Regulation of a high-affinity diamine transport system in Trypanosoma cruzi epimastigotes.
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ABSTRACT:
SUBMITTER: Le Quesne SA
PROVIDER: S-EPMC1217375 | biostudies-other | 1996 Jun
REPOSITORIES: biostudies-other
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Project description:Whereas mammalian cells and most other organisms can synthesize polyamines from basic amino acids, the protozoan parasite Trypanosoma cruzi is incapable of polyamine biosynthesis de novo and therefore obligatorily relies upon putrescine acquisition from the host to meet its nutritional requirements. The cell surface proteins that mediate polyamine transport into T. cruzi, as well as most eukaryotes, however, have by-in-large eluded discovery at the molecular level. Here we report the identification and functional characterization of two polyamine transporters, TcPOT1.1 and TcPOT1.2, encoded by alleles from two T. cruzi haplotypes. Overexpression of the TcPOT1.1 and TcPOT1.2 genes in T. cruzi epimastigotes revealed that TcPOT1.1 and TcPOT1.2 were high-affinity transporters that recognized both putrescine and cadaverine but not spermidine or spermine. Furthermore, the activities and subcellular locations of both TcPOT1.1 and TcPOT1.2 in intact parasites were profoundly influenced by extracellular putrescine availability. These results establish TcPOT1.1 and TcPOT1.2 as key components of the T. cruzi polyamine transport pathway, an indispensable nutritional function for the parasite that may be amenable to therapeutic manipulation.
| S-EPMC3183094 | biostudies-literature
Project description:The properties and regulation of the pstSCAB-encoded Pi uptake system from the alfalfa symbiont Sinorhizobium meliloti are reported. We present evidence that the pstSCAB genes and the regulatory phoUB genes are transcribed from a single promoter that contains two PhoB binding sites and that transcription requires PhoB. S. meliloti strain 1021 (Rm1021) and its derivatives were found to carry a C deletion frameshift mutation in the pstC gene (designated pstC1021) that severely impairs activity of the PstSCAB Pi transport system. This mutation is absent in RCR2011, the parent of Rm1021. Correction of the pstC1021 mutation in Rm1021 by site-directed mutagenesis revealed that PstSCAB is a Pi-specific, high-affinity (Km, 0.2 microM), high-velocity (Vmax, 70 nmol/min/mg protein) transport system. The pstC1021 allele was shown to generate a partial pho regulon constitutive phenotype, in which transcription is activated by PhoB even under Pi-excess conditions that render PhoB inactive in a wild-type background. The previously reported symbiotic Fix- phenotype of phoCDET mutants was found to be dependent on the pstC1021 mutation, as Rm1021 phoCDET mutants formed small white nodules on alfalfa that failed to reduce N2, whereas phoCDET mutant strains with a corrected pstC allele (RmP110) formed pink nodules on alfalfa that fixed N2 like the wild type. Alfalfa root nodules formed by the wild-type RCR2011 strain expressed the low-affinity orfA-pit-encoded Pi uptake system and neither the pstSCAB genes nor the phoCDET genes. Thus, metabolism of alfalfa nodule bacteroids is not Pi limited.
| S-EPMC1347321 | biostudies-literature
Project description:BackgroundThe three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.ResultsWe constructed a platform with a flexible structure allowing the exchange of various elements, such as promoters, fusion tags, intergenic regions or resistance markers. This platform is based on Gateway® technology, to ensure a fast and efficient cloning system. We obtained plasmid vectors carrying genes for fluorescent proteins (green, cyan or yellow), and sequences for the c-myc epitope, and tandem affinity purification or polyhistidine tags. The vectors were verified by successful subcellular localization of two previously characterized proteins (TcRab7 and PAR 2) and a putative centrin. For the tandem affinity purification tag, the purification of two protein complexes (ribosome and proteasome) was performed.ConclusionsWe constructed plasmids with an efficient cloning system and suitable for use across various applications, such as protein localization and co-localization, protein partner identification and protein expression. This platform also allows vector customization, as the vectors were constructed to enable easy exchange of its elements. The development of this high-throughput platform is a step closer towards large-scale trypanosome applications and initiatives.
