Integrated in silico-wet biology approach for improving the annotation of Babesia bigemina perforin

ROMINA PETRIGH; NATALIA REGO; BEATRIZ VALENTINI; IGNACIO ECHAIDE; HUGO NAYA; MARISA FARBER

Montevideo

Congreso; ISCB Latin America 2010; 2010

ISCB

Integrated in silico-wet biology approach for improving the annotation of Babesia bigemina perforin familyin silico-wet biology approach for improving the annotation of Babesia bigemina perforin family Romina Petrigh1, Natalia Rego2, Beatriz Valentini3, Ignacio Echaide3, Hugo Naya2, Marisa Farber1 1Instituto de Biotecnología, CICVyA, INTA Castelar, CP 1712 Buenos Aires, Argentina.1, Natalia Rego2, Beatriz Valentini3, Ignacio Echaide3, Hugo Naya2, Marisa Farber1 1Instituto de Biotecnología, CICVyA, INTA Castelar, CP 1712 Buenos Aires, Argentina.Instituto de Biotecnología, CICVyA, INTA Castelar, CP 1712 Buenos Aires, Argentina. 2Unidad de Bioinformática, Instituto Pasteur de Montevideo, CP 11400, Montevideo, Uruguay.Unidad de Bioinformática, Instituto Pasteur de Montevideo, CP 11400, Montevideo, Uruguay. 3Sanidad animal, EEAA -Rafaela, INTA Rafaela, CP 2300, Santa Fe, ArgentinaSanidad animal, EEAA -Rafaela, INTA Rafaela, CP 2300, Santa Fe, Argentina Background Babesia bigemina is an apicomplexan haemoparasite that causes babesiosis in cattle, a tick-transmitted disease worldwide distributed. Some of the steps involved in host cell-infection depends on secreted proteins localized into organelles called micronemas. Previous studies in Plasmodium yoelli reported a secreted proteins family containing membrane- attack complex/ perforin (MACPF) - like domains, known as Perforing like Proteins (PLP), one of them localized into micronemas of sporozoites [1]. With the aim to understand PLPs importance foris an apicomplexan haemoparasite that causes babesiosis in cattle, a tick-transmitted disease worldwide distributed. Some of the steps involved in host cell-infection depends on secreted proteins localized into organelles called micronemas. Previous studies in Plasmodium yoelli reported a secreted proteins family containing membrane- attack complex/ perforin (MACPF) - like domains, known as Perforing like Proteins (PLP), one of them localized into micronemas of sporozoites [1]. With the aim to understand PLPs importance forPlasmodium yoelli reported a secreted proteins family containing membrane- attack complex/ perforin (MACPF) - like domains, known as Perforing like Proteins (PLP), one of them localized into micronemas of sporozoites [1]. With the aim to understand PLPs importance for Babesia transmission, we searched for plp genes repertories in Babesia spp. and studied their phylogenetic relationships with previously known apicomplexa PLPs. Considering that B. bigemina genome assembly and annotation is still in progress (http://www.sanger.ac.uk/Projects/Protozoa/), we used both in silico and biochemical tools to perform the plp genes annotation .transmission, we searched for plp genes repertories in Babesia spp. and studied their phylogenetic relationships with previously known apicomplexa PLPs. Considering that B. bigemina genome assembly and annotation is still in progress (http://www.sanger.ac.uk/Projects/Protozoa/), we used both in silico and biochemical tools to perform the plp genes annotation .B. bigemina genome assembly and annotation is still in progress (http://www.sanger.ac.uk/Projects/Protozoa/), we used both in silico and biochemical tools to perform the plp genes annotation .http://www.sanger.ac.uk/Projects/Protozoa/), we used both in silico and biochemical tools to perform the plp genes annotation .plp genes annotation . Methods MACPF domains from related B. bovis plp genes were used to perform TBLASTN searches on B. bigeminaB. bovis plp genes were used to perform TBLASTN searches on B. bigemina contigs. The selected ones were annotated using gene prediction software based on HMM: FGENESH and Glimmer HMM, trained with P. falciparum and B. bovis respectively. Coding sequence from putative plp genes was checked in SMART for signal peptide and MACPF domain presence. Resulting data was used to design sequencing and RT-PCR experiments in order to confirm in-silico predictions and to study PLPs expression patterns. Structural alignments of the MACPFdomains were used as starting point for a maximum likelihood phylogenetic reconstruction of the family in Piroplasmida (Babesia spp. and Theileria spp.) and Haemosporida (Plasmodium spp.)P. falciparum and B. bovis respectively. Coding sequence from putative plp genes was checked in SMART for signal peptide and MACPF domain presence. Resulting data was used to design sequencing and RT-PCR experiments in order to confirm in-silico predictions and to study PLPs expression patterns. Structural alignments of the MACPFdomains were used as starting point for a maximum likelihood phylogenetic reconstruction of the family in Piroplasmida (Babesia spp. and Theileria spp.) and Haemosporida (Plasmodium spp.)in-silico predictions and to study PLPs expression patterns. Structural alignments of the MACPFdomains were used as starting point for a maximum likelihood phylogenetic reconstruction of the family in Piroplasmida (Babesia spp. and Theileria spp.) and Haemosporida (Plasmodium spp.)Babesia spp. and Theileria spp.) and Haemosporida (Plasmodium spp.)Plasmodium spp.) Results and discussion We determined seven coding sequences with MACPF domain in B. bigemina partial assembly. As in B. bovis, some of them are intron-free. RT-PCR revealed the expression of all plp genes in the B. bigeminaB. bigemina partial assembly. As in B. bovis, some of them are intron-free. RT-PCR revealed the expression of all plp genes in the B. bigeminaB. bigemina intraerythrocytic stage. According to the phylogenetic tree obtained, evolutionary history has been complex in this family with some duplication events occurred before Piroplasmida and Haemosporida divergence while others occurred more recently. As evolutionary distances are high and we have found some putative rearranges in Babesia PLPs, further experimental studies are currently being conducted in this area.Babesia PLPs, further experimental studies are currently being conducted in this area. Conclusion In the present work, we combined bioinformatic tools and experimental approaches to annotate a new protein family of B. bigemina potentially involved in parasite invasion mechanism to different cells. Future studies will help us to reveal the function of this family.B. bigemina potentially involved in parasite invasion mechanism to different cells. Future studies will help us to reveal the function of this family. Acknowledgment Dr. Arnab Pain from Sanger Institut supported us regarding gene prediction and valuable comments on the assembly version currently available. References 1. Kaiser K, Camargo N, Coppens I, Morrisey JM, Vaidya AB, Kappe SH: A member of a conserved Plasmodium protein family with membrane-attack complex/perforin (MACPF)-like domains localizes to the micronemes of sporozoites. Mol Biochem Parasitol. 2004, 133(1):15-26A member of a conserved Plasmodium protein family with membrane-attack complex/perforin (MACPF)-like domains localizes to the micronemes of sporozoites. Mol Biochem Parasitol. 2004, 133(1):15-26Mol Biochem Parasitol. 2004, 133(1):15-26