The G9a Histone Methyltransferase Inhibitor BIX-01294 Modulates Gene Expression during Plasmodium falciparum Gametocyte Development and Transmission.

Transmission of the malaria parasite Plasmodium falciparum from the human to the mosquito is initiated by specialized sexual cells, the gametocytes. In the human, gametocytes are formed in response to stress signals and following uptake by a blood-feeding Anopheles mosquito initiate sexual reproduction. Gametocytes need to fine-tune their gene expression in order to develop inside the mosquito to continue life-cycle progression. Previously, we showed that post-translational histone acetylation controls gene expression during gametocyte development and transmission. However, the role of histone methylation remains poorly understood. We here use the histone G9a methyltransferase inhibitor BIX-01294 to investigate the role of histone methylation in regulating gene expression in gametocytes. In vitro assays demonstrated that BIX-01294 inhibits intraerythrocytic replication with a half maximal inhibitory concentration (IC50) of 13.0 nM. Furthermore, BIX-01294 significantly impairs gametocyte maturation and reduces the formation of gametes and zygotes. Comparative transcriptomics between BIX-01294-treated and untreated immature, mature and activated gametocytes demonstrated greater than 1.5-fold deregulation of approximately 359 genes. The majority of these genes are transcriptionally downregulated in the activated gametocytes and could be assigned to transcription, translation, and signaling, indicating a contribution of histone methylations in mediating gametogenesis. Our combined data show that inhibitors of histone methylation may serve as a multi-stage antimalarial.

The development of malaria parasites in the mosquito midgut.

The mosquito midgut stages of malaria parasites are crucial for establishing an infection in the insect vector and to thus ensure further spread of the pathogen. Parasite development in the midgut starts with the activation of the intraerythrocytic gametocytes immediately after take-up and ends with traversal of the midgut epithelium by the invasive ookinetes less than 24 h later. During this time period, the plasmodia undergo two processes of stage conversion, from gametocytes to gametes and from zygotes to ookinetes, both accompanied by dramatic morphological changes. Further, gamete formation requires parasite egress from the enveloping erythrocytes, rendering them vulnerable to the aggressive factors of the insect gut, like components of the human blood meal. The mosquito midgut stages of malaria parasites are unprecedented objects to study a variety of cell biological aspects, including signal perception, cell conversion, parasite/host co-adaptation and immune evasion. This review highlights recent insights into the molecules involved in gametocyte activation and gamete formation as well as in zygote-to-ookinete conversion and ookinete midgut exit; it further discusses factors that can harm the extracellular midgut stages as well as the measures of the parasites to protect themselves from any damage.

The Plasmodium falciparum blood stages acquire factor H family proteins to evade destruction by human complement.

The acquisition of regulatory proteins is a means of blood-borne pathogens to avoid destruction by the human complement. We recently showed that the gametes of the human malaria parasite Plasmodium falciparum bind factor H (FH) from the blood meal of the mosquito vector to assure successful sexual reproduction, which takes places in the mosquito midgut. While these findings provided a first glimpse of a complex mechanism used by Plasmodium to control the host immune attack, it is hitherto not known, how the pathogenic blood stages of the malaria parasite evade destruction by the human complement. We now show that the human complement system represents a severe threat for the replicating blood stages, particularly for the reinvading merozoites, with complement factor C3b accumulating on the surfaces of the intraerythrocytic schizonts as well as of free merozoites. C3b accumulation initiates terminal complement complex formation, in consequence resulting in blood stage lysis. To inactivate C3b, the parasites bind FH as well as related proteins FHL-1 and CFHR-1 to their surface, and FH binding is trypsin-resistant. Schizonts acquire FH via two contact sites, which involve CCP modules 5 and 20. Blockage of FH-mediated protection via anti-FH antibodies results in significantly impaired blood stage replication, pointing to the plasmodial complement evasion machinery as a promising malaria vaccine target.

Transcriptional Profiling Defines Histone Acetylation as a Regulator of Gene Expression during Human-to-Mosquito Transmission of the Malaria Parasite Plasmodium falciparum.

Transmission of the malaria parasite Plasmodium falciparum from the human to the mosquito is mediated by the intraerythrocytic gametocytes, which, once taken up during a blood meal, become activated to initiate sexual reproduction. Because gametocytes are the only parasite stages able to establish an infection in the mosquito, they are crucial for spreading the tropical disease. During gametocyte maturation, different repertoires of genes are switched on and off in a well-coordinated sequence, pointing to regulatory mechanisms of gene expression. While epigenetic gene control has been studied during erythrocytic schizogony of P. falciparum, little is known about this process during human-to-mosquito transmission of the parasite. To unveil the potential role of histone acetylation during gene expression in gametocytes, we carried out a microarray-based transcriptome analysis on gametocytes treated with the histone deacetylase inhibitor trichostatin A (TSA). TSA-treatment impaired gametocyte maturation and lead to histone hyper-acetylation in these stages. Comparative transcriptomics identified 294 transcripts, which were more than 2-fold up-regulated during gametocytogenesis following TSA-treatment. In activated gametocytes, which were less sensitive to TSA, the transcript levels of 48 genes were increased. TSA-treatment further led to repression of ~145 genes in immature and mature gametocytes and 7 genes in activated gametocytes. Up-regulated genes are mainly associated with functions in invasion, cytoadherence, and protein export, while down-regulated genes could particularly be assigned to transcription and translation. Chromatin immunoprecipitation demonstrated a link between gene activation and histone acetylation for selected genes. Among the genes up-regulated in TSA-treated mature gametocytes was a gene encoding the ring finger (RING)-domain protein PfRNF1, a putative E3 ligase of the ubiquitin-mediated signaling pathway. Immunochemistry demonstrated PfRNF1 expression mainly in the sexual stages of P. falciparum with peak expression in stage II gametocytes, where the protein localized to the nucleus and cytoplasm. Pfrnf1 promoter and coding regions associated with acetylated histones, and TSA-treatment resulted in increased PfRNF1 levels. Our combined data point to an essential role of histone acetylation for gene regulation in gametocytes, which can be exploited for malaria transmission-blocking interventions.