Nucleoside diphosphate kinases (NDPK) are key enzymes involved in the intracellular nucleotide maintenance in all living organisms, especially in trypanosomatids which are unable to synthesise purines de novo. Four putative NDPK isoforms were identified in the Trypanosoma cruzi Chagas, 1909 genome but only two of them were characterised so far. In this work, we studied a novel isoform from T. cruzi called TcNDPK3. This enzyme presents an atypical N-terminal extension similar to the DM10 domains. In T. cruzi, DM10 sequences targeted other NDPK isoform (TcNDPK2) to the cytoskeleton, but TcNDPK3 was localised in glycosomes despite lacking a typical peroxisomal targeting signal. In addition, TcNDPK3 was found only in the bloodstream trypomastigotes where glycolytic enzymes are very abundant. However, TcNDPK3 mRNA was also detected at lower levels in amastigotes suggesting regulation at protein and mRNA level. Finally, 33 TcNDPK3 gene orthologs were identified in the available kinetoplastid genomes. The characterisation of new glycosomal enzymes provides novel targets for drug development to use in therapies of trypanosomatid associated diseases.

• Klíčová slova
• Chagas disease, energy metabolism, glycosomes, trypanosomatids, trypomastigotes,
• MeSH
• Chagasova nemoc parazitologie   MeSH
• energetický metabolismus * MeSH
• fylogeneze MeSH
• izoenzymy MeSH
• mikrotělíska enzymologie   MeSH
• nukleosiddifosfátkinasa genetika   MeSH
• proteinové domény MeSH
• protozoální proteiny genetika   MeSH
• stadia vývoje MeSH
• Trypanosoma cruzi enzymologie   genetika   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• izoenzymy MeSH
• nukleosiddifosfátkinasa MeSH
• protozoální proteiny MeSH

Chagas disease (CD) is a neglected disease caused by Trypanosoma cruzi Chagas, 1909. Causative treatment can be achieved with two drugs: benznidazole or Nifurtimox. There are some gaps that hinder progress in eradicating the disease. There is no test that can efficiently assess cure control after treatment. Currently, the decline in anti-T. cruzi antibody titres is assessed with conventional serological tests, which can take years. However, the search for new markers of cure must continue to fill this gap. The present study aimed to evaluate the decline in serological titres using chimeric proteins after treatment with benznidazole in chronic patients diagnosed with CD. It was a prospective cross-sectional cohort study between 2000 and 2004 of T. cruzi-positive participants from the Añatuya region (Argentina) treated with benznidazole. Serum samples from ten patients were collected before treatment (day zero) and after the end of treatment (2, 3, 6, 12, 24 and 36 months). For the detection of anti-T. cruzi antibodies, an indirect ELISA was performed using two chimeric recombinant proteins (IBMP-8.1 and IBMP-8.4) as antigens. The changes in reactivity index within the groups before and after treatment were evaluated using the Friedman test. All participants experienced a decrease in serological titres after treatment with benznidazole, especially IBMP-8.1. However, due to the small number of samples and the short follow-up period, it is premature to conclude that this molecule serves as a criterion for sustained cure. Further studies are needed to validate tests based on these or other biomarkers to demonstrate parasitological cure.

• Klíčová slova
• Chagas disease, antibody titre, benznidazole, biomarker, seroconversion, serological test,
• MeSH
• Chagasova nemoc * farmakoterapie   MeSH
• lidé MeSH
• nitroimidazoly * MeSH
• prospektivní studie MeSH
• průřezové studie MeSH
• rekombinantní fúzní proteiny terapeutické užití   MeSH
• Trypanosoma cruzi * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• benzonidazole MeSH Prohlížeč
• nitroimidazoly * MeSH
• rekombinantní fúzní proteiny MeSH

The cytotoxicity of 18 new 1,2,6-thiadiazin-3,5-dione 1,1-dioxides was evaluated. This group of products was previously assayed against epimastigotes of Trypanosoma cruzi and some of them showed a high antiprotozoal activity. Thereafter 13 compounds with a high anti-epimastigote activity and low cytotoxicity were selected to be assayed against amastigotes. Some of the products showed the same or even lower cytotoxicity than nifurtimox and benznidazole, but most of them were very toxic for macrophages at 100 microg/ml. Only one of the compounds had an anti-amastigote activity similar to that of reference drugs at 10 microg/ml, but unfortunately this disappeared at lower concentrations.

