Project description:We sequenced total RNA from Dirofilaria immitis in order to generate the first tissue-specific gene expression profile of a filarial nematode and its Wolbachia endosymbiont.
Project description:The filarial nematodes Brugia malayi, Wuchereria bancrofti and Onchocerca volvulus cause elephantiasis, dermatitis and blindness, resulting in severe morbidity in developing countries. 1.3 billion people are at risk of infection. Targeting the essential Wolbachia endobacteria of filarial nematodes with doxycycline has proven to be an effective therapy, resulting in a block in embryogenesis and worm development, and macrofilaricidal effects. However, doxycycline is contraindicated for a large portion of the at-risk population. To identify new targets for anti-wolbachial therapy, understanding the molecular basis of the Wolbachia-filaria symbiosis is required. We performed cross-species hybridization by using the Brugia malayi microarray to identify differentially expressed genes in the rodent filaria Litomosoides sigmodontis after depletion of Wolbachia which therefore might have a role in symbiosis. Female adult Litomosoides sigmodontis from patent infections were treated with tetracycline to deplete endosymbiotic Wolbachia bacteria. RNA from tetracycline-treated Litomosoides sigmodontis was compared to untreated age-matched control worms. This experiment was performed for three different timepoints: day 6, 15 and 36 of tetracycline treatment. One biological replicate was performed each with two technical replicates (dye-flip replicates).
Project description:We sequenced total RNA from Dirofilaria immitis in order to generate the first tissue-specific gene expression profile of a filarial nematode and its Wolbachia endosymbiont. Examination of transcript levels in 7 different Dirofilaria immitis tissues, in duplicate, using Illumina GAIIx.
Project description:The filarial nematodes Brugia malayi, Wuchereria bancrofti and Onchocerca volvulus cause elephantiasis, dermatitis and blindness, resulting in severe morbidity in developing countries. 1.3 billion people are at risk of infection. Targeting the essential Wolbachia endobacteria of filarial nematodes with doxycycline has proven to be an effective therapy, resulting in a block in embryogenesis and worm development, and macrofilaricidal effects. However, doxycycline is contraindicated for a large portion of the at-risk population. To identify new targets for anti-wolbachial therapy, understanding the molecular basis of the Wolbachia-filaria symbiosis is required. We performed cross-species hybridization by using the Brugia malayi microarray to identify differentially expressed genes in the rodent filaria Litomosoides sigmodontis after depletion of Wolbachia which therefore might have a role in symbiosis.
Project description:Female worms (Brugia malayi) were collected from infected jirds treated with 2.5 mg/ml tetracycline in drinking water for 7, 14, or 21 days to eliminate the worm's endosymbiont, Wolbachia.<br>Control age matched female worms were recovered from infected jirds given normal water for drinking.<br>The Filarial Nematode Oligonucleotide Array (version 2) was used in hybridization analyses on cDNA generated from extracted total RNA.<br>Each microarray was hybridized with a mixture of control and experimental cDNA differentially labeled with Cy3 and Cy5 in a flip-dye experiment.<br>Gridding and analysis of images were performed using ScanArray v3.0, each spot defined pixel-by-pixel using a modified Mann-Whitney test, and the resulting values processed with Gene-Spring 7.1 software.
Project description:Filarial nematodes (superfamily Filarioidea) are responsible for an annual global health burden of approximately 6.3 million disability-adjusted life-years, which represents the greatest single component of morbidity attributable to helminths affecting humans. No vaccine exists for the major filarial diseases, lymphatic filariasis and onchocerciasis; in part because research on protective immunity against filariae has been constrained because the human-parasitic species cannot complete their lifecycles in laboratory mice. However, the rodent filaria Litomosoides sigmodontis has become a popular experimental model, as BALB/c mice are fully permissive for its development and reproduction. Here, we provide a comprehensive analysis of excretory-secretory products from L. sigmodontis across five lifecycle stages. Applying intensity-based quantification, we determined the abundance of 302 unique excretory-secretory proteins, of which 64.6% were present in quantifiable amounts only from gravid adult female nematodes. This lifecycle stage, together with immature first-stage larvae (microfilariae), released four proteins that have not previously been evaluated as vaccine candidates: a predicted 28.5 kDa filaria-specific protein, a zonadhesin and SCO-spondin-like protein, a vitellogenin, and a protein containing six metridin-like ShK toxin domains. Female nematodes also released two proteins derived from the obligate Wolbachia symbiont. Notably, excretory-secretory products from all parasite stages contained several uncharacterised members of the transthyretin-like protein family. Furthermore, biotin labelling revealed that redox proteins and enzymes involved in purinergic signalling were enriched on the adult nematode cuticle. Comparison of the L. sigmodontis adult secretome with that of the human–infective filarial nematode Brugia malayi (reported previously in three independent published studies) identified differences that suggest a considerable underlying diversity of potential immunomodulators. The molecules identified in L. sigmodontis excretory-secretory products show promise not only for vaccination against filarial infections, but for the amelioration of allergy and autoimmune diseases.
Project description:Brugia malayi is a parasitic nematode that causes lymphatic filariasis in humans. A total of 178 novel microRNA were identified from short read transcriptional data, which when combined with known Brugia microRNAs yielded a total of 284 microRNA. Of these, 123 microRNA sequences (43%) are differentially expressed over the mammalian life stages of B. malayi that we examined. Putative targets of these microRNA were identified from inversely expressed target clusters that contain valid seed sequences for the corresponding microRNAs. The largest identified cluster is downregulated in adult females and enriched in zinc finger domains, helicase domains, and DNA binding domains suggesting this microRNA cluster may have regulatory control over a large proportion of adult female specific mRNA genes. MicroRNA-like molecules are identified as produced by the Wolbachia endosymbiont, providing evidence for direct nucleic acid-based interdomain communication between filarial nematodes and their bacterial obligate endosymbiont.
Project description:Lymphatic filarial nematodes maintain a mutualistic association with the intracellular bacterium Wolbachia. Wolbachia populations expand following infection of the mammalian host, to support larval growth and development. Utilizing transcriptomic data from Brugia malayi over the first two weeks post-infection, we present an analysis of the biochemical pathways that are involved in Wolbachia population growth and regulation in support of larval development. In Wolbachia, we observe coordinated regulation of carbon metabolism with an alternating pattern of glycolysis and TCA cycle pathways reminiscent of the ‘Warburg effect’. Wolbachia's purine, pyrimidine and haem biosynthesis and Type IV secretion pathways are also upregulated and correlate with the upregulation of the nematode’s DNA replication pathway. In the nematode we observe up-regulation of the autophagy pathway, a key regulator of Wolbachia populations. These findings support a key role for nucleotide and haem provisioning from Wolbachia in support of the larval growth and development of its nematode host.