Function Analysis Of Plasmodium Yoelii Tubulin Binding Cofactor A

Malaria is an infectious disease transmitted by Anopheles.It causes hundreds of millions of infections and hundreds of thousands of deaths every year all over the world.Plasmodium has a complex life cycle and two reproductive modes,asexual reproduction and sexual reproduction.Cytoskeleton protein is expressed in all stages of life of Plasmodium,and is a potential target for anti-malaria.A few of the asexual cells of Plasmodium have become male and female gametocytes.When gametocytes enter Anopheles mosquitoes,they will be stimulated then become male and female gametes.The marker of male gametocytes activation is the assembly and extension of flagellate filaments,which involves the assembly of microtubules.Although it was reported that tubulin binding cofactor A(TBCA)is involved in the assembly of microtubules,there is no direct evidence to show that TBCA protein is associated with the activation of male gametocytes of Plasmodium.Based on the functional analysis of the gene tbca,this study revealed that TBCA protein participates in the activation of male gametocytes.Firstly,the Plasmodium yoelii TBCA protein was in mutiple sequence alightment with different kinds of Plasmodium,and it is found that there is a conservative TBCA domain in it.We constructed an endogenous tagged strain of tbca gene and analyzed the expression of TBCA protein in the life cycle of Plasmodium.The results showed that TBCA protein was expressed in every life cycle of Plasmodium,and partially co-localized with Tubulin protein at gametocyte stage.Three TBCA knockout strains were obtained by three independent knockouts.The results showed that the gene tbca knockout strains could not establish malaria infection in new healthy mice through mosquito transmission,and the phenotypic defects could be restored by rescued experiment.Further experiments showed that the male gametocyte exgellation rate is induced compared to wild type.Immunofluorescence results showed that gene tbca knockout did not affect the ability to rupture erythrocyte mem branes and parasitophorous vacuole membrane during gametocyte activation.Through mosquito hybridization and immunofluorescence labeling P28 protein of activated female gametocyte,it was verified that the phenotypic defects caused by gene tbca knockout results in male gametophyte defects only,and the female gametocytes was normalIn this paper,we use 17XNL of Plasmodium yoelii as a model to study TBCA by CRISPR/Cas9 system,investigating TBCA in gametocyte activation,and try to reveal the relationship between TBCA and Tubulin ?/?.This study lays a foundation for further study of TBCA protein and provides a new target for drug research and development of antimalarial targets.