Isolation of viable Babesia bovis merozoites to study parasite invasion

Abstract

Babesia parasite invades exclusively red blood cell (RBC) in mammalian host and induces alterations to host cell for survival. Despite the importance of Babesia in livestock industry and emerging cases in humans, their basic biology is hampered by lack of suitable biological tools. In this study, we aimed to develop a synchronization method for Babesia bovis which causes the most pathogenic form of bovine babesiosis. Initially, we used compound 2 (C2), a specific inhibitor of cyclic GMP-dependent protein kinase (PKG), and a derivative of C2, ML10. While both inhibitors were able to prevent B. bovis egress from RBC and increased percentage of binary forms, removal of inhibitors from culture did not result in a synchronized egress of parasites. Because using PKG inhibitors alone was not efficient to induce a synchronized culture, we isolated viable and invasive B. bovis merozoites and showed dynamics of merozoite invasion and development in RBCs. Using isolated merozoites we showed that BbVEAP, VESA1-export associated protein, is essential for parasite development in the RBC while has no significant role in invasion. Given the importance of invasion for the establishment of infection, this study paves the way for finding novel antigens to be used in control strategies against bovine babesiosis.

Recent Advances in Molecular Genetic Tools for Babesia

“…The knockdown of BbVEAP in the presence of inducer was ~90% at protein level (Figure 3B) [26]. This reduction confirmed the role of BbVEAP in parasite development in the RBC, VESA1 export and cytoadhesion of iRBCs to endothelial cells [26,60]. Riboswitch system could be simply employed by insertion of glmS sequence at 3' non-coding region of the gene of interest open reading frame, downstream of the stop codon and is a promising method to be used for mRNA knockdown of Babesia.…”

“…Currently, iRBCs are being used for transfection. Large-scale preparation of parasite merozoites is available and may improve the efficiency of transfection [60].…”

Advances in understanding red blood cell modifications by Babesia

“…Mammalian RBCs are terminally differentiated and anucleate and lack cellular machinery and metabolism. Babesia parasites have a fast metabolism and require nucleic acids and proteins, and the relatively short doubling time (10 to 12 h for B. bovis) and DNA replication (2.9 h for B. bovis) [65] necessitate uptake of metabolic precursors from the host plasma, such as sugars, purines, and amino acids. For P. falciparum iRBCs, studies using osmotic lysis, patch-clamp methods, and radioactive labeled substrates have documented increases in permeabilities to various nutrients, called the new permeability pathway or plasmodial surface anion channel (PSAC) [70,71].…”

“…Given that BbVEAP knockdown did not affect SBP4 export, BbVEAP expression is necessary for the export and correct localization of a subset of proteins including VESA1 and ridge-forming proteins. Additionally, it was shown that BbVEAP is indispensable for parasite development in the RBC, making it the only known essential exportome protein [ 33 , 65 ]. The existence of VEAP among piroplasma parasites indicates a piroplasma-specific conserved function.…”

“…bovis ) and DNA replication (2.9 h for B . bovis ) [ 65 ] necessitate uptake of metabolic precursors from the host plasma, such as sugars, purines, and amino acids. For P .…”