• MeSH
• antiprotozoální látky farmakologie   MeSH
• Chagasova nemoc farmakoterapie   MeSH
• makrofágy účinky léků   parazitologie   MeSH
• myši MeSH
• parazitické testy citlivosti MeSH
• thiadiaziny farmakologie   MeSH
• Trypanosoma cruzi účinky léků   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antiprotozoální látky MeSH
• thiadiaziny MeSH

Trypanosomatids regulate gene expression mainly at the post-transcriptional level through processing, exporting and stabilising mRNA and control of translation. In most eukaryotes, protein synthesis is regulated by phosphorylation of eukaryotic initiation factor 2 (eIF2) at serine 51. Phosphorylation halts overall translation by decreasing availability of initiator tRNAmet to form translating ribosomes. In trypanosomatids, the N-terminus of eIF2α is extended with threonine 169 the homologous phosphorylated residue. Here, we evaluated whether eIF2α phosphorylation varies during the Trypanosoma cruzi life cycle, the etiological agent of Chagas' disease. Total levels of eIF2α are diminished in infective and non-replicative trypomastigotes compared with proliferative forms from the intestine of the insect vector or amastigotes from mammalian cells, consistent with decreased protein synthesis reported in infective forms. eIF2α phosphorylation increases in proliferative intracellular forms prior to differentiation into trypomastigotes. Parasites overexpressing eIF2αT169A or with an endogenous CRISPR/Cas9-generated eIF2αT169A mutation were created and analysis revealed alterations to the proteome, largely unrelated to the presence of μORF in epimastigotes. eIF2αT169A mutant parasites produced fewer trypomastigotes with lower infectivity than wild type, with increased levels of sialylated mucins and oligomannose glycoproteins, and decreased galactofuranose epitopes and the surface protease GP63 on the cell surface. We conclude that eIF2α expression and phosphorylation levels affect proteins relevant for intracellular progression of T. cruzi.

• Klíčová slova
• Trypanosoma cruzi, differentiation, eIF2, phosphorylation, translation, virulence,
• MeSH
• buněčné linie MeSH
• Chagasova nemoc parazitologie   MeSH
• CRISPR-Cas systémy MeSH
• eukaryotický iniciační faktor 2 genetika   metabolismus   MeSH
• fosforylace MeSH
• lidé MeSH
• mutace MeSH
• nádorové buněčné linie MeSH
• parazitemie MeSH
• proteom metabolismus   MeSH
• proteosyntéza MeSH
• protozoální proteiny analýza   biosyntéza   metabolismus   MeSH
• regulace genové exprese MeSH
• stadia vývoje MeSH
• Trypanosoma cruzi růst a vývoj   metabolismus   patogenita   MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• eukaryotický iniciační faktor 2 MeSH
• proteom MeSH
• protozoální proteiny MeSH

Trypanosoma cruzi is a unicellular protistan parasitic species that is comprised of strains and isolates exhibiting high levels of genetic and metabolic variability. In the insect vector, it is known to be highly responsive to starvation, a signal for progression to a life stage in which it can infect mammalian cells. Most mRNAs encoded in its mitochondrion require the targeted insertion and deletion of uridines to become translatable transcripts. This study defined differences in uridine-insertion/deletion RNA editing among three strains and established the mechanism whereby abundances of edited (and, thus, translatable) mitochondrial gene products increase during starvation. Our approach utilized our custom T-Aligner toolkit to describe transcriptome-wide editing events and reconstruct editing products from high-throughput sequencing data. We found that the relative abundance of mitochondrial transcripts and the proportion of mRNAs that are edited varies greatly between analyzed strains, a characteristic that could potentially impact metabolic capacity. Starvation typically led to an increase in overall editing activity rather than affecting a specific step in the process. We also determined that transcripts CR3, CR4, and ND3 produce multiple open reading frames that, if translated, would generate different proteins. Finally, we quantitated the inherent flexibility of editing in T. cruzi and found it to be higher relative to that in a related trypanosomatid lineage. Over time, new editing domains or patterns could prove advantageous to the organism and become more widespread within individual transcriptomes or among strains.

