Mutant/mutation
The mutant contains a mutated clamp gene with two LoxN sites. The clamp open reading frame (ORF) is C-terminally tagged with a 3xHA epitopeI. It also contains a gfp , cas9 and dicre expression cassettes, introduced into the neutral 230p locus (from the parent line PbCasDiCre-GFP; RMgm-5567).

In order to validate the Cas9 and DiCre combination, we used the PbCasDiCre-GFP line (RMgm-5567) to conditionally delete the gene encoding the Claudin-like apicomplexan microneme protein (CLAMP, PBANKA_0514200). A one-step strategy to introduce a first LoxP site inside an artificial intron, as described in P. falciparum, and a second LoxP site immediately downstream of the STOP codon. A triple HA tag was introduced before the STOP codon for C-terminal tagging of CLAMP. Using this strategy, CRE-mediated recombination should result in immediate and complete suppression of CLAMP expression. Two guide RNAs were selected in the clamp locus, in the 5’ and 3’ regions of the coding sequence, respectively, and cloned into a single plasmid containing PbU6 and PfU6 promoters. The donor DNA template carrying a modified clamp gene with an artificial intron taken from the P. berghei slarp gene (PBANKA_0902100) and containing the first LoxP site, a 3xHA tag and a second LoxP site, was provided as a synthetic gene and linearized before transfection.
Following the transfection of PbCasDiCre-GFP parasites with the double sgRNA plasmid targeting clamp and the linearized donor template DNA, integration of the repair construct by double homologous recombination results in the generation of parasites containing a floxed clamp gene with a C-terminal 3xHA tag. Recombinant parasites were selected with pyrimethamine.

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2024.10.14.618196

Protein (function)
Invasion of host cells by apicomplexan parasites such as Toxoplasma and Plasmodium spp requires the sequential secretion of the parasite apical organelles, the micronemes and the rhoptries. The claudin-like apicomplexan microneme protein (CLAMP) is a conserved protein that plays an essential role during invasion in Toxoplasma gondii tachyzoites and Plasmodium falciparum merozoites. Topology predictions indicate structural similarities between CLAMP and the mammalian tight-junction proteins claudin-15 and claudin-19. CLAMP is also expressed in Plasmodium sporozoites, the mosquito transmitted forms of the malaria parasite. CLAMP is essential for Plasmodium blood stage growth and is refractory to conventional gene deletion.

Phenotype
No phenotype different from wild type parasites.
Analysis of clampcKO-HA merozoites by immunofluorescence using anti-HA antibodies revealed a punctate distribution of the protein which was often predominant at one pole of the parasite, reminiscent of the apical accumulation of the protein.

After rapamycin treatment of parasites the clamp gene (and the GFP gene) is excised. See Additional Information below.

Additional information
Conditional deletion of the clamp gene by rapamycin treatment of blood stage parasites resulted in the following phentotype:
Rapamycin-induced excision of clamp reduced parasitaemia to nearly zero in a single cycle (<24 hours), as compared to untreated parasites, in agreement with an essential role for CLAMP in asexual blood stages. To assess the invasive capacity of CLAMP-deficient merozoites, clampcKO merozoites were produced in culture in the presence or absence of rapamycin, and injected intravenously into mice. Following inoculation, untreated merozoites efficiently invaded mouse erythrocytes and established a blood stage infection, as evidenced by flow cytometry, while rapamycin-exposed merozoites failed to invade erythrocytes, as evidenced by the absence of detectable parasitaemia after inoculation into mice.

Other mutants