title: Stage-regulated GFP Expression in Trypanosoma cruzi: applications from host-parasite interactions to drug screening.
abstract: Trypanosoma cruzi is the etiological agent of Chagas disease, an illness that affects about 10 million people, mostly in South America, for which there is no effective treatment or vaccine. In this context, transgenic parasites expressing reporter genes are interesting tools for investigating parasite biology and host-parasite interactions, with a view to developing new strategies for disease prevention and treatment. We describe here the construction of a stably transfected fluorescent T. cruzi clone in which the GFP gene is integrated into the chromosome carrying the ribosomal cistron in T. cruzi Dm28c. This fluorescent T. cruzi produces detectable amounts of GFP only at replicative stages (epimastigote and amastigote), consistent with the larger amounts of GFP mRNA detected in these forms than in the non replicative trypomastigote stages. The fluorescence signal was also strongly correlated with the total number of parasites in T. cruzi cultures, providing a simple and rapid means of determining the growth inhibitory dose of anti-T.cruzi drugs in epimastigotes, by fluorometric microplate screening, and in amastigotes, by the flow cytometric quantification of T. cruzi-infected Vero cells. This fluorescent T. cruzi clone is, thus, an interesting tool for unbiased detection of the proliferating stages of the parasite, with multiple applications in the genetic analysis of T. cruzi, including analyses of host-parasite interactions, gene expression regulation and drug development.
authors.affiliation: Instituto Carlos Chagas, FIOCRUZ, Curitiba, Paraná, Brazil.
authors.forename: Rafael Luis
authors.initials: RL
authors.lastname: Kessler
authors.forename: Daniela Fiori
authors.initials: DF
authors.lastname: Gradia
authors.forename: Rita de Cássia
authors.initials: Rde C
authors.lastname: Pontello Rampazzo
authors.forename: Édio Elígio
authors.initials: ÉE
authors.lastname: Lourenço
authors.forename: Nilson José
authors.initials: NJ
authors.lastname: Fidêncio
authors.forename: Lauro
authors.initials: L
authors.lastname: Manhaes
authors.forename: Christian Macagnan
authors.initials: CM
authors.lastname: Probst
authors.forename: Andréa Rodrigues
authors.initials: AR
authors.lastname: Ávila
authors.forename: Stenio Perdigão
authors.initials: SP
authors.lastname: Fragoso
mesh_terms: D000818:Animals; D002522:Chlorocebus aethiops; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005434:Flow Cytometry; D005470:Fluorometry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis* / Q000235:genetics; D006790:Host-Parasite Interactions; D020128:Inhibitory Concentration 50; D050296:Microbial Viability; D009593:Nitroimidazoles / Q000494:pharmacology; D030781:Organisms, Genetically Modified / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D014344:Trypanocidal Agents / Q000494:pharmacology; D014349:Trypanosoma cruzi / Q000187:drug effects / Q000502:physiology*; D014709:Vero Cells
chemical_list: D009593:Nitroimidazoles; D014344:Trypanocidal Agents; D049452:Green Fluorescent Proteins; C009999:benzonidazole