Production of Recombinant Trypanosoma cruzi Antigens in Leishmania tarentolae.

Trypanosomatids are unicellular organisms that colonize a wide diversity of environments and hosts. For instance, Trypanosoma cruzi is a human pathogen responsible for Chagas diseases, while Leishmania tarentolae infects amphibians and became a biotechnological tool suitable for recombinant protein expression. T. cruzi antigens are needed for the development of improved epitope-based methods for diagnosis and treatment of Chagas disease. Molecular cloning for the production of recombinant proteins offers the possibility to obtain T. cruzi antigens at high yield and purity. L. tarentolae appears as the ideal expression host to obtain recombinant T. cruzi antigens with a structure and posttranslational modifications typical of trypanosomatids. In this chapter, we present a protocol for the analytical to mid-scale production of recombinant T. cruzi antigens, using L. tarentolae as expression host (LEXSY® inducible system).

The ubiquitin-conjugating enzyme CDC34 is essential for cytokinesis in contrast to putative subunits of a SCF complex in Trypanosoma brucei.

The ubiquitin-proteasome system is a post-translational regulatory pathway for controlling protein stability and activity that underlies many fundamental cellular processes, including cell cycle progression. Target proteins are tagged with ubiquitin molecules through the action of an enzymatic cascade composed of E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, and E3 ubiquitin ligases. One of the E3 ligases known to be responsible for the ubiquitination of cell cycle regulators in eukaryotes is the SKP1-CUL1-F-box complex (SCFC). In this work, we identified and studied the function of homologue proteins of the SCFC in the life cycle of Trypanosoma brucei, the causal agent of the African sleeping sickness. Depletion of trypanosomal SCFC components TbRBX1, TbSKP1, and TbCDC34 by RNAi resulted in decreased growth rate and contrasting cell cycle abnormalities for both procyclic (PCF) and bloodstream (BSF) forms. Depletion of TbRBX1 in PCF cells interfered with kinetoplast replication, whilst depletion of TbSKP1 arrested PCF and BSF cells in the G1/S transition. Silencing of TbCDC34 in BSF cells resulted in a block in cytokinesis and caused rapid clearance of parasites from infected mice. We also show that TbCDC34 is able to conjugate ubiquitin in vitro and in vivo, and that its activity is necessary for T. brucei infection progression in mice. This study reveals that different components of a putative SCFC have contrasting phenotypes once depleted from the cells, and that TbCDC34 is essential for trypanosome replication, making it a potential target for therapeutic intervention.

Characterization of TcCYC6 from Trypanosoma cruzi, a gene with homology to mitotic cyclins.

Trypanosoma cruzi, the etiologic agent of Chagas disease, is a protozoan parasite with a life cycle that alternates between replicative and non-replicative forms, but the components and mechanisms that regulate its cell cycle are poorly described. In higher eukaryotes, cyclins are proteins that activate cyclin-dependent kinases (CDKs), by associating with them along the different stages of the cell cycle. These cyclin-CDK complexes exert their role as major modulators of the cell cycle by phosphorylating specific substrates. For the correct progression of the cell cycle, the mechanisms that regulate the activity of cyclins and their associated CDKs are diverse and must be controlled precisely. Different types of cyclins are involved in specific phases of the eukaryotic cell cycle, preferentially activating certain CDKs. In this work, we characterized TcCYC6, a putative coding sequence of T. cruzi which encodes a protein with homology to mitotic cyclins. The overexpression of this sequence, fused to a tag of nine amino acids from influenza virus hemagglutinin (TcCYC6-HA), showed to be detrimental for the proliferation of epimastigotes in axenic culture and affected the cell cycle progression. In silico analysis revealed an N-terminal segment similar to the consensus sequence of the destruction box, a hallmark for the degradation of several mitotic cyclins. We experimentally determined that the TcCYC6-HA turnover decreased in the presence of proteasome inhibitors, suggesting that TcCYC6 degradation occurs via ubiquitin-proteasome pathway. The results obtained in this study provide first evidence that TcCYC6 expression and degradation are finely regulated in T. cruzi.

Colchicine treatment reversibly blocks cytokinesis but not mitosis in Trypanosoma cruzi epimastigotes.

