Unraveling the complexity of the rhomboid serine protease 4 family of Babesia bovis by bioinformatics and experimental studies

Authors: GALLENTI, ROMINA - Instituto De Clima Y Agua (INTA); HUSSEIN, HALA - Washington State University; ALZAN, HEBA - Washington State University; Suarez, Carlos; Ueti, Massaro; ASURMENDI, SEBASTIAN - Instituto De Clima Y Agua (INTA); BENITEZ, DANIEL - Instituto De Clima Y Agua (INTA); ARAUJO, FLABIO - Washington State University; ROLLS, PETER - Biosecurity Queensland, Department Of Employment, Economic Development & Innovation; SIBEKO-MATJILA, KGMOTSO - Onderstepoort Veterinary Institute; SCHNITTGER, LEONHARD - Instituto De Clima Y Agua (INTA); FLORIN-CHRISTENSEN, MONICA - Instituto De Clima Y Agua (INTA)

Submitted to: Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/6/2022
Publication Date: 3/12/2022
Citation: Gallenti, R., Hussein, H.E., Alzan, H.F., Suarez, C.E., Ueti, M.W., Asurmendi, S., Benitez, D., Araujo, F.R., Rolls, P., Sibeko-Matjila, K., Schnittger, L., Florin-Christensen, M. 2022. Unraveling the complexity of the rhomboid serine protease 4 family of Babesia bovis by bioinformatics and experimental studies. Pathogens. 11(3) Article 344. https://doi.org/10.3390/pathogens11030344.
DOI: https://doi.org/10.3390/pathogens11030344

Interpretive Summary: Babesia bovis mis a tick-transmitted apicomplexan protozoon that infects cattle in tropical and subtropical regions around the world. In this study we characterize a family of five genes encoding proteins named rhomboid serine protease 4 (ROM4) that conserved among apicomplexans and play important roles during the process of invasion of erythrocytes by Babesia bovis parasites. Interestingly, we found that some of the members of these gene family are highly expressed in tick stages of the parasite lifecycle, which provide important information for improving our understanding of the biology of the parasite and can be used for developing alternative transmission blocking vaccine approaches. Importantly, the sequences of these genes is highly conserved among distinct strains. However, sequence polymorphismswere found restricted to a variable number of a tandemly repeated 29-amino acid sequence in one of these proteins, namely ROM4.5. This sequence repeat is highly conserved and predicted as antigenic. B. bovis. In conclusion, ROMs likely participate in relevant host-pathogen interactions and are possibly useful targets for the development of new control strategies against bovine babesiosis such as novel subunit vaccines and babecisidal drugs.

Technical Abstract: Babesia bovis, a tick-transmitted apicomplexan protozoon, infects cattle in tropical and subtropical regions around the world. In the apicomplexans Toxoplasma gondii and Plasmodium falciparum, rhomboid serine protease 4 (ROM4) fulfills an essential role in host cell invasion. We thus inves-tigated B. bovis ROM4 coding genes, their genomic organization, expression in in vitro cultured asexual (AS) and sexual stages (SS), and strain polymorphism. B. bovis contains five rom4 paralogous genes in chromosome 2, which we have named rom4.1, 4.2, 4.3, 4.4 and 4.5, with moderate degrees of sequence identity between them, and rom4.3 and 4.4 being almost identical copies. RT-qPCR analysis showed that rom4.1 and rom4.3/4.4 respectively display 18-fold and 218-fold significant increased (p<0.01) levels of transcription in SS than in AS, suggesting a role in gametogene-sis-related processes. In contrast, transcription of rom4.4 and 4.5 differed non-significantly between both stages. ROM4 polymorphism among geographic isolates was essentially restricted to a variable number of a tandemly repeated 29-amino acid sequence in ROM4.5. This sequence repeat is highly conserved and predicted as antigenic. B. bovis ROMs likely participate in relevant host-pathogen interactions and are possibly useful targets for the development of new control strategies against bovine babesiosis.