Critical role of Babesia bovis spherical body protein 3 in ridge formation on infected red blood cells

Babesia bovis, an apicomplexan intraerythrocytic protozoan parasite, causes serious economic loss to cattle industries around the world. Infection with this parasite leads to accumulation of infected red blood cells (iRBCs) in the brain microvasculature that results in severe clinical complications known as cerebral babesiosis. Throughout its growth within iRBCs, the parasite exports various proteins to the iRBCs that lead to the formation of protrusions known as "ridges" on the surface of iRBCs, which serve as sites for cytoadhesion to endothelial cells. Spherical body proteins (SBPs; proteins secreted from spherical bodies, which are organelles specific to Piroplasmida) are exported into iRBCs, and four proteins (SBP1-4) have been reported to date. In this study, we elucidated the function of SBP3 using an inducible gene knockdown (KD) system. Localization of SBP3 was assessed by immunofluorescence assay, and only partial colocalization was detected between SBP3 and SBP4 inside the iRBCs. In contrast, colocalization was observed with VESA-1, which is a major parasite ligand responsible for the cytoadhesion. Immunoelectron microscopy confirmed localization of SBP3 at the ridges. SBP3 KD was performed using the glmS system, and effective KD was confirmed by Western blotting, immunofluorescence assay, and RNA-seq analysis. The SBP3 KD parasites showed severe growth defect suggesting its importance for parasite survival in the iRBCs. VESA-1 on the surface of iRBCs was scarcely detected in SBP3 KD parasites, whereas SBP4 was still detected in the iRBCs. Moreover, abolition of ridges on the iRBCs and reduction of iRBCs cytoadhesion to the bovine brain endothelial cells were observed in SBP3 KD parasites. Immunoprecipitation followed by mass spectrometry analysis detected the host Band 3 multiprotein complex, suggesting an association of SBP3 with iRBC cytoskeleton proteins. Taken together, this study revealed the vital role of SBP3 in ridge formation and its significance in the pathogenesis of cerebral babesiosis. Author summaryBabesia bovis causes a high-mortality complication called cerebral babesiosis in cattle, similar to cerebral malaria in humans. Both complications are caused by the cytoadhesion of infected red blood cells (iRBCs) to the host brain endothelial cells. These parasites export numerous proteins to the host iRBCs and produce protrusions on the iRBCs that are called ridges for B. bovis and knobs for Plasmodium falciparum. Ridges and knobs play an important role in cytoadhesion as they are the sites of adherence; however, the molecules responsible for ridge formation remain unknown. In this study, we showed that SBP3 is a crucial protein for ridge formation. The SBP3 knockdown parasites showed severe growth defects and abolition of ridges on the iRBCs, and cytoadhesion of iRBCs to the bovine brain endothelial cells was significantly reduced. An immunoprecipitation experiment suggested an association of SBP3 with the host Band 3 multiprotein complex. Although there is no similarity in amino acid sequences, we suggest SBP3 to be a functional ortholog of KAHRP in P. falciparum. In summary, our results shed light on the molecular mechanism of ridge formation and the pathogenesis of B. bovis.