A proteomic approach to the investigation of cymelarsan resistance in Trypanosoma brucei

Foucher, Aude Lise (2003) A proteomic approach to the investigation of cymelarsan resistance in Trypanosoma brucei. PhD thesis, University of Glasgow.

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Abstract

Two parasitic trypanosomatids, T. b. gambieme and T. b. rhodesieme, are responsible for around 150,000 death a year by Human African Trypanosomiasis (HAT). Prevalence values in some villages reaches up to 50%, making HAT the leading cause of mortality. There are currently only four drugs available for treating HAT, but problems with availability, administration, disease stage specificity and severe side effects have hindered their uses. The most used drug is the arsenical, Melarsoprol, but resistance is increasingly reported in the field. Some reports have suggested that point mutations within the TbAT1, encoding the P2 transporter, were associated with drug resistance in both laboratory-derived strains and field isolates. However, analysis of a TbAT1 cell showed a low level of resistance and 50% of field isolates had a non-mutated P2 transporter. Consequently, it seems likely that multiple mechanisms can confer a resistance phenotype but these have been largely undescribed to date. The advantage of a global proteomic expression profiling study of drug sensitive and drug resistant isogenic T. brucei lines is that, in principle, all possible mechanisms could be identified without pre-supposing a specific mechanism. The release of an almost complete T. brucei genome database, the advances in 2DE technology and mass spectrometry techniques, and the availability of drug sensitive and resistant isogenic T. brucei lines rendered this analysis feasible. Subtractive analysis of the whole proteome of the drug sensitive and resistant isogenic line of STIB 386 identified two proteins whose loss of expression was associated with Cymelarsan resistance. Cymelarsan is a drug that is structurally and functionally closely related to Melarsoprol. Tandem mass spectrometry analysis matched the first protein onto either open reading frame (ORF) 274 or 277, present as identical tandem repeats on chromosome DC. This protein spot was shown to be an isoform of a spot located a similar molecular weight but with a more basic pi. As both ORPs have identical predicted amino acid sequences, are transcribed and no other homologues were found in the T. brucei genome, it is likely that the missing protein is an isoform of the adjacent protein which arose due to a post-translational modification. I have named this protein the Cymelarsan Associated Resistance (CAR) protein. The absence of the protein was not associated with Cymelarsan resistance in a pair of isogenic drug sensitive and resistant lines of 247. The second protein spot associated with Cymelarsan resistance was identified as an arginine kinase from either ORF 342 or 343. These two ORF, present as tandem repeat on chromosome IX are not identical. However, the degree of sequencing obtained from the Tandem mass spectrometry analysis did not enable to differentiate between product of each ORF. Finally, a diminished reduction of the Alamar Blue dye was formed associated with Cymelarsan resistance in both pairs of isogenic lines analysed. An attempt was made to determine the mechanism that impaired the reduction of the Alamar blue dye in the drug resistant lines. The transport and efflux of the dye were analysed and did not account for the difference in reduction of the Alamar Blue dye. The dye was found to concentrate in vesicle-like structures thought to be acidocalcisomes.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Mike Turner
Keywords: Pharmacology, Parasitology
Date of Award: 2003
Depositing User: Enlighten Team
Unique ID: glathesis:2003-71423
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 10 May 2019 10:49
Last Modified: 10 May 2019 10:49
URI: http://theses.gla.ac.uk/id/eprint/71423

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