Mutant/mutation
The mutant lacks expression of PBANKA_0100800

Protein (function)
See below

Phenotype
Normal numbers of female and male gametocytes are produced. 90% reduction in the number of exflagellation centers formed in male gametocytes. No ookinete formation. No oocyst formation.

Additional information
Previous studies have shown that the phosphatase inhibitor BVT-948 has good transmission blocking activity. To evaluate the effectiveness of using the phosphatase inhibitor BVT948 to screen for functional proteins involved in P. berghei sexual development, we selected proteins whose phosphorylation levels increased after the administration of BVT-948 and whose functions were unknown. The gene IDs corresponding to these 8 proteins were PBANKA_0100800, PBANKA_0314900, PBANKA_0408400, PBANKA_0704700, PBANKA_0836800, PBANKA_0927700, PBANKA_1000600 and PBANKA_1320100. Domain prediction of these proteins indicated that they had a variety of structures and suggested their diverse functions.
We first evaluated gametocyte development in these 8 KO lines. The PBANKA_0100800 KO, PBANKA_0704700 KO, and PBANKA_0836800 KO clones showed no significant differences from WT parasites. However, PBANKA_0314900, PBANKA_0408400, PBANKA_0927700 and PBANKA_1320100 KO significantly affected gametocytogenesis, and the PBANKA_1000600 KO lines showed significantly higher gametocyte density than the WT parasites. Notably, PBANKA_0100800, PBANKA_0704700, PBANKA_0836800 and PBANKA_1000600 did not have essential roles in gametocyte sex differentiation.
To determine whether knocking out PBANKA_0100800, PBANKA_0704700, PBANKA_0836800 or PBANKA_1000600 affected subsequent sexual development, we performed in vitro ookinete culture using the WT and KO clones. In vitro analysis revealed an over 90% reduction in the number of exflagellation centers formed in male gametocytes of both PBANKA_0100800 KO lines compared with those in WT control gametocytes. There were no defects in the egress of male gametes in the PBANKA_0704700, PBANKA_0836800 or PBANKA_1000600 KO lines. An in vitro ookinete culture assay showed that PBANKA_0100800 was essential for ookinete formation. Deletion of PBANKA_0100800 completely reduced the ookinete number compared to that in the WT line. The PBANKA_0704700 and PBANKA_1000600 deletion lines also showed significant reductions in ookinete formation, with ~10% and ~40% reductions in ookinete numbers, respectively.
In direct mosquito feeding assays, complete reductions in the percentage of infected mosquitoes and oocyst density per mosquito midgut were observed in those feeding on PBANKA_0100800 KO-infected mice compared to the WT control. Mosquitoes fed on mice infected with the PBANKA_0704700 KO lines showed modest reductions in the infection rate compared to those fed on mice infected with the WT parasites.
There were slight reductions in the mean number of oocysts per midgut in mosquitoes fed on mice infected with the PBANKA_0704700 and PBANKA_1000600 KO lines compared to those infected with the WT strain. However, the differences in these two indices were not significant between these two KO lines and the WT control.

As the deletion of PBANKA_0100800 significantly interrupted P. berghei sexual development and blocked transmission, we performed a bioinformatics analysis of PBANKA_0100800 to explore its possible role in P. berghei sexual development. To predict the underlying mechanism, we first analysed the interaction protein network of the protein encoded by PBANKA_0100800. The interaction network showed that PBANKA_0100800 might interact with PBANKA_0713800, PBANKA_0922200 and PBANKA_1141700 and their associated genes. GO analysis revealed that the biological processes related to PBANKA_0100800 were mainly peptide biosynthetic processes. In terms of molecular function, the processes predominantly related to PBANKA_0100800 were structural constituents of ribosomes and structural molecule activity. In terms of cellular components, components associated with ribosomes, especially the intracellular ribonucleoprotein complex, were the most enriched. To evaluate the reference value of this functional analysis of PBANKA_0100800 in P. berghei for other Plasmodium species that infect humans, we analysed the conservation of the C3H1 domain (66–92 aa) of the protein encoded by PBANKA_0100800 in P. berghei with P. falciparum and P. vivax. Multiple sequence alignment revealed that this protein was highly conserved among these Plasmodium species and that the C3H1 domain was completely the same in all three.

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