Trypanosoma cruzi Fibrillarins: Two paralogous proteins with non-identical signals for nuclear transport.

Trypanosoma cruzi is a parasitic protozoa causative of Chagas disease. As part of our interest in studying the basic biology of this microorganism, this work reports our observations related to the characterization of motifs and structural domains present in two fibrillarin isoforms (TcFib1 and TcFib2) that were found to be necessary for the nuclear targeting of these nucleolar proteins. Previous characterization of these proteins indicated that they share 68.67% of identical amino acids and are both expressed as nucleolar proteins in T. cruzi epimastigotes. Using an approach based on the transfection of recombinant genes encoding fluorescent fibrillarin-EGFP fusion proteins, this study found evidence for the presence of 4 motifs or protein domains that help target these proteins to the nucleus: The GAR domain and carboxyl terminus in both TcFibs, as well as two lysines and a computationally predicted cNLS in TcFib1. As a distinctive feature, the GAR domain of TcFib2 proved to be essential for the nuclear localization of this protein paralog. Such a difference between TcFib1 and Tcfib2 nuclear localization signals can be explained as the presence of two partially related nuclear import pathways for the two fibrillarin homologues in this organism.

Trypanosoma cruzi Importin α: ability to bind to a functional classical nuclear localization signal of the bipartite type.

Importin α, a transport factor in the classical pathway of nuclear transport of proteins in eukaryotes, has not been experimentally studied in trypanosomatids. A chimeric fluorescent version of this protein (TcImportin α-EGFP) expressed in transfected epimastigotes of Trypanosoma cruzi is characterized here. Initially, the cellular localization of the tagged protein was analysed in exponentially growing and non-growing quiescent cells in a stationary phase. In growing epimastigotes, the fluorescence signal appeared to be mostly localized in the nucleolus, with additional minor fluorescent dots observed close to the nuclear periphery. In the stationary phase, both aged epimastigotes and metacyclic trypomastigotes presented with dispersed fluorescence of a granular form within the nucleoplasm of the cells that predominantly localized in poorly DAPI-stained regions. On the other hand, the ability of a tagged (6×His) version of TcImportin α to bind the nuclear protein cargo TcRPA31 (TcRPA31-EGFP) was determined by pull-down assays of co-transfected cultures. In addition, the results from the in vitro analyses with these tagged recombinant proteins showed that the functional nuclear localization signal (NLS) previously mapped to TcRPA31 was sufficient to sustain binding to TcImportin α. Moreover, the second cluster of basic amino acids within this bipartite NLS (formerly termed element B) was found to be essential for complex formation, as previously described for the nuclear translocation of these fluorescent chimeras. To our knowledge, this approach is the first in which Importin α was experimentally researched in kinetoplastids. The ability of TcImportin α to bind the NLS motif analysed here, is an essential feature expected for its potential functional role as a soluble transport factor.

An in vitro characterisation of the Trichomonas vaginalis TATA box-binding proteins (TBPs).

The protozoan parasite Trichomonas vaginalis is a common human pathogen from one of the earliest-diverging eukaryotic lineages. At the transcriptional level, the highly conserved Inr element of RNA pol II-transcribed genes surrounds the transcription start site and is recognised by IBP39, a protein exclusive of T. vaginalis. Typical TATA boxes have not been identified in this organism but, in contrast, BLAST analyses of the T. vaginalis genome identified two genes encoding putative TATA-binding proteins (herein referred to as TvTBP1 and TvTBP2). The goal of this work was to characterise these two proteins at the molecular level. Our results show that both TvTBPs theoretically adopt the saddle-shaped structure distinctive to TBPs and both Tvtbp genes are expressed in T. vaginalis. TvTBP1 did not complement a Saccharomyces cerevisiae mutant lacking TBP; however, TvTBP1 and TvTBP2 proteins bound T. vaginalis DNA promoter sequences in EMSA assays. We propose that TvTBP1 may be part of the preinitiation transcription complex in T. vaginalis since TvTBP1 recombinant protein was able to bind IBP39 in vitro. This work represents the first approach towards the characterisation of general transcription factors in this early divergent organism.

