IDENTIFICACIÓN DE UN GEN QUE CODIFICA PARA UNA PROTEÍNA ROMBOIDAL EN EL HEMOPARÁSITO BOVINO BABESIA BOVIS

MESPLET M; SUAREZ C; ECHAIDE I; DOMINGUEZ M; FERRERI L; FARBER M; BENITEZ D; MOSQUEDA J; FLORIN CHRISTENSEN, M

Cordoba – Argentina

Congreso; XI Congreso Argentino de Microbiologia; 2007

Sociedad Argentina de Microbiología

The identification of apicomplexan proteases that belong to the invasion machinery can provide important information for the development of novel therapeutic strategies against the diseases caused by this important group of pathogens. Rhomboids form a family of polytopic membrane proteases conserved throughout evolution and found in bacteria, yeast, plants and animals. The founding member of the family, Drosophila Rhomboid-1, was characterised as an intramembrane serine protease, which cleaves the epidermal growth factor (EGF)-like substrates Spitz, resulting in the secretion of the soluble factors from the cell. Rhomboids have been suggested to participate in the invasive step of the apicomplexan protozoa Toxoplasma sp. and Plasmodium sp. We have searched in the recently sequenced genome of the bovine hemoparasite Babesia bovis (B bovis) for homologous genes to those codifying for rhomboid proteins in other apicomplexan parasites. A 1.4 kb putative ORF with 31.5 % identity with the serine protease gene of Theileria equi was found present in B bovis chromosome 4. Bioinformatic analysis predicted a 52 kDa protein with a pI of 5.88, a N-terminal hydrophyllic extracellular region followed by a hydrophobic “rhomboid region“ consisting of 6 transmembrane domains and a conserved protease active site domain in the C´ terminus. Comparison of the rhomboid region with that of other apicomplexans showed the highest degree of matches with Toxoplasma gondii rhomboid 3 (gi50845220). The complete ORF of this gene was amplified by PCR from DNA extracted from seven B bovis isolates from Argentina, Brazil, Uruguay and Mexico. Sequence analysis of the PCR amplicons showed a high degree of conservation among geographical isolates (97.2% overall similarity). The transcript of the putative rhomboid gene was amplified by RT-PCR from total RNA extracted from in vitro cultures of B bovis merozoites (S2P Argentine strain), followed by cloning in pCR2.1 and sequencing. Sequence analysis showed no difference with the DNA amplicon thus demonstrating that the rhomboid-like gene is transcribed in the merozoite stage of B bovis and that it does not contain introns. A His6-tagged recombinant form of the complete rhomboid-like ORF was produced in E. coli Rossetta strain using the pRSET-B expression system. Studies are under way to analyze the expression of the putative rhomboid protein in vitro and in vivo, as well as to study its physiological relevance. A better understanding of the parasite pathogenic mechanisms and the identification of potential vaccine candidates that can be useful in a subunit vaccine can lead to improved control strategies for this parasite.