RESUMO
This study aims to evaluate the antitrypanosomiasis activity of a synthetic dichloro-substituted aminochalcone via in vitro assays against infected cell cultures, as well as a theoretical characterization of pharmacokinetics and pharmacodynamics against the protein targets of the evolutionary cycle of T. cruzi. The in vitro evaluation of parasite proliferation inhibition was performed via cytotoxicity analysis on mammalian host cells, effect on epimastigote and trypomastigote forms, and cell death analysis, while computer simulations characterized the electronic structure of (2E)-1-(4-aminophenyl)-3-(2,4-dichlorophenyl)prop-2-en-1-one (DCl), the mechanism of action against the proteins of the evolutionary cycle of T. cruzi: Cruzain, Trypanothione reductase, TcGAPDH, and CYP51 by molecular docking and dynamics and predictive pharmacokinetics by MPO-based ADMET. The in vitro tests showed that the DCl LC50 in order of 178.9 ± 23.9 was similar to the BZN, evidencing the effectiveness of chalcone against Trypomastigotes. Molecular docking and dynamics simulations suggest that DCl acts on the active site of the CYP51 receptor, with hydrogen interactions that showed a high degree of occupation, establishing a stable complex with the target. MPO analysis and ADMET prediction tests suggest that the compound presents an alignment between permeability and hepatic clearance, although it presents low metabolic stability. Chalcone showed stable pharmacodynamics against the CYP51 target, but can form reactive metabolites from N-conjugation and C = C epoxidation, as an indication of controlled oral dose, although the estimated LD50 rate > 500 mg/kg is a indicative of low incidence of lethality by ingestion, constituting a promising therapeutic strategy.
Assuntos
Chalconas , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Tripanossomicidas , Trypanosoma cruzi , Tripanossomicidas/farmacologia , Tripanossomicidas/química , Trypanosoma cruzi/efeitos dos fármacos , Animais , Chalconas/farmacologia , Chalconas/química , Proteínas de Protozoários/metabolismo , Humanos , NADH NADPH Oxirredutases/antagonistas & inibidores , NADH NADPH Oxirredutases/metabolismo , Teoria Quântica , Camundongos , Doença de Chagas/tratamento farmacológicoRESUMO
Chagas' is a neglected disease caused by the eukaryotic kinetoplastid parasite, Trypanosoma cruzi. Currently, approximately 8 million people are infected worldwide, most of whom are in the chronic phase of the disease, which involves cardiac, digestive, or neurologic manifestations. There is an urgent need for a vaccine because treatments are only effective in the initial phase of infection, which is generally underdiagnosed. The selection and combination of antigens, adjuvants, and delivery platforms for vaccine formulations should be designed to trigger mixed humoral and cellular immune responses, considering that T. cruzi has a complex life cycle with both intracellular and bloodstream circulating parasite stages in vertebrate hosts. Here, we report the effectiveness of vaccination with a T. cruzi-specific protein family (TcTASV), employing both recombinant proteins with aluminum hydroxide and a recombinant baculovirus displaying a TcTASV antigen at the capsid. Vaccination stimulated immunological responses by producing lytic antibodies and antigen-specific CD4+ and CD8+ IFNÉ£ secreting lymphocytes. More than 90% of vaccinated animals survived after lethal challenges with T. cruzi, whereas all control mice died before 30 days post-infection. Vaccination also induced a strong decrease in chronic tissue parasitism and generated immunological memory that allowed vaccinated and infected animals to control both the reactivation of the infection after immunosuppression and a second challenge with T. cruzi. Interestingly, inoculation with wild-type baculovirus partially protected the mice against T. cruzi. In brief, we demonstrated for the first time that the combination of the baculovirus platform and the TcTASV family provides effective protection against Trypanosoma cruzi, which is a promising vaccine for Chagas disease.
Assuntos
Doença de Chagas , Parasitos , Vacinas Protozoárias , Trypanosoma cruzi , Vacinas , Humanos , Animais , Camundongos , Baculoviridae/genética , Antígenos de Protozoários/genética , Doença de Chagas/parasitologia , Trypanosoma cruzi/genética , Vacinação , Vacinas Protozoárias/genéticaRESUMO
This work investigated the mechanical transmission of Trypanosoma vivax by Stomoxys calcitrans to cattle in a region without a cyclic vector. The study involved two experiments, one with calves experimentally infected with T. vivax, in the acute phase of trypanosomosis (Experiment 1) and the other in the chronic phase (Experiment 2). In both experiments, two transmission methods were used with flies that had not fed for 24 h or had never fed: (i) Method 1: flies released freely in cattle pens (≈3,300 flies/pen for 10 days); and (ii) Method 2: flies placed in a feeding chamber (12 flies/animal). To develop Method 1 in the two experiments (acute and chronic phases), T. vivax-positive animals were kept with T. vivax-negative animals. Periodically, the Brener method, Woo method, blood smears, cPCR, ELISA, IFAT, and Imunoteste® were performed to detect T. vivax in the animals. We also recorded the animals' head tossing and hoof stomping and the number of flies near the pens' inner walls. Subsequently, biological testing was performed using lambs. For Method 2 in both experiments, flies inside the feeding chamber first fed on T. vivax-positive animals and later on negative animals. In both experiments and methods, we examined the flies for the presence of T. vivax through blood smears and cPCR of the proboscis and abdomen. In Experiment 2 (chronic phase), a test was conducted to determine how long trypomastigotes forms could survive on the blood of animals with different levels of parasitemia. None of the animals (calves and lambs) became infected with T. vivax or showed antibodies against it. During the evaluation period, the animals in the presence of the flies exhibited more hoof stomping and head tossing compared to those without flies (control). Additionally, there was an increase in the number of flies in the pens during the experiment. Only in Experiment 1 (acute phase) were T. vivax trypomastigotes and DNA found in the abdomen of the flies but not in the proboscis. In Experiment 2 (chronic phase), higher concentrations of trypomastigotes per milliliter of blood were associated with a shorter the lifespan of this stage of the parasite. In conclusion, under the variable conditions of the experiments (hosts, number of flies, and level of parasitemia), S. calcitrans was unable to mechanically transmit T. vivax to cattle.
