Identification of a Babesia spp protein family that contains MACPF-domain

ROMINA PETRIGH; ROBERTO NEUMANN; NATALIA REGO; ROSALIA MORETTA; DANIELA TOSTO; JUAN MOSQUEDA; HUGO NAYA; MARISA FARBER

Buenos Aires, Argentina

Conferencia; VI Internacional Conference on Ticks and Ticks- 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 parasite 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 identify 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 the speciation process. To gain some insight into PLP functional role we chose Babesia bigemina as a model for experimental studies. Using the unfinished B. bigemina (Bbi) genome data we are performing partial annotation so as to identify all the family genes and analyze 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 obtain a polyclonal antiserum. Ongoing experiments are being undertaken for verifying protein expression and determining subcellular localization. Acknowledgments: This work was supported by ANPCyT (PICTO-0812920), INTA y CONICET