A novel protein phosphatase 2A (PP2A) is involved in the transformation of human protozoan parasite Trypanosoma cruzi.
Here we provide evidence for a critical role of PP2As (protein phosphatase 2As) in the transformation of Trypanosoma cruzi. In axenic medium at pH 5.0, trypomastigotes rapidly transform into amastigotes, a process blocked by okadaic acid, a potent PP2A inhibitor, at concentrations as low as 0.1 microM. 1-Norokadaone, an inactive okadaic acid analogue, did not affect the transformation. Electron microscopy studies indicated that okadaic acid-treated trypomastigotes had not undergone ultrastructural modifications, reinforcing the idea that PP2A inhibits transformation. Using a microcystin-Sepharose affinity column we purified the native T. cruzi PP2A. The enzyme displayed activity against 32P-labelled phosphorylase a that was inhibited in a dose-dependent manner by okadaic acid. The protein was also submitted to MS and, from the peptides obtained, degenerate primers were used to clone a novel T. cruzi PP2A enzyme by PCR. The isolated gene encodes a protein of 303 amino acids, termed TcPP2A, which displayed a high degree of homology (86%) with the catalytic subunit of Trypanosoma brucei PP2A. Northern-blot analysis revealed the presence of a major 2.1-kb mRNA hybridizing in all T. cruzi developmental stages. Southern-blot analysis suggested that the TcPP2A gene is present in low copy number in the T. cruzi genome. These results are consistent with the mapping of PP2A genes in two chromosomal bands by pulsed-field gel electrophoresis and chromoblot hybridization. Our studies suggest that in T. cruzi PP2A is important for the complete transformation of trypomastigotes into amastigotes during the life cycle of this protozoan parasite.
Parasitology Unit, Department of Medical Technology, University of Antofagasta, Antofagasta, PO Box 170, Chile. jgonzalez@uantof.cl
Jorge
J
González
Alberto
A
Cornejo
Marcia R M
MR
Santos
Esteban M
EM
Cordero
Bessy
B
Gutiérrez
Patricio
P
Porcile
Renato A
RA
Mortara
Hernán
H
Sagua
José Franco
JF
Da Silveira
Jorge E
JE
Araya
D000595:Amino Acid Sequence; D000818:Animals; D005591:Chemical Fractionation; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry; D005407:Flagella / Q000737:chemistry; D017125:Genes, Protozoan; D016678:Genome; D006131:Growth Inhibitors / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D019319:Okadaic Acid / Q000737:chemistry; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation / Q000187:drug effects; D054648:Protein Phosphatase 2; D014158:Transcription, Genetic; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development* / Q000648:ultrastructure
D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D006131:Growth Inhibitors; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2