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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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Ultrastructural distribution ofWolbachiaVirB6 in lateral cord ofBrugia malayi. Immuno-transmission electron microphotographs showing distribution of Wolbachia VirB6 in lateral cord of Brugia malayi and a mosquito cell line. A, B – localisation of VirB6 in a bacterium-bacterium connection in cytoplasm of lateral cord of adult B. malayi, C, D – a single complex of VirB6 detected on the bacterial pole in developing microfilaria, E, F – VirB6 on wAlbB bacterial membrane in cytoplasm of mosquito cells (C6/36 cell line).
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Fig3: Ultrastructural distribution ofWolbachiaVirB6 in lateral cord ofBrugia malayi. Immuno-transmission electron microphotographs showing distribution of Wolbachia VirB6 in lateral cord of Brugia malayi and a mosquito cell line. A, B – localisation of VirB6 in a bacterium-bacterium connection in cytoplasm of lateral cord of adult B. malayi, C, D – a single complex of VirB6 detected on the bacterial pole in developing microfilaria, E, F – VirB6 on wAlbB bacterial membrane in cytoplasm of mosquito cells (C6/36 cell line).

Mentions: Antibodies raised to wBmVirB6, were unsuitable for western blot analysis due to non-specific binding. Immunogold ultrastructural localisation of VirB6 in host cells was determined using immuno-TEM analysis of B. malayi females and C6/36 (wAlbB) cells. Wolbachia VirB6 protein localised as a single discreet cluster(s) on the Wolbachia membranes (Figure 3A, B). In addition, occasional immunogold labelling was also observed localised within the bacterial matrix (Figure 3C, D), which could represent a precursor of the protein synthesised in bacteria. This pattern of protein distribution was similar in somatic cells and developing embryos of B. malayi (Figure 3A-D). Serial sections of bacteria determined that the VirB6 protein clusters as a single complex established in bacterial membranes and on some sections near the pole of bacteria. In some cases Wolbachia had two clusters, which were located on the opposite poles prior to division.Figure 3


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)

Ultrastructural distribution ofWolbachiaVirB6 in lateral cord ofBrugia malayi. Immuno-transmission electron microphotographs showing distribution of Wolbachia VirB6 in lateral cord of Brugia malayi and a mosquito cell line. A, B – localisation of VirB6 in a bacterium-bacterium connection in cytoplasm of lateral cord of adult B. malayi, C, D – a single complex of VirB6 detected on the bacterial pole in developing microfilaria, E, F – VirB6 on wAlbB bacterial membrane in cytoplasm of mosquito cells (C6/36 cell line).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Ultrastructural distribution ofWolbachiaVirB6 in lateral cord ofBrugia malayi. Immuno-transmission electron microphotographs showing distribution of Wolbachia VirB6 in lateral cord of Brugia malayi and a mosquito cell line. A, B – localisation of VirB6 in a bacterium-bacterium connection in cytoplasm of lateral cord of adult B. malayi, C, D – a single complex of VirB6 detected on the bacterial pole in developing microfilaria, E, F – VirB6 on wAlbB bacterial membrane in cytoplasm of mosquito cells (C6/36 cell line).
Mentions: Antibodies raised to wBmVirB6, were unsuitable for western blot analysis due to non-specific binding. Immunogold ultrastructural localisation of VirB6 in host cells was determined using immuno-TEM analysis of B. malayi females and C6/36 (wAlbB) cells. Wolbachia VirB6 protein localised as a single discreet cluster(s) on the Wolbachia membranes (Figure 3A, B). In addition, occasional immunogold labelling was also observed localised within the bacterial matrix (Figure 3C, D), which could represent a precursor of the protein synthesised in bacteria. This pattern of protein distribution was similar in somatic cells and developing embryos of B. malayi (Figure 3A-D). Serial sections of bacteria determined that the VirB6 protein clusters as a single complex established in bacterial membranes and on some sections near the pole of bacteria. In some cases Wolbachia had two clusters, which were located on the opposite poles prior to division.Figure 3

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