Location and expression kinetics of Tc24 in different life stages of Trypanosoma cruzi.
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ABSTRACT:
SUBMITTER: Versteeg L
PROVIDER: S-EPMC8415617 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Trypanosoma cruzi causes life-long disease after infection and leads to cardiac disease in 30% of infected individuals. After infection, the parasites are readily detectable in the blood during the first few days before disseminating to infect numerous cell types. Preliminary data suggested that the Tc24 protein that localizes to the T. cruzi membrane during all life stages possesses B-cell superantigenic properties. These antigens facilitate immune escape by interfering with antibody-mediated responses, particularly the avoidance of catalytic antibodies. These antibodies are an innate host defense mechanism present in the naive repertoire, and catalytic antibody-antigen binding results in hydrolysis of the target. We tested the B-cell superantigenic properties of Tc24 by comparing the degree of Tc24 hydrolysis by IgM purified from either Tc24 unexposed or exposed mice and humans. Respective samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, silver stained, and the degree of hydrolysis was measured. Data presented in this report suggest that the T. cruzi Tc24 is a B-cell superantigen based on the observations that 1) Tc24 was hydrolyzed by IgM present in serum of unexposed mice and humans and 2) exposure to Tc24 eliminated catalytic activity as early as 4 days after T. cruzi infection.
| S-EPMC4710414 | biostudies-literature
Project description:BACKGROUND: The kinetoplast DNA (kDNA) of trypanosomatids consists of an unusual arrangement of circular molecules catenated into a single network. The diameter of the isolated kDNA network is similar to that of the entire cell. However, within the kinetoplast matrix, the kDNA is highly condensed. Studies in Crithidia fasciculata showed that kinetoplast-associated proteins (KAPs) are capable of condensing the kDNA network. However, little is known about the KAPs of Trypanosoma cruzi, a parasitic protozoon that shows distinct patterns of kDNA condensation during their complex morphogenetic development. In epimastigotes and amastigotes (replicating forms) the kDNA fibers are tightly packed into a disk-shaped kinetoplast, whereas trypomastigotes (non-replicating) present a more relaxed kDNA organization contained within a rounded structure. It is still unclear how the compact kinetoplast disk of epimastigotes is converted into a globular structure in the infective trypomastigotes. RESULTS: In this work, we have analyzed KAP coding genes in trypanosomatid genomes and cloned and expressed two kinetoplast-associated proteins in T. cruzi: TcKAP4 and TcKAP6. Such small basic proteins are expressed in all developmental stages of the parasite, although present a differential distribution within the kinetoplasts of epimastigote, amastigote and trypomastigote forms. CONCLUSION: Several features of TcKAPs, such as their small size, basic nature and similarity with KAPs of C. fasciculata, are consistent with a role in DNA charge neutralization and condensation. Additionally, the differential distribution of KAPs in the kinetoplasts of distinct developmental stages of the parasite, indicate that the kDNA rearrangement that takes place during the T. cruzi differentiation process is accompanied by TcKAPs redistribution.
| S-EPMC2700280 | biostudies-literature
Project description:IntroductionChagas disease, caused by the protozoan parasite Trypanosoma cruzi, is a major public health problem in the Americas, and existing drugs have severe limitations. In this context, a vaccine would be an attractive alternative for disease control. One of the difficulties in developing an effective vaccine lies in the high genetic diversity of T. cruzi. In this study, we evaluated the level of sequence diversity of the leading vaccine candidate Tc24 in multiple parasite strains.Methods and resultsWe quantified its level of polymorphism within and between T. cruzi discrete typing units (DTUs) and how this potential polymorphism is structured by different selective pressures. We observed a low level of polymorphism of Tc24 protein, weakly associated with parasite DTUs, but not with the geographic origin of the strains. In particular, Tc24 was under strong purifying selection pressure and predicted CD8+ T-cell epitopes were mostly conserved. Tc24 strong conservation may be associated with structural/functional constrains to preserve EF hand domains and their calcium-binding loops, and Tc24 is likely important for the parasite fitness.DiscussionTogether, these results show that a vaccine based on Tc24 is likely to be effective against a wide diversity of parasite strains across the American continent, and further development of this vaccine candidate should be a high priority.
