Functional characterization of Babesia bovis PLP1 through hemolysis assays and generation of a knock out strain

MARTINA PAOLETTA; PAUL LACY; MARISA FARBER; JOSÉ MANUEL JARAMILLO ORTIZ; CARLOS SUAREZ; LUDMILA LÓPEZ ARIAS; JACOB LAUGHERY; SILVINA E. WILKOWSKY

Virtual

Conferencia; The Australian Society for Parasitology online conference; 2020

The Australian Society for Parasitology

Bovine babesiosis is a tick-borne disease caused by Babesia parasites affecting livestock production worldwide. We have previously identified in B. bovis a family of Perforin-Like Proteins (PLP) involved in pore formation and erythrocyte damage. One member, PLP1, is exposed to the host immune system during infection. This study aims to determine the function of PLP1 and its contribution to parasite?s pathogenesis.The recombinant MACPF domain (responsible for pore formation in PLPs) of PLP1 was expressed and hemolysis assays were done incubating this protein with bovine RBCs. High hemolysis levels were obtained in a wide range of conditions.A B. bovis knock out (KO) strain was generated by disruption of plp1. Parasites were transfected with a plasmid to guide replacement of plp1 with an egfp-bsd fusion gene, which was confirmed by PCR and sequencing. The in vitro replication of the KO in bovine RBC cultures was evaluated. Results showed a decreased growth rate compared to the wild type strain and a peculiar phenotype consisting of multiple parasites within a single RBC which suggests that the lack of PLP1 has a negative impact on the parasite?s egress and in its capacity to proliferate. Further studies are aimed to determine if the replication defect results in an attenuated phenotype in vivo.