Charakterisierung der Aquaglyceroporine von Trypanosoma brucei

89 6 Abstract The protozoan parasite Trypanosoma brucei causes human sleeping sickness, Nagana in domestic animals and depends on the tsetse fly for dissemination. In its complex life cycle two dividing stages can be differentiated, the slender form in blood, lymphatic fluid, and brain of the mammalian host and the procyclic form in the midgut of the fly vector. For energy production bloodstream parasites rely on glucose, but alternatively they can also metabolize glycerol. Glycolysis in trypanosomes differs markedly from other eukaryotic cells, because the first seven reactions of the pathway are compartmentalized to a special organelle, called glycosome. Bloodstream form trypanosomes, because of the absence of a lactate dehydrogenase, use oxygen to maintain the NAD/NADH balance in the glycosome and throw out pyruvate. Under low oxygen conditions, the parasite can utilize a glycosomal glycerol kinase to maintain NAD/NADH homeostasis and produce glycerol as waste. The procyclic forms can feed on glucose too, but mainly use proline as an energy source; they have a functional tricarboxylic acid cycle and oxidative phosphorylation capabilities. In its life cycle the trypanosome is also subjected to considerable variations of osmolarity. To cope with water and glycerol transport across membranes, they have three aquaglyceroporins, which are furthermore permeable for small uncharged solutes such as ammonia, arsenic or antimony. Peptide-derived antibodies directed against each individual aquaglyceroporin were purified by affinity chromatography utilizing the synthesized peptides. Immunofluorescence staining showed AQP1 only in the flagellar membrane, whereas AQP3 was exclusively localized to the plasma membrane. For determining the AQP2 localization an AQP2 overexpressing trypanosome cell line was created to counter the low affinity of the Anti-AQP2 antibody. From the respective immunofluorescence micrographs, only a non-specific intracellular membrane localization could be concluded. Besides that aquaglyceroporin-deficiant RNA interference cell lines were used to backup and complement these results. A heritable and inducible RNAi vector was cloned for each individual aquaglyceroporin knockdown and one for a simultaneous triple knockdown. The plasmids were used to create corresponding procyclic and in bloodstream form knockdown cell lines. After verifying the success of the knockdown clones via Northern blot, the cell lines were tested phenotype changes like growth inhibition or their response to a hypo-osmotic shock. The procyclic and bloodstream form AQP1 knockdown-rates were 99 % and 83 %, respectively. These cells showed no growth inhibition. They were capable to recover from a hypo-osmotic shock of 150 mosm and displayed, in accordance with AQP1 expression levels and the AQP1 flagellar membrane localization, a prolonged time to swell to the maximum size. The procyclic form AQP3 knockdown transcribed AQP3 knockdown RNA but was not able to reduce the already low AQP3 transcript level, so accordingly no phenotype changes could be found. Bloodstream form trypanosomes have more than fourfold more AQP3 transcript than procyclic forms; when the bloodstream form AQP3 knockdown was induced the AQP3 mRNA amount was reduced to a sixth. The lack of AQP3 in the plasma membrane led to no growth inhibition and normal recovery from hypo-osmotic stress conditions. It also led to a profoundly prolonged time for cell swelling, as would be anticipated.