A successful strategy for the recovering of active P21, an insoluble recombinant protein of Trypanosoma cruzi.
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ABSTRACT:
SUBMITTER: dos Santos MA
PROVIDER: S-EPMC3941101 | biostudies-other | 2014
REPOSITORIES: biostudies-other
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Project description:BackgroundP21 is a secreted protein expressed in all developmental stages of Trypanosoma cruzi. The aim of this study was to determine the effect of the recombinant protein based on P21 (P21-His(6)) on inflammatory macrophages during phagocytosis.FindingsOur results showed that P21-His(6) acts as a phagocytosis inducer by binding to CXCR4 chemokine receptor and activating actin polymerization in a way dependent onthe PI3-kinase signaling pathway.ConclusionsThus, our results shed light on the notion that native P21 is a component related to T. cruzi evasion from the immune response and that CXCR4 may be involved in phagocytosis. P21-His(6) represents an important experimental control tool to study phagocytosis signaling pathways of different intracellular parasites and particles.
| S-EPMC3519637 | biostudies-literature
Project description:Trypanosoma cruzi P21 protein (P21) is a putative secreted and immunomodulatory molecule with potent bioactive properties such as induction of phagocytosis and actin cytoskeleton polymerization. Despite the bioactive properties described so far, the action of P21 on parasite replication in muscle cell lineage or T. cruzi parasitism during acute experimental infection is unclear. We observed that recombinant P21 (rP21) decreased the multiplication of T. cruzi in C2C12 myoblasts, phenomenon associated with greater actin polymerization and IFN-γ and IL-4 higher expression. During experimental infection, lower cardiac nests, inflammatory infiltrate and fibrosis were observed in mice infected and treated with rP21. These results were correlated with large expression of IFN-γ counterbalanced by high levels of IL-10, which was consistent with the lower cardiac tissue injury found in these mice. We have also observed that upon stress, such as that induced by the presence of the IFN-γ cytokine, T. cruzi produced more P21. The effect of P21 in controlling the replication of T. cruzi, may indicate an evolutionary mechanism of survival developed by the parasite. Thus, when subjected to different stress conditions, the protozoan produces more P21, which induces T. cruzi latency in the host organism, enabling the protozoan to evade the host's immune system.
| S-EPMC7325895 | biostudies-literature
Project description:IntroductionToxoplasma gondii is the etiologic agent of toxoplasmosis, a disease that affects about one-third of the human population. Most infected individuals are asymptomatic, but severe cases can occur such as in congenital transmission, which can be aggravated in individuals infected with other pathogens, such as HIV-positive pregnant women. However, it is unknown whether infection by other pathogens, such as Trypanosoma cruzi, the etiologic agent of Chagas disease, as well as one of its proteins, P21, could aggravate T. gondii infection.MethodsIn this sense, we aimed to investigate the impact of T. cruzi and recombinant P21 (rP21) on T. gondii infection in BeWo cells and human placental explants.ResultsOur results showed that T. cruzi infection, as well as rP21, increases invasion and decreases intracellular proliferation of T. gondii in BeWo cells. The increase in invasion promoted by rP21 is dependent on its binding to CXCR4 and the actin cytoskeleton polymerization, while the decrease in proliferation is due to an arrest in the S/M phase in the parasite cell cycle, as well as interleukin (IL)-6 upregulation and IL-8 downmodulation. On the other hand, in human placental villi, rP21 can either increase or decrease T. gondii proliferation, whereas T. cruzi infection increases T. gondii proliferation. This increase can be explained by the induction of an anti-inflammatory environment through an increase in IL-4 and a decrease in IL-6, IL-8, macrophage migration inhibitory factor (MIF), and tumor necrosis factor (TNF)-α production.DiscussionIn conclusion, in situations of coinfection, the presence of T. cruzi may favor the congenital transmission of T. gondii, highlighting the importance of neonatal screening for both diseases, as well as the importance of studies with P21 as a future therapeutic target for the treatment of Chagas disease, since it can also favor T. gondii infection.
