Visualisation of PAL and VirB6 in whole worm mounts ofB. malayi. A, B, Wolbachia (small red foci, bacteria DNA stained by propidium iodide, large red spot, nematode nuclei, see Methods) and PAL (green, secondary antibody labeled by FITC) in lateral cord of filarial nematode, C, merged A and B. D, E, VirB6 (green) and Wolbachia (small red foci) in B. malayi (wBm). F, merged D and E. G, H, negative control – Wolbachia-free B. malayi treated with antibiotic (lack of PAL reactivity shown as representation of both antibodies). I, merged G and H.
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Fig4: Visualisation of PAL and VirB6 in whole worm mounts ofB. malayi. A, B, Wolbachia (small red foci, bacteria DNA stained by propidium iodide, large red spot, nematode nuclei, see Methods) and PAL (green, secondary antibody labeled by FITC) in lateral cord of filarial nematode, C, merged A and B. D, E, VirB6 (green) and Wolbachia (small red foci) in B. malayi (wBm). F, merged D and E. G, H, negative control – Wolbachia-free B. malayi treated with antibiotic (lack of PAL reactivity shown as representation of both antibodies). I, merged G and H.

Mentions: To determine whether the protein localisation was associated with Wolbachia location in whole organisms or cells, we used whole mount confocal microscopy. The location of antibodies to both wBmPAL and wBmVirB6 were associated with Wolbachia (stained by propidium iodide, Figure 4A-F) in the hypodermal cord cells of B. malayi. Antibody reactivity was absent from tetracycline treated B. malayi (Figure 4G-I).Figure 4


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.

Voronin D, Guimarães AF, Molyneux GR, Johnston KL, Ford L, Taylor MJ - Parasit Vectors (2014)

Visualisation of PAL and VirB6 in whole worm mounts ofB. malayi. A, B, Wolbachia (small red foci, bacteria DNA stained by propidium iodide, large red spot, nematode nuclei, see Methods) and PAL (green, secondary antibody labeled by FITC) in lateral cord of filarial nematode, C, merged A and B. D, E, VirB6 (green) and Wolbachia (small red foci) in B. malayi (wBm). F, merged D and E. G, H, negative control – Wolbachia-free B. malayi treated with antibiotic (lack of PAL reactivity shown as representation of both antibodies). I, merged G and H.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4197220&req=5

Fig4: Visualisation of PAL and VirB6 in whole worm mounts ofB. malayi. A, B, Wolbachia (small red foci, bacteria DNA stained by propidium iodide, large red spot, nematode nuclei, see Methods) and PAL (green, secondary antibody labeled by FITC) in lateral cord of filarial nematode, C, merged A and B. D, E, VirB6 (green) and Wolbachia (small red foci) in B. malayi (wBm). F, merged D and E. G, H, negative control – Wolbachia-free B. malayi treated with antibiotic (lack of PAL reactivity shown as representation of both antibodies). I, merged G and H.
Mentions: To determine whether the protein localisation was associated with Wolbachia location in whole organisms or cells, we used whole mount confocal microscopy. The location of antibodies to both wBmPAL and wBmVirB6 were associated with Wolbachia (stained by propidium iodide, Figure 4A-F) in the hypodermal cord cells of B. malayi. Antibody reactivity was absent from tetracycline treated B. malayi (Figure 4G-I).Figure 4

Bottom Line: 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.In whole worm mounts, antibody labelling of both lipoproteins were associated with Wolbachia.Both lipoproteins localised to bacterial membranes with wBmVirB6 present as a single cluster suggesting a single Type IV Secretory System on each Wolbachia cell.

View Article: PubMed Central - PubMed

Affiliation: Department of Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, UK. voronind@gmail.com.

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.

Show MeSH
Related in: MedlinePlus