Hufnagel, Linda [faculty advisor, Department of Cell and Molecular Biology]
Tetrahymena thermophila; Ciliophora; tBLAST; vaccine; molecular biology
Tetrahymena thermophila is a member of the phylum Ciliophora (the ciliated protozoa) and is currently of great interest due to its unique qualities that make it useful as a model for research directed towards understanding how eukaryotic cells function. Recently, the T. thermophila genome was sequenced, and made available online. Since Tetrahymena is a member of the Alveolata, the major eukaryotic clade that also contains the apicomplexan parasites that cause a number of serious human and animal diseases, we sought to determine whether the genes coding for apicomplexan vaccine candidates were represented in the genome of Tetrahymena. In addition, we sought to find out whether there was any data available online to support the expression of the vaccine candidate orthologues in Tetrahymena.
By using the tBLAST functions at the NCBI database (National Center of Biotechnology Information) and the Tetrahymena Genome Database, the amino acid sequences of the apicomplexan vaccine candidates were compared to the predicted protein sequences derived from the Tetrahymena genome. By doing so, the homologue of a vaccine candidate, Phosphoprotein p0, was found and identified as a predicted protein coded by the gene TTHERM_ 00636970. The predicted Tetrahymena phosphoprotein is 324 amino acids long. This protein had already been identified as a 60S Acidic Ribosomal Phosphoprotein in an unrelated study on ribosomal proteins of ciliated protists. In apicomplexans as well as other organisms such as yeast and mammals, phosphoprotein p0 is is also known to localize on the cell surface as well as intracellularly, although it has no transmembrane domain. When apicomplexan parasites are exposed to antibodies against p0, invasion of the host is blocked, explaining why it has become a target for vaccine development. From a recently published microarray survey of gene expression in Tetrahymena, we have determined that the Tetrahymena p0 is expressed at 250x background continuously during the life cycle of T.Thermophila. The results from the BLAST yielded e-values of 2.7x10-35, 5.4x10-37, 1.8x10-36 for the apicomplexans Plasmodium falciparum, Babesia microti, and Toxoplasma gondii respectfully. Though not apicomplexans, species of other protozoan parasites, including Leishmania, Trypanosoma, Neospora, and cryptosporidium, also contain phosphoprotein p0.
We used Clustal W to align all the parasite p0 sequences with the p0 of Tetrahymena (Ttp0) and found domains of strong homology with Ttp0. These domains in Ttp0 are currently under further analysis and we are further analyzing the Ttp0 sequence for other functional domains that might provide evidence for localization to the cell surface. We also plan to conduct immunocytochemical studies to determine whether the putative p0 protein of Tetrahymena is found at the cell surface, as it is in apicomplexan parasites and other protist cells. If so, Tetrahymena may be a useful cell in which to discover the mechanisms that direct p0 to the cell surface rather than to the ribosomes.