Geno- and phenotypic characteristics of a transfected babesia bovis 6-Cys-E knockout clonal line

Authors: ALZAN, AF - Washington State University; SILVA, MG - Washington State University; DAVIS, WC - Washington State University; Herndon, David; Schneider, David; Suarez, Carlos

Submitted to: Parasites & Vectors
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/19/2017
Publication Date: 5/2/2017
Citation: Alzan, A., Silva, M., Davis, W., Herndon, D.R., Schneider, D.A., Suarez, C.E. 2017. Geno- and phenotypic characteristics of a transfected babesia bovis 6-Cys-E knockout clonal line. Parasites & Vectors. 10(1):214.

Interpretive Summary: Babesia bovis is an intra-erythrocytic tick transmitted apicomplexan protozoan parasite. It has a complex life style including asexual replication in the mammalian host and sexual replication occurring in the midgut of host tick vector, typically, Rhipicephalus microplus. Previous evidence showed that certain B. bovis genes, including members of 6-Cys gene family are differentially expressed during tick and mammalian stages of the parasite’s life cycle. Moreover, the 6-Cys E gene is differentially expressed in the T3Bo strain of B. bovis tick stages, and anti 6-Cys E antibodies were shown to be able to inhibit in vitro growth of B. bovis Mo7clonal line. In this study, the 6-Cys E gene of B. bovis T3Bo strain was disrupted by transfection. The resulting, biologically cloned mutated strain was designated EKO-cln . Full genome sequencing of 6-Cys EKO-cln parasites demonstrated single insertion of egfp-bsd gene that disrupts the integrity of 6-Cys gene E. Undistinguishable growth rate of 6-Cys EKO-cln line compared to wild type 6-Cys E intact T3Bo B. bovis strain in in vitro cultures indicates that expression of gene 6-Cys E is not essential for blood stage replication in this strain. In vitro inhibition assays using anti 6-Cys E antibodies confirmed inhibition the ability of anti-6 Cys E antibodies to inhibit the growth of the wild type Mo7 and T3Bo B. bovis parasites, but no significant inhibition was found for 6-Cys EKO-cln line parasites. Overall, the data suggests that the anti-6 Cys E antibody neutralizing effect on the wild type strains is likely due to mechanical hindrance, or cross-reactivity, rather than due to functional requirements of 6-Cys gene E product for survival and development of the erythrocyte stages. Further investigation is underway to determine if the 6-Cys E protein is required for replication and sexual stage development of B. bovis during tick stages.

Technical Abstract: Babesia bovis is an intra-erythrocytic tick transmitted apicomplexan protozoan parasite. It has a complex life style including asexual replication in the mammalian host and sexual replication occurring in the midgut of host tick vector, typically, Rhipicephalus microplus. Previous evidence showed that certain B. bovis genes, including members of 6-Cys gene family are differentially expressed during tick and mammalian stages of the parasite’s life cycle. Moreover, the 6-Cys E gene is differentially expressed in the T3Bo strain of B. bovis tick stages, and anti 6-Cys E antibodies were shown to be able to inhibit in vitro growth of B. bovis Mo7clonal line. In this study, the 6-Cys E gene of B. bovis T3Bo strain was disrupted by transfection using a plasmid containing 6-Cys gene E 5’ and 3’ regions to guide homologous recombination, and the egfp-bsd fusion gene under control of a ef-1' promoter, yielding a B. bovis clonal line designated 6-Cys EKO-cln. Full genome sequencing of 6-Cys EKO-cln parasites demonstrated single insertion of egfp-bsd gene that disrupts the integrity of 6-Cys gene E. Undistinguishable growth rate of 6-Cys EKO-cln line compared to wild type 6-Cys E intact T3Bo B. bovis strain in in vitro cultures indicates that expression of gene 6-Cys E is not essential for blood stage replication in this strain. In vitro inhibition assays using anti 6-Cys E antibodies confirmed inhibition the ability of anti-6 Cys E antibodies to inhibit the growth of the wild type Mo7 and T3Bo B. bovis parasites, but no significant inhibition was found for 6-Cys EKO-cln line parasites. Overall, the data suggests that the anti-6 Cys E antibody neutralizing effect on the wild type strains is likely due to mechanical hindrance, or cross-reactivity, rather than due to functional requirements of 6-Cys gene E product for survival and development of the erythrocyte stages. Further investigation is underway to determine if the 6-Cys E protein is required for replication and sexual stage development of B. bovis during tick stages.