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.

Authors: PAOLETTA, MARTINA - Instituto De Clima Y Agua (INTA); LAUGHERY, JACOB - Washington State University; LÓPEZ ARIAS, LUDMILA - Instituto De Clima Y Agua (INTA); JARAMILLO ORTIZ, JOSÉ - Instituto De Clima Y Agua (INTA); MONTENEGRO, VALERIA - Instituto De Clima Y Agua (INTA); PETRIGH, ROMINA - Instituto De Clima Y Agua (INTA); Ueti, Massaro; Suarez, Carlos; FARBER, MARISA - Instituto De Clima Y Agua (INTA); WILKOWSKY, SILVINA - Instituto De Clima Y Agua (INTA)

Submitted to: International Journal for Parasitology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/3/2020
Publication Date: 3/20/2021
Citation: Paoletta, M.S., Laughery, J.M., Arias, L.S.L., Ortiz, J.M.J., Montenegro, V.N., Petrigh, R., Ueti, M.W., Suarez, C.E., Farber, M.D., Wilkowsky, S.E. 2021. 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. International Journal for Parasitology. 51(8):643-658. https://doi.org/10.1016/j.ijpara.2020.12.010.
DOI: https://doi.org/10.1016/j.ijpara.2020.12.010

Interpretive Summary: Bovine babesiosis is a tick-transmitted disease caused by apicomplexan parasites of the Babesia genus that represents a major constraint to livestock production worldwide. Our goal is identifying vaccine candidate antigens for developing new and effective vaccines against B. bovis that can substitute the current vaccines based on live parasites. The perforin-like protein (PLP) family has been identified as a key player in cell traversal and egress in related apicomplexans and it was also identified in Babesia, but its function in this parasite remains unknown. The aim of this work was to define the PLP family in Babesia and functionally characterize PLP1, a representative member of the family in B. bovis. Bioinformatic analyses demonstrate a variable number of plp genes (3 to 8) in the genomes of six different Babesia species and conservation of the family members at structure levels. We demonstrate here that Babesia PLPs contain the critical domains present in other apicomplexan PLPs to display the lytic capacity. Functional gene KO analysis indicates that lack of PLP1 has a negative impact on the mechanism of egression of the parasite and therefore in its capacity to proliferate. It is possible that PLP1 is associated with other proteins in the processes of invasion and egress of the parasite. As such it may be a valid candidate for inclusion in a recombinant vaccine against bovine babesiosis.

Technical Abstract: Bovine babesiosis is a tick-transmitted disease caused by apicomplexan parasites of the Babesia genus that represents a major constraint to livestock production worldwide. Our goal is identifying vaccine candidate antigens for developing new and effective vaccines against B. bovis that can substitute the current vaccines based on live parasites. The perforin-like protein (PLP) family has been identified as a key player in cell traversal and egress in related apicomplexans and it was also identified in Babesia, but its function in this parasite remains unknown. The aim of this work was to define the PLP family in Babesia and functionally characterize PLP1, a representative member of the family in B. bovis. Bioinformatic analyses demonstrate a variable number of plp genes (3 to 8) in the genomes of six different Babesia species and conservation of the family members at the secondary and tertiary structure levels. We demonstrate here that Babesia PLPs contain the critical domains present in other apicomplexan PLPs to display the lytic capacity. We then focused on the functional characterization of the PLP1 protein of B. bovis both in vitro and in vivo. PLP1 is expressed and exposed to the host immune system during infection and has high hemolytic capacity in a wide range of conditions in vitro. A B bovis plp1 knock out line displayed decreased growth rate in vitro compared to the wild type strain and a peculiar phenotype consisting of multiple parasites within a single RBC, although in low frequency. This phenotype suggests that the lack of PLP1 has a negative impact on the mechanism of egression of the parasite and therefore in its capacity to proliferate. It is possible that PLP1 is associated with other proteins in the processes of invasion and egress, which were found to have redundant mechanisms in related apicomplexans. Future work will be focused on unravelling the network of proteins involved in these essential parasite functions.