Malaria is a vector-borne infectious disease caused by protozoan parasites of the genus Plasmodium. Infections with P. falciparum are responsible for the vast majority of all malaria-related morbidity and mortality in humans. Over the last two decades concerted intervention efforts targeting both the insect vector and the parasite led to a remarkable decline in malaria cases worldwide 1. However, progress has come to a standstill in the past few years and in 2018 malaria was still accountable for 228 million clinical cases and 405’000 deaths, primarily in sub-Saharan Africa 2. To further reduce the spread of malaria, intervention strategies will not only have to overcome the widespread resistance of mosquitoes and parasites to insecticides and first-line antimalarial drugs, respectively, but will also have to include efficient tools that interrupt parasite transmission from the human host to the mosquito vector 3,4.
People get infected with malaria parasites when P. falciparum-infested female Anopheles mosquitoes inject sporozoites into their skin. After reaching the liver via the bloodstream, sporozoites multiply within hepatocytes to release thousands of merozoites into circulation. Merozoites invade red blood cells (RBCs) and develop through the ring and trophozoite stage into a multi-nucleated schizont. After daughter cell formation, up to 32 merozoites egress from each infected RBC (iRBC) to invade and replicate inside new RBCs. Consecutive rounds of these intra-erythrocytic developmental cycles (IDCs) are responsible for all disease symptoms and chronic infection. Importantly, however, during each replication cycle a small proportion of schizonts produce sexually committed ring stage progeny that differentiate into gametocytes 5. When taken up by a mosquito, terminally differentiated gametocytes egress from the iRBC and develop into gametes. After fertilization, the zygote transforms into an ookinete that traverses the midgut epithelium to initiate sporogony, which ultimately renders the mosquito infectious to other humans. Hence, as the only forms of the parasite able to infect mosquitoes, gametocytes are highly specialized cells that secure human-to-human malaria transmission 6.
In P. falciparum, the process of sexual differentiation takes 10–12 days during which the parasite undergoes drastic changes in cellular morphology. Sexual ring stages (day 1) develop into spherical stage I gametocytes (day 2) that continuously elongate into lemon-shaped stage II (day 4), D-shaped stage III (day 6) and spindle-shaped stage IV cells (day 8), before falciform mature stage V gametocytes are formed (day 10+) 7,8. These morphological transitions are linked to the gradual expansion of the inner membrane complex, an endomembrane system underlying the parasite plasma membrane, and microtubule and actin cytoskeleton networks underneath that are disassembled again at the stage IV to V transition 9–13. Stage I to IV gametocytes are sequestered away from circulation, primarily in the parenchyma of the bone marrow and spleen 6,14. The mechanisms involved in gametocyte homing to and sequestration in these extravascular niches are poorly understood. However, the high rigidity of stage I to IV gametocyte-infected RBCs 9,15,16, established through parasite-induced alterations of the RBC membrane and underlying cytoskeletal networks 15,17−19, appears to play a primary role in gametocyte retention. Reversal of these modifications at the stage IV to V transition confers increased deformability 9,15−19, which likely allows for the release of stage V gametocytes into the bloodstream 6,20. Once in circulation, stage V gametocytes remain competent for transmission to the mosquito for days/weeks 21. The enormous transmission reservoir represented by the hundreds of millions of infected people in endemic areas, and the failure of almost all currently licensed antimalarial drugs except primaquine to efficiently kill mature gametocytes, pose major obstacles to malaria control and elimination efforts 4,22,23. Furthermore, the development of transmission-blocking drugs and vaccines is compromised by our poor understanding of the molecular mechanisms underlying essential gametocyte biology and the lack of reliable and readily applicable experimental tools.
Parasites commit to gametocytogenesis during the IDC preceding gametocyte differentiation. This finding was made based on experiments showing that all ring stage descendants derived from a single schizont have the same fate; they either all undergo another round of intracellular replication or they all differentiate into either female or male gametocytes 24,25. In addition to this ‘next cycle sexual conversion (NCC)’ process, recent studies reported ‘same cycle sexual conversion (SCC)’ where ring stages directly commit to sexual development 26,27. Irrespective of the NCC or SCC routes, sexual conversion is triggered by an epigenetic switch that activates expression of the master transcription factor AP2-G 28–31. In asexual parasites, heterochromatin-dependent silencing of ap2-g prevents AP2-G expression 28,30−32. In a small subset of trophozoites (NCC) or ring stages (SCC), however, the ap2-g locus gets activated by molecular mechanisms that are still largely unknown 27–31,33−35. Gametocyte development 1 (GDV1), a nuclear protein essential for gametocytogenesis in P. falciparum 36, plays a key role in the NCC process 34. GDV1 is specifically expressed in sexually committed parasites, where it displaces heterochromatin protein 1 (HP1) from the pfap2-g locus thereby licensing PfAP2-G expression 34. Consistent with the epigenetic control mechanisms regulating pfap2-g expression, sexual commitment rates vary in response to various environmental conditions 5. In particular, depletion of the host serum lipid lysophosphatidylcholine (LysoPC) triggers sexual commitment and this response is channeled via induction of GDV1 and PfAP2-G expression 34,35. Once expressed, PfAP2-G initiates a specific transcriptional programme that drives sexual conversion and subsequent gametocyte differentiation 5. While the PfAP2-G-dependent transcriptional changes in sexually committed schizonts are minor, a more pronounced gene expression signature emerges in the sexually committed ring stage progeny, where several dozen genes are specifically induced or repressed compared to asexual ring stages 28,30,31,33,34,37,38. Most of these upregulated genes are directly targeted by PfAP2-G 33 and encode proteins implicated in iRBC remodeling, while some others have expected roles in regulating gene expression during gametocyte maturation 28,30,31,33,34,37.