| S-EPMC10617204 | biostudies-literature
Project description:Trypanosoma cruzi P21 is a protein secreted by the parasite that plays biological roles directly involved in the progression of Chagas disease. The recombinant protein (rP21) demonstrates biological properties, such as binding to CXCR4 receptors in macrophages, chemotactic activity of immune cells, and inhibiting angiogenesis. This study aimed to verify the effects of rP21 interaction with CXCR4 from non-tumoral cells (MCF-10A) and triple-negative breast cancer cells (MDA-MB-231). Our data showed that the MDA-MB-231 cells expressed higher levels of CXCR4 than did the non-tumor cell lines. Besides, cytotoxicity assays using different concentrations of rP21 showed that the recombinant protein was non-toxic and was able to bind to the cell membranes of both cell lineages. In addition, rP21 reduced the migration and invasion of MDA-MB-231 cells by the downregulation of MMP-9 gene expression. In addition, treatment with rP21 blocked the cell cycle, arresting it in the G1 phase, mainly in MDA-MB-231 cells. Finally, rP21 prevents the chemotaxis and proliferation induced by CXCL12. Our data showed that rP21 binds to the CXCR4 receptors in both cells, downregulates CXCR4 gene expression, and decreases the receptors in the cytoplasm of MDA-MB-231 cells, suggesting CXCR4 internalization. This internalization may explain the desensitization of the receptors in these cells. Thus, rP21 prevents migration, invasion, and progression in MDA-MB-231 cells.
| S-EPMC7604327 | biostudies-literature
Project description:Chagas disease, which is caused by the parasite Trypanosoma cruzi, is an important cause of cardiomyopathy in Latin America. It is estimated that 10%-30% of all infected individuals will acquire chronic chagasic cardiomyopathy (CCC). The etiology of CCC is multifactorial and involves parasite genotype, host genetic polymorphisms, immune response, signaling pathways and autoimmune progression. Herein we verified the impact of the recombinant form of P21 (rP21), a secreted T. cruzi protein involved in host cell invasion, on progression of inflammatory process in a polyester sponge-induced inflammation model. Results indicated that rP21 can recruit immune cells induce myeloperoxidase and IL-4 production and decrease blood vessels formation compared to controls in vitro and in vivo. In conclusion, T. cruzi P21 may be a potential target for the development of P21 antagonist compounds to treat chagasic cardiomyopathy.
| S-EPMC4648062 | biostudies-literature
Project description:The ribosomal P proteins are located on the stalk of the ribosomal large subunit and play a critical role during the elongation step of protein synthesis. The single chain recombinant antibody C5 (scFv C5) directed against the C-terminal region of the Trypanosoma cruzi P2? protein (TcP2?) recognizes the conserved C-terminal end of all T. cruzi ribosomal P proteins. Although this region is highly conserved among different species, surface plasmon resonance analysis showed that the scFv C5 possesses very low affinity for the corresponding mammalian epitope, despite having only one single amino-acid change. Crystallographic analysis, in silico modelization and NMR assays support the analysis, increasing our understanding on the structural basis of epitope specificity. In vitro protein synthesis experiments showed that scFv C5 was able to specifically block translation by T. cruzi and Crithidia fasciculata ribosomes, but virtually had no effect on Rattus norvegicus ribosomes. Therefore, we used the scFv C5 coding sequence to make inducible intrabodies in Trypanosoma brucei. Transgenic parasites showed a strong decrease in their growth rate after induction. These results strengthen the importance of the P protein C terminal regions for ribosomal translation activity and suggest that trypanosomatid ribosomal P proteins could be a possible target for selective therapeutic agents that could be derived from structural analysis of the scFv C5 antibody paratope.
| S-EPMC3343115 | biostudies-literature
Project description:The protozoan Trypanosoma cruzi is the causative agent of Chagas' disease, a major public health problem in Latin America and of growing concern in the United States as the number of infected immigrants increases. There is currently no testing of U.S. blood products for T. cruzi infection, and the best tests available, although highly sensitive, are not of high enough specificity to be useful for widespread screening of the blood supply in this country. Among the parasite antigens detected by sera of infected humans and mice, those in the range of 24 to 26 kDa are particularly reactive. With an aim of developing a sensitive, specific, recombinant antigen-based serologic test for T. cruzi infection, we used two antibody reagents specific for these 24- to 26-kDa antigens to isolate cDNA clones from a T. cruzi expression library. One clone was found to encode a previously characterized T. cruzi antigen, a 24-kDa flagellar calcium-binding protein (FCaBP). Recombinant FCaBP was found to be a sensitive, specific reagent for distinguishing T. cruzi-infected individuals from uninfected persons, and it therefore could potentially be used for screening purposes, especially if combined with other recombinant T. cruzi antigens that have similarly high degrees of diagnostic sensitivity and specificity.
