This study is about Theileria equi is a widely distributed apicomplexan parasite that causes severe hemolytic anemia in equid species. There is currently no effective vaccine for control of the parasite and understanding the mechanism thatT. equi utilizes to invade host cells may be crucial for vaccine development. Unlike most apicomplexan species studied to date, the role of micronemes in T. equi invasion of host cells is unknown. We therefore assessed the role of the T. equi claudin-like apicomplexan microneme protein (CLAMP) in the invasion of equine erythrocytes as a first step towards understanding the role of this organelle in the parasite. Our findings show that CLAMP is expressed in the merozoite and intra-erythrocytic developmental stages of T. equi and in vitro neutralization experiments suggest that the protein is involved in erythrocyte invasion. Proteomic analyses indicate that CLAMP interacts with the equine erythrocyte α-and β- spectrin chains in the initial stages of T. equi invasion and maintains these interactions while also associating with the anion-exchange protein, tropomyosin 3, band 4.1 and cytoplasmic actin 1 after invasion.
Fig: T. equi CLAMP contains immunogenic epitopes, and the gene is transcribed and expressed by T. equi merozoites. (a) CLAMP’s transmembrane topology as predicted and annotated by Phobius and Protter algorithms, respectively. Peptides 1, 2 and 3 indicate the protein’s predicted immunogenic peptides. (b) A 1% agarose gel showing amplified clamp transcript at ~ 1226 bp. The T. equi equine merozoite antigen-1 (ema-1) transcript was amplified as a positive control, and rt- (negative control) represents amplified merozoites’ RNA without addition of reverse transcriptase. L indicates the Thermo Scientific GeneRuler 1 kb DNA Ladder. (c) Immunoblot showing CLAMP expressed by T. equimerozoites (test) at ~ 39 kDa. Probing of merozoites lysate with pre-immunization serum and the polyclonal anti-RAP-1a antibody were used as negative control (NC) and positive control (PC) respectively. L represents the ProteinSimple Wes Ladder.
Additionally, serological analyses show that T. equi-infected horses mount robust antibody responses against CLAMP indicating that the protein is immunogenic and therefore represents a potential vaccine candidate.
Onzere, C.K., Fry, L.M., Bishop, R.P. et al. Theileria equi claudin like apicomplexan microneme protein contains neutralization-sensitive epitopes and interacts with components of the equine erythrocyte membrane skeleton. Sci Rep 11, 9301 (2021). https://doi.org/10.1038/s41598-021-88902-4