This work analyzes the effect of the alkaloid colchicine on the growth of Trypanosoma cruzi epimastigotes, using immunofluorescence microscopy and flow cytometry techniques. We found that colchicine reversibly inhibited cytokinesis but not synthesis or segregation of nuclear and kinetoplastid DNA, in a concentration-dependent manner. We showed that, once colchicine was removed from the growth medium, cytokinesis was restored but abnormal segregation of kinetoplasts and nuclei generated zoids and parasites with two nuclei and one kinetoplast, among other aberrant cells. After drug removal, we also observed a few anucleated cells carrying two kinetoplasts in a stage compatible with the end of cytokinesis. The anomalous subcellular localization of the kinetoplast and flagellum observed in treated parasites suggests that the effect of colchicine and its interaction with T. cruzi microtubules is cell cycle dependent. The crosstalk between nuclear and kinetoplastid mitosis and its incidence on flagellum growth and parasite cell division regulation are discussed.

Functional characterization of TcCYC2 cyclin from Trypanosoma cruzi.

In eukaryotes, an oscillating network of protein kinase activities drives the order and timing of the cell cycle progression. Complexes formed by cyclins associated to cyclin-dependent kinases (CDKs) are the central components of this network. Cyclins act as the activating subunits and their abundance is regulated by different mechanisms in order to promote or prevent kinase activity. Protein synthesis, proteasomal degradation and/or differential subcellular compartmentalization modulate cyclin expression levels along the cell cycle. We describe in this work the characterization of Trypanosoma cruzi Cyclin 2 (TcCYC2), which contributes to a better understanding of the cell cycle regulation in this protozoan parasite. We found TcCYC2 exhibited cyclin function in a yeast complementation assay and over-expression of hemagglutinin tagged TcCYC2-HA rendered shorter duplication times and smaller cell sizes in the epimastigote form of the parasite. Analysis of synchronized cultures showed that over-expression of TcCYC2-HA altered the timing epimastigotes pass through G2/M boundary or cytokinesis. Taken together, our results showed that TcCYC2 is a functional cyclin whose over-expression modifies the dynamics of the cell cycle as well as the morphology of epimastigote forms of T. cruzi, suggesting it plays an important role in the cell cycle regulation machinery.

Characterization of StABF1, a stress-responsive bZIP transcription factor from Solanum tuberosum L. that is phosphorylated by StCDPK2 in vitro.

ABF/AREB bZIP transcription factors mediate plant abiotic stress responses by regulating the expression of stress-related genes. These proteins bind to the abscisic acid (ABA)-responsive element (ABRE), which is the major cis-acting regulatory sequence in ABA-dependent gene expression. In an effort to understand the molecular mechanisms of abiotic stress resistance in cultivated potato (Solanum tuberosum L.), we have cloned and characterized an ABF/AREB-like transcription factor from potato, named StABF1. The predicted protein shares 45-57% identity with A. thaliana ABFs proteins and 96% identity with the S. lycopersicum SlAREB1 and presents all of the distinctive features of ABF/AREB transcription factors. Furthermore, StABF1 is able to bind to the ABRE in vitro. StABF1 gene is induced in response to ABA, drought, salt stress and cold, suggesting that it might be a key regulator of ABA-dependent stress signaling pathways in cultivated potato. StABF1 is phosphorylated in response to ABA and salt stress in a calcium-dependent manner, and we have identified a potato CDPK isoform (StCDPK2) that phosphorylates StABF1 in vitro. Interestingly, StABF1 expression is increased during tuber development and by tuber-inducing conditions (high sucrose/nitrogen ratio) in leaves. We also found that StABF1 calcium-dependent phosphorylation is stimulated by tuber-inducing conditions and inhibited by gibberellic acid, which inhibits tuberization.

Characterization of StPPI1, a proton pump interactor from Solanum tuberosum L. that is up-regulated during tuber development and by abiotic stress.

Plasma membrane proton pumps (PM H(+)-ATPases) are involved in several physiological processes, such as growth and development, and abiotic stress responses. The major regulators of the PM H(+)-ATPases are proteins of the 14-3-3 family, which stimulate its activity. In addition, a novel interaction partner of the AHA1 PM H(+)-ATPase, named PPI1 (proton pump interactor, isoform 1), was identified in Arabidopsis thaliana. This protein stimulates the activity of the proton pump in vitro. In this work, we report the characterization of an A. thaliana PPI1 homolog in potato (Solanum tuberosum L.) named StPPI1. The full-length coding sequence of StPPI1 was obtained. The open reading frame (ORF) encodes a protein of 629 amino acids showing 50% identity with A. thaliana PPI1 protein. The StPPI1 ORF is divided into seven exons split by six introns. Southern blot analysis suggests that StPPI1 belongs to a family of related genes. Recombinant StPPI1 stimulates H(+)-ATPase activity in vitro. Basal levels of StPPI1 transcripts are observed in all tissues, however, StPPI1 expression is higher in proliferative regions (shoot apex and flower buds), flowers and leaves than in shoots and roots. StPPI1 mRNA levels significantly increase during tuber development. StPPI1 is induced by salt stress and cold. Drought and mechanical wounding slightly increase StPPI1 transcript levels. In addition, the expression of SlPPI1, the tomato homolog of StPPI1, was determined under adverse environmental conditions in tomato plants. SlPPI1 mRNA levels are increased by drought and cold, but are unaffected by salt stress. Mechanical wounding slightly increases SlPPI1 expression.