Regarding the complex process of sexual differentiation, mRNA and protein expression profiling studies conducted mainly on late stage gametocytes 39–43, but also on early stages 42,43 or across gametocyte maturation 44,45, identified hundreds of genes and proteins specifically up- or downregulated compared to asexual blood stages, or between female and male gametocytes 46–49. Stage I/II gametocytes express exported proteins involved in sexual stage-specific iRBC remodeling 42. Enzymes of the oxidative tricarboxylic acid (TCA) cycle are upregulated in gametocytes 39,44,45, consistent with their increased dependence on the TCA cycle for energy production 50. Factors involved in protein synthesis, mitochondrial function and translational repression are upregulated in females 46–49, in line with the increased abundance of ribosomes 12, enlarged mitochondrion 12,51 and large number of translationally repressed transcripts in these stages 46. Proteins linked to DNA replication and axoneme formation are enriched in males to prepare for the three rapid rounds of genome duplication and generation of eight motile microgametes during male gametogenesis 46–48. Gametocytes also show increased expression of transcription factors and chromatin modifiers 28,30,33,39,44,45, expanded subtelomeric heterochromatin domains 32,52, distinct histone post-translational modification profiles 53 and altered three-dimensional chromosome organisation 52. Furthermore, earlier studies investigating gametocyte morphology at the ultrastructural level described an intriguing nuclear dimorphism between female and male gametocytes 12,54. These nuclear processes and features have not been further explored in any great detail but are likely linked to the control of gametocyte and sex-specific differentiation. These and many additional studies 5,55 provided invaluable insight into gametocyte- and sex-specific biology. However, a functional and mechanistic understanding of the underlying molecular and cellular processes is often lacking since only a relatively small number of genes has been investigated by reverse genetics approaches in P. falciparum gametocytes.
Our limited knowledge about gametocyte biology is primarily due to the difficulty in generating large numbers of synchronous gametocyte stages for experimental studies: (i) in vitro cultured P. falciparum parasites show low sexual commitment rates (< 10%) (reflected as the proportion of sexually committed ring stages among all ring stage parasites) 5,6; (ii) gametocytes are non-proliferative cells and thus rapidly overgrown by asexual parasites; and (iii) sexual commitment occurs during each consecutive IDC, which results in asynchronous gametocyte populations. Currently used approaches to increase sexual commitment rates for in vitro gametocyte production are based on exposing parasite cultures to poorly defined stress conditions imposed for instance by high parasitaemia and/or nutrient starvation (spent/conditioned medium) 8,56,57. Several different protocols relying on this strategy exist and reach reported sexual commitment rates of 10–30% 58–64. However, most of these protocols use cumbersome experimental workflows, rely on large culture volumes, are expensive, are difficult to reproduce and/or produce asynchronous gametocyte populations. The recently developed approach employing LysoPC- or choline-depleted minimal fatty acid medium35 achieves sexual commitment rates in the range of 15–60%, but growth under these nutrient-restricted conditions leads to lower numbers of sexually committed progeny produced per schizont 34,35,65,66.
Here, we used CRISPR/Cas9-based genome editing to engineer transgenic P. falciparum lines suitable for the routine mass production of synchronous gametocytes. Through the targeted induction of GDV1 expression these lines consistently achieve sexual conversion rates of 75% and generate synchronous gametocyte populations at high yield across all stages of gametocytogenesis. Furthermore, we demonstrate that these gametocytes undergo sexual reproduction and oocyst formation in female Anopheles mosquitoes. Lastly, by generating iGP parasites expressing an mScarlet-tagged version of the nuclear pore protein NUP313 we show that further genetic engineering of iGP lines is straightforward and enables the generation of large numbers of gametocyte mutants for the targeted investigation of P. falciparum transmission stage biology.