Molecular characterization of calcineurin B from the non-virulent Trypanosoma rangeli kinetoplastid indicates high gene conservation

Abstract

Calcineurin B, the regulatory subunit of calcineurin, a serine/threonine protein phosphatase, is highly conserved throughout the evolutionary scale including trypanosomatids such as Trypanosoma cruzi, and Leishmania major. Thus, in these flagellates the protein is required for mammalian host cell invasion and virulence and stress responses. With the aim of determining the presence of calcineurin B in Trypanosoma rangeli, a non-virulent trypanosome for mammals, the respective gene was amplified by PCR, cloned and sequenced. Two sequences of 531 bp in length showing a nucleotide polymorphism (314A>C) were obtained in spite of a single-copy gene was revealed by Southern blot. These sequences, probably the alleles from the gene, showed a 79% of identity with those from T. cruzi and clustered as the sister group of this trypanosome species in a Maximum Parsimony analysis. Deduced amino acid sequence comparison with trypanosomatids and other organisms through the phylogenetic scale as well as the obtained protein structural homology model suggested the presence of the four potential EF-hand regions and the corresponding calcium binding sites of the last three of these domains. Having assessed the expression of this protein in T. rangeli epimastigotes, and taking into account the following facts: (i) calcineurin inhibitors have inhibitory effect on the in vitro replication of T. cruzi, (ii) L. major promastigote growth is inhibited by chelating agents, and (iii) T. rangeli does not seem to productively infect mammalian cells, it is hypothesized herein that the function of this protein in T. rangeli is required for epimastigote growth.

Calcineurin-Crz1 Signaling in Lower Eukaryotes

“…However, in the last decade calcineurin has also been investigated more extensively in filamentous fungi (reviewed in reference 3) as well as in other lower eukaryotes such as the social amoeba Dictyostelium discoideum (4,5), the parasites Plasmodium falciparum (6), Trypanosoma cruzi (7,8), Trypanosoma rangeli (9), Schistosoma mansoni (10), and Leishmania major (11), and in Paramecium tetraurelia (12). Furthermore, many target proteins of this phosphatase have been identified in different organisms (reviewed in reference 13).…”

In Silico Characterization of Calcineurin from Pathogenic Obligate Intracellular Trypanosomatids: Potential New Biological Roles

“…These structural characteristics suggest a differential activation mechanism for their corresponding catalytic subunits, which could be closely related to the associated functional roles, such as invasion, proliferation, response to stress, and motility, among others [ 32 , 33 , 34 , 35 ]. Interestingly, the analysis of the domain architecture in Trypanosoma rangeli , a non-virulent trypanosome for mammals, suggests the presence of four EF-hand motifs with a potential role in the growth of epimastigotes, and of these four EF-hand motifs, only three fulfill the expected pattern of amino acid residues involved in coordinating Ca 2+ [ 79 ]. In the case of Hs CaNB, the analysis using the Prosite program attributes a lower score to the EH-hand motives towards N-term, while these are higher towards C-term, according to what has already been evidenced [ 80 ].…”

Identification of a type I nitroreductase gene in non-virulent Trypanosoma rangeli

“…To investigate whether T. rangeli type I NTR is expressed at the mRNA level and undergoes maturation, reverse transcription (RT)-PCR was performed using cytoplasmic mRNA from T. rangeli Tre epimastigotes (Montenegro et al 2013). cDNA was synthesised using the oligo (dT) 15 primer and NTR-FW- and NTR-RV-specific primers to amplify the NTR-coding sequence or the SL/TcTr (5’-AAC TAA CGC TAT TGA TAC AGT T-3’) and NTRTr/430 RV (5’-ACC GGC AAA CAC AAT TGT CA-3’) primer pair to obtain the 5’UTR sequence.…”