Expression of GPI-anchored vaccine candidate antigens of Babesia bovis in the free-living ciliate Tetrahymena thermophila

MONTES MG; ELGUERO ME; RODRIGUEZ AE; FLORES D; FLORIN-CHRISTENSEN M; SCHNITTGER L; NUSBLAT AD

Conferencia; 9th Tick and Tick-borne Pathogen Conference & 1st Asia Pacific Rickettsia Conference; 2017

Glycosylphosphatidylinositol (GPI)-anchored proteins are abundantly expressed on the surface of parasitic protozoa and involved in the invasion of host cells, thus constituting attractive vaccine candidates. Free-living protozoa also express GPI-anchored proteins as has been shown for the ciliate Tetrahymena thermophila. This protozoan has attractive features that make it an optimal eukaryotic expression system for heterologous proteins, and has already been used as expression platform for GPI-anchored vaccine candidates for malaria and fish white dot disease. In this work, we have selected two promising GPI-anchored vaccine candidates of the tick-transmitted cattle hemoparasite Babesia bovis for expression in T. thermophila: MSA-2c and GPI-4. Both are immunodominant and have surface-exposed neutralization-sensitive B cell epitopes. Sequences were optimized to the codon usage frequency of T. thermophila and cloned downstream of the metallothionein promoter 1 (Mtt1) of pICY-gtw plasmid. The 3? ends containing encoded GPI-anchor signals were removed, while the original 5? signal peptide-encoding regions were conserved. Recombinant plasmids were introduced in T. thermophila ex-conjugants by electroporation and transformed clones were selected by paromomycin resistance. Episomal transformation with both plasmids was verified by PCR. In the case of MSA-2c, recombinant protein expression was achieved by induction with cadmium chloride and verified by immunoblot and HPLC-MS. Importantly, the protein was detected in Triton X-114 cell membrane extracts, indicating recognition of the B. bovis signal peptide by the T. thermophila secretory machinery. Application of the developed protocols to GPI-4 expression is under way. Financed by MINCyT (PICT 2013-1708) and INTA (PNBIO 1131034), Argentina.