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Background: The human filarial parasites cause diseases that are among the most important causes of morbidity in the developing world. The elimination programs targeting these infections rely on a limited number of drugs, making the identification of new chemotherapeutic agents a high priority. The study of these parasites has lagged due to the lack of reverse genetic methods., Methodology/principal Findings: We report a novel co-culture method that results in developmentally competent infective larvae of one of the human filarial parasites (Brugia malayi) and describe a method to efficiently transfect the larval stages of this parasite. We describe the production of constructs that result in integrative transfection using the piggyBac transposon system, and a selectable marker that can be used to identify transgenic parasites. We describe the production and use of dual reporter plasmids containing both a secreted luciferase selectable marker and fluorescent protein reporters that will be useful to study temporal and spatial patterns of gene expression., Conclusions/significance: The methods and constructs reported here will permit the efficient production of integrated transgenic filarial parasite lines, allowing reverse genetic technologies to be applied to all life cycle stages of the parasite., Competing Interests: The authors have declared that no competing interests exist.

The human filarial parasite Brugia malayi harbors an endosymbiotic bacterium of the genus Wolbachia. The Wolbachia represent an attractive target for the control of filarial induced disease as elimination of the bacteria affects molting, reproduction and survival of the worms. The molecular basis for the symbiotic relationship between Wolbachia and their filarial hosts has yet to be elucidated. To identify proteins involved in this process, we focused on the Wolbachia surface proteins (WSPs), which are known to be involved in bacteria-host interactions in other bacterial systems. Two WSP-like proteins (wBm0152 and wBm0432) were localized to various host tissues of the B. malayi female adult worms and are present in the excretory/secretory products of the worms. We provide evidence that both of these proteins bind specifically to B. malayi crude protein extracts and to individual filarial proteins to create functional complexes. The wBm0432 interacts with several key enzymes involved in the host glycolytic pathway, including aldolase and enolase. The wBm0152 interacts with the host cytoskeletal proteins actin and tubulin. We also show these interactions in vitro and have verified that wBm0432 and B. malayi aldolase, as well as wBm0152 and B. malayi actin, co-localize to the vacuole surrounding Wolbachia. We propose that both WSP protein complexes interact with each other via the aldolase-actin link and/or via the possible interaction between the host's enolase and the cytoskeleton, and play a role in Wolbachia distribution during worm growth and embryogenesis.