Nuclear distribution of the Trypanosoma cruzi RNA Pol I subunit RPA31 during growth and metacyclogenesis, and characterization of its nuclear localization signal.

Trypanosoma cruzi is the aetiologic agent of Chagas disease. Our research group studies ribosomal RNA (rRNA) gene transcription and nucleolus dynamics in this species of trypanosomes. RPA31 is an essential subunit of RNA polymerase I (Pol I) whose presence is apparently restricted to trypanosomes. Using fluorescent-tagged versions of this protein (TcRPA31-EGFP), we describe its nuclear distribution during growth and metacyclogenesis. Our findings indicate that TcRPA31-EGFP alters its nuclear presence from concentrated nucleolar localization in exponentially growing epimastigotes to a dispersed granular distribution in the nucleoplasm of stationary epimastigotes and metacyclic trypomastigotes. These changes likely reflect a structural redistribution of the Pol I transcription machinery in quiescent cellular stages where downregulation of rRNA synthesis is known to occur. In addition, and related to the nuclear internalization of this protein, the presence of a classical bipartite-type nuclear localization signal was identified towards its C-terminal end. The functionality of this motif was demonstrated by its partial or total deletion in recombinant versions of the tagged fluorescent protein. Moreover, ivermectin inhibited the nuclear localization of the labelled chimaera, suggesting the involvement of the importin α/ß transport system.

Characterization of cyclin-dependent kinases and Cdc2/Cdc28 kinase subunits in Trichomonas vaginalis.

Cyclin-dependent kinases (CDKs) have important roles in regulating key checkpoints between stages of the cell cycle. Their activity is tightly regulated through a variety of mechanisms, including through binding with cyclin proteins and the Cdc2/Cdc28 kinase subunit (CKS), and their phosphorylation at specific amino acids. Studies of the components involved in cell cycle control in parasitic protozoa are limited. Trichomonas vaginalis is the causative agent of trichomoniasis in humans and is therefore important in public health; however, some of the basic biological processes used by this organism have not been defined. Here, we characterized proteins potentially involved in cell cycle regulation in T. vaginalis. Three genes encoding protein kinases were identified in the T. vaginalis genome, and the corresponding recombinant proteins (TvCRK1, TvCRK2, TvCRK5) were studied. These proteins displayed similar sequence features to CDKs. Two genes encoding CKSs were also identified, and the corresponding recombinant proteins were found to interact with TvCRK1 and TvCRK2 by a yeast two-hybrid system. One putative cyclin B protein from T. vaginalis was found to bind to and activate the kinase activities of TvCRK1 and TvCRK5, but not TvCRK2. This work is the first characterization of proteins involved in cell cycle control in T. vaginalis.

Differential Effects of Two Widely Used Solvents, DMSO and Ethanol, on the Growth and Recovery of Trypanosoma cruzi Epimastigotes in Culture.

Trypanosoma cruzi is the etiological agent of Chagas disease. Epimastigote forms of T. cruzi can be readily cultured in axenic conditions. Ethanol and dimethyl sulfoxide (DMSO) are commonly used solvents employed as vehicles for hydrophobic compounds. In order to produce a reference plot of solvent dependent growth inhibition for T. cruzi research, the growth of epimastigotes was analyzed in the presence of different concentrations of ethanol (0.1-4.0%) and DMSO (0.5-7.5%). The ability of the parasites to resume growth after removal of these solvents was also examined. As expected, both ethanol and DMSO produced a dose-dependent inhibition of cellular growth. Parasites could recover normal growth after 9 days in up to 2% ethanol or 5% DMSO. Since DMSO was better tolerated than ethanol, it is thus recommended to prefer DMSO over ethanol in the case of a similar solubility of a given compound.

The yeasts phosphorelay systems: a comparative view.

