Malaria transmission requires the production of male and female gametocytes in the human host followed by fertilization and sporogonic development in the mosquito midgut. Although essential for the spread of malaria through the population, little is known about the initiation of gametocytogenesis in vitro or in vivo. Using a gametocyte-defective parasite line and genetic complementation, we show that Plasmodium falciparum gametocyte development 1 gene (Pfgdv1), encoding a peri-nuclear protein, is critical for early sexual differentiation. Transcriptional analysis of Pfgdv1 negative and positive parasite lines identified a set of gametocytogenesis early genes (Pfge) that were significantly down-regulated (>10 fold) in the absence of Pfgdv1 and expression was restored after Pfgdv1 complementation. Progressive accumulation of Pfge transcripts during successive rounds of asexual replication in synchronized cultures suggests that gametocytes are induced continuously during asexual growth. Comparison of Pfge gene transcriptional profiles in patient samples divided the genes into two groups differing in their expression in mature circulating gametocytes and providing candidates to evaluate gametocyte induction and maturation separately in vivo. The expression profile of one of the early gametocyte specific genes, Pfge1, correlated significantly with asexual parasitemia, which is consistent with the ongoing induction of gametocytogenesis during asexual growth observed in vitro and reinforces the need for sustained transmission-blocking strategies to eliminate malaria.

SummaryMalaria transmission requires that the parasites differentiate into gametocytes prior to ingestion by a mosquito during a blood meal. Once in the mosquito midgut the gametocytes emerge from red blood cells (RBCs), fertilize, develop into ookinetes and finally infectious sporozoites. Gamete surface antigen, Pfs230, is an important malaria transmissionblocking vaccine candidate, but its function has remained unclear. Two clones with distinct Pfs230 gene disruptions (D1.356 and D2.560) and a clone with a disruption of Pfs48/45 were used to evaluate the role of Pfs230 in the mosquito midgut. Pfs230 disruptants successfully emerge from RBCs and male gametes exflagellate producing microgametes. However, exflagellating Pfs230-minus males, in the presence or absence of Pfs48/45, are unable to interact with RBCs and form exflagellation centres. Oocyst production and mosquito infectivity is also significantly reduced, 96-92% and 76-71% respectively. In contrast, in the Pfs230 disruptants the expression and localization of other known sexual stage-specific antigens, including Pfs48/45, Pfs47, the Pfs230 paralogue (PfsMR5), Pfs16 or Pfs25, were not altered and the Pfs230-minus gametes retained resistance to the alternative pathway of human complement. These results suggest that Pfs230 is the surface molecule on males that mediates RBC binding and plays an important role in oocyst development, a critical step in malaria transmission.

Plasmodium sexual differentiation is required for malaria transmission, yet much remains unknown about its regulation. Here, we quantify early gametocyte-committed ring (gc-ring) stage, P. falciparum parasites in 260 uncomplicated malaria patient blood samples 10 days before maturation to transmissible stage V gametocytes using a gametocyte conversion assay (GCA). Seventy six percent of the samples have gc-rings, but the ratio of gametocyte to asexual-committed rings (GCR) varies widely (0–78%). GCR correlates positively with parasitemia and is negatively influenced by fever, not hematocrit, age or leukocyte counts. Higher expression levels of GDV1-dependent genes, ap2-g , msrp1 and gexp5 , as well as a gdv1 allele encoding H 217 are associated with high GCR, while high plasma lysophosphatidylcholine levels are associated with low GCR in the second study year. The results provide a view of sexual differentiation in the field and suggest key regulatory roles for clinical factors and gdv1 in gametocytogenesis in vivo.

Malaria continues to be a major global health problem, but only a limited arsenal of effective drugs is available. None of the antimalarial compounds commonly used clinically kill mature gametocytes, which is the form of the parasite that is responsible for malaria transmission. The parasite that causes the most virulent human malaria, Plasmodium falciparum, has a 48-h asexual cycle, while complete sexual differentiation takes 10 to 12 days. Once mature, stage V gametocytes circulate in the peripheral blood and can be transmitted for more than a week. Consequently, if chemotherapy does not eliminate gametocytes, an individual continues to be infectious for several weeks after the clearance of asexual parasites. The work reported here demonstrates that nanomolar concentrations of the proteasome inhibitor epoxomicin effectively kill all stages of intraerythrocytic parasites but do not affect the viability of human or mouse cell lines. Twenty-four hours after treatment with 100 nM epoxomicin, the total parasitemia decreased by 78%, asexual parasites decreased by 86%, and gametocytes decreased by 77%. Seventy-two hours after treatment, no viable parasites remained in the 100 or 10 nM treatment group. Epoxomicin also blocked oocyst production in the mosquito midgut. In contrast, the cysteine protease inhibitors epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester and N-acetyl-L-leucyl-Lleucyl-L-methioninal blocked hemoglobin digestion in early gametocytes but had no effect on later stages. Moreover, once the cysteine protease inhibitor was removed, sexual differentiation resumed. These findings provide strong support for the further development of proteasome inhibitors as antimalaria agents that are effective against asexual, sexual, and mosquito midgut stages of P. falciparum.The current recommended treatments for malaria caused by Plasmodium falciparum, including artemisinin combination therapy, eliminate intraerythrocytic asexual parasites that are responsible for the clinical symptoms. However, these treatments do not kill mature intraerythrocytic gametocytes that are required for the transmission of the parasite (24). In contrast to the 2-day asexual cycle of P. falciparum, the production of a mature stage V gametocyte takes 10 to 12 days. Once mature gametocytes are taken up by a mosquito during a blood meal, fertilization is stimulated. The resulting zygotes develop into oocysts where thousands of sporozoites are produced that can be transmitted to humans during a subsequent blood meal. The prolonged period required for P. falciparum gametocyte maturation in the human host suggests that malaria can be transmitted for several weeks after asexual parasites are eliminated (23). Thus, the development of drugs that are effective against both asexual-stage parasites and gametocytes may directly decrease malaria morbidity and mortality and reduce the spread of the disease.Cysteine protease and proteasome inhibitors have been found to affect asexual intraerythrocytic parasites and are being evaluated as possible an...

SummaryCysteine proteases are currently targets for drug development in a number of parasitic diseases, including malaria. In Plasmodium falciparum , the parasite responsible for the most virulent form of human malaria, there are four members of the cathepsin L-like family of cysteine proteases. Three of these (falcipains 2A, 2B and 3) are thought to be primarily involved in haemoglobin digestion, whereas falcipain 1 has recently been linked to erythrocyte invasion. Neither their expression nor their role in P. falciparum gametocytogenesis, which is required for malaria transmission, has been evaluated. In this study, RNA transcripts for the falcipain family members were identified as the parasite developed through all five stages of gametocytogenesis. Falcipain 1 transcript was upregulated in gametocytes, while levels of falcipain 2A/2B decreased in late-stage gametocytes and gametes. To evaluate the function of falcipain 1, the gene was disrupted, and clones from independent transformations were isolated. The asexual growth of the falcipain 1 minus clones was not overtly affected, and they produced morphologically normal gametocytes and gametes. However, when falcipain 1 minus parasites were fed to a mosquito, oocyst production was reduced by 70-90%, suggesting an important role for falcipain 1 during parasite development in the mosquito midgut.