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Exploring the fundamental biology of microsporidian mitosomes

Final Report Summary - MICMIT (Exploring the fundamental biology of microsporidian mitosomes)

The Fellow investigated the biology of highly reduced mitochondria called mitosomes that are found in obligate intracellular microsporidian parasites (Trachipleistophora hominis) and in the mucosal parasite Giardia lamblia. Both parasites are important human pathogens. The Fellow carried out proteomic analyses of mitosomes from both species isolated using a variety of treatments including a novel method of organelle immuno-capture. This work identified new candidate mitosomal proteins for both species but also demonstrated that most of the proteins detected for Trachipleistophora hominis were from the host cells in which the parasites live. The fractionation methods developed by the Fellow for purifying mitosomes and the new antibodies developed to track organelle enrichment and purity, are now invaluable techniques and reagents for ongoing work in the host laboratory in to which the Fellow will contribute – extending and building upon the Fellowship. The Fellow isolated microsporidian mitosomes that, for the first time, show demonstrable in vitro activity using assays for cysteine desulphurase, a known mitosomal enzyme. Using bioinformatics analyses the Fellow demonstrated that Giardia and microsporidian mitosomes have strong similarities in their functional proteomes (for example the absence of mitochondrial carrier family proteins), and hence Giardia mitosomes provide a tractable alternative model system for studying and developing organelle proteomics. The Fellow made excellent progress on this part of the project. A number of candidate transporter proteins for the Giardia mitosome were identified and the presence of the Fe-S cluster biosynthesis pathway, potentially its only function, were confirmed in organelle fractions. Giardia mitosomes isolated by the Fellow have been used to investigate in vitro biosynthesis of Fe-S clusters for cytosolic and nuclear proteins. During this part of the Fellowship the Fellow developed skills in parasite co-culture, advanced microscopy, differential centrifugation for organelle purification and characterization and bioinformatics and was able to gain and further refine advanced skills in molecular biology. The Fellow also established collaborations with two international laboratories outside of his host laboratory.

The Fellow used bioinformatic analyses to identify putative mitosomal transport proteins, collaborating with scientists in the host laboratory to achieve this work and by doing so gaining further valuable training in bioinformatics and microscopy. Three homologues of the key mitochondrial ABC transporter ATM1 (yeast nomenclature), were detected in the genome of the microsporidian Trachipleistophora hominis, and the expressed proteins were used to make antibodies for immunofluorescence microscopy localization studies. Interestingly, none of the 3 candidate T. hominis ATM1 transporters localized to the mitosomes of Trachipleistophora hominis. This suggests that either the T. hominis mitosome uses a different type of transporter to export the products of Fe-S cluster biosynthesis, or that active transport function has been lost during reductive evolution of the parasite. Based upon the proteomics of Giardia mitosomes the Fellow identified eight potential mitosomal ABC transporters for this mitosome. These are currently being studied further in the host laboratory in collaboration with the Fellow.

Based upon analyses of the genomic data for microsporidians and Giardia and careful independent reading of the literature the Fellow formulated a novel hypothesis for a crucial role for GTP in mitosomal Fe-S cluster biosynthesis pathway. The key enzyme hypothesised to be inolved is a nucleotide diphosphate kinase that is called Ynk1 in yeast. The Fellow identified homologues of Ynk1 in microsporidians and Giardia and localized them in both species. He also functionally characterized the T. hominis enzyme. The main pools of Ynk1 in T. hominis and Giardia appear to be cytosolic rather than mitosomal but there appears to be a small pool in both species that is protected from proteinase digestion of enriched mitosome material. The Fellow’s analyses of the genome of T. hominis suggests that the YnK1 homologue has a critical role to play in parasite DNA and RNA biosynthesis. Like many parasites, microsporidians have lost ATP expensive pathways for de novo synthesis of purines and pyrimidines. Instead they steal key early substrates from the host cells in which they live and carry out interconversions to make DNA and RNA. Based upon the Fellow’s analyses it appears that Ynk1 may mediate many of these interconversions. An important role for Ynk1 in parasite biology is supported by recently published proteomics data that demonstrate that it is a major protein detected in purified spores.

In summary, the Fellow has made significant progress in most areas of his project and has generated novel and publishable data of biomedical and fundamental scientific interest. The Fellow has developed new and active collaborations, and is currently writing up papers from the Fellowship. The Fellow gained new skills in bioinformatics and evolutionary analysis as well as advanced training in a variety of modern and relevant experimental techniques during the Fellowship. These achievements and training have been instrumental in allowing the next step towards independence, as the Fellow successfully applied for a post in a third country to continue working on parasite molecular biology, a field in which the Fellow plans to build an independent career. Contact details for the scientist supervising the project are Prof. Martin Embley (martin.embley@ncl.ac.uk).