Assuntos
Muscidae , Animais , Ovinos , Bovinos , Trypanosoma vivax , Parasitemia , Carneiro Doméstico , AnticorposRESUMO
The subclass naphthoquinone represents a substance group containing several compounds with important activities against various pathogenic microorganisms. Accordingly, we evaluated O-allyl-lawsone (OAL) antiparasitic and antifungal activity free and encapsulated in 2-hydroxypropyl-ß-cyclodextrin (OAL MKN) against Trypanosoma cruzi and Sporothrix spp. OAL and OAL MKN were synthesized and characterized by physicochemical methods. The IC50 values of OAL against T. cruzi were 2.4 µM and 96.8 µM, considering epimastigotes and trypomastigotes, respectively. At the same time, OAL MKN exhibited a lower IC50 value (0.5 µM) for both trypanosome forms and low toxicity for mammalian cells. Additionally, the encapsulation showed a selectivity index approximately 240 times higher than that of benznidazole. Regarding antifungal activity, OAL and OAL MKN inhibited Sporothrix brasiliensis growth at 16 µM, while Sporothrix schenckii was inhibited at 32 µM. OAL MKN also exhibited higher selectivity toward fungus than mammalian cells. In conclusion, we described the encapsulation of O-allyl-lawsone in 2-hydroxypropyl-ß-cyclodextrin, increasing the antiparasitic activity compared with the free form and reducing the cytotoxicity and increasing the selectivity towardSporothrix yeasts and the T. cruzi trypomastigote form. This study highlights the potential development of this inclusion complex as an antiparasitic and antifungal agent to treat neglected diseases.
Assuntos
Doença de Chagas , Naftoquinonas , Trypanosoma cruzi , Animais , 2-Hidroxipropil-beta-Ciclodextrina/farmacologia , Antifúngicos/farmacologia , Antifúngicos/química , Antifúngicos/uso terapêutico , Antiparasitários/uso terapêutico , Doença de Chagas/tratamento farmacológico , Mamíferos , Naftoquinonas/uso terapêuticoRESUMO
Chagas disease is a neglected infectious disease caused by the protozoan Trypanosoma cruzi, primarily transmitted by triatomine vectors, and it threatens approximately seventy-five million people worldwide. This parasite undergoes a complex life cycle, transitioning between hosts and shifting from extracellular to intracellular stages. To ensure its survival in these diverse environments, T. cruzi undergoes extreme morphological and molecular changes. The metacyclic trypomastigote (MT) form, which arises from the metacyclogenesis (MTG) process in the triatomine hindgut, serves as a crucial link between the insect and human hosts and can be considered the starting point of Chagas disease. This review provides an overview of the current knowledge regarding the parasite's life cycle, molecular pathways, and mechanisms involved in metabolic and morphological adaptations during MTG, enabling the MT to evade the immune system and successfully infect human cells.
Assuntos
Doença de Chagas , Trypanosoma cruzi , HumanosRESUMO
Na+/H+ exchanger isoform 1 (NHE1), a member of a large family of integral membrane proteins, plays a role in regulating the cortical actin cytoskeleton. Trypanosoma cruzi, the agent of Chagas disease, depends on F-actin rearrangement and lysosome mobilization to invade host cells. To determine the involvement of NHE1 in T. cruzi metacyclic trypomastigote (MT) internalization, the effect of treatment in cells with NHE1 inhibitor amiloride or of NHE1 depletion was examined in human epithelial cells. MT invasion decreased in amiloride-treated and NHE1-depleted cells. The phosphorylation profile of diverse protein kinases, whose activation is associated with remodeling of actin fibers, was analyzed in amiloride-treated and NHE1-depleted cells. In amiloride-treated cells, the phosphorylation levels of protein kinase C (PKC), focal adhesion kinase (FAK) and Akt were similar to those of untreated cells, whereas those of extracellular signal-regulated protein kinases (ERK1/2) increased. In NHE1-deficient cells, with marked alteration in the actin cytoskeleton architecture and in lysosome distribution, the levels of phospho-PKC and phospho-FAK decreased, whereas those of phospho-Akt and phospho-ERK1/2 increased. These data indicate that NHE1 plays a role in MT invasion, by maintaining the activation status of diverse protein kinases in check and preventing the inappropriate F-actin arrangement that affects lysosome distribution.
RESUMO
Chagas