Genetic and Functional Characterization of the Type IV Secretion System inWolbachia

Abstract

A type IV secretion system (T4SS) is used by many symbiotic and pathogenic intracellular bacteria for the successful infection of and survival, proliferation, and persistence within hosts. In this study, the presence and function of the T4SS in Wolbachia strains were investigated by a combination of genetic screening and immunofluorescence microscopy. Two operons of virB-virD4 loci were found in the genome of Wolbachia pipientis strain wAtab3, from the Hymenoptera Asobara tabida, and strain wRi, infecting Drosophila simulans. One operon consisted of five vir genes (virB8, virB9, virB10, virB11, and virD4) and the downstream wspB locus. The other operon was composed of three genes (virB3, virB4, and virB6) and included four additional open reading frames (orf1 to orf4) orientated in the same direction. In cell culture and insect hosts infected with different Wolbachia strains, the bona fide vir genes were polycistronically transcribed, together with the downstream adjacent loci, notably, as virB8 to virD4 and wspB and as virB3, virB4, virB6, and orf1 to orf4. Two peptides encompassing conserved C and N termini of the Wolbachia VirB6 protein were used for the production of polyclonal antibodies. Anti-VirB6 antibodies could detect the corresponding recombinant protein by chemifluorescence on Western blots of total proteins from Escherichia coli transformants and Wolbachia strains cultured in cell lines. Using immunofluorescence microscopy, we further demonstrated that the VirB6 protein was produced by Wolbachia strains in ovaries of insects harboring wAtab3 or wRi and cell lines infected with wAlbB or wMelPop. As VirB6 is known to associate with other VirB proteins to form a membrane-spanning structure, this finding suggests that a T4SS may function in Wolbachia.

Phylogenomics Reveals a DiverseRickettsialesType IV Secretion System

“…Our analysis, as well as the findings of prior studies (55,106,108), supports the vertical transmission of the rvh T4SS after the split of "Candidatus Pelagibacter" from the Rickettsiales ancestor. The most parsimonious recreation of an LGT event that equipped this ancestor with a P-T4SS involves a transporter containing 12 genes (rvhB1 to rvhB11 and rvhD4), with ancestral deletion, recombination, and duplication events creating a modified transporter comprising 18 genes (rvhB1 to rvhB3, rvhB4a and rvhB4b, rvhB6a to rvhB6e, rvhB7, rvhB8a and rvhB8b, rvhB9a and rvhB9b, rvhB10, rvhB11, and rvhD4).…”

Composition of Bacterial Communities Associated with Natural and Laboratory Populations of Asobara tabida Infected with Wolbachia

“…However, only Wolbachia was detected in ovaries by FISH, a technique applied for the first time in A. tabida. The location of Wolbachia in oocyte poles is in agreement with published data (19,57). Probes specific for Brevibacillus and Pseudomonas did not produce clear signals in entire ovaries, suggesting that PCR products or isolates obtained from homogenates might be dissection contaminants from other organs of the insect body, such as the gut, which is known to contain such bacteria (72).…”

“…No significant difference in the overall density of bacteria was found between treated and untreated lines. However, multi-infected individuals harbored a significantly higher proportion of Wolbachia, consistent with previous data confirming that the strain (18), gypsy moths (11), and the mosquito Culex quinquefasciatus (57). However, only Wolbachia was detected in ovaries by FISH, a technique applied for the first time in A. tabida.…”

“…4B), extending to the subpole area, than in Pi(3) oocytes (Fig. 4C), and the location was usual for Wolbachia strains in these lines (19,57). No fluorescent signals were detected in ovaries with probes targeting the genera Brevibacillus and Pseudomonas, although fluorescent dots were observed in cultures from both bacteria (not shown).…”

The bacteriaWolbachiain filariae, a biological Russian dolls’ system: new trends in antifilarial treatments

“…Protein secretion plays an important role in the virulence of these bacteria (Burns, 1999). in Wolbachia, genes encoding t4SS proteins have been identified and characterised at two separate operons (rances et al, 2008(rances et al, , Wu et al, 2004. the sequences and organisation of these operons are strictly conserved among numerous arthropod Wolbachia strains (Masui et al, 2000b;Pichon et al, 2009).…”