Cells contain signal transduction pathways that mediate communication between the extracellular environment and the cell interior. These pathways control transcriptional programs and posttranscriptional processes that modify cell metabolism in order to maintain homeostasis. One type of these signal transduction systems are the so-called Two Component Systems (TCS), which conduct the transfer of phosphate groups between specific and conserved histidine and aspartate residues present in at least two proteins; the first protein is a sensor kinase which autophosphorylates a histidine residue in response to a stimulus, this phosphate is then transferred to an aspartic residue located in a response regulator protein. There are classical and hybrid TCS, whose difference consists in the number of proteins and functional domains involved in the phosphorelay. The TCS are widespread in bacteria where the sensor and its response regulator are mostly specific for a given stimulus. In eukaryotic organisms such as fungi, slime molds, and plants, TCS are present as hybrid multistep phosphorelays, with a variety of arrangements (Stock et al. in Annu Rev Biochem 69:183-215, 2000; Wuichet et al. in Curr Opin Microbiol 292:1039-1050, 2010). In these multistep phosphorelay systems, several phosphotransfer events take place between different histidine and aspartate residues localized in specific domains present in more than two proteins (Thomason and Kay, in J Cell Sci 113:3141-3150, 2000; Robinson et al. in Nat Struct Biol 7:626-633, 2000). This review presents a brief and succinct description of the Two-component systems of model yeasts, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans, Cryptococcus neoformans and Kluyveromyces lactis. We have focused on the comparison of domain organization and functions of each component present in these phosphorelay systems.

Functional analysis of sequence motifs involved in the polyadenylation of Trichomonas vaginalis mRNAs.

Synthesis of functional mRNA in eukaryotes involves processing of precursor transcripts, including the addition of a poly(A) tail at the 3' end. A multiprotein complex recognizes a polyadenylation signal, generally the hexanucleotide AAUAAA in metazoans, to direct processing of the pre-mRNA. Based on sequence analysis of several cDNAs, we have previously suggested that the UAAA tetranucleotide (which may include the UAA translation stop codon) could be the polyadenylation signal in Trichomonas vaginalis, a parasitic protozoon that causes human trichomoniasis. This proposal is analyzed here with the aid of a transient-expression system of a reporter gene (cat flanked by T. vaginalis actin noncoding sequences). When cells were transfected with a plasmid bearing the original 3' untranslated region (UTR) sequence containing the UAAA motif, the resulting cat mRNA was polyadenylated similarly to the endogenous actin mRNA. Base changes in the UAAA sequence produced alterations to the polyadenylation site of the reporter mRNAs, while nucleotide substitutions at either side of UAAA did not. Furthermore, relocation of the UAAA motif redirected the processing and polyadenylation of the reporter mRNA. In addition, a pre-mRNA cleavage site for polyadenylation was defined. Interaction of T. vaginalis proteins with the UAAA motif was shown by electrophoretic mobility shift assays. Based on our findings, we provide evidence that in T. vaginalis the UAAA tetranucleotide has a role equivalent to that of the metazoan consensus AAUAAA polyadenylation signal.

Stationary phase in Trypanosoma cruzi epimastigotes as a preadaptive stage for metacyclogenesis.

Trypanosoma cruzi is a species of parasitic protozoa that causes American trypanosomiasis or Chagas disease. These parasites go through a complex life cycle in Triatominae insects and vertebrate hosts. Epimastigotes are replicative forms that colonize the digestive tract of the vector and can be cultured in axenic media. The growth curve of epimastigotes allows assessment of differences in cells undergoing growth rate transitions from an exponential growth to a stationary phase. Since the classical descriptions of T. cruzi, it has been noted that the growth curve of epimastigotes in culture can give rise, in the stationary phase, to nonreplicating forms of metacyclic trypomastigotes. Metacyclogenesis therefore regards to the development process by which epimastigote transform into infective metacyclic trypomastigotes. In nature, these metacyclic forms allow the spread of Chagas disease when transmitted from an infected vector to a vertebrate host. This work reviews cellular phenomena that occur during the growth rate transitions of epimastigotes in culture, which may be related to very early physiological conditions for metacyclogenesis. Many of these events have not been thoroughly investigated. Their analysis can stimulate new hypotheses and future research in an important area not fully exploited.

