Exploring the Role of Wolbachia Endobacteria in the Biology of Filarial Nematode Parasites

Filarial nematodes are vector borne parasitic worms that cause a variety of disfiguring and disabling diseases, including lymphatic filariasis and onchocerciasis. Many filarial species require Wolbachia endobacteria: family: Rickettsiaceae) to carry out their life cycle. Studies using antibiotics to target the endobacteria, thereby interfering with worm fertility and viability, have generated interest in using Wolbachia as an antifilarial drug target. However, the exact mechanisms underpinning this interesting mutualistic interaction are poorly understood. Wolbachia-dependence is not ubiquitous in the filarial family. Some species are able to survive in the absence of an endosymbiont. The inconsistent patterns of Wolbachia-dependence and independence seen in filarial nematodes may be explained by two hypotheses. Following infection with the endobacteria, reductive evolution could have removed redundant genes or pathways present in both partners. Thus, deletions in the worm\u27s genome would render it dependent on Wolbachia for vital gene products. Conversely, Wolbachia-dependent species could re-acquire vital genes from the endosymbiont by horizontal gene transfer, rendering the bacteria expendable. Mitochondria and Wolbachia are co-transmitted vertically from mother to offspring, therefore the mitochondrial genome: mtDNA) is particularly sensitive to evolutionary pressures exerted by the endosymbiont. Wolbachia is also thought to be closely related to the mitochondrial progenitor, so they may overlap in function: e.g., energy production). In order to address our first hypothesis, we sequenced the mitochondrial genomes of several species of Wolbachia-dependent and independent filarial nematodes in hopes of finding some degeneracy in the mtDNA of the Wolbachia-dependent species. Our studies have shown that the mtDNA of all examined species encodes the same 12 protein coding genes, 2 ribosomal RNA genes and 22 transfer RNA genes. Despite a careful analysis, no sequence-level differences were observed between the mtDNA of infected and uninfected species. In order to address our second hypothesis, we surveyed the genomes of two Wolbachia-independent filarial species, Acanthocheilonema viteae and Onchocerca flexuosa, in search of evidence of horizontal gene transfer from Wolbachia. Many genomic fragments containing regions with high homology to Wolbachia sequences were identified. Follow-up transcriptomic and proteomic analyses in O. flexuosa have shown that Wolbachia-like sequnces are expressed at the RNA and protein levels. Imaging studies indicate that Wolbachia-like RNAs are mainly produced in tissues known to harbor Wolbachia in infected species, while a Wolbachia-like protein was found nearby but not in the same tissue. This project has produced a vast amount of data that will be useful to the filariasis research community, including the mtDNA sequences of five filarial species, genomic sequences from A. viteae and O. flexuosa, transcriptomic sequences from O. flexuosa and a survey of the O. flexuosa adult worm proteome. Our results have verified a longstanding hypothesis that the ancestor(s) of many Wolbachia-free filarial nematode species was colonized in the distant past despite the present lack of endobacteria. Future studies may prove that horizontally transferred bacterial genes are necessary for the survival of Wolbachia-free filarial worms that would otherwise require Wolbachia for reproduction and development