Dataset Information

Phenotypic and molecular analysis of the effect of 20-hydroxyecdysone on the human filarial parasite Brugia malayi.

ABSTRACT: A homologue of the ecdysone receptor has been identified and shown to be responsive to 20-hydroxyecdysone in Brugia malayi. However, the role of this master regulator of insect development has not been delineated in filarial nematodes. Gravid adult female B. malayi cultured in the presence of 20-hydroxyecdysone produced significantly more microfilariae and abortive immature progeny than control worms, implicating the ecdysone receptor in regulation of embryogenesis and microfilarial development. Transcriptome analyses identified 30 genes whose expression was significantly up-regulated in 20-hydroxyecdysone-treated parasites compared with untreated controls. Of these, 18% were identified to be regulating transcription. A comparative proteomic analysis revealed 932 proteins to be present in greater amounts in extracts of 20-hydroxyecdysone-treated adult females than in extracts prepared from worms cultured in the absence of the hormone. Of the proteins exhibiting a greater than two-fold difference in the 20-hydroxyecdysone-treated versus untreated parasite extracts, 16% were involved in transcriptional regulation. RNA interference (RNAi) phenotype analysis of Caenorhabditis elegans orthologs revealed that phenotypes involved in developmental processes associated with embryogenesis were significantly over-represented in the transcripts and proteins that were up-regulated by exposure to 20-hydroxyecdysone. Taken together, the transcriptomic, proteomic and phenotypic data suggest that the filarial ecdysone receptor may play a role analogous to that in insects, where it serves as a regulator of egg development.

SUBMITTER: Mhashilkar AS

PROVIDER: S-EPMC4936774 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Phenotypic and molecular analysis of the effect of 20-hydroxyecdysone on the human filarial parasite Brugia malayi.

Mhashilkar Amruta S AS Adapa Swamy R SR Jiang Rays H Y RH Williams Steven A SA Zaky Weam W Slatko Barton E BE Luck Ashley N AN Moorhead Andrew R AR Unnasch Thomas R TR

International journal for parasitology 20160216 5-6

A homologue of the ecdysone receptor has been identified and shown to be responsive to 20-hydroxyecdysone in Brugia malayi. However, the role of this master regulator of insect development has not been delineated in filarial nematodes. Gravid adult female B. malayi cultured in the presence of 20-hydroxyecdysone produced significantly more microfilariae and abortive immature progeny than control worms, implicating the ecdysone receptor in regulation of embryogenesis and microfilarial development. ...[more]

PMID: 26896576

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Project description:Lymphatic filariasis (LF) is a socio-economically devastating mosquito-borne Neglected Tropical Disease caused by parasitic filarial nematodes. The interaction between the parasite and host, both mosquito and human, during infection, development and persistence is dynamic and delicately balanced. Manipulation of this interface to the detriment of the parasite is a promising potential avenue to develop disease therapies but is prevented by our very limited understanding of the host-parasite relationship. Exosomes are bioactive small vesicles (30-120 nm) secreted by a wide range of cell types and involved in a wide range of physiological processes. Here, we report the identification and partial characterization of exosome-like vesicles (ELVs) released from the infective L3 stage of the human filarial parasite Brugia malayi. Exosome-like vesicles were isolated from parasites in culture media and electron microscopy and nanoparticle tracking analysis were used to confirm that vesicles produced by juvenile B. malayi are exosome-like based on size and morphology. We show that loss of parasite viability correlates with a time-dependent decay in vesicle size specificity and rate of release. The protein cargo of these vesicles is shown to include common exosomal protein markers and putative effector proteins. These Brugia-derived vesicles contain small RNA species that include microRNAs with host homology, suggesting a potential role in host manipulation. Confocal microscopy shows J774A.1, a murine macrophage cell line, internalize purified ELVs, and we demonstrate that these ELVs effectively stimulate a classically activated macrophage phenotype in J774A.1. To our knowledge, this is the first report of exosome-like vesicle release by a human parasitic nematode and our data suggest a novel mechanism by which human parasitic nematodes may actively direct the host responses to infection. Further interrogation of the makeup and function of these bioactive vesicles could seed new therapeutic strategies and unearth stage-specific diagnostic biomarkers.

Project description:We have previously shown that the microfilarial (mf) stage of Brugia malayi can inhibit the mammalian target of rapamycin (mTOR; a conserved serine/threonine kinase critical for immune regulation and cellular growth) in human dendritic cells (DC) and we have proposed that this mTOR inhibition is associated with the DC dysfunction seen in filarial infections. Extracellular vesicles (EVs) contain many proteins and nucleic acids including microRNAs (miRNAs) that might affect a variety of intracellular pathways. Thus, EVs secreted from mf may elucidate the mechanism by which the parasite is able to modulate the host immune response during infection. EVs, purified from mf of Brugia malayi and confirmed by size through nanoparticle tracking analysis, were assessed by miRNA microarrays (accession number GSE157226) and shown to be enriched (>2-fold, p-value<0.05, FDR = 0.05) for miR100, miR71, miR34, and miR7. The microarray analysis compared mf-derived EVs and mf supernatant. After confirming their presence in EVs using qPCR for these miRNA targets, web-based target predictions (using MIRPathv3, TarBAse and MicroT-CD) predicted that miR100 targeted mTOR and its downstream regulatory protein 4E-BP1. Our previous data with live parasites demonstrated that mf downregulate the phosphorylation of mTOR and its downstream effectors. Additionally, our proteomic analysis of the mf-derived EVs revealed the presence of proteins commonly found in these vesicles (data are available via ProteomeXchange with identifier PXD021844). We confirmed internalization of mf-derived EVs by human DCs and monocytes using confocal microscopy and flow cytometry, and further demonstrated through flow cytometry, that mf-derived EVs downregulate the phosphorylation of mTOR in human monocytes (THP-1 cells) to the same degree that rapamycin (a known mTOR inhibitor) does. Our data collectively suggest that mf release EVs that interact with host cells, such as DC, to modulate host responses.

Dataset Information

Phenotypic and molecular analysis of the effect of 20-hydroxyecdysone on the human filarial parasite Brugia malayi.

Publications

Phenotypic and molecular analysis of the effect of 20-hydroxyecdysone on the human filarial parasite Brugia malayi.

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