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Abstract: The Brugia malayi endosymbiont Wolbachia has recently been shown to be essential for its host’s survival and development. However, relatively little is known about Wolbachia proteins that interact with the filarial host and which might be important in maintaining the obligate symbiotic relationship. The Wolbachia surface proteins (WSPs) are members of the outer membrane protein family and we hypothesise that they might be involved in the Wolbachia–Brugia symbiotic relationship. Notably, immunolocalisation studies of two WSP members, WSP-0432 and WSP-0284 in B. malayi female adult worms showed that the corresponding proteins are not only present on the surface of Wolbachia but also in the host tissues, with WSP-0284 more abundant in the cuticle, hypodermis and the nuclei within the embryos. These results confirmed that WSPs might be secreted by Wolbachia into the worm’s tissue. Our present studies focus on the potential involvement of WSP-0284 in the symbiotic relationship of Wolbachia with its filarial host. We show that WSP-0284 binds specifically to B. malayi crude protein extracts. Furthermore, a fragment of the hypothetical B. malayi protein (Bm1_46455) was found to bind WSP-0284 by panning of a B. malayi cDNA library. The interaction of WSP-0284 and this protein was further confirmed by ELISA and pull-down assays. Localisation by immunoelectron microscopy within Wolbachia cells as well as in the worm’s tissues, cuticle and nuclei within embryos established that both proteins are present in similar locations within the parasite and the bacteria. Identifying such specific interactions between B. malayi and Wolbachia proteins should lead to a better understanding of the molecular basis of the filarial nematode and Wolbachia symbiosis. [Copyright &y& Elsevier]

Abstract: Transient transfection of isolated Brugia malayi embryos by biolistics has proven to be useful in defining promoter structure and function in this parasite. However, isolated transfected embryos are developmentally incompetent. A method of producing developmentally competent transfected parasites is therefore needed. We report that L3 parasites can be chemically transfected in situ in the peritoneal cavity of a gerbil with a construct consisting of a secreted luciferase reporter gene containing a promoter, the 3′ untranslated region and first intron derived from the B. malayi 70kDa heat shock protein gene. The in situ chemically transfected parasites are developmentally competent, producing adult parasites with an efficiency similar to that obtained from implanted untreated L3s. Cultured adult parasites and progeny microfilariae (mf) derived from L3s transfected with this construct secreted luciferase into the culture medium. When the transfected mf were fed to mosquitoes and the resulting L3s collected, the L3s also secreted luciferase into the culture medium. Progeny mf from transgenic adult parasites contained transgenic DNA, and the transgenic mRNA produced in these parasites was found to be correctly cis- and trans-spliced. In situ chemical transformation thus results in developmentally competent transfected B. malayi in which the transgenic sequences remain transcriptionally active in all life cycle stages and are present in the subsequent generation. [Copyright &y& Elsevier]