Final Report Summary - TRAHOMGEN (Reductive evolution of parasite genomes with a focus on the microsporidian Trachipleistophora hominis) I annotated and analysed the unpublished genome of Trachipleistophora hominis, an obligate intracellular eukaryotic parasite infecting man, to investigate its genome structure and evolution and to better understand how Trachipleistophora hominis exploits its host. I also reconstructed a putative proteome for the Trachipleistophora hominis remnant mitochondrion (mitosome), focusing particularly on formulating and testing hypotheses of candidate transport proteins, including localisation studies and functional characterisation. My training needs were fully during the experience of formulating, participating in, and leading, the multidisciplinary team needed to meet my goals. All of the main objectives of my project have been met. Comparative analysis of the non-coding genomic region between Trachipleistophora hominis and Encephalitozoon cuniculi demonstrated drastic differences; whereas Encephalitozoon cuniculi has lost hallmark features of eukaryotic genomes such as a TATA box or long stretches of intergenic regions, the genome of Trachipleistophora hominis resembles other known fungal genomes. Additional comparison with Nosema ceranae, another large microsporidian genome, confirmed that the high reduction of the Encephalitozoon cuniculi intergenic regions are a feature specific for this organism and not for the Microsporidia in general. The interpretation of the metabolic pathways of Trachipleistophora hominis gave insight into its highly reduced metabolic potential and identified essential substrates which have to be obtained from the host cell. The analysis of the metabolome was complemented by an in-depth analysis of the transport potential of Trachipleistophora hominis. We detected a high number of Microporidia- and Trachipleistophora hominis-specific transport proteins, which shows that despite the trend towards a drastic reduction of the genomes, some expansion seems to have occured as well. Another main focus of this project was the detailed analysis of the the transport proteins (NTT) used to import ATP from the host cell, as Microsporidia have lost the ability to generate ATP by oxidative phosphorylation. These transporters may also be involved in nucleotide import, as the Microsporidia have lost the ability to de novo synthesise purines and pyrimidines. The NTTs furthermore provide the highly reduced mitosomes with the ATP necessary to synthesize Fe-S clusters. To confirm the presence of one (or more) mitosomal NTTs in Trachipleistophora hominis, peptide antibodies against each of the identified NTTs were generated. Immunofluorescence analysis demonstrated the presence of two of the four proteins at the plasma membrane, the analysis of the other two NTTs is still ongoing. Immuno-electron microscopy confirmed plasma membrane locations of all four NTT proteins; in addition, two of them also show signals at the mitosomes, indicating a dual targeting of these two proteins; this is of high interest and stands in contrast to the clear distinction between three plasma-membrane and one mitosomally located NTTs in Encephalitozoon cuniculi. Functional characterisation of the transporters is currently ongoing. In addition to this, computational and lab work, the on-the-job training of the fellow has resulted in her contribution to two analyses of complex phylogenetic studies as well as her contribution to an analysis of the conserved mitochondrial proteome and to an analysis of Giardia lamblia mitosomal transport proteins, as well as a study focusing on the functional characterisation of the Trachipleistophora hominis mitosomes. Research on the topics covered by the fellowship is relevant to understanding the biology of a human parasite and, as the model is a highly reduced parasite, for identifying the essential components of eukaryotic cells. It also speaks directly to the Seventh Framework Programme (FP7) Health Priority Systems Biology (HEALTH-2007-2.1.2) to combine and integrate data from biological pathways in unicellular eukaryotic organisms to human cells and organs. The project website is: http://research.ncl.ac.uk/microbial_eukaryotes/evaheinz.html.