ves1α genes expression is the major determinant of Babesia bovis-infected erythrocytes cytoadhesion to endothelial cells

Babesia bovis causes the most pathogenic form of babesiosis in cattle, resulting in high mortality in naive adults. This parasite invades red blood cells (RBCs) within the bovine hosts where they multiply and produce clinical disease. Babesia bovis exports numerous proteins into invaded RBCs changing its properties. Thus, the infected RBCs (iRBCs) are capable to cytoadhere in the microvasculature of internal organs and brain, leading to respiratory distress, neurologic signs, and mortality. Variant Erythrocyte Surface Antigen 1 (VESA1) is one of those exported proteins by B. bovis which represents a major virulence factor due to its central role in immune evasion by antigenic variation and intravascular parasite sequestration. VESA1 is a heterodimer protein encoded by ves1 and ves1{beta} multigene family and localized on the ridges, the focal point for cytoadhesion. To gain further insights into the molecular mechanisms of cytoadhesion of B. bovis, we panned the parasites with bovine brain microvasculature endothelial cells, which resulted in obtaining several clones with different cytoadherence abilities. The transcriptome analysis of 2 high and 2 low cytoadherent clones revealed that ves1 sequences were diversified, likely resulting from genomic recombination. On the other hand, ves1{beta} sequences were almost identical among these 4 clones. Insertion and expression of ves1 of a clone with high binding into ef-1 locus of a low binging clone increased cytoadherence confirming the role of ves1 suggested by our transcriptome data. Whole genome sequencing of cytoadherent clones revealed active locus of ves1 on chromosome 2. These results suggest that VESA1a proteins encoded by ves1 genes determine the cytoadherence specificity and/or cytoadherence strength of B. bovis and they are in the active site for recombination. Author summaryBabesia bovis is an apicomplexan intraerythrocytic protozoan parasite which causes the most pathogenic form of babesiosis in cattle. This pathogenicity is the result of parasite multiplication and cytoadherence of infected red blood cells (iRBCs) in the microvasculature of brain and internal organs and is mediated by B. bovis surface exposed ligand, Variant Erythrocyte Surface Antigen 1 (VESA1). Here using parasite panning assay, transcriptomics, and genetic tools, we showed that VESA1a is the main determinant of B. bovis cytoadhesion. VESA1 are large hypervariable proteins (>100kDa) consisting of VESA1a and VESA1b subunits encoded by ves1 and ves1{beta} multigene family. Panning B. bovis with bovine brain endothelial cells resulted in obtaining cytoadherent parasite clones with different binding abilities. Comparative transcriptome analysis revealed diversification of ves1 sequences. Insertion and expression of ves1 of a clone with high-binding ability in the genome of a low-binding clone increased cytoadherence confirming the role of ves1. Mapping RNA-seq on the genome of cytoadherent clones revealed the locus of active transcription and this locus was suggested to be the active site for recombination which promoted the production of variants of ves1 with different binding abilities. Altogether, our results provide new insights into B. bovis cytoadhesion and VESA1 biology.