ABSTRACT
We characterized a gene encoding an YchF-related protein, TcYchF, potentially associated with the protein translation machinery of Trypanosoma cruzi. YchF belongs to the translation factor-related (TRAFAC) class of P-loop NTPases. The coding region of the gene is 1185bp long and encodes a 44.3kDa protein. BlastX searches showed TcYchF to be very similar (45-86%) to putative GTP-binding proteins from eukaryotes, including some species of trypanosomatids (Leishmania major and Trypanosoma brucei). A lower but significant level of similarity (38-43%) was also found between the predicted sequences of TcYchF and bacterial YyaF/YchF GTPases of the Spo0B-associated GTP-binding protein (Obg) family. Some of the most important features of the G domain of this family of GTPases are conserved in TcYchF. However, we found that TcYchF preferentially hydrolyzed ATP rather than GTP. The function of YyaF/YchF is unknown, but other members of the Obg family are known to be associated with ribosomal subunits. Immunoblots of the polysome fraction from sucrose gradients showed that TcYchF was associated with ribosomal subunits and polysomes. Immunoprecipitation assays showed that TcYchF was also associated with the proteasome of T. cruzi. Furthermore, inactivation of the T. brucei homolog of TcYchF by RNA interference inhibited the growth of procyclic forms of the parasite. These data suggest that this protein plays an important role in the translation machinery of trypanosomes.
Subject(s)
Adenosine Triphosphatases/genetics , Genes, Protozoan , Trypanosoma brucei brucei/genetics , Trypanosoma cruzi/genetics , Adenosine Triphosphatases/metabolism , Animals , Base Sequence , GTP Phosphohydrolases/metabolism , GTP-Binding Proteins/genetics , Male , Molecular Sequence Data , RNA, Protozoan/metabolism , RabbitsABSTRACT
We have identified two zinc finger proteins of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease in humans. These proteins, named tcZFP1 and tcZFP2, share the unusual zinc finger motif (CCCH) found in a diverse range of RNA-binding proteins involved in various aspects of the control of cell homeostasis and differentiation. We report here the functional expression of a recombinant tcZFP1, and the relative affinity and stability of the specific complexes formed between the protein and synthetic oligoribonucleotides containing C-rich sequences.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Oligoribonucleotides/chemistry , Protozoan Proteins/chemistry , Protozoan Proteins/metabolism , Trypanosoma cruzi/metabolism , Amino Acid Sequence , Animals , Blotting, Northern , Cloning, Molecular , Dose-Response Relationship, Drug , Green Fluorescent Proteins , Kinetics , Luminescent Proteins/chemistry , Molecular Sequence Data , Polyribosomes/chemistry , Protein Binding , Protein Structure, Tertiary , RNA-Binding Proteins/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Proteins/chemistry , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Time Factors , Zinc FingersABSTRACT
An indirect enzyme-linked immunosorbent assay (ELISA) using a crude antigen was evaluated for its performance to detect Babesia bigemina antibodies. The sensitivity and specificity were 98.0 per cent and 99.0 per cent, respectively. In agreement with the high specificity, no cross-reactions were verified with sera from calves inoculated three times with 10 7(subscribe) Babesia bovis organisms. With regard to the comparison of ELISA and indirect fluorescent antibody test (IFAT) in detecting antibodies against B. bigemina in calves experimentally infected with five Brazilian geographical isolates of this hemoparasite, IFAT was able to detect antibodies one day earlier in most of the calves' sera. There was a good agreement between results shown by ELISA and IFAT with sera from an enzootically stable area (k=0.61). However, there was no agreement between these serological tests with sera from an enzootically unstable area (k=0.33). The ELISA was employed in an epidemiological survey using with 1,367 sera from four counties in the Pantanal of Mato Grosso do Sul and characterized this region as an enzootically stable area, since the prevalence ranged from 87.7 to 98.9 per cent. Therefore, this ELISA with high sensitivity, specificity and performance similar to IFAT can be employed in serological diagnosis of B. bigemina
