The effect of over-expression of the alternative oxidase in the procyclic forms of Trypanosoma brucei.

Trypanosome alternative oxidase (TAO) is the cyanide-resistant but SHAM-sensitive terminal oxidase of the mitochondrial electron transport chain in African trypanosomes. The bloodstream forms of Trypanosoma brucei lack cytochromes and respire exclusively via TAO. On the other hand, the insect, or procyclic form possesses a fully developed cytochrome system, and down regulates TAO several folds by reducing the stability of the TAO transcript. We expressed an ectopic copy of TAO in the procyclic form from a tetracycline regulated stable expression vector, in which the TAO 3'-UTR was replaced by T. brucei aldolase 3'-UTR. The TAO transcript produced from the ectopic copy was stably accumulated in the procyclic form. Upon induction with doxycycline, TAO protein level was gradually increased about five-fold within 72 h. TAO over-expression did not show any effect on the growth of the parasite. The rate of respiration and the SHAM-sensitive respiratory pathway capacity was increased about two- and five-fold, respectively, and the cytochrome-mediated respiratory pathway capacity was reduced two- to three-folds within 5 days after induction of TAO. Doxycycline induced TAO+ cells preferentially utilized CN-resistant, SHAM-sensitive pathway of respiration, whereas, in the control cells 70-80% of total respiration was via the CN-sensitive pathway. Moreover, we have found that increased expression of TAO caused about two-fold down regulation of cytochrome oxidase subunit IV, and cytochrome c1 protein level and also caused a four-fold up-regulation of the expression of the surface coat protein, GPEET procyclin in the procyclic form. This suggests that the expression of two terminal oxidases and the coat protein is linked in T. brucei.