Probing the localisation and function of a novel kinetoplast-associated protein in evolutionarily divergent protists

In evolutionary terms, the origin of the kinetoplast remains ambiguous. However, interestingly some components of the TAC are found in the genome of the free-living Bodo Saltans. This includes the protein designated Tb9260, which is important in kinetoplast organisation during cell division a fragment of the Bodo saltans Tb9260 orthologue, named Trett, will be expressed as recombinant protein in E.coli in order to raise a BsTrett antibody. This will be utilised for the in vivo targeting by confocal and super-resolution microscopy of BsTrett within Bodo saltans in order to ascertain its localisation. Additionally, Bodo saltans TAC65, TAC60, TAC40 and Trett orthologues will be cloned into a pNUS-GFPcH expression vector and transfected into Crithidia fasciculata. The localisations of these orthologous proteins are shown here with some surprising phenotypes. In addition, a cross species bioinformatic analysis of the Bodo saltans transcriptome against peptide sequences of putative proteins identified within the TbMitoCarta will aim to identify possible conserved orthologues and future protein targets. Bodo saltans is a divergent free-living ancestor of the parasitic trypanosomatids, including species of trypanosomes and Leishmania. Similarities between the Kinetoplastids have demonstrated the importance of understanding the evolutionary process which has led to parasitism and the question of whether it evolved from, or separately, to its free-living ancestor remains unanswered. Here I show the localisations of the aforementioned proteins and their similarities between the free-living and parasitic organisms.