Bovipain-2,the falcipain-2 ortholog,is expressed in intraerythrocytic stages of the tick-transmitted hemoparasite Babesia bovis

Background: cysteine proteases have been shown to be highly relevant for apicomplexan parasites. in the case of babesia bovis,a tick-transmitted hemoparasite of cattle,inhibitors of these enzymes were shown to hamper intraerythrocytic replication of the parasite,underscoring their importance for survival. results. four papain-like cysteine proteases were found to be encoded by the b. bovis genome using the merops database. one of them,the ortholog of plasmodium falciparum falcipain-2,here named bovipain-2,was further characterized. bovipain-2 is encoded in b. bovis chromosome 4 by an orf of 1.3 kb,has a predicted molecular weight of 42 kda,and is hydrophilic with the exception of a transmembrane region. it has orthologs in several other apicomplexans,and its predicted amino acid sequence shows a high degree of conservation among several b. bovis isolates from north and south america. synteny studies demonstrated that the bovipain-2 gene has expanded in the genomes of two related piroplasmids,theileria parva and t. annulata,into families of 6 and 7 clustered genes respectively. the bovipain-2 gene is transcribed in in vitro cultured intra-erythrocyte forms of a virulent and an attenuated b. bovis strain from argentina,and has no introns,as shown by rt-pcr followed by sequencing. antibodies against a recombinant form of bovipain-2 recognized two parasite protein bands of 34 and 26 kda,which coincide with the predicted sizes of the pro-peptidase and mature peptidase,respectively. immunofluorescence studies showed an intracellular localization of bovipain-2 in the middle-rear region of in vitro cultured merozoites,as well as diffused in the cytoplasm of infected erythrocytes. anti-bovipain-2 antibodies also reacted with b. bigemina-infected erythrocytes giving a similar pattern,which suggests cross-reactivity among these species. antibodies in sera of two out of six b. bovis-experimentally infected bovines tested,reacted specifically with recombinant bovipain-2 in immunoblots,thus demonstrating expression and immunogenicity during bovine-infecting stages. conclusions. overall,we present the characterization of bovipain-2 and demonstrate its in vitro and in vivo expression in virulent and attenuated strains. given the involvement of apicomplexan cysteine proteases in essential parasite functions,bovipain-2 constitutes a new vaccine candidate and potential drug target for bovine babesiosis. © 2010 mesplet et al; licensee biomed central ltd.