| S-EPMC7691455 | biostudies-literature
Project description:Chronic chagasic cardiomyopathy is a debilitating and frequently fatal outcome of human infection with the protozoan parasite, Trypanosoma cruzi. Microarray analysis of gene expression during the T. cruzi life-cycle could be a valuable means of identifying drug and vaccine targets based on their appropriate expression patterns, but results from previous microarray studies in T. cruzi and related kinetoplastid parasites have suggested that the transcript abundances of most genes in these organisms do not vary significantly between life-cycle stages.In this study, we used whole genome, oligonucleotide microarrays to globally determine the extent to which T. cruzi regulates mRNA relative abundances over the course of its complete life-cycle. In contrast to previous microarray studies in kinetoplastids, we observed that relative transcript abundances for over 50% of the genes detected on the T. cruzi microarrays were significantly regulated during the T. cruzi life-cycle. The significant regulation of 25 of these genes was confirmed by quantitative reverse-transcriptase PCR (qRT-PCR). The T. cruzi transcriptome also mirrored published protein expression data for several functional groups. Among the differentially regulated genes were members of paralog clusters, nearly 10% of which showed divergent expression patterns between cluster members.Taken together, these data support the conclusion that transcript abundance is an important level of gene expression regulation in T. cruzi. Thus, microarray analysis is a valuable screening tool for identifying stage-regulated T. cruzi genes and metabolic pathways.
| S-EPMC2907688 | biostudies-literature
Project description:To generate a high quality, annotated gene expression database of T. cruzi trypomastigotes(TRP), amastigotes (AMA), epimastigotes (EPI), and metacyclic trypomastigotes (MET). The global assessment of transcript abundances in each life-cycle stage of T. cruzi will identify stage-regulated genes and provide an essential element of an integrated database of T. cruzi genomic, proteomic, and transcriptomic data. RNAs from 3 biological replicates from a single time point in each stage will be subjected to DNA microarrays containing probes for the complete, annotated T. cruzi genome, as it is currently known. The arrays for this project will be provided by the Pathogen Functional Genomics Resource Center (PFGRC) at The Institute for Genomic Research (TIGR) sponsored by the National Institute of Allergy and Infectious Diseases (NIAID), and will contain long oligonucleotides complementary to every annotated gene in the newly sequenced T. cruzi genome. The resulting data and analysis results will be deposited in TcruziDB (http://TcruziDB.org). Keywords: Gene expression comparisons between the four life-cycle stages of T. cruzi
2009-08-01 | GSE14641 | GEO
Project description:Pep5 (WELVVLGKL) is a fragment of cyclin D2 that exhibits a 2-fold increase in the S phase of the HeLa cell cycle. When covalently bound to a cell-penetrating peptide (Pep5-cpp), the nonapeptide induces cell death in several tumor cells, including breast cancer and melanoma cells. Additionally, Pep5-cpp reduces the in vivo tumor volume of rat glioblastoma. Chagas disease, which is caused by the flagellated parasite Trypanosoma cruzi, is a neglected disease that occurs mainly in the Americas, where it is considered an important public health issue. Given that there are only two options for treating the disease, it is exceptionally crucial to search for new molecules with potential pharmacological action against the parasites. In this study, we demonstrate that Pep5-cpp induces cell death in epimastigote, trypomastigote, and amastigote forms of T. cruzi The Pep5-cpp peptide was also able to decrease the percentage of infected cells without causing any detectable toxic effects in mammalian host cells. The infective, i.e., trypomastigote form of T. cruzi pretreated with Pep5-cpp was unable to infect LLC-MK2 monkey kidney cells. Also, Pep5-binding proteins were identified by mass spectrometry, including calmodulin-ubiquitin-associated protein, which is related to the virulence and parasitemia of T. cruzi Taken together, these data suggest that Pep5 can be used as a novel alternative for the treatment of Chagas disease.
| S-EPMC6496079 | biostudies-literature
Project description:BackgroundTrypanosoma cruzi, a flagellate protozoan, is the etiological agent of Chagas disease, a chronic illness that causes irreversible damage to heart and digestive tract in humans. Previous 2-DE analyses of T. cruzi proteome have not focused on basic proteins, possibly because of inherent difficulties for optimizing 2-DE in the alkaline pH range. However, T. cruzi wide pH range 2-DE gels have shown few visible spots in the alkaline region, indicating that the parasite either did not have an appreciable amount of alkaline proteins or that these proteins were underrepresented in the 2-DE gels.ResultsDifferent IEF conditions using 6-11 pH gradient strips were tested for separation of T. cruzi alkaline proteins. The optimized methodology described here was performed using anodic "paper bridge" sample loading supplemented by increased concentration of DTT and Triton X-100 on Multiphor II (GE Healthcare) equipment and an electrode pad embedded in DTT- containing solution near the cathode in order to avoid depletion of reducing agent during IEF. Landmark proteins were identified by peptide mass fingerprinting allowing the production of an epimastigote 2-DE map. Most identified proteins corresponded to metabolic enzymes, especially those related to amino acid metabolism. The optimized 2-DE protocol was applied in combination with the "two-in-one gel" method to verify the relative expression of the identified proteins between samples from epimastigote and trypomastigote life stages.ConclusionHigh resolution 2-DE gels of T. cruzi life forms were achieved using the optimized methodology and a partial epimastigote alkaline 2-DE map was built. Among 700 protein spots detected, 422 were alkaline with a pI above 7.0. The "two-in-one gel" method simplified the comparative analysis between T. cruzi life stages since it minimized variations in spot migration and silver-stained spot volumes. The comparative data were in agreement with biological traits of T. cruzi life forms and also corroborated previous T. cruzi proteomic studies. For instance, enzymes related to amino acid metabolism and dehydrogenases were more abundant in epimastigote 2-DE gel whilst trans-sialidase and a paraflagellar protein were found specifically in the trypomastigote 2-DE profile.
