Nirdosh, N. | Search bioRxiv

bioRxiv2025

Plasmodium actin-like proteins are essential for DNA segregation during male gametogenesis and malaria transmission

Protozoan parasites of the genus Plasmodium cause malaria and involve infection of multiple hosts and cell types during the life cycle. Producing sexually fit gametocytes is essential for transmitting the Plasmodium parasite into an anopheline mosquito vector. After the uptake of malaria parasites, male gametocytes undergo three rounds of DNA replication to produce eight nucleated flagellar gametes. Here, we report that the actin-like proteins Alp5a and Alp5b are involved in DNA segregation during male gametogenesis. The Plasmodium-specific paralogous genes Alp5a and Alp5b superimposed on human Arp2 and Arp3, localize to the nucleus, and interact with each other. Alp5a and Alp5b are dispensable for the development of P. berghei blood-stages individually but indispensable simultaneously. In agreement with genetic studies, the inhibitory activity of the Arp2/3 complex inhibitor in Plasmodium supports an essential role for this complex during the blood stage. Deletion of Alp5a or Alp5b did not affect actin nucleation, parasite growth, or gametocytemia in the blood. The knockout parasites successfully invaded the midgut and developed into oocysts, but their size was significantly reduced, and they failed to survive. Genetic crosses revealed defects in male gamete integrity. We found that the reduced oocyst development was due to impaired DNA segregation during male gametogenesis. This study provides molecular insight into the fundamental requirements of the Alps in Plasmodium, which are essential for malaria transmission.

microbiology↗

bioRxiv2024

A novel micronemal protein, Scot1, is essential for apicoplast biogenesis and liver stage development in Plasmodium berghei

Plasmodium sporozoites invade hepatocytes, transform into liver stages, and replicate into thousands of merozoites that infect erythrocytes and cause malaria. Proteins secreted from micronemes play an essential role in hepatocyte invasion, and unneeded micronemes are subsequently discarded for replication. The liver-stage parasites are potent immunogens that prevent malarial infection. Late liver stage-arresting genetically attenuated parasites (GAPs) exhibit greater protective efficacy than early GAP. However, the number of late liver-stage GAPs for generating GAPs with multiple gene deletions is limited. Here, we identified Scot1 (Sporozoite Conserved Orthologous Transcript 1), which was previously shown to be upregulated in sporozoites, and by endogenous tagging with mCherry, we demonstrated that it is expressed in the sporozoite and liver stages in micronemes. Using targeted gene deletion in Plasmodium berghei, we showed that Scot1 is essential for late liver-stage development. Scot1 KO sporozoites grew normally into liver stages but failed to initiate blood-stage infection in mice due to impaired apicoplast biogenesis and merozoite formation. Bioinformatic studies suggested that Scot1 is a metal/small molecule carrier protein. Remarkably, supplementation with metals in the culture of infected Scot1 KO cells did not rescue their phenotype. Immunization with Scot1 KO sporozoites in C57BL/6 mice confers protection against a malaria challenge via infection. These proof-of-concept studies will enable the generation of P. falciparum Scot1 mutants that could be exploited to generate GAP malaria vaccines. ImportanceMalaria parasites experience significant bottlenecks as transmitted to the mammalian host during a mosquito bite. Sporozoites invade liver cells, reproducing into thousands of merozoites, which are released after liver cell ruptures. The specific arrest of sporozoites during liver stage development acts as a powerful immunogen and provides sterile protection against sporozoite infection. GAP leading to an arrest in late liver stage development offers superior protection. Here, we report that a micronemal protein, Scot1, is essential for parasite maturation in the liver. Deletion of Scot1 resulted in impaired apicoplast biogenesis and merozoite formation. Vaccination with Scot1 KO sporozoites protects against malaria challenge. We have identified a late arresting GAP that will aid in developing new as well as safeguarding existing whole parasite vaccines.

microbiology↗