Babesia microti immunoreactive rhoptry-associated protein-1 paralogs are ancestral members of the piroplasmid-confined RAP-1 family

Authors: BASTO, REGINALDO - Washington State University; THEKKINIATH, JOSE - Nutrigenetics Unlimited, Inc; MAMOUN, CHOUKRI - Yale School Of Medicine; FULLER, LEE - Nutrigenetics Unlimited, Inc; MOLESTINA, ROBERT - American Type Culture Collections; FLORIN-CHRISTENSEN, MONICA - Instituto De Clima Y Agua (INTA); SCHNITTGER, LEONHARD - Instituto De Clima Y Agua (INTA); ALZAN, HEBA - Washington State University; Suarez, Carlos

Submitted to: Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/23/2021
Publication Date: 10/26/2021
Citation: Basto, R.G., Thekkiniath, J., Mamoun, C.B., Fuller, L., Molestina, R.E., Florin-Christensen, M., Schnittger, L., Alzan, H.F., Suarez, C.E. 2021. Babesia microti immunoreactive rhoptry-associated protein-1 paralogs are ancestral members of the piroplasmid-confined RAP-1 family. Pathogens. 10(11). Article 1384. https://doi.org/10.3390/pathogens10111384.
DOI: https://doi.org/10.3390/pathogens10111384

Interpretive Summary: Babesia microti is responsible for babesiosis in humans and wild life animals, and is an emergent disease in the US. The members of the Babesia RAP-1 gene family are candidates for novel vaccines and diagnostic tools. However, members of this gene family remained unidentified in B. microti. In this study we identified putative members of the RAP-1 family in B. microti by performing sequence and genome analysis. We thus demonstrated the relationship among two B. microti genes and members of the RAP-1 gene family. The two B. microti putative RAP-1 proteins were expressed as recombinant proteins and tested for their interaction with antibodies in infected humans. Both protein are recognized by antibodies in sera of B. microti infected patients, thus demonstrating their potential as candidates for candidates for developing novel vaccines and diagnostic methods.

Technical Abstract: Babesia, Cytauxzoon and Theileria are tick-borne apicomplexan parasites members of the order Piroplasmida, responsible for causing diseases in humans and animals. The rhoptry-associated protein-1 (RAP-1) is a unique family of piroplasmid-specific proteins not homolog to Plasmodium RAP-1, with which they only share the designation. RAP-1 proteins appear to be required for erythrocyte invasion and may contribute to the establishment of persistent infections and evasion of the host immune system. The piroplasmid RAP-1 genes have so far not be identified in this sensu lato Babesia sp. Hereby we propose that the closely linked B. microti genes annotated as BMR1_03g00947 and BMR1_03g00960 encode for two paralogue RAP-1-like proteins, the first of which was previously denominated BmIPA48 and for the second we propose the name Bm960. The genes are tandemly arranged head to tail, highly expressed in blood stage parasites, syntenic to rap-1 genes of other piroplasmids, and share large portions of an almost identical, ~225 bp repeated region located in their 5’ putative regulatory regions. Also, both B. microti-paralogs contain a signal peptide and show a very low sequence identity (below than 13%) to other piroplasmid RAP-1 proteins. Altogether, these observations, in addition to significant sequence alignments of their Cys-rich domains, supports the assumption that both B. microti RAP-1-like proteins are members of the RAP-1 family in piroplasmids, but they also have unique features, such as transmembrane domains. Diversification of the piroplasmid-confined rap-1 family is characterized by amplification of genes, protein domains, and a high sequence polymorphism. This suggests a functional involvement of RAP-1 at the parasite-host interface, possibly by parasite adhesion, attachment, and/or evasion of the host immune defenses. Both, BmIPA48 and Bm960, are recognized by antibodies in sera from humans infected with B. microti suggesting they might be candidates for developing novel serodiagnosis and vaccines.