Anand Srivastava

Placental malaria is characterized by the massive accumulation and sequestration of infected erythrocytes in the placental intervillous blood spaces, causing severe birth outcomes. The variant surface antigen VAR2CSA is associated with Plasmodium falciparum sequestration in the placenta via its capacity to adhere to chondroitin sulfate A. We have previously shown that the extracellular region of VAR2CSA is phosphorylated on several residues and that the phosphorylation enhances the adhesive properties of CSA-binding infected erythrocytes. Here, we aimed to identify the kinases mediating this phosphorylation. We report that Human and Plasmodium falciparum Casein Kinase 2α are involved in the phosphorylation of the extracellular region of VAR2CSA. We notably show that both CK2α can phosphorylate the extracellular region of recombinant and immunoprecipitated VAR2CSA. Mass spectrometry analysis of recombinant VAR2CSA phosphorylated by recombinant Human and P. falciparum CK2α combined with site-directed mutagenesis led to the identification of residue S1068 in VAR2CSA, which is phosphorylated by both enzymes and is associated with CSA binding. Furthermore, using CRISPR/Cas9 we generated a parasite line in which phosphoresidue S1068 was changed to alanine. This mutation strongly impairs infected erythrocytes adhesion by abolishing VAR2CSA translocation to the surface of infected erythrocytes. We also report that two specific CK2 inhibitors reduce infected erythrocytes adhesion to CSA and decrease the phosphorylation of the recombinant extracellular region of VAR2CSA using either infected erythrocytes lysates as a source of kinases or recombinant Human and P. falciparum casein kinase 2. Taken together, these results undoubtedly demonstrate that host and P. falciparum CK2α phosphorylate the extracellular region of VAR2CSA and that this post-translational modification is important for VAR2CSA trafficking and for infected erythrocytes adhesion to CSA.

Buparvaquone (BQO) is used for treatment of bovine theileriosis, a tickborne disease caused by parasites of the Theileria genus. Studies on T. annulata have linked the mechanism of BQO resistance predominantly to genetic variations in the parasite cytochrome b (cytb) gene. In addition, cryptic mechanisms of resistance involving the parasite peptidyl-prolyl isomerase (pin1) and dihydroorotate dehydrogenase (dhodh) genes require assessment. In India, where bovine theileriosis is endemic, and BQO is widely used for treatment, monitoring and establishing the link between genetic variations in cytb, dhodh and pin1 genes and BQO resistance is essential. In this study, multiplexed PCR amplification and nanopore sequencing approaches were used for genotyping the complete gene loci of the target genes. Analysis of 420 T. annulata field samples collected from seven different states of India revealed the presence of previously reported variations S129G, A146T and P253S in cytb and A53P in pin1, which are linked to BQO resistance. The role of A146T, a highly prevalent variation which mostly co-occurred with I203V, in BQO resistance needs to be evaluated. From 60 samples having T. orientalis infection, the genetic variations identified from the three genes were found to be mostly natural variations based on the reference genotypes and were distinct from T. annulata variations. This study has revealed the presence of BQO resistance-linked cytb gene mutations in T. annulata infecting dairy cattle in India and establishes a nanopore sequencing method for molecular surveillance of genetic variation in field samples. Author Summary Buparvaquone (BQO) is the most effective drug that is available for the treatment of cattle theileriosis caused by parasites of the Theileria genus. However, treatment failure due to drug resistance is reported, and gene mutations linked to BQO resistance have been reported in African and Middle Eastern countries. These mutations occur in the cytochrome b (cytb) and peptidyl-prolyl isomerase (pin1) genes in the parasite. In this study, genetic variations occurring in these two genes and the parasite dihydroorotate dehydrogenase (dhodh) gene have been mapped using PCR amplification and Nanopore sequencing from field samples collected from seven different states of India. At least three previously reported mutations linked to BQO resistance were found in the T. annulata cytb gene sequence obtained from field samples. While one of these mutations (A146T) was highly prevalent, the other two were found to occur in only a few samples. Similarly, the BQO resistance linked to A53P mutation in the T. annulata pin1 gene was present in only a few samples. Despite their low frequency, the presence of these mutations reveals the existence of BQO resistance due to genetic variations in the parasite population present in India. This is the first comprehensive report of BQO resistance-conferring mutations occurring in Theileria parasites affecting dairy cattle in India and establishes a scalable method for large-scale molecular surveillance studies.