A cytoplasmic new catalytic subunit of calcineurin in Trypanosoma cruzi and its molecular and functional characterization.
Parasitological cure for Chagas disease is considered extremely difficult to achieve because of the lack of effective chemotherapeutic agents against Trypanosoma cruzi at different stages of infection. There are currently only two drugs available. These have several limitations and can produce serious side effects. Thus, new chemotherapeutic targets are much sought after. Among T. cruzi components involved in key processes such as parasite proliferation and host cell invasion, Ca(2+)-dependent molecules play an important role. Calcineurin (CaN) is one such molecule. In this study, we cloned a new isoform of the gene coding for CL strain catalytic subunit CaNA (TcCaNA2) and characterized it molecularly and functionally. There is one copy of the TcCaNA2 gene per haploid genome. It is constitutively transcribed in all T. cruzi developmental forms and is localized predominantly in the cytosol. In the parasite, TcCaNA2 is associated with CaNB. The recombinant protein TcCaNA2 has phosphatase activity that is enhanced by Mn(2+)/Ni(2+). The participation of TcCaNA2 in target cell invasion by metacyclic trypomastigotes was also demonstrated. Metacyclic forms with reduced TcCaNA2 expression following treatment with morpholino antisense oligonucleotides targeted to TcCaNA2 invaded HeLa cells at a lower rate than control parasites treated with morpholino sense oligonucleotides. Similarly, the decreased expression of TcCaNA2 following treatment with antisense morpholino oligonucleotides partially affected the replication of epimastigotes, although to a lesser extent than the decrease in expression following treatment with calcineurin inhibitors. Our findings suggest that the calcineurin activities of TcCaNA2/CaNB and TcCaNA/CaNB, which have distinct cellular localizations (the cytoplasm and the nucleus, respectively), may play a critical role at different stages of T. cruzi development, the former in host cell invasion and the latter in parasite multiplication.
Department of Medical Technology, University of Antofagasta, Antofagasta, Chile.
Patricio R
PR
Orrego
Biomedical Department, University of Antofagasta, Antofagasta, Chile.
Héctor
H
Olivares
Department of Microbiology, Immunology and Parasitology, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil.
Esteban M
EM
Cordero
Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, São Paulo, Brazil.
Albert
A
Bressan
Department of Parasitology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, São Paulo, Brazil.
Mauro
M
Cortez
Department of Medical Technology, University of Antofagasta, Antofagasta, Chile.
Hernán
H
Sagua
Department of Medical Technology, University of Antofagasta, Antofagasta, Chile.
Ivan
I
Neira
Department of Medical Technology, University of Antofagasta, Antofagasta, Chile.
Jorge
J
González
Department of Microbiology, Immunology and Parasitology, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil.
José Franco
JF
da Silveira
Department of Microbiology, Immunology and Parasitology, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, São Paulo, Brazil.
Nobuko
N
Yoshida
Department of Medical Technology, University of Antofagasta, Antofagasta, Chile.
Jorge E
JE
Araya
D000953:Antigens, Protozoan; D019703:Calcineurin / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D049109:Cell Proliferation; D003001:Cloning, Molecular; D004705:Endocytosis; D020536:Enzyme Activators / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D009532:Nickel / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014349:Trypanosoma cruzi / Q000235:genetics / Q000378:metabolism*
D000953:Antigens, Protozoan; D020536:Enzyme Activators; D015800:Protozoan Proteins; C060967:cytoplasmic antigen, Trypanosoma; D008345:Manganese; D009532:Nickel; D019703:Calcineurin; D010744:Phosphoric Monoester Hydrolases