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An assay to probe Plasmodium falciparum growth, transmission stage formation and early gametocyte development.

ABSTRACT: Conversion from asexual proliferation to sexual differentiation initiates the production of the gametocyte, which is the malaria parasite stage required for human-to-mosquito transmission. This protocol describes an assay designed to probe the effect of drugs or other perturbations on asexual replication, sexual conversion and early gametocyte development in the major human malaria parasite Plasmodium falciparum. Synchronized asexually replicating parasites are induced for gametocyte production by the addition of conditioned medium, and they are then exposed to the treatment of interest during sexual commitment or at any subsequent stage of early gametocyte development. Flow cytometry is used to measure asexual proliferation and gametocyte production via DNA dye staining and the gametocyte-specific expression of a fluorescent protein, respectively. This screening approach may be used to identify and evaluate potential transmission-blocking compounds and to further investigate the mechanism of sexual conversion in malaria parasites. The full protocol can be completed in 11 d.

SUBMITTER: Brancucci NM

PROVIDER: S-EPMC4581880 | biostudies-other | 2015 Aug

REPOSITORIES: biostudies-other

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An assay to probe Plasmodium falciparum growth, transmission stage formation and early gametocyte development.

Brancucci Nicolas M B NM Goldowitz Ilana I Buchholz Kathrin K Werling Kristine K Marti Matthias M

Nature protocols 20150702 8

Conversion from asexual proliferation to sexual differentiation initiates the production of the gametocyte, which is the malaria parasite stage required for human-to-mosquito transmission. This protocol describes an assay designed to probe the effect of drugs or other perturbations on asexual replication, sexual conversion and early gametocyte development in the major human malaria parasite Plasmodium falciparum. Synchronized asexually replicating parasites are induced for gametocyte production ...[more]

PMID: 26134953

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Project description:BackgroundThe Plasmodium sexual gametocyte stages are the only transmissible form of the malaria parasite and are thus responsible for the continued transmission of the disease. Gametocytes undergo extensive functional and morphological changes from commitment to maturity, directed by an equally extensive control program. However, the processes that drive the differentiation and development of the gametocyte post-commitment, remain largely unexplored. A previous study reported enrichment of H3K36 di- and tri-methylated (H3K36me2&3) histones in early-stage gametocytes. Using chromatin immunoprecipitation followed by high-throughput sequencing, we identify a stage-specific association between these repressive histone modifications and transcriptional reprogramming that define a stage II gametocyte transition point.ResultsHere, we show that H3K36me2 and H3K36me3 from stage II gametocytes are associated with repression of genes involved in asexual proliferation and sexual commitment, indicating that H3K36me2&3-mediated repression of such genes is essential to the transition from early gametocyte differentiation to intermediate development. Importantly, we show that the gene encoding the transcription factor AP2-G as commitment master regulator is enriched with H3K36me2&3 and actively repressed in stage II gametocytes, providing the first evidence of ap2-g gene repression in post-commitment gametocytes. Lastly, we associate the enhanced potency of the pan-selective Jumonji inhibitor JIB-04 in gametocytes with the inhibition of histone demethylation including H3K36me2&3 and a disruption of normal transcriptional programs.ConclusionsTaken together, our results provide the first description of an association between global gene expression reprogramming and histone post-translational modifications during P. falciparum early sexual development. The stage II gametocyte-specific abundance of H3K36me2&3 manifests predominantly as an independent regulatory mechanism targeted towards genes that are repressed post-commitment. H3K36me2&3-associated repression of genes is therefore involved in key transcriptional shifts that accompany the transition from early gametocyte differentiation to intermediate development.

Dataset Information

An assay to probe Plasmodium falciparum growth, transmission stage formation and early gametocyte development.

Publications

An assay to probe Plasmodium falciparum growth, transmission stage formation and early gametocyte development.

Similar Datasets

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