Ookluc: A Plasmodium berghei Line for Identifying Transmission-blocking Compounds

Malaria remains a world health challenge; in 2023, 263 million cases and 597,000 deaths were reported around the globe. The parasite's life cycle is complex, and targeted interventions at critical developmental stages are needed to effectively control the disease. Transmission-blocking (TB) strategies aim to interrupt Plasmodium transmission by targeting either the mosquito vector or the parasite's sexual stages through vaccines and drugs. TB interventions can focus on gametocytes in the human host or other forms in the mosquito midgut, such as gametes, zygotes, ookinetes, and oocysts. The development of new TB approaches faces significant challenges. Studying the parasite's sexual stages is inherently difficult, and high-throughput screening (HTS) for gametocytocidal compounds relies primarily on P. falciparum cultures. However, robust in vitro tools for assessing interventions against later sexual stages remain limited. To accelerate the discovery of TB compounds, our group developed a novel HTS assay for gametocyte activation and fertilization using P. berghei, a murine model of Plasmodium infection. The transgenic parasite, Ookluc, expresses nanoluciferase exclusively after zygote formation, with peak luminescence during ookinete development. This luminescence-based assay provides a quantitative readout of gamete to zygote and zygote to ookinete differentiation, where increased luminescence directly correlates with ookinete formation. This video article presents the methods for screening malaria TB compounds using Ookluc.