Protein phosphatases type 2A mediate tuberization signaling in Solanum tuberosum L. leaves.

Tuber formation in potato (Solanum tuberosum L.) is regulated by hormonal and environmental signals that are thought to be integrated in the leaves. The molecular mechanisms that mediate the responses to tuberization-related signals in leaves remain largely unknown. In this study we analyzed the roles of protein phosphatase type 2A catalytic subunits (PP2Ac) in the leaf responses to conditions that affect tuberization. The responses were monitored by analyzing the expression of the "tuber-specific" genes Patatin and Pin2, which are induced in tubers and leaves during tuber induction. Experiments using PP2A inhibitors, together with PP2Ac expression profiles under conditions that affect tuberization indicate that high sucrose/nitrogen ratio, which promotes tuber formation, increases the transcript levels of Patatin and Pin2, by increasing the activity of PP2As without affecting PP2Ac mRNA or protein levels. Gibberellic acid (GA), a negative regulator of tuberization, down-regulates the transcription of catalytic subunits of PP2As from the subfamily I and decreases their enzyme levels. In addition, GA inhibits the expression of Patatin and Pin2 possibly by a PP2A-independent mechanism. PP2Ac down-regulation by GA may inhibit tuberization signaling downstream of the inductive effects of high sucrose/nitrogen ratio. These results are consistent with the hypothesis that PP2As of the subfamily I may positively modulate the signaling pathways that lead to the transcriptional activation of "tuber-specific" genes in leaves, and act as molecular switches regulated by both positive and negative modulators of tuberization.

Serine/threonine protein phosphatases type 2A and their roles in stress signaling.

Serine/threonine protein phosphatases are ubiquitous enzymes in all eukaryotes but many of their physiological roles in plants remain unknown. The available results have demonstrated critical functions for these enzymes in the regulation of adaptive stress responses, and recent studies have directed attention to the functional roles of Ser/Thr phosphatases type 2A (PP2A) as components of stress signaling pathways. This review is focused primarily on plant PP2As and their participation in the control of biotic and abiotic stress responses.

Characterization of potato (Solanum tuberosum) and tomato (Solanum lycopersicum) protein phosphatases type 2A catalytic subunits and their involvement in stress responses.

Protein phosphorylation/dephosphorylation plays critical roles in stress responses in plants. This report presents a comparative characterization of the serine/threonine PP2A catalytic subunit family in Solanum tuberosum (potato) and S. lycopersicum (tomato), two important food crops of the Solanaceae family, based on the sequence analysis and expression profiles in response to environmental stress. Sequence homology analysis revealed six isoforms in potato and five in tomato clustered into two subfamilies (I and II). The data presented in this work show that the expression of different PP2Ac genes is regulated in response to environmental stresses in potato and tomato plants and suggest that, in general, mainly members of the subfamily I are involved in stress responses in both species. However, the differences found in the expression profiles between potato and tomato suggest divergent roles of PP2A in the plant defense mechanisms against stress in these closely related species.

Wounding increases salt tolerance in tomato plants: evidence on the participation of calmodulin-like activities in cross-tolerance signalling.

Cross-tolerance is the phenomenon by which a plant resistance to a stress results in resistance to another form of stress. It has previously been shown that salt stress causes the accumulation of proteinase inhibitors and the activation of other wound-related genes in tomato plants (Solanum lycopersicum). However, very little is known about how different stresses interact with one another, and which are the signalling components that interrelate the responses triggered by different stress types. In the present work, it is shown that mechanical wounding increases salt-stress tolerance in tomato plants through a mechanism that involves the signalling peptide systemin and the synthesis of JA. Data are also provided indicating that calmodulin-like activities are necessary for the downstream signalling events that lead to cross-tolerance between wounding and salt stress. Finally, evidence was gathered supporting the hypothesis that LeCDPK1, a Ca2+ -dependent protein kinase from tomato previously described in our laboratory, could participate in this cross-tolerance mechanism interrelating the signalling responses to wounding and salt stress.