• Klíčová slova
• Chagas disease, RNA editing, electron transport chain, epimastigote, metabolism,
• MeSH
• editace RNA MeSH
• messenger RNA genetika   metabolismus   MeSH
• protozoální proteiny genetika   metabolismus   MeSH
• RNA mitochondriální genetika   metabolismus   MeSH
• RNA protozoální genetika   metabolismus   MeSH
• RNA metabolismus   MeSH
• savci genetika   MeSH
• transkriptom MeSH
• Trypanosoma brucei brucei * genetika   MeSH
• Trypanosoma cruzi * genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• messenger RNA MeSH
• protozoální proteiny MeSH
• RNA mitochondriální MeSH
• RNA protozoální MeSH
• RNA MeSH

BACKGROUND: Infection with Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, results in chronic infection that leads to cardiomyopathy with increased mortality and morbidity in endemic regions. In a companion study, our group found that a high-fat diet (HFD) protected mice from T. cruzi-induced myocardial damage and significantly reduced post-infection mortality during acute T. cruzi infection. METHODS: In the present study metabolic syndrome was induced prior to T. cruzi infection by feeding a high fat diet. Also, mice were treated with anti-diabetic drug metformin. RESULTS: In the present study, the lethality of T. cruzi (Brazil strain) infection in CD-1 mice was reduced from 55% to 20% by an 8-week pre-feeding of an HFD to induce obesity and metabolic syndrome. The addition of metformin reduced mortality to 3%. CONCLUSIONS: It is an interesting observation that both the high fat diet and the metformin, which are known to differentially attenuate host metabolism, effectively modified mortality in T. cruzi-infected mice. In humans, the metabolic syndrome, as presently construed, produces immune activation and metabolic alterations that promote complications of obesity and diseases of later life, such as myocardial infarction, stroke, diabetes, Alzheimer's disease and cancer. Using an evolutionary approach, we hypothesized that for millions of years, the channeling of host resources into immune defences starting early in life ameliorated the effects of infectious diseases, especially chronic infections, such as tuberculosis and Chagas disease. In economically developed countries in recent times, with control of the common devastating infections, epidemic obesity and lengthening of lifespan, the dwindling benefits of the immune activation in the first half of life have been overshadowed by the explosion of the syndrome's negative effects in later life.

• Klíčová slova
• Trypanosoma cruzi, high-fat diet, infectious disease, metabolic syndrome, metformin, mortality,
• MeSH
• adipozita účinky léků   MeSH
• analýza přežití MeSH
• bílá tuková tkáň účinky léků   imunologie   metabolismus   parazitologie   MeSH
• buněčné linie MeSH
• Chagasova nemoc krev   imunologie   metabolismus   parazitologie   MeSH
• cytokiny krev   metabolismus   MeSH
• energetický metabolismus účinky léků   MeSH
• hypoglykemika farmakologie   terapeutické užití   MeSH
• inbrední kmeny myší MeSH
• leptin krev   metabolismus   MeSH
• lidé MeSH
• metabolický syndrom farmakoterapie   etiologie   imunologie   parazitologie   MeSH
• metformin farmakologie   terapeutické užití   MeSH
• modely imunologické * MeSH
• náhodné rozdělení MeSH
• obezita krev   imunologie   metabolismus   patofyziologie   MeSH
• předkožka účinky léků   imunologie   metabolismus   parazitologie   MeSH
• srdeční komory účinky léků   imunologie   metabolismus   parazitologie   MeSH
• Trypanosoma cruzi účinky léků   imunologie   izolace a purifikace   patogenita   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• srovnávací studie MeSH
• Názvy látek
• cytokiny MeSH
• hypoglykemika MeSH
• leptin MeSH
• metformin MeSH

The TcK2 protein kinase of Trypanosoma cruzi, the causative agent of Chagas disease, is structurally similar to the human kinase PERK, which phosphorylates the initiation factor eIF2α and, in turn, inhibits translation initiation. We have previously shown that absence of TcK2 kinase impairs parasite proliferation within mammalian cells, positioning it as a potential target for treatment of Chagas disease. To better understand its role in the parasite, here we initially confirmed the importance of TcK2 in parasite proliferation by generating CRISPR/Cas9 TcK2-null cells, albeit they more efficiently differentiate into infective forms. Proteomics indicates that the TcK2 knockout of proliferative forms expresses proteins including trans-sialidases, normally restricted to infective and nonproliferative trypomastigotes explaining decreased proliferation and better differentiation. TcK2 knockout cells lost phosphorylation of eukaryotic initiation factor 3 and cyclic AMP responsive-like element, recognized to promote growth, likely explaining both decreased proliferation and augmented differentiation. To identify specific inhibitors, a library of 379 kinase inhibitors was screened by differential scanning fluorimetry using a recombinant TcK2 encompassing the kinase domain and selected molecules were tested for kinase inhibition. Only Dasatinib and PF-477736, inhibitors of Src/Abl and ChK1 kinases, showed inhibitory activity with IC50 of 0.2 ± 0.02 mM and 0.8 ± 0.1, respectively. In infected cells Dasatinib inhibited growth of parental amastigotes (IC50 = 0.6 ± 0.2 mM) but not TcK2 of depleted parasites (IC50 > 34 mM) identifying Dasatinib as a potential lead for development of therapeutics for Chagas disease targeting TcK2.