| S-EPMC2553069 | biostudies-literature
Project description:Chagas disease is caused by the protozoan Trypanosoma cruzi, affecting around 8 million people worldwide. After host cell invasion, the infective trypomastigote form remains 2-4 hours inside acidic phagolysosomes to differentiate into replicative amastigote form. In vitro acidic-pH-induced axenic amastigogenesis was used here to study this step of the parasite life cycle. After three hours of trypomastigote incubation in amastigogenesis promoting acidic medium (pH 5.0) or control physiological pH (7.4) medium samples were subjected to three rounds of centrifugation followed by ultrafiltration of the supernatants. The resulting exoproteome samples were trypsin digested and analysed by nano flow liquid chromatography coupled to tandem mass spectrometry. Computational protein identification searches yielded 271 and 483 protein groups in the exoproteome at pH 7.4 and pH 5.0, respectively, with 180 common proteins between both conditions. The total amount and diversity of proteins released by parasites almost doubled upon acidic incubation compared to control. Overall, 76.5% of proteins were predicted to be secreted by classical or non-classical pathways and 35.1% of these proteins have predicted transmembrane domains. Classical secretory pathway analysis showed an increased number of mucins and mucin-associated surface proteins after acidic incubation. However, the number of released trans-sialidases and surface GP63 peptidases was higher at pH 7.4. Trans-sialidases and mucins are anchored to the membrane and exhibit an enzyme-substrate relationship. In general, mucins are glycoproteins with immunomodulatory functions in Chagas disease, present mainly in the epimastigote and trypomastigote surfaces and could be enzymatically cleaved and released in the phagolysosome during amastigogenesis. Moreover, evidence for flagella discard during amastigogenesis are addressed. This study provides the first comparative analysis of the exoproteome during amastigogenesis, and the presented data evidence the dynamism of its profile in response to acidic pH-induced differentiation.
| S-EPMC6874342 | biostudies-literature
Project description:The effect of a parasite on the life history of its vector is important for understanding and predicting disease transmission. Chagas disease agent Trypanosoma cruzi is a generalist parasite that is diverse across scales from its genetic diversity to the 100s of mammal and vector species it infects. Its vertebrate hosts show quite variable responses to infection, however, to date there are no studies looking at how T. cruzi variability might result in variable outcomes in its invertebrate host. Therefore, we investigated the effect of different T. cruzi I strains on Rhodnius prolixus survival and development. We found significant variation between insects infected with different strains, with some strains having no effect, as compared with uninfected insects, and others with significantly lower survival and development. We also found that different variables had varying importance between strains, with the effect of time postinfection and the blood:weight ratio of the infective meal significantly affecting the survival of insects infected with some strains, but not others. Our results suggest that T. cruzi can be pathogenic not only to its vertebrate hosts but also to its invertebrate hosts.
| S-EPMC4559699 | biostudies-literature
Project description:To generate a high quality, annotated gene expression database of T. cruzi trypomastigotes(TRP), amastigotes (AMA), epimastigotes (EPI), and metacyclic trypomastigotes (MET). The global assessment of transcript abundances in each life-cycle stage of T. cruzi will identify stage-regulated genes and provide an essential element of an integrated database of T. cruzi genomic, proteomic, and transcriptomic data. RNAs from 3 biological replicates from a single time point in each stage will be subjected to DNA microarrays containing probes for the complete, annotated T. cruzi genome, as it is currently known. The arrays for this project will be provided by the Pathogen Functional Genomics Resource Center (PFGRC) at The Institute for Genomic Research (TIGR) sponsored by the National Institute of Allergy and Infectious Diseases (NIAID), and will contain long oligonucleotides complementary to every annotated gene in the newly sequenced T. cruzi genome. The resulting data and analysis results will be deposited in TcruziDB (http://TcruziDB.org). Keywords: Gene expression comparisons between the four life-cycle stages of T. cruzi Six hybridizations were performed for each life-cycle stage. The hybridizations consisted of three dye-swap experiments from three independent samples (biological replicates). In each case, the experimental sample was from a single life-cycle stage and the control sample was an equal mixture of all four life-cycle stages. A total of 24 hybridizations were preformed.
2010-05-15 | E-GEOD-14641 | biostudies-arrayexpress