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Title: Identification and functional characterization of putative mitochondrial iron transporters from the human pathogen Trypanosoma brucei
Author: Zheng, Fuli
ISNI:       0000 0004 8499 2202
Awarding Body: University of Hull
Current Institution: University of Hull
Date of Award: 2017
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Abstract:
Trypanosoma brucei is a medically important protozoan parasite causing African sleeping sickness in humans. Treatment of this disease is possible, but is hindered by the limited availability of effective drugs as well as the rapid emergence of drug resistance. It is therefore of importance to identify novel drug targets for the future development of more effective drugs. Mitochondrial carrier family (MCF) proteins transport a wide range of different key metabolites across the mitochondrial inner membrane and are important for the maintenance of key metabolic pathways in all eukaryotic cells. In particular, MCF proteins implicated in the mitochondrial import of iron are suggested to be essential for cell function and survival by providing iron required for iron-sulfur cluster assembly, mitochondrial electron transport and associated ATP production, and the defence against oxidative stress. Because of these important physiological roles, mitochondrial iron transporters could be considered as potential novel drug targets. The main aim of this thesis is the identification and functional characterization of putative mitochondrial iron transporters in T. brucei. Sequence analysis revealed that the T. brucei genome contains 3 MCF proteins, e.g. TbMCP12, TbMCP17 and TbMCP23, for which a potential role in mitochondrial iron transport could be predicted based on their homology to functionally characterized MCF proteins known to transport iron. Of these TbMCPs, TbMCP17 has the highest sequence homology with functionally characterized mitochondrial iron transporters from other eukaryotes, like for example MRS3/4 from Saccharomyces cerevisiae. Sequence analysis predicted further that TbMCP12 is more closely related to mitochondrial di/tri-carboxylate transporters, such as DIC1 from S. cerevisiae, whilst TbMCP23 is closely related to mitochondrial pyrimidine transporters, such as for example S. cerevisiae RIM2. Both mitochondrial di/tri-carboxylate and pyrimidine transporters have been suggested previously to potentially catalyse the mitochondrial co-transport of iron with their main metabolic substrates, here di/tri-carboxylic acids and pyrimidines, respectively. In addition to the identified TbMCPs, another potential mitochondrial iron transporter, here TbSFNX, was predicted to be potentially involved in mitochondrial iron transport. Sequence analysis revealed significant sequence homology of TbSFNX with members of the mammalian sideroflexin protein family. The exact physiological role of sideroflexins has yet to be determined, but a potential role in the mitochondrial co-transport of iron with citrate has previously been suggested. Immunofluorescence microscopy experiments using recombinant tagged protein versions of the identified 3 TbMCPs and TbSFNX, and/or polyclonal antibodies raised against these proteins, confirmed that all identified putative iron transporters are exclusively localized in the mitochondrion of T. brucei, suggesting that they indeed perform a mitochondrial transport function. Whether the expression of the 3 TbMCPs and TbSFNX is essential for cell survival, was investigated by the generation of conventional/conditional gene knockout and/or knockdown of expression (RNA interference) T. brucei cell lines and the subsequent analysis of cell growth. Results showed that the expression of TbMCP17, TbMCP23 and TbSFNX is essential for trypanosome survival, whereas the knockout/down of TbMCP12 did not show any growth phenotype in T. brucei. These results indicate that TbMCP17, TbMCP23 and TbSFNX fulfill key roles in the transport of essential metabolites, while TbMCP12 plays a less important transport role or is redundant in transport function due to the presence of other MCF proteins with similar transport roles. The putative transport function of the different TbMCPs was investigated by a combination of different experiments, including (1) functional complementation studies using specific Saccharomyces cerevisiae deletion strains lacking a specific MCF protein, and (2) mitochondrial ATP production assays using isolated mitochondria from wildtype and knockout/down T. brucei cell lines for which the expression of a specific MCF protein has been ablated or down-regulated. In S. cerevisiae, DIC is involved in the mitochondrial transport of dicarboxylates such as malate and oxoglutarate. Functional complementation studies using the S. cerevisiae deletion strain DIC revealed that TbMCP12 expression was able to restore growth of this strain on the non-fermentable carbon source glycerol. In addition, mitochondrial ATP production assays revealed that TbMCP12-depleted T. brucei mitochondria were not able to produce ATP from dicarboxylic acids, such as malate and oxoglutarate, and the tricarboxylic acid citrate. These results confirmed that TbMCP12 most likely functions as a di/tri-carboxylate transporter in T. brucei. Further experimentation revealed that, compared to the wildtype cell line, the TbMCP12-depleted cell line has become sensitive to oxidative stress in the form of the application of hydrogen peroxide to the culture medium. Measurement of the cellular NADP+/NADPH balance in the TbMCP12-depleted cell line revealed a significant shift towards NADP+ accumulation. These results clearly indicate that TbMCP12 also plays an important role in cellular oxidative stress defence in T. brucei, which is in agreement with its role as a mitochondrial di/tri-carboxylate transporter and the potential co-transport of iron. A similar investigative approach was used for TbMCP17. In S. cerevisiae, MRS3/4 is essential for the mitochondrial transport of iron across the mitochondrial inner membrane, but only under iron-limiting conditions. Functional complementation studies using the S. cerevisiae deletion strain MRS3/4 revealed that TbMCP17 expression was able to restore growth of this strain under iron-limiting conditions. In addition, growth experiments using a TbMCP17-depleted T. brucei cell line revealed that this cell line was not able to grow under iron-limiting conditions, however the ectopic over-expression of a recombinant version of TbMCP17 under the same conditions restored T. brucei growth. These results confirmed that TbMCP17 most likely functions as a mitochondrial iron transporter in T. brucei. Unfortunately, the same investigative approach could not be followed for TbMCP23 and TbSFNX. This is mainly due to the lack of suitable S. cerevisiae deletion strains and an associated detectable phenotype, and the extremely low/non-detectable expression levels of both TbMCPs. Further experiments revealed that not only the knockdown of TbSFNX, but also its overexpression resulted in severe growth defects in T. brucei. In addition, measurement of substrate consumption (glucose, proline) and product formation (acetate, succinate) revealed a substantially increased metabolic flux in both the TbSFNX knockdown and overexpression T. brucei cell lines. These results clearly suggest that TbSFNX plays a key role in the T. brucei energy metabolism. However, a specific transport function cannot be attributed to TbSFNX at this point. For TbMCP23, there was unfortunately not enough time left to further investigate its potential role in mitochondrial iron transport. In conclusion, 4 different potential mitochondrial iron transporters have been identified in T. brucei, e.g. TbMCP12, TbMCP17, TbMCP23, and TbSFNX. Of the identified transporters, only TbMCP17 was confirmed to function as a mitochondrial iron transporter, while TbMCP12 was shown to function as a mitochondrial di/tri-carboxylate transporter. Further research will be required to determine the specific mitochondrial transport function of the remaining candidates. The findings provide vital information on the mitochondrial metabolites transport of T. brucei and provide a solid foundation for future novel drug invention with TbMCP17 as the most suitable target.
Supervisor: Voncken, Frank ; Ettelaie, Camille Sponsor: University of Hull ; China Scholarship Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.788897  DOI: Not available
Keywords: Biological sciences
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