A NIMA-related Protein Kinase Is Essential for Completion of the Sexual Cycle of Malaria Parasites

Abstract

The molecular mechanisms regulating the sexual development of malaria parasites from gametocytes to oocysts in their mosquito vector are still largely unexplored. In other eukaryotes, NIMA-related kinases (Neks) regulate cell cycle progression and have been implicated in the regulation of meiosis. Here, we demonstrate that Nek-4, a new Plasmodium member of the Nek family, is essential for completion of the sexual cycle of the parasite. Recombinant Plasmodium falciparum Nek-4 possesses protein kinase activity and displays substrate preferences similar to those of other Neks. Nek-4 is highly expressed in gametocytes, yet disruption of the nek-4 gene in the rodent malaria parasite P. berghei has no effect on gamete formation and subsequent fertilization. However, further differentiation of zygotes into ookinetes is abolished. Measurements of nuclear DNA content indicate that zygotes lacking Nek-4 fail to undergo the genome replication to the tetraploid level that precedes meiosis. Cell cycle progression in the zygote is identified as a likely precondition for its morphological transition to the ookinete and for the successful establishment of a malaria infection in the mosquito.Malaria remains a devastating disease in most tropical and subtropical regions. The problem has been exacerbated over the last decades of the twentieth century by the emergence and spread of resistance of the causative agents, parasitic protists of the genus Plasmodium, to available antimalarials (Plasmodium falciparum is the species responsible for the vast majority of lethal cases) (1). Infection of the human host is initiated by the bite of an infected Anopheles mosquito, which delivers sporozoites into the bloodstream. The sporozoites rapidly gain the liver, where they invade hepatocytes and undergo a first round of schizogony. The merozoites produced in this process are released into the bloodstream and invade erythrocytes, where they undergo recurrent and synchronized schizogony. This is the phase of the parasite's life cycle that is responsible for malaria pathogenesis. A proportion of merozoites, upon invasion of a new red blood cell, do not enter schizogony, but arrest their cell cycle and develop into male or female gametocytes, the only forms capable of infecting the mosquito vector. Ingestion of gametocytes during a blood meal triggers their further development into gametes, a process, which for the male gametocyte, involves three rounds of genome replication and generation of eight flagellated male gametes. Fertilization is followed by nuclear fusion, one round of genome replication, and meiosis, which occurs within 3 h (2, 3). The nuclear envelope remains intact throughout this process, and meiosis is not followed by nuclear division. As a consequence the ookinete, a motile form that develops from the zygote and exits the mosquito midgut lumen, is tetraploid. The ookinete establishes an oocyst at the basal lamina, which produces several thousand sporozoites. These accumulate in the insect salivary glands and render the mosquito...

The development of malaria parasites in the mosquito midgut

“…berghei resulted in male sterility and blocked fertilization (Hirai et al ., 2008; Liu et al ., 2008). Following gamete fusion, nuclear fusion ensues, and over the next 3 h, meiosis occurs, and the zygote becomes tetraploid (Janse et al ., 1986), a process involving the NIMA‐related kinases Nek‐2 and Nek‐4 (Reininger et al ., 2005, 2009). Parasite tetraploidy persists throughout the ookinete stage until sporozoite budding in the oocyst restores the haploid state (Janse et al ., 1986) (Table 1).…”

An evolutionary perspective on the kinome of malaria parasites

“…The roles of individual protein kinases were first investigated following a reverse genetics approach in the rodent malaria model Plasmodium berghei. This revealed essential functions of a MAPK (Pbmap-2) and a CDPK (PbCDPK4) in male gametogenesis [29,46,47], of two NIMA-related kinases in meiosis in the mosquito vector [48,49], and of a CDK in asexual proliferation in erythrocytes [50]. This approach culminated in a kinome-wide study demonstrating that 23 P. berghei ePKs are redundant for asexual erythrocytic parasite development in mice and identifying phenotypes in sexual development for a number of these 23 ePKs [51].…”

“…The expansion of the NEK family is seen to a lesser extent in Trichomonas vaginalis [83] and trypanosomes [93], which are also parasitic excavates with elaborate flagellar systems. Three of the four Plasmodium NEK family members are expressed predominantly in sexual stages [48,49,94], which include the only flagellated cell form in the Plasmodium life cycle, the male gamete; however, only one Pfnek has been clearly shown to be male-specific [95,96], whereas two others are associated with female development and appear to be involved in meiosis [48,49].…”

Signalling in malaria parasites – The MALSIG consortium

“…Interestingly, the orthologue in P. falciparum, Pfmap-2, is required for asexual growth (Dorin-Semblat et al, 2007). The initial steps of sporogony following fertilisation in P. berghei require Pbnek-4, a NIMA-related kinase with a role in meiosis (Reininger et al, 2005), and a second enzyme of this family was recently shown to also be essential for meiosis in both P. berghei and P. falciparum (Reininger et al, 2009). PbCDPK3 is required for traversal of the mosquito midgut epithelium (Ishino et al, 2006;SidenKiamos et al, 2006).…”

“…The protein kinases CDPK4 and Pbmap-2 have been associated with the production of male gametes (Billker et al, 2004;Khan et al, 2005;Rangarajan et al, 2005), Pbnek-4 and Pbnek-2 with ookinete maturation (Reininger et al, 2005;Reininger et al, 2009), and PbCDPK3 and guanylate cyclise beta with ookinete physiology (Ishino et al, 2006;SidenKiamos et al, 2006;Hirai et al, 2006). The role of signalling molecules in several other phases of sexual differentiation is however completely unknown, despite the fact that some of these processes, such as the establishment of the gametocyte parasitophorous vacuole, or gamete emergence are intrinsically complex cellular rearrangements, which proceed in response to specific cellular and environmental signals.…”