Autophagy is a means which allows eukaryotic cells to selectively self-digest cellular components or whole compartments in order to adapt to new environmental conditions, to re-organize during differentiation or to overcome times of lack of nutrients. It seems that an evolutionary simple autophagic system has been conserved in trypanosomes, the causative agent of African sleeping sickness. Out of two known systems, which are re- sponsible for the expansion and completion of autophagosomes, apparantly only one, namely the ATG8-based system, seems to be present in T. brucei.
Three potential ATG8 orthologues have been identified in T. brucei, two of them being nearly identical. In this work an inducible knockdown of all three ATG8 genes has been established by RNA interference. Therefore single marker bloodstream form trypanoso- mes have been used, which express T7 RNA polymerase and tetracyclin repressor. The RNAi vector contained two T7 promotor and Tet operator sites in opposite directions, al- lowing dsRNA to be expressed under tetracyclin induction.
At first, ATG8.1 A/B double knockdown and ATG8.2 single knockdown constructs were cloned into E. coli. Afterwards, the ATG8.1 A/B and ATG8.2 PCR products were cou- pled by PCR ligation technique and cloned, leading to a triple knockdown construct. The correct composition of all RNAi constructs was confirmed by restriction analysis and DNA sequencing. SMB cells were transfected with the p2T7.ATG8.1 A/B-ATG8.2 triple knockdown construct and p2T7.alpha-tubulin as positive control. Foremost, the electro- poration of bloodstream forms had to be established as a new method in the laboratory. Therefore several parameters, including the best suited amount of DNA, convenient con- centration of selective antibiotics, measurement of cell density control and variation of medium supplements, had to be optimized. In order to elucidate ATG8 mRNA levels by Northern blotting, specific probes had to be constructed. This implicated the need to de- termine the length of the ATG8 5-untranslated regions by several methods, including 5-RACE PCR. The knockdown strains were analysed by Northern blotting and it was tested which induction parameters lead to the best results. The effect of autophagy and its potential knockdown was visualized in growth curves. An explicit analysis of the respec- tive phenotypes was not part of this work.