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Title: Coordination of cell differentiation and mitochondrial development in Trypanosoma brucei
Author: Amegatcher, Gloria
ISNI:       0000 0004 7969 0808
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2019
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Trypanosomes are unicellular parasites of humans and livestock, characterised by a single mitochondrion and a highly complex mitochondrial DNA network, the kinetoplast. Trypanosoma brucei causes human African trypanosomiasis (HAT, or sleeping sickness) and Nagana in animals, often lethal diseases transmitted by tsetse flies in sub-Saharan Africa. The trypanosome mitochondrial respiratory system involved in energy production comprises of the five classical complexes (I-V); and in addition, a type 2 NADH:ubiquinone oxidoreductase, a glycerol-3-phosphate dehydrogenase and a trypanosome alternative oxidase. Some subunits for respiratory complexes I, III, IV and V are encoded in the kinetoplast DNA. T. brucei encodes genes for complex I; null mutants of complex I (cI) in monomorphic bloodstream form background and RNAi in procyclic cells have not shown any growth defects. Akinetoplastic stumpy cells have shorter life span which could be due to absence of mitochondrially encoded subunits and thus cannot express functional NADH dehydrogenase. Moreover, BF parasites in adipose tissue appear to upregulate pathway for beta-oxidation, which is expected to increase demand for NADH dehydrogenase activity. Little is known about the metabolism and bioenergetics of cI in stumpy forms. Mitochondrial activity changes across the life cycle stages of the parasite. The parasite life stages include slender and stumpy forms (found in the mammalian bloodstream) and procyclic form (found in the tsetse fly). The mammalian bloodstream stage parasite does not express respiratory complexes III and IV and generates most if not all ATP via glycolysis; the insect stage parasite generates most ATP mitochondrially. Thus, coordination of the expression patters for nuclearly and mitochondrially encoded respiratory chain subunits is essential for parasite life cycle progression. In organisms such as yeast, mammals and plants, 'mitochondrial retrograde signalling' pathways, including the 'unfolded protein response (UPRmt), convey information on the functional status of this organelle to the nucleus and modulate expression of nuclear genes accordingly. It is not known if similar signalling pathways exist in trypanosomes. This thesis investigated two questions: 1. Does complex I play an important role in stumpy and/or in cells residing in adipose tissue? 2. Does retrograde signalling pathway exists in T. brucei, lack of mitochondrial genome results in changes in nuclear gene expression? To address the first question, the goal was to generate genetic null mutants for key nuclearly encoded cI subunits (NUBM or NUKM) in pleomorphic (i.e. differentiation competent) Trypanosoma brucei brucei cell line EATRO 1125. Next, the ability of these knockout cell lines to differentiate and to reside in adipose issue would be monitored. Unfortunately, I could not generate a cI null mutant in pleomorphic background as I subsequently determined that the parental cell line used was not fully pleomorphic in mice. Even though the null mutant generated for complex I were not fully pleomorphic, I still investigated their survival in adipose tissue. Results showed that these null mutants were able to reside in adipose tissue. I further probed this by using pleomorphic cell lines devoid of mitochondrial DNA (akinetoplastic cell lines (AK)), which are also cI deficient as all seven mitochondrially encoded subunits of this complex are absent. These AK were able to differentiate in both blood and adipose tissue. The AK seems to present more in the adipose tissue when compared with the wild type. Coinfection with AK and WT together in mice showed that they both migrate from blood into adipose tissue. I observed that the ratio of WT/AK in blood is much higher than in adipose tissue. Moreover, blood/AT balance in AK is shifted towards AT, compared to WT. To explore potential retrograde signalling pathways in T. brucei, we compared the nuclear transcriptome of a wild type strain with an AK mutant, before and after differentiation from the slender to the stumpy form (a transitional stage on route to differentiation to the insect stage). This was done by RNA-sequencing. In WT and AK parasites, genes showed significant upregulation in stumpy forms (we considered a difference of ≥ 2-fold with a p-value ≤ 0.05 as significant) in comparison to WT. Most of these genes were hypothetical proteins. Genes involved in the glycolytic pathway were generally downregulated in stumpy cells. We also observed robust downregulation in stumpy cells of numerous histones and of two genes involved in kinetoplast maintenance, mitochondrial DNA ligase LIG k alpha (Tb927.7.610) and cysteine peptidase PNT1 (Tb927.11.6550). Other changes in akinetoplastic stumpy cells concerned a hypothetical protein (~3-fold upregulated) and a putative adenylosuccinate lyase (~3-fold downregulated), but overall, we observed only a limited number of robust changes (13 up, 9 down). This study suggests that the absence of the mitochondrial genome has a surprisingly limited effect on the levels of nuclearly encoded messenger RNA needed to make proteins in bloodstream stage of T. brucei. In summary, this will provide a comprehensive view of the potential effects of mitochondrial dysfunction on nuclear gene expression in these parasites. Also, my work will give understanding of metabolism and bioenergetics of cI in stumpy and adipose tissue forms of T. brucei.
Supervisor: Schnaufer, Achim ; Matthews, Keith Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Trypanosoma brucei ; T. brucei. ; nuclear transcriptome ; kinetoplast ; stumpy ; slender ; pleomorphic cell lines ; akinetoplastic cell lines