Identification of a Babesia spp. protein family that contains MACPR-Domain

PETRIGH R; NEUMANN R; REGO N; MORETTA R; TOSTO D; MOSQUEDA J; NAYA H; FARBER M

Buenos Aires, Argentina

Conferencia; VI International Conference on Ticks and Tick-borne Pathogens; 2008

The phylum Apicomplexa is an early branching eukaryotic lineage. Life cycles include a number of stages that invade distinct cell types within the arthropod vector and vertebrate host. Up to now, little is known about parasites proteins involved in cell-cell interactions. A previous study has reported a family of five related genes in the genome of Plasmodium yoelii (Py) encoding secreted proteins containing membrane-attack complex/perforin (MACPF)-like domain. The perforin-like protein (PLP) is localized into micronemas of sporozoites, organelles involved in host cell-infection. With the aim to identified PLPs in other apicomplexan parasites, we searched in the available complete genomes of Piroplasma: Babesia bovis (Bbo), Theileria parva (Tp) and Theileria annulata (Ta). BLASTP analysis led to the identification of six proteins with MACPF- like domain in each of them. In order to study the relationship among apicomplexa PLPs we performed sequences alignment  and phylogenetic analysis using Parsimony. The cladogram showed one cluster formed by Plasmodium falciparum and Py orthologous and another one by Bbo and the two Theileria orthologous genes. This result is in agreement with the phylogenetic relationship obtained using concatenated housekeeping genes. Moreover, phylogenetic analysis of Piroplama PLPs confirmed orthology between PLP1 to PLP5 in the three analyzed species. Consistently, pairwise alignments between orthologous genes from Bbo, Ta and Tp revealed higher sequence similarity than the similarity obtained when comparing paralogous genes. Evolutive history of perforin protein family in the analysed Apicomplexa suggests that gene duplication happened in the most recent common ancestor of each clade before especiation process. To gain some insight into PLP functional role we chose Babesia. bigemina as a model for experimental studies. Using the  the unfinished Babesia bigemina (Bbi) genome data we are performing partial annotation so as to  identify all the family genes and analyzed their genomic organization. We selected  a region of one of the identified  Bbi plp genes for performing gene expression analysis. Transcription of the macpf region in blood and sexual stages was verified by RT-PCR assays. A recombinant fragment coding for MACPF domain was used to obtained a polyclonal antiserum. Ongoing experiments are being undertaken for verifying protein expression and determine subcellular localization.