Trypanosoma cruzi histone H1 is phosphorylated in a typical cyclin dependent kinase site accordingly to the cell cycle.

Histone H1 of most eukaryotes is phosphorylated during the cell cycle progression and seems to play a role in the regulation of chromatin structure, affecting replication and chromosome condensation. In trypanosomatids, histone H1 lacks the globular domain and is shorter when compared with the histone of other eukaryotes. We have previously shown that in Trypanosoma cruzi, the agent of Chagas' disease, histone H1 is phosphorylated and this increases its dissociation from chromatin. Here, we demonstrate using mass spectrometry analysis that T. cruzi histone H1 is only phosphorylated at the serine 12 in the sequence SPKK, a typical cyclin-dependent kinase site. We also found a correlation between the phosphorylation state of histone H1 and the cell cycle. Hydroxyurea and lactacystin, which, respectively, arrest parasites at the G1/S and G2/M stages of the cell cycle, increased the level of histone H1 phosphorylation. Cyclin-dependent kinase-related enzymes TzCRK3, and less intensely the TzCRK1 were able to phosphorylate histone H1 in vitro. Histone H1 dephosphorylation was prevented by treating the parasites with okadaic acid but not with calyculin A. These findings suggest that T. cruzi histone H1 phosphorylation is promoted by cyclin dependent kinases, present during S through G2 phase of the cell cycle, and its dephosphorylation is promoted by specific phosphatases.

Regulation of CDPK isoforms during tuber development.

CDPK activities present during tuber development were analysed. A high CDPK activity was detected in the soluble fraction of early stolons and a lower one was detected in soluble and particulate fractions of induced stolons. The early and late CDPK activities displayed diverse specificity for in vitro substrates and different subcellular distribution. Western blot analysis revealed two CDPKs of 55 and 60 kDa that follow a precise spatial and temporal profile of expression. The 55 kDa protein was only detected in early-elongating stolons and the 60 kDa one was induced upon stolon swelling, correlating with early and late CDPK activities. A new member of the potato CDPK family, StCDPK3, was identified from a stolon cDNA library. Gene specific RT-PCR demonstrated that this gene is only expressed in early stolons, while the previously identified StCDPK1 is expressed upon stolon swelling. This expression profile suggests that StCDPK3 could correspond to the 55 kDa isoform while StCDPK1 could encode the 60 kDa isoform present in swelling stolons. StCDPK1 has myristoylation and palmitoylation consensus possibly involved in its dual intracellular localization. Transient expression studies with wild-type and mutated forms of StCDPK1 fused to GFP were used to show that subcellular localization of this isoform is controlled by myristoylation and palmitoylation. Altogether, our data suggest that sequential activation of StCDPK3 and StCDPK1 and the subcellular localisation of StCDPK1 might be critical regulatory steps of calcium signalling during potato tuber development.

A calcium-dependent protein kinase is systemically induced upon wounding in tomato plants.

A full-length cDNA clone (LeCDPK1) from tomato (Lycopersicon esculentum) encoding a calcium-dependent protein kinase (CDPK) was isolated by screening a cDNA library from tomato cell cultures exposed to Cladosporium fulvum elicitor preparations. The predicted amino acid sequence of the cDNA reveals a high degree of similarity with other members of the CDPK family. LeCDPK1 has a putative N-terminal myristoylation sequence and presents a possible palmitoylation site. The in vitro translated protein conserves the biochemical properties of a member of the CDPK family. In addition, CDPK activity was detected in soluble and particulate extracts of tomato leaves. Basal levels of LeCDPK1 mRNA were detected by northern-blot analysis in roots, stems, leaves, and flowers of tomato plants. The expression of LeCDPK1 was rapidly and transiently enhanced in detached tomato leaves treated with pathogen elicitors and H2O2. Moreover, when tomato greenhouse plants were subjected to mechanical wounding, a transient increase of LeCDPK1 steady-state mRNA levels was detected locally at the site of the injury and systemically in distant non-wounded leaves. The increase observed in LeCDPK1 mRNA upon wounding correlates with an increase in the amount and in the activity of a soluble CDPK detected in extracts of tomato leaves, suggesting that this kinase is part of physiological plant defense mechanisms against biotic or abiotic attacks.