Post-genomic, functional characterization of filarial genes regulated in response to the loss of their Wolbachia endosymbionts
Zusammenfassung der Projektergebnisse
Filarial nematodes infect more than 150 million people in developing countries of the tropics and sub-tropics, causing severe and disfiguring pathologies with high morbidity. Most of the filarial nematodes infecting and causing disease in humans contain obligate essential endobacteria of the genus Wolbachia. These endosymbionts are required for oogenesis, embryogenesis, larval molting and ultimately adult worm survival. Therefore, they are appealing targets for discovering new therapeutics for treating the infections. In addition to practical aspects of drug development, understanding the symbiosis between filarial worms and their Wolbachia will elucidate scientific questions about the biology of these worms and endobacteria, especially as they differ from their close relatives that are found in many arthropod species. Thus, this project aimed to elucidate the molecular basis of the symbiosis using a murine model of filarial infection by Litomosoides sigmodontis. The two aims were to 1) continue work showing an up-regulation of a filarial phosphate permease (PPE) at the protein level and its localization in relation to Wolbachia within the worm, and 2) determine other genes/pathways involved in the symbiosis using the B. malayi microarray. We successfully showed that PPE is up-regulated with Wolbachia depletion at the protein level in L. sigmodontis and Onchocerca volvulus, a human filarial worm. Additionally, immunohistology provided images that showed PPE was up-regulated in the same worm tissues and compartments as Wolbachia are found, further supporting our hypothesis that PPE is involved in the symbiosis. The microarray experiments revealed >300 genes that are up- or down-regulated when the Wolbachia are depleted. Quantitative PCR confirmed the change in expression for 60% of 20 selected genes. Additionally, it was found that mitochondrial encoded proteins of the respiratory chain were also up-regulated in response to the loss of the endobacteria. This upregulation was specific for Wolbachia as tetracycline treatment did not induce these genes in the Wolbachia-free filarial species Acanthocheilonema viteae. Many of the genes regulated in response to Wolbachia depletion are specific for filarial worms or poorly characterized in other nematode species. Because our original method of RNAi is difficult and expensive, we invested time to develop a method of ex vivo culturing L. sigmodontis. With this method we will now be able to more easily test different methods of RNAi for each gene of interest, e.g. dsRNA versus siRNA. In summary, we have shown that PPE is regulated at the protein level as well as the previously seen mRNA level and shown that it is expressed in regions of the worm where Wolbachia are found, further supporting the hypothesis that PPE provided phosphate to the endobacteria for the production of nucleotides, metabolites that filarial worms cannot synthesize. Furthermore, we have identified many genes that are regulated in response to Wolbachia depletion, including mitochondrial encoded proteins of the respiratory chain. We hypothesize that these are the result of a decrease in heme available to the worms which then reduces the number of complexes respiratory chain complexes that are functional since most require heme for the transfer of electrons and thus the generation of energy for the worms. As a direct result, our research reveals that Wolbachia in filarial nematodes are necessary for more than one metabolic pathway. Future work will elucidate these and other interaction in the symbiosis, perhaps providing a new target for antibiotic treatment of filarial infections.
Projektbezogene Publikationen (Auswahl)
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(2007). A niche for Wolbachia. Trends Parasitol 23, 5-7
Pfarr, K.M., and Hoerauf, A.
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(2007). It Takes Two: Lessons From the First Nematode Wolbachia Genome Sequence. In: Wolbachia: A Bug's Life in another Bug, A. Hoerauf, and R.U. Rao, eds. (Basel, Karger), pp. 52-65
Pfarr, K., Foster, J., and Slatko, B.
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(2007). On the taxonomic status of the intracellular bacterium Wolbachia pipientis: should this species name include the intracellular bacteria of filarial nematodes? Int J Syst Evol Microbiol 57, 1677-1678
Pfarr, K., Foster, J., Slatko, B., Hoerauf, A., and Eisen, J.A.
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(2007). Wolbachia endosymbionts: An Achilles' Heel of filarial nematodes. In: Wolbachia: a bug's life in another bug, A. Hoerauf, and R.U. Rao, eds. (Basel, Karger), pp. 15-30
Hoerauf, A., and Pfarr, K.
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(2008). Infection of the intermediate mite host with Wolbachia-depleted Litomosoides sigmodontis microfilariae: Impaired L1 to L3 development and subsequent sex-ratio distortion in adult worms. Int J Parasitol 38, 981-987
Arumugam, S., Pfarr, K.M., and Hoerauf, A.
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(2008). Mitochondrial encoded genes are up-regulated in response to depletion of essential Wolbachia endobacteria from Brugia malayi. Molecular and Cellular Biology of Helminth Parasites V, Hydra, Greece: 12-17 September 2008
Pfarr, K., Strübing, U., and Hoerauf, A.
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(2008). The mitochondrial heat shock protein 60 (HSP60) is up-regulated in Onchocerca volvulus after the depletion of Wolbachia. Parasitology 135, 529-538
Pfarr, K.M., Heider, U., Schmetz, C., Buttner, D.W., and Hoerauf, A.
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(2009). Filariasis and lymphoedema. Parasite Immunol 31, 664-672
Pfarr, K.M., Debrah, A.Y., Specht, S., and Hoerauf, A.
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(2010). Mitochondrial genes for hemedependent respiratory chain complexes are up-regulated after depletion of Wolbachia from filarial nematodes. Int J Parasitol 40, 1193-1202
Strübing, U., Lucius, R., Hoerauf, A., and Pfarr, K.M.
