Phenotype of recombinant Leishmania donovani and Trypanosoma cruzi which over-express trypanothione reductase

Sensitivity towards agents that are thought to induce oxidative stress

Authors


Correspondence to J. M. Kelly, London School of Hygiene and Tropical Medicine, Keppel Street, London, England, WC1E 7HT

Abstract

Trypanothione reductase is thought to be important in maintaining an intracellular reducing environment in trypanosomatids. To investigate the role of trypanothione reductase we transfected Leishmania donovani and Trypanosoma cruzi with an expression vector containing the L. donovani trypanothione reductase gene and achieved over-expression of enzyme activity (10–14-fold) in transformed cells. Following treatment of L. donovani cells with the thiol-oxidizing agent diamide, the ability to regenerate dihydrotrypanothione from trypanothione disulphide was considerably enhanced in cells which over-expressed trypanothione reductase. However, the growth of transformed and control cells was equally sensitive to inhibition by nifurtimox, nitrofurazone and gentian violet, drugs that are thought to act by inducing oxidative damage. Likewise, growth of transformed and control cells were equally susceptible to inhibition by hydrogen peroxide, and control and transformed L. donovani promastigotes metabolized hydrogen peroxide at comparable rates. Thus, these experiments suggest that the ability to regenerate dihydrotrypanothione from trypanothione disulphide is not a rate-limiting step in the metabolism of hydrogen peroxide.

Abbreviations
GSH

glutathione

GSSG

glutathione disulphide

PCR

polymerase chain reaction

GspdSH

glutathionylspermidine

T(SH)2

dihydrotrypanothione

T(S)2

trypanothione disulphide

IC50

concentration which inhibits growth by 50%

Enzymes
 

Glutathione reductase (EC 1.6.4.2)

 

trypanothione reductase (EC 1.6.4.8)

 

trypanothione peroxidase (EC 1.11.1.–)

Ancillary