The key to egress? Babesia bovis perforin-like protein 1 (PLP1) with hemolytic capacity is required for blood stage replication and is involved in the exit of the parasite from the host cell

PAOLETTA M; JARAMILLO ORTIZ, JOSÉ M.; LOPEZ ARIAS L; LACY; LAUGHERY J; SUAREZ, C; FARBER M; WILKOWSKY S. E

Evento virtual global

Congreso; XXXI Annual Molecular Parasitology Meeting; 2020

Genetics Society of America

The key to egress? Babesia bovis perforin-like protein 1 (PLP1) with hemolytic capacity is required for blood stage replication and is involved in the exit ofthe parasite from the host cell. Martina Paoletta1, Jose Jaramillo Ortiz1, Ludmila Lopez Arias1, Paul Lacy2, Jacob M. Laughery3, Carlos Suarez2,3, Marisa Farber1,Silvina Wilkowsky1 1) Instituto de Agrobiotecnologia y Biologia Molecular (IABIMO) - INTA-CONICET, Argentina; 2) Animal Disease Research Unit, USDA-ARS,Washington State University, 3003 ADBF, Pullman, WA, USA; 3) Department of Veterinary Microbiology and Pathology, Washington State University, Pullman,WA, USA..Bovine babesiosis is a tick-borne disease caused by Babesia parasites affecting livestock production worldwide. Perforin-like proteins (PLP) are apicomplexan proteins with the capacity of generating pores in lipid bilayers and, although they have been described as key factors in host-pathogen interaction in related pathogens, their role in Babesia remains unknown. We have previously identified in B. bovis a family of six PLPs that might be involved in pore formation and red blood cell (RBC) damage, and demonstrated that one member, PLP1, is expressed and exposed to the host immune system during infection.The aim of this study was 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 thisprotein with bovine RBCs. Cell lysis was expressed as a percentage of maximum hemoglobin release with Triton X-100 treatment. High hemolysis levels (> 80%) were obtained at [rMACPF] > 80 nM, and pH > 5, which supports the pore forming function of the protein. The hemolysis activity was not affected by changes in [Ca2+], and a dose-response curve reflected a cooperative-positive response of the protein.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 that acts as a reporter and a selectable marker. The efficient replacement of plp1 was confirmed by PCR and sequencing, and a clonal line was generated by FACS assays. 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 suggesting that the lack of PLP1 has a negative impact on theparasite?s egress and in its capacity to proliferate.We conclude that PLP1 is a pore forming protein involved in the egress of the parasite from the host cell and, even though it is not essential, plays an important role in B. bovis blood stages. Further studies will be focused on determining if the replication defect results in an attenuated phenotype in vivo and the potential use of this strain as a genetically attenuated vaccine