| S-EPMC3020659 | biostudies-literature
Project description:Potassium is the major intracellular cation in S. cerevisiae, which can be concentrated up to 200-300 mM even from relatively low potassium (< 1 mM) environments. This is achieved by mean of a high affinity K+-transport system encoded by the genes TRK1 and TRK2. Recently, our group became interested in the effects of sudden shortage of extracellular potassium. Transcriptomic analysis indicates that lack of potassium drastically alters sulfur metabolism (mainly Met and Cys metabolism), triggers an oxidative stress response and activates the mitochondrial retrograde pathway. We also observe a dramatic halt in the expression of genes required for ribosome biogenesis and translation, as well as decrease in expression of diverse genes (cyclins, protein kinases) required for progression through the cell cycle. Only subsets of these changes were observed in a strain deleted for the TRK1 and TRK2 genes growing in the presence of sufficient potassium (50 mM). Research involving molecular genetics and metabolomic approaches aiming to clarify the primary targets for potassium requirements is currently ongoing. We compare the expression profile of two yeast strains: WT and trk1 trk2 double mutant, growing in a Translucent K-free medium containing 50 mM KCl until OD600 = 0.6. Two independent experiments were performed, and for each experiment a dye-swap was carried out. Total number of chips analyzed: 4.
2012-10-13 | E-GEOD-24711 | biostudies-arrayexpress
Project description:Trypanosoma cruzi is incapable of synthesizing putrescine or cadaverine de novo, and, therefore, salvage of polyamines from the host milieu is an obligatory nutritional function for the parasite. A high-affinity diamine transporter (TcPOT1) from T. cruzi has been identified previously that recognizes both putrescine and cadaverine as ligands. In order to assess the functional role of TcPOT1 in intact parasites, a ?tcpot1 null mutant was constructed by targeted gene replacement and characterized. The ?tcpot1 mutant lacked high-affinity putrescine-cadaverine transport capability but retained the capacity to transport diamines via a non-saturable, low-affinity mechanism. Transport of spermidine and arginine was not impacted by the ?tcpot1 lesion. The ?tcpot1 cell line exhibited a significant but not total defect in its ability to subsist in Vero cells, although initial infection rates were not affected by the lesion. These findings reveal that TcPOT1 is the sole high-affinity diamine permease in T. cruzi, that genetic obliteration of TcPOT1 impairs the ability of the parasite to maintain a robust infection in mammalian cells, and that a secondary low-affinity uptake mechanism for this key parasite nutrient is operative but insufficient for optimal infection.
| S-EPMC4822861 | biostudies-literature
Project description:Potassium is the major intracellular cation in S. cerevisiae, which can be concentrated up to 200-300 mM even from relatively low potassium (< 1 mM) environments. This is achieved by mean of a high affinity K+-transport system encoded by the genes TRK1 and TRK2. Recently, our group became interested in the effects of sudden shortage of extracellular potassium. Transcriptomic analysis indicates that lack of potassium drastically alters sulfur metabolism (mainly Met and Cys metabolism), triggers an oxidative stress response and activates the mitochondrial retrograde pathway. We also observe a dramatic halt in the expression of genes required for ribosome biogenesis and translation, as well as decrease in expression of diverse genes (cyclins, protein kinases) required for progression through the cell cycle. Only subsets of these changes were observed in a strain deleted for the TRK1 and TRK2 genes growing in the presence of sufficient potassium (50 mM). Research involving molecular genetics and metabolomic approaches aiming to clarify the primary targets for potassium requirements is currently ongoing.
2012-10-13 | GSE24711 | GEO
Project description:Resistance to benznidazole in certain strains of Trypanosoma cruzi may be caused by the increased production of enzymes that act on the oxidative metabolism, such as mitochondrial tryparedoxin peroxidase which catalyses the reduction of peroxides. This work presents cytotoxicity assays performed with ferrocenyl diamine hydrochlorides in six different strains of T. cruzi epimastigote forms (Y, Bolivia, SI1, SI8, QMII, and SIGR3). The last four strains have been recently isolated from triatominae and mammalian host (domestic cat). The expression of mitochondrial tryparedoxin peroxidase was analyzed by the Western blotting technique using polyclonal antibody anti mitochondrial tryparedoxin peroxidase obtained from a rabbit immunized with the mitochondrial tryparedoxin peroxidase recombinant protein. All the tested ferrocenyl diamine hydrochlorides were more cytotoxic than benznidazole. The expression of the 25.5kDa polypeptide of mitochondrial tryparedoxin peroxidase did not increase in strains that were more resistant to the ferrocenyl compounds (SI8 and SIGR3). In addition, a 58kDa polypeptide was also recognized in all strains. Ferrocenyl diamine hydrochlorides showed trypanocidal activity and the expression of 25.5kDa mitochondrial tryparedoxin peroxidase is not necessarily increased in some T. cruzi strains. Most likely, other mechanisms, in addition to the over expression of this antioxidative enzyme, should be involved in the escape of parasites from cytotoxic oxidant agents.
