Unhale, T. | Search bioRxiv

bioRxiv2026

The CLAMP-Linked Invasion Protein (CLIP) plays an essential role in Plasmodium berghei zoites

Apicomplexan parasites such as Toxoplasma and Plasmodium spp. rely on the sequential secretion of parasite apical organelles, called micronemes and rhoptries, to invade host cells. The claudin-like apicomplexan microneme protein (CLAMP) is a conserved protein that plays an essential role during host cell invasion in Toxoplasma and Plasmodium zoites. Previous studies have shown that CLAMP is essential in Plasmodium merozoites for erythrocyte invasion and also in sporozoites for the invasion of the mosquito vector salivary glands and of mammalian host hepatocytes. In Toxoplasma gondii tachyzoites, CLAMP forms a complex with two other microneme proteins, the Secreted Protein with an Altered Thrombospondin Repeat (SPATR) and the CLAMP-Linked Invasion Protein (CLIP). Both SPATR and CLIP are also expressed in Plasmodium sporozoites, and downregulation of SPATR impacts sporozoite infectivity in P. berghei. In contrast, the role of CLIP in sporozoites remains unknown. To study the function of CLIP, we used a CRISPR-assisted conditional genome editing strategy based on the dimerisable Cre recombinase in the rodent malaria model parasite P. berghei. Deletion of clip in P. berghei blood stages impaired parasite growth and prevented erythrocyte invasion by merozoites. Upon deletion of clip gene in P. berghei transmission stages, sporozoite development in mosquitoes was not affected, but invasion of the mosquito salivary glands was dramatically reduced. In addition, CLIP-deficient sporozoites were impaired in cell traversal and productive invasion of mammalian hepatocytes, associated with a defect in gliding motility, recapitulating the phenotype of CLAMP-deficient parasites. Collectively, our data demonstrate that CLIP plays an essential role in host cell invasion by P. berghei merozoites and sporozoites, and support a conserved role of the CLAMP-CLIP-SPATR complex in invasive stages of apicomplexan parasites.

microbiology↗

bioRxiv2024

Constitutive expression of Cas9 and rapamycin-inducible Cre recombinase facilitates conditional genome editing in Plasmodium berghei

Malaria is caused by protozoan parasites of the genus Plasmodium and remains a global health concern. The parasite has a highly adaptable life cycle comprising successive rounds of asexual replication in a vertebrate host and sexual maturation in the mosquito vector Anopheles. Genetic manipulation of the parasite has been instrumental for deciphering the function of Plasmodium genes. Conventional reverse genetic tools cannot be used to study essential genes of the asexual blood stages, thereby necessitating the development of conditional strategies. Among various such strategies, the rapamycin-inducible dimerisable Cre (DiCre) recombinase system emerged as a powerful approach for conditional editing of essential genes in human-infecting P. falciparum and in the rodent malaria model parasite P. berghei. We previously generated a DiCre-expressing P. berghei line and validated it by conditionally deleting several essential asexual stage genes, revealing their important role also in sporozoites. The advent of CRISPR enabled targeted genome editing with higher accuracy and specificity and greatly advanced genome engineering in Plasmodium spp. Here, we developed new P. berghei parasite lines by integrating the DiCre cassette and a fluorescent marker in parasites constitutively expressing Cas9. Owing to the dual integration of CRISPR-Cas9 and DiCre, these new lines allow unparalleled levels of gene modification and conditional regulation simultaneously. To illustrate the versatility of this new tool, we conditionally knocked-out the essential gene encoding the claudin-like apicomplexan micronemal protein (CLAMP) in P. berghei and confirm the role of CLAMP during invasion of erythrocytes.

microbiology↗