Descriptive and functional analyses of four cyclin proteins in Trichomonas vaginalis.

Trichomonas vaginalis is an early divergent protozoan parasite that causes trichomoniasis, the most common non-viral sexually transmitted infection. In metazoans, there is abundant and detailed research on the cell cycle and the components involved in the regulation mechanisms. Regulators such as the cyclin-dependent kinases (CDKs) and cyclins activate the highly regulated processes of cell division. While CDKs have important roles in the phosphorylation of specific substrates, cyclins are important activating-components of CDKs that allow orderly passage through the different stages of the cell cycle. Cell cycle cyclins are characterized by showing drastic changes in their concentration during the cell cycle progression. However, in protists such as T. vaginalis, some biological processes such as cell cycle regulation remain less well studied. In an attempt to gain insight into cell cycle regulation in T. vaginalis, as an initial approach we characterized four proteins with features of cyclins. The genes encoding these putative cyclins were cloned to produce the recombinant proteins TvCYC1, TvCYC2, TvCYC3, and TvCYC4. The functional activity of TvCYC2, TvCYC3, and TvCYC4 was assessed through their complementation of a yeast cln1,2,3Δ mutant strain; TvCYC1 was not able to complement this mutant. Furthermore, our results suggest that TvCYC1, TvCYC2, and TvCYC3, are able to interact with and activate the kinase activity of TvCRK1, a kinase previously characterized by our group. The present study represents the first characterization of cyclins potentially involved in cell cycle regulation in T. vaginalis.

Saliva is a reliable and accessible source for the detection of SARS-CoV-2.

OBJECTIVES: The aim of this study was to investigate the feasibility of saliva sampling as a non-invasive and safer tool to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to compare its reproducibility and sensitivity with nasopharyngeal swab samples (NPS). The use of sample pools was also investigated. METHODS: A total of 2107 paired samples were collected from asymptomatic healthcare and office workers in Mexico City. Sixty of these samples were also analyzed in two other independent laboratories for concordance analysis. Sample processing and analysis of virus genetic material were performed according to standard protocols described elsewhere. A pooling analysis was performed by analyzing the saliva pool and the individual pool components. RESULTS: The concordance between NPS and saliva results was 95.2% (kappa 0.727, p = 0.0001) and 97.9% without considering inconclusive results (kappa 0.852, p = 0.0001). Saliva had a lower number of inconclusive results than NPS (0.9% vs 1.9%). Furthermore, saliva showed a significantly higher concentration of both total RNA and viral copies than NPS. Comparison of our results with those of the other two laboratories showed 100% and 97% concordance. Saliva samples are stable without the use of any preservative, and a positive SARS-CoV-2 sample can be detected 5, 10, and 15 days after collection when the sample is stored at 4 °C. CONCLUSIONS: The study results indicate that saliva is as effective as NPS for the identification of SARS-CoV-2-infected asymptomatic patients. Sample pooling facilitates the analysis of a larger number of samples, with the benefit of cost reduction.

Identification and characterization of a surface-associated, subtilisin-like serine protease in Trichomonas vaginalis.

SUMMARY: Trichomonas vaginalis is a protozoan parasite causing trichomonosis, a sexually transmitted infection in humans. This parasite has numerous proteases, most of which are cysteine proteases that appear to be involved in adherence and cytotoxicity of host cells. In this report we identify and characterize a putative subtilisin-like serine protease (SUB1). The sub1 gene encodes a 101-kDa protein. In silico analyses predict signal and pro-peptides at the N-terminus, and a transmembrane helix at the carboxy-terminal region. The sub1 gene was found as single copy by Southern analysis, albeit additional serine protease related genes are annotated in the T. vaginalis genome. The expression of sub1 could only be detected by RT-PCR and Ribonuclease Protection Assays, suggesting a low abundant mRNA. The sub1 gene transcription start site was correctly assigned by RPA. The transcript abundance was found to be modulated by the availability of iron in the growth medium. Antibodies raised to a specific SUB1 peptide recognized a single protein band (approximately 82 kDa) in Western blots, possibly representing the mature form of the protein. Immunofluorescence showed SUB1 on the trichomonad surface, and in dispersed vesicles throughout the cytoplasm. A bioinformatic analysis of genes annotated as serine proteases in the T. vaginalis genome is also presented. To our knowledge this is the first putative serine protease experimentally described for T. vaginalis.