• Klíčová slova
• T.cruzi EIF2AK2, chagas disease, chemical inhibitor, eIF2α, invasion, protein kinase, proteome, recombinant protein,
• MeSH
• Chagasova nemoc * farmakoterapie   parazitologie   MeSH
• dasatinib MeSH
• kinasa eIF-2 genetika   metabolismus   MeSH
• lidé MeSH
• paraziti * MeSH
• proliferace buněk MeSH
• savci metabolismus   MeSH
• Trypanosoma cruzi * genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dasatinib MeSH
• kinasa eIF-2 MeSH

Nowadays, there are no simple techniques for mimicking in vitro the life cycle of the kinetoplasmtid Trypanosoma cruzi Chagas, 1909, causative agent of Chagas disease, especially for parasite strains maintained as epimastigotes for many years. In the present study, we propose a method for obtaining metacyclic trypomastigotes, which were capable of infecting mammalian cells by simply lowering pH media. The collected amastigotes and trypomastigotes were differentiated into epimastigotes closing T. cruzi life cycle in vitro. Metacyclogenesis rates and infectivity were enhanced in cycled parasites. Finally, using this method, we were able to infect cells with transgenic parasites obtaining trypomastigotes and amastigotes using a neomycin-resistant cell line.

• MeSH
• Cercopithecus aethiops MeSH
• geneticky modifikované organismy MeSH
• stadia vývoje MeSH
• Trypanosoma cruzi genetika   fyziologie   MeSH
• Vero buňky MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Horizontal gene transfer involving kinetoplast DNA minicircles between Trypanosoma cruzi and its mammalian hosts has recently been proposed as a usual consequence of infection (Hecht et al., 2010). However, we have found no sequences longer than 29 bp perfectly matching minicircles of T. cruzi in the unassembled reads from Colombian and Peruvian human populations provided by the 1,000 Genome project (129 individuals in total, coverage from 1.4× to 36.3×, read length from 42 to 101 bp). The weak sequence matches that were identified are shared with a Finnish population used as a control from a non-endemic area.

• MeSH
• genom lidský * MeSH
• kinetoplastová DNA genetika   MeSH
• lidé MeSH
• rekombinace genetická * MeSH
• sekvenční analýza DNA MeSH
• sekvenční homologie nukleových kyselin MeSH
• Trypanosoma cruzi genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Kolumbie MeSH
• Peru MeSH
• Názvy látek
• kinetoplastová DNA MeSH

Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from Trypanosoma cruzi (LAPTc) in particular is a promising target for Chagas disease. To exploit LAPTc as a potential target, it is essential to develop potent and selective inhibitors. To achieve this, we report a high-throughput screening method for LAPTc. Two methods were developed and optimized: a Leu-7-amido-4-methylcoumarin-based fluorogenic assay and a RapidFire mass spectrometry (RapidFire MS)-based assay using the LSTVIVR peptide as substrate. Compared with a fluorescence assay, the major advantages of the RapidFire MS assay are a greater signal-to-noise ratio as well as decreased consumption of enzyme. RapidFire MS was validated with the broad-spectrum LAP inhibitors bestatin (IC50 = 0.35 μM) and arphamenine A (IC50 = 15.75 μM). We suggest that RapidFire MS is highly suitable for screening for specific LAPTc inhibitors.

• Klíčová slova
• M17-leucyl aminopeptidase, RapidFire mass spectrometry, arphamenine, bestatin, high-throughput screening,
• MeSH
• Chagasova nemoc diagnóza   enzymologie   parazitologie   MeSH
• hmotnostní spektrometrie MeSH
• kinetika MeSH
• leucylaminopeptidasa genetika   izolace a purifikace   MeSH
• lidé MeSH
• rychlé screeningové testy * MeSH
• sekvence aminokyselin genetika   MeSH
• substrátová specifita MeSH
• Trypanosoma cruzi enzymologie   izolace a purifikace   patogenita   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• leucylaminopeptidasa MeSH