The use of transgenic Trypanosoma brucei to identify compounds inducing the differentiation of bloodstream forms to procyclic forms.
The expression of procyclins is the earliest known marker of differentiation of bloodstream forms of Trypanosoma brucei to procyclic forms. We have generated transgenic bloodstream and procyclic forms in which the coding region of one procyclin gene was replaced by E. coli beta-glucuronidase (GUS). GUS activity can be monitored in a simple one-step colour reaction in microtitre plates; this assay is potentially suitable for large-scale screening for compounds that influence differentiation. GUS was stage-specifically expressed in procyclic forms and its synthesis occurred in parallel with that of procyclin when bloodstream forms were triggered to differentiate by the addition of cis-aconitate. GUS could also be induced by brief treatment with the proteases trypsin, pronase or thermolysin, but not with pepsin or thrombin. Interestingly, a combination of one of the active proteases with cis-aconitate resulted in increased GUS activity relative to either trigger alone. In contrast to cis-aconitate, protease treatment resulted in considerable cell death. Experiments with the pleomorphic strain AnTat 1.1 showed that long slender bloodstream forms were rapidly killed by proteases, whereas stumpy forms were largely resistant. Stumpy forms treated with trypsin differentiated synchronously and expressed procyclin with faster kinetics than when they were triggered by cis-aconitate. As predicted by the GUS assay, differentiation was even more rapid when both inducers were used simultaneously, with all cells expressing maximal levels of procyclin within 3 h.
Sbicego
S
S
Departement für Chemie und Biochemie, Universität Bern, Switzerland.
Vassella
E
E
Kurath
U
U
Blum
B
B
Roditi
I
I
D000156:Aconitic Acid / Q000494:pharmacology; D000818:Animals; D030801:Animals, Genetically Modified; D002951:Citrates / Q000494:pharmacology; D010450:Endopeptidases / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005434:Flow Cytometry; D018507:Gene Expression Regulation, Developmental; D005966:Glucuronidase / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D015800:Protozoan Proteins; D019076:Transgenes; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development*
D002951:Citrates; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; C053277:procyclic acidic repetitive protein, Trypanosoma; D000156:Aconitic Acid; D005966:Glucuronidase; D010450:Endopeptidases