| S-EPMC9427605 | biostudies-literature
Project description:Trypanosoma cruzi is the etiologic agent for Chagas disease, which affects 6-7 million people worldwide. The biological diversity of the parasite reflects on inefficiency of benznidazole, which is a first choice chemotherapy, on chronic patients. ABC transporters that extrude xenobiotics, metabolites, and mediators are overexpressed in resistant cells and contribute to chemotherapy failure. An ABCC-like transport was identified in the Y strain and extrudes thiol-conjugated compounds. As thiols represent a line of defense towards reactive species, we aimed to verify whether ABCC-like transport could participate in the regulation of responses to stressor stimuli. In order to achieve this, ABCC-like activity was measured by flow cytometry using fluorescent substrates. The present study reveals the participation of glutathione and ceramides on ABCC-like transport, which are both implicated in stress. Hemin modulated the ABCC-like efflux which suggests that this protein might be involved in cellular detoxification. Additionally, all strains evaluated exhibited ABCC-like activity, while no ABCB1-like activity was detected. Results suggest that ABCC-like efflux is not associated with natural resistance to benznidazole, since sensitive strains showed higher activity than the resistant ones. Although benznidazole is not a direct substrate, ABCC-like efflux increased after prolonged drug exposure and this indicates that the ABCC-like efflux mediated protection against cell stress depends on the glutathione biosynthesis pathway.
| S-EPMC8227891 | biostudies-literature
Project description:Trypanosoma cruzi, a flagellated protozoan, is the causative agent of Chagas’ disease, a chronic and potentially fatal disease that causes irreversible damage to heart and digestive tract in humans. Like all trypanosomes, the protein coding genes of T. cruzi are arranged into large polycistronic gene clusters transcribed by polymerase II (Pol II). Therefore, trypanosomes presumably rely on post-transcriptional process to regulate gene expression. Pol II promoters have not been identified and there is no evidence for regulated gene expression at the transcriptional level. However, the presence of the hyper-modified DNA base J, Beta-D-glucosyl hydroxymethyluracil, at regions flanking the polycistronic units (PTU) in T. brucei suggested its involvement in regulating Pol II transcription. We now demonstrate that base J is localized at PTU flanking regions in T. cruzi and levels are differentially regulated by the thymidine hydroxylases, JBP1 and JBP2, involved in J biosynthesis. Microarray analysis of the JBP1dKO and JBP2dKO indicate the up and down regulation of several hundred genes distributed throughout the genome. We show a large increase in Pol II transcription rate following the decrease in base J at the PTU flanks. Changes in gene expression include virulence genes and parasites are defective in host cell invasion and egress. There is a direct correlation between the reduction in base J levels, number of genes affected and strength of the virulence phenotype. These studies indicate that base J represents an epigenetic factor regulating Pol II transcription initiation in kinetoplastids and provides a biological role of the only hyper-modified DNA base in eukaryotes.
2011-01-01 | GSE23855 | GEO
Project description:Autophagy is a cellular process required for the removal of aged organelles and cytosolic components through lysosomal degradation. All types of eukaryotic cells from yeasts to mammalian cells have the machinery to activate autophagy as a result of many physiological and pathological situations. The most frequent stimulus of autophagy is starvation and the result, in this case, is the fast generation of utilizable food (e.g. amino acids and basic nutrients) to maintain the vital biological processes. In some organisms, starvation also triggers other associated processes such as differentiation. The protozoan parasite Trypanosoma cruzi undergoes a series of differentiation processes throughout its complex life cycle. Although not all autophagic genes have been identified in the T. cruzi genome, previous works have demonstrated the presence of essential autophagic-related proteins. Under starvation conditions, TcAtg8, which is the parasite homolog of Atg8/LC3 in other organisms, is located in autophagosome-like vesicles. In this work, we have characterized the autophagic pathway during T. cruzi differentiation from the epimastigote to metacyclic trypomastigote form, a process called metacyclogenesis. We demonstrated that autophagy is stimulated during metacyclogenesis and that the induction of autophagy promotes this process. Moreover, with exception of bafilomycin, other classical autophagy modulators have similar effects on T. cruzi autophagy. We also showed that spermidine and related polyamines can positively regulate parasite autophagy and differentiation. We concluded that both polyamine metabolism and autophagy are key processes during T. cruzi metacyclogenesis that could be exploited as drug targets to avoid the parasite cycle progression.
| S-EPMC5683653 | biostudies-literature