| S-EPMC228339 | biostudies-other
Project description:Protein geranylgeranyltransferase type I (PGGT-I) and protein farnesyltransferase (PFT) occur in many eukaryotic cells. Both consist of two subunits, the common alpha subunit and a distinct beta subunit. In the gene database of protozoa Trypanosoma cruzi, the causative agent of Chagas' disease, a putative protein that consists of 401 amino acids with approximately 20% amino acid sequence identity to the PGGT-I beta of other species was identified, cloned, and characterized. Multiple sequence alignments show that the T. cruzi ortholog contains all three of the zinc-binding residues and several residues uniquely conserved in the beta subunit of PGGT-I. Co-expression of this protein and the alpha subunit of T. cruzi PFT in Sf9 insect cells yielded a dimeric protein that forms a tight complex selectively with [(3)H]geranylgeranyl pyrophosphate, indicating a key characteristic of a functional PGGT-I. Recombinant T. cruzi PGGT-I ortholog showed geranylgeranyltransferase activity with distinct specificity toward the C-terminal CaaX motif of protein substrates compared to that of the mammalian PGGT-I and T. cruzi PFT. Most of the CaaX-containing proteins with X=Leu are good substrates of T. cruzi PGGT-I, and those with X=Met are substrates for both T. cruzi PFT and PGGT-I, whereas unlike mammalian PGGT-I, those with X=Phe are poor substrates for T. cruzi PGGT-I. Several candidates for T. cruzi PGGT-I or PFT substrates containing the C-terminal CaaX motif are found in the T. cruzi gene database. Among five C-terminal peptides of those tested, a peptide of a Ras-like protein ending with CVLL was selectively geranylgeranylated by T. cruzi PGGT-I. Other peptides with CTQQ (Tcj2 DNAJ protein), CAVM (TcPRL-1 protein tyrosine phosphatase), CHFM (a small GTPase like protein), and CQLF (TcRho1 GTPase) were specific substrates for T. cruzi PFT but not for PGGT-I. The mRNA and protein of the T. cruzi PGGT-I beta ortholog were detected in three life-cycle stages of T. cruzi. Cytosol fractions from trypomastigotes (infectious mammalian stage) and epimastigotes (insect stage) were shown to contain levels of PGGT-I activity that are approximately 100-fold lower than PFT activity. The CaaX mimetics known as PGGT-I inhibitors show very low potency against T. cruzi PGGT-I compared to the mammalian enzyme, suggesting the potential to develop selective inhibitors against the parasite enzyme.
| S-EPMC2246023 | biostudies-literature
Project description:Cellular invasion by Trypanosoma cruzi metacyclic trypomastigotes (MTs) or tissue culture trypomastigotes (TCTs) is a complex process involving host-parasite cellular and molecular interactions. Particularly, the involvement of host cell actin cytoskeleton during trypomastigote invasion is poorly investigated, and still, the results are controversial. In the present work, we compare side by side both trypomastigote forms and employ state-of-the-art live-cell imaging showing for the first time the dynamic mobilization of host cell actin cytoskeleton to MT and TCT invasion sites. Moreover, cytochalasin D, latrunculin B, and jasplakinolide-pretreated cells inhibited MT and TCT invasion. Furthermore, our results demonstrated that TCT invasion decreased in RhoA, Rac1, and Cdc-42 GTPase-depleted cells, whereas MT invasion decreased only in Cdc42-and RhoA-depleted cells. Interestingly, depletion of the three studied GTPases induced a scattered lysosomal distribution throughout the cytosol. These observations indicate that GTPase depletion is sufficient to impair parasite invasion despite the importance of lysosome spread in trypomastigote invasion. Together, our results demonstrate that the host cell actin cytoskeleton plays a direct role during TCT and MT invasion.
| S-EPMC9314811 | biostudies-literature
Project description:Comparative genomic analysis of T. cruzi CLB vs Trypanosoma rangeli (strains SC, Choachí, C23, H14, R1625 and PIT10) and Trypanosoma conorhini
2023-04-11 | GSE229354 | GEO