Dataset Information

A dynamic and combinatorial histone code drives malaria parasite asexual and sexual development

ABSTRACT: Histone post-translational modifications (PTMs) are frequently co-occurring on the same chromatin domains or even the same molecule. It is now established that these ‘histone codes’ are the result of cross-talk between enzymes that catalyse multiple PTMs with univocal readout as compared to these PTMs in isolation. Here, we performed a comprehensive identification and quantification of histone codes of the malaria parasite, Plasmodium falciparum. We used advanced quantitative middle-down proteomics to identify combinations of PTMs in both the proliferative, asexual stages and transmissible, sexual gametocyte stages of P. falciparum. We provide an updated, high-resolution compendium of 72 PTMs on H3 and H3.3, of which 30 newly identified. Several co-existing PTMs with unique stage distinction was identified, indicating that many of these combinatorial PTMs are associated to specific stages of the parasite life cycle. We focused on the code H3R17me2K18acK23ac for its unique presence in mature gametocytes; chromatin proteomics identified a gametocyte-specific SAGA-like effector complex including the transcription factor AP2-G2 which we associated to this specific histone code, as involved in regulating gene expression in mature gametocytes. Ultimately, this study unveils previously undiscovered histone PTMs and their functional relationship with co-existing partners. These results highlight that investigating chromatin regulation in the parasite using single histone PTM assays might overlook higher order gene regulation for distinct proliferation and differentiation processes.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Plasmodium Falciparum V1

TISSUE(S): Blood Cell, Cell Suspension Culture, Monocyte, Cell Culture

SUBMITTER: Simone Sidoli

LAB HEAD: Simone Sidoli

PROVIDER: PXD030181 | Pride | 2022-05-19

REPOSITORIES: Pride

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Publications

A Dynamic and Combinatorial Histone Code Drives Malaria Parasite Asexual and Sexual Development.

von Grüning Hilde H Coradin Mariel M Mendoza Mariel R MR Reader Janette J Sidoli Simone S Garcia Benjamin A BA Birkholtz Lyn-Marié LM

Molecular & cellular proteomics : MCP 20220117 3

Histone posttranslational modifications (PTMs) frequently co-occur on the same chromatin domains or even in the same molecule. It is now established that these "histone codes" are the result of cross talk between enzymes that catalyze multiple PTMs with univocal readout as compared with these PTMs in isolation. Here, we performed a comprehensive identification and quantification of histone codes of the malaria parasite, Plasmodium falciparum. We used advanced quantitative middle-down proteomics ...[more]

PMID: 35051657

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Project description:During intra-erythrocytic development, late asexually replicating Plasmodium falciparum parasites sequester from peripheral circulation. This facilitates chronic infection and is linked to severe disease and organ-specific pathology including cerebral and placental malaria. Immature gametocytes M-bM-^@M-^S sexual stage precursor cells M-bM-^@M-^S likewise disappear from circulation. Recent work has demonstrated that these sexual stage parasites are located in the hematopoietic system of the bone marrow before mature gametocytes are released into the blood stream to facilitate mosquito transmission. However, as sequestration occurs only in vivo and not during in vitro culture, the mechanisms by which it is regulated and enacted (particularly by the gametocyte stage) remain poorly understood. We generated the most comprehensive P. falciparum functional gene network to date by integrating global transcriptional data from a large set of asexual and sexual in vitro samples, patient-derived in vivo samples, and a new set of in vitro samples profiling sexual commitment. We defined more than 250 functional modules (clusters) of genes that are co-expressed primarily during the intra-erythrocytic parasite cycle, including 35 during sexual commitment and gametocyte development. Comparing the in vivo and in vitro datasets allowed us, for the first time, to map the time point of asexual parasite sequestration in patients to 22 hours post invasion, confirming previous in vitro observations on the dynamics of host cell modification and cytoadherence. Moreover, we were able to define the properties of gametocyte sequestration, demonstrating the presence of two circulating gametocyte populations: gametocyte rings between 0 and ~30 hours post invasion and mature gametocytes after around 7 days post invasion. We used 164/TdTom, a transgenic parasite line expressing a red fluorescent protein reporter under a gametocyte-specific promoter to generate schizont samples. Schizonts were subsequently isolated from both the fluorescent and non-fluorescent population by FACS and prepared for microarray analysis. Two biological replicates were produced for both the fluorescent and the non-fluorescent samples.

Project description:For malaria transmission, the parasite must undergo sexual differentiation into mature gametocytes. However, the molecular basis for this critical transition in the parasites life cycle is unknown. Six previously uncharacterized genes, Pfg14.744, Pfg14.745, Pfg14.748, Pfg14.763, Pfg14.752 and Pfg6.6 that are members of a 36 gene Plasmodium falciparum-specific subtelomeric superfamily were found to be expressed in parasites that are committed to sexual development as suggested by co-expression of Pfs16 and Pfg27. Northern blots demonstrated that Pfg14.744 and Pfg14.748 were first expressed before the parasites differentiated into morphologically distinct gametocytes, transcription continued to increase until stage II gametocytes were formed and then rapidly decreased. Immunofluorescence assays indicated that both proteins were only produced in the subpopulation of ring stage parasites that are committed to gametocytogenesis and both localized to the parasitophorous vacuole (PV)b of the early ring stage parasites. As the parasites continued to develop Pfg14.748 remained within the parasitophorous vacuole, while Pfg14.744 was detected in the erythrocyte. The 5' flanking region of either gene alone was sufficient to drive early gametocyte specific expression of green fluorescent protein (GFP). In parasites transfected with a plasmid containing the Pfg14.748 5' flanking region immediately upstream of GFP, fluorescence was observed in a small number of schizonts the cycle before stage I gametocytes were observed. This expression pattern is consistent with commitment to sexual differentiation prior to merozoite release and erythrocyte invasion. Further investigation into the role of these genes in the transition from asexual to sexual differentiation could provide new strategies to block malaria transmission. Microarray analysis was used to compare two clones derived from Plasmodium falciparum strain 3D7 parasites that differ in their ability to undergo gametocytogenesis. Clone G+ produces gametocytes and clone G- produces very few if any gametocytes. RNA was harvested from the cultures when the asexual parasitemia was 0.9-1.48% (day 4) (n=4) after setting up the gametocyte cultures and 5.2-5.58% (day 6) (n=4) prior to the appearance of morphologically distinct gametocytes and used to generate cDNA that was labeled with Cy3 or Cy5 and hybridized to the Plasmodium falciparum 70 mer oligonucleotide microarray developed by DeRisi and co-workers.

Dataset Information

A dynamic and combinatorial histone code drives malaria parasite asexual and sexual development

Publications

A Dynamic and Combinatorial Histone Code Drives Malaria Parasite Asexual and Sexual Development.

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