Localization and functional difference of MSA-2 paralogs in Babesia bovis merozoites

Abstract Babesia bovis merozoite surface antigen 2 (MSA-2) are proteins encoded by msa multigene family and implicated in erythrocyte recognition and invasion, yet their roles remain only partially understood. We investigated spatiotemporal differences of three MSA-2 paralogs (MSA-2a1, MSA-2a2, and MSA-2b) by using epitope-specific rabbit antisera. Alignment of amino acid sequences of B. bovis T2Bo strain and Texas strain C1 clone showed that MSA-2a1 is highly conserved (99.6% identity) among the parasites, whereas MSA-2a2 (84%) and MSA-2b (94%) are more variable; notably, the least conserved paralog, MSA-2a2, harbored a contiguous 24 amino acid deletion. Indirect immunofluorescence assays using the antisera revealed apical localization of all three proteins in intraerythrocytic parasite. Following merozoite purification and brief incubation, localization of MSA-2a1 was observed on the merozoite surface before the attachment, whereas MSA-2a2 and MSA-2b remained at the apical end. Moreover, growth-inhibition assays, antiserum Ab-1 recognizing MSA-2a2 and MSA-2b inhibited 90.4% at 48 h incubation, whereas Ab-2 recognizing MSA-2a1 and MSA-2a2 inhibited only 13.1%. These results suggest functional difference among MSA-2 paralogs: MSA-2a1 was secreted to the surface before attachment, while MSA-2a2 and MSA-2b remain inside the merozoite at the onset of attachment. Taken together, MSA-2a1, MSA-2a2, and MSA-2b appear structurally similar, yet functionally distinct proteins that may contribute to the coordinated control of erythrocyte invasion.