Drug-Resistance and Genetic Exchange in Trypanosoma brucei

Scott, Alan (1995) Drug-Resistance and Genetic Exchange in Trypanosoma brucei. PhD thesis, University of Glasgow.

Abstract

The aims of the work described in this thesis were to generate well characterised stable cymelarsan (melCy)-resistant and suramin-resistant lines of Trypanosoma brucei, and to use these lines to investigate the biochemical and genetic basis of drug-resistance. During these studies, different methods were used to assay drug-resistance in the cloned lines and these methods were critically compared to develop a rapid method for determining drug-resistance. MelCy-resistance was investigated by analysing the pattern of cross-resistance to other melaminophenyl arsenicals and two diamidines, and the role of altered drug uptake in the mechanism of melCy-resistance. Genetic exchange between drug-resistant lines provided information on the genetic basis of melCy-resistance and suramin-resistance. Four drug-resistant lines were generated from two cloned stocks of Trypanosoma brucei (STIB 247 and STIB 386) by passaging trypanosomes in immunosuppressed mice in the presence of gradually increasing sub-curative drug doses. Drug-resistant populations were recloned, producing two melCy- resistant lines (247MelCyR and 386MelCyR) and two suramin-resistant lines (247SurR and 386SurR). In vivo assays in mice indicated that the levels of melCy-resistance had increased to greater than 40mg/Kg in both 247MelCyR (130-fold increase) and 386MelCyR (20-fold increase). Similarly, suramin- resistance was measured at 70mg/Kg for both 247SurR (47-fold increase) and 386SurR (35-fold increase). No cross-resistance between the two drugs was observed. The drug-resistant phenotypes in all four lines were stable upon passaging in mice for a number of weeks in the absence of drug pressure. Three lines (247MelCyR, 247SurR and 386MelCyR) were transmitted through tsetse flies, without attenuation of their levels of drug-resistance. The levels of sensitivity/resistance to each drug were determined in the two unselected stocks (247 and 386) in vitro in procyclic culture forms and in short-term axenic bloodstream form cultures. Results were expressed in terms of the effective concentration that inhibited growth by 50% over twenty-four hours (EC50). MelCy-resistance was expressed in procyclic and bloodstream culture forms. Suramin-resistance was not expressed in procyclic culture forms, with EC50 values of approximately 0.1 mM for both the unselected and suramin- resistant lines, although suramin-resistance was expressed in bloodstream culture forms. There was no convincing evidence for the existence of cross-resistance between melCy and suramin in any of the drug-resistant lines in vitro. It was concluded that the short-term bloodstream culture was a suitable method for the rapid assessment of drug-resistance. oth melCy-resistant lines expressed cross-resistance to melarsoprol (melB) and trimelarsen (melW) in vivo at the maximum doses tolerated in mice of 40mg/Kg. Procyclic culture forms of 247MelCyR expressed resistance to melW and melarsen oxide (melOx), with increases in drug-resistance in vitro of 770-fold and 290-fold respectively. However, cross-resistance with melB was considerably lower in procyclic culture forms, with an increase in resistance from 247 to 247MelCyR of only 2.3-fold. A similar pattern was seen with 386 and 386MelCyR, with the melCy-resistant line expressing resistance to both melW (79-fold increase) and melOx (310-fold increase) but not to melB (1.3-fold increase) in procyclic culture forms. Furthermore, the melCy-resistant lines did not express melB-resistance in short-term bloodstream culture forms. It was concluded that melCy-resistance and melB-resistance may be brought about through different mechanisms. The 247MelCyR line was characterised with respect to its mechanism(s) of arsenical uptake, the relationship between arsenical transport and purine and diamidine transport, and the aherations in uptake associated with melCy- resistance. Cross-resistance was observed in the 247MelCyR line to the two diamidines, berenil and pentamidine, in vivo. The possibility that melCy, melB and melOx, together with berenil and pentamidine, shared a common adenosine transporter for entry into the trypanosome was investigated in vitro. Berenil and pentamidme were able to inhibit lysis by all three melaminophenyl arsenicals in a concentration-dependent manner. MelOx and melCy-induced lysis of 247 were inhibited in a concentration-dependent manner by adenosine and adenine, in agreement with the proposal that these arsenical drugs compete with these purines for transport (Carter and Fairlamb 1993). However, melB-induced lysis could not be blocked by adenosine or adenine, suggesting that melB did not share this adenosine transporter. (Abstract shortened by ProQuest.).

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Mike Turner
• Keywords: Parasitology, Genetics
• Date of Award: 1995
• Depositing User: Enlighten Team
• Unique ID: glathesis:1995-74931
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 27 Sep 2019 15:07
• Last Modified: 27 Sep 2019 15:07
• URI: https://theses.gla.ac.uk/id/eprint/74931