Rhomboid serine proteases type 4/5 of Babesia bovis: Polymorphism and pattern of expression in asexual and sexual parasite stages

GALLENTI ROMINA; FLORIN-CHRISTENSEN, M; SCHNITTGER, LEONHARD

buenos aires

Congreso; International Congress on Tropical Veterinary Medicine 2nd joint AITVM-STVM Meeting; 2018

Rhomboid serine proteases (ROM) constitute a large family of at least ten integral membrane proteins with an active site immersed in the lipid bilayer. They are present in both prokaryotic and eukaryotic organisms, highlighting their importance in various basic biological functions.The ROM4/5 family has been intensively studied, and it has been demonstrated, both in Toxoplasma gondii and Plasmodium falciparum, that these proteases cleave certain parasite adhesins during invasion, facilitating entry into host cells, an essential step for parasite development and survival. In previous bioinformatic analyses, we grouped the 8 rhomboids present in the Texas strain T2Bo of Babesia bovis into 4 families (Rom4/5, Rom6, Rom7 and Rom8), and showed an expansion of the Rom4/5 family with respect to Plasmodium falciparum to 5 paralogs, which we named Rom4.1, 4.2, 4.3, 4.4 and 4.5. Rom4.1, 4.2 and 4.3 are organized in tandem head to tail in chromosome 2, while Rom 4.4 and 4.5 are located respectively 19 and 27 kbp downstream in the same chromosome. The aims of this work are focused in examining the polymorphism of B. bovis Rom4/5 genes in different geographical isolates and their patterns of expression in both, sexual and asexual parasite stages. Rom4.2 and 4.3 are identical copies and cannot be differentiated from each other, so they were treated together. Sequence analysis of PCR amplification products showed a high degree of conservation among isolates along all genes, with the exception of the amino-terminal hydrophilic region of ROM4.1 predicted protein. Interestingly, polymorphism in ROM4.1 is restricted to the number of tandem repetitions of a 29 amino acid segment that is represented between 2.5 and 8.5 times in isolates from Argentina, Brazil, Mexico, USA, Israel and Australia. Several conserved predicted B-cell epitopes are present in the extracellular hydrophilic regions of ROM4/5 peptides, including one that coincides with the ROM4.1 repetitive segment, providing potential targets for immunotherapeutic interventions. RT-PCR analysis demonstrated that all B. bovis Rom4/5 genes are transcribed in both, in vitro cultured merozoites, and in xanthurenic acid-in vitro induced sexual stages of B. bovis. Moreover, RNAseq analysis of hemolymph derived from B. bovis-infected ticks and blood from a B. bovis-infected cow revealed differential patterns of expression among all Rom4 genes in bovine and tick stages. Notably, Rom4.5 transcripts were particularly abundant in the latter, suggesting a relevant role for Rom4.5 in gametogenesis, transmission or other processes undergone by the parasite in the tick milieu, and a target for developing transmission blocking interventions against B. bovis. This work was supported by MINCyT (PICT 2013-1249) and INTA (PNBIO 1131034), Argentina, and USDA-ARS Project Numbers: 2090-32000-039-00-D, 2090-32000-039-12-I, and2090-32000-039-20-T.