The highly diverse TATA box-binding proteins among protists: A review.

Transcription is the first step of gene expression regulation and is a fundamental mechanism for establishing the viability and development of a cell. The TATA box-binding protein (TBP) interaction with a TATA box in a promoter is one of the best studied mechanisms in transcription initiation. TBP is a transcription factor that is highly conserved from archaea to humans and is essential for the transcription initiated by each of the three RNA polymerases. In addition, the discovery of TBP-related factor 1 (TRF1) and other factors related to TBP shed light on the variability among transcription initiation complexes, thus demonstrating that the compositions of these complexes are, in fact, more complicated than originally believed. Despite these facts, the majority of studies on transcription have been performed on animal, plant and fungal cells, which serve as canonical models, and information regarding protist cells is relatively scarce. The aim of this work is to review the diversity of the TBPs that have been documented in protists and describe some of the specific features that differentiate them from their counterparts in higher eukaryotes.

Comparative analyses among the Trichomonas vaginalis, Trichomonas tenax, and Tritrichomonas foetus 5S ribosomal RNA genes.

The 5S ribosomal RNA (5S rRNA) is an essential component of ribosomes. Throughout evolution, variation is found among 5S rRNA genes regarding their chromosomal localization, copy number, and intergenic regions. In this report, we describe and compare the gene sequences, motifs, genomic copy number, and chromosomal localization of the Trichomonas vaginalis, Trichomonas tenax, and Tritrichomonas foetus 5S rRNA genes. T. vaginalis and T. foetus have a single type of 5S rRNA-coding region, whereas two types were found in T. tenax. The sequence identities among the three organisms are between 94 and 97%. The intergenic regions are more divergent in sequence and size with characteristic species-specific motifs. The T. foetus 5S rRNA gene has larger and more complex intergenic regions, which contain either an ubiquitin gene or repeated sequences. The 5S rRNA genes were located in Trichomonads chromosomes by fluorescent in situ hybridization.

Nuclear localization signals in trypanosomal proteins.

The nuclear import of proteins in eukaryotic cells is a fundamental biological process. While it has been analysed to different extents in model eukaryotic organisms, this event has rarely been studied in the early divergent protozoa of the order Kinetoplastida. The work presented here represents an overview of nuclear import in these important species of human pathogens. Initially, an in silico study of classical nuclear localization signals within the published nuclear proteomes of Trypanosoma brucei and Trypanosoma cruzi was carried out. The basic amino acids that comprise the monopartite and bipartite classical nuclear localization signals (cNLS) in trypanosomal proteins are similar to the consensus sequences observed for the nuclear proteins of yeasts, animals and plants. In addition, a summarized description of published studies that experimentally address the NLS of nuclear proteins in trypanosomatids is presented, and the clear occurrence of non-classical NLS (NLS that lack the consensus motifs of basic amino acids) in the analysed reports indicate a complex scenario for the types of receptors in these species. In general, the information presented here agrees with the hypothetical appearance of mechanisms for the recognition of nuclear proteins in early eukaryotic evolution.

Potential regulatory elements in the Trypanosoma cruzi rRNA gene promoter.

The Trypanosoma cruzi rRNA gene promoter was characterized by deletion and point mutation analyses. A core of 89 bp was identified as the minimal region with full promoter activity. This core region is flanked upstream by a control element that stimulates its activity, and downstream by a novel down regulating region of about 200 bp. A point mutation analysis of the transcription start region evidenced 7 contiguous nucleotides where individual substitutions produced in all cases a defective promoter. It is generally accepted that the anciently speciated trypanosomatids lack strict promoters for protein coding genes transcribed by RNA polymerase II. The occurrence of a well structured rRNA gene promoter in these species suggests an early appearance of the RNA polymerase I promoters in the evolution of eukaryotic cells.

A simple model to explain three-base periodicity in coding DNA.

A simple model is put forward to explain the long-known three-base periodicity in coding DNA. We propose the concept of same-phase triplet clustering, i.e. a condition wherein a triplet appears several times in one phase without interruption by the two other possible phases. For instance, in the sequence (i): NTT_GNN_NTT_GNN_NTT_GNN_NNN_NTT_GNN (where N is any nucleotide but combinations producing TTG are excluded) there would be clustering of same-phase TTG because this triplet appears uninterruptedly in phase 2. In contrast, in the sequence (ii): TTG_NTT_GNN_NNT_TGN_NNN_NTT_GNN there is no same-phase clustering because neighboring TTGs are all in different phases. Observe also that in sequence (i) TTG triplets are separated by 3, 3 and 6 nucleotides (3n distances), while in sequence (ii) they are separated by 1, 4 and 5 nucleotides (non-3n distances). In this work, we demonstrate that in coding DNA the 3n distances generated by (i)-type sequences proportionally outnumber the non-3n distances generated by (ii)-type sequences, this condition would be the basis of three-base periodicity. Randomized sequences had (i)- and (ii)-type sequences too but clustering was statistically different. To prove our model we generated (i)-type sequences in a randomized sequence by inducing clustering of same-phase triplets. In agreement with the model this sequence displayed three-base periodicity. Furthermore, two- and four-base periodicities could also be induced by artificially inducing clustering of duplets and tetraplets.

The stabilization of housekeeping transcripts in Trypanosoma cruzi epimastigotes evidences a global regulation of RNA decay during stationary phase.

The relative steady state concentration of mRNAs of four housekeeping single-copy type Trypanosoma cruzi genes (actin, triosephosphate isomerase, trypanothion reductase and the ribosomal protein S4) was analyzed throughout the growth curve. A distinguishable pattern was observed with maximal levels occurring at the logarithmic phase of growth and minimum levels occurring at the stationary phase. The half-lives of all analyzed messenger RNAs, and also of three molecular species of immature ribosomal RNAs were increased in cells isolated from stationary phase. These results suggest the occurrence of a novel global regulation mechanism that might protect transcripts from degradation in stationary epimastigotes, probably as a strategy to perpetuate through this quiescent stage.

Separable putative polyadenylation and cleavage motifs in Trichomonas vaginalis mRNAs.

3' Untranslated region processing and polyadenylation in Trichomonas vaginalis was analyzed by 3' rapid amplification of cDNA ends and sequence analysis of T. vaginalis mRNAs. A putative polyadenylation signal with the sequence UAAA was found 11-30 nucleotides upstream from the cleavage site. The motif pyrimidine( downward arrow)(A)(0-3)AAUU is proposed to be the cleavage site for polyadenylation of transcripts. This potential sequence defining the cleavage site for polyadenylation in eukaryotes is a novel finding. As in other eukaryotes, runs of several U's downstream from the cleavage site were identified. A working hypothesis is proposed which couples the UAA translation stop codon with the signaling for the 3'end processing of transcripts in this early divergent parasitic protozoa.

Evidence supporting a major promoter in the Trypanosoma cruzi rRNA gene.

Two clearly separated transcription start points (tsp) have been reported within the Trypanosoma cruzi rDNA (DNA encoding rRNA) gene spacer region. These sites are separated by 270 bp, a distance compatible with the occurrence of two core promoters. To characterize the individual participation of these two elements, a deletion analysis was carried out. Different versions of the promoter regions were assayed in a transient transfection analysis of epimastigotes, using the chloramphenicol acetyl transferase gene (cat) as a reporter. The data indicate that the so-called distal tsp-associated region (relative to the small subunit rRNA 5' terminus coding region) comprises most (80%) if not all of the observed activity. In addition, an associated locus specific repeated element showed a modest upregulating activity, since its presence stimulated the cat reporter gene by about 20%. The data here presented should be valuable in the design of expression vectors for T. cruzi, where the rRNA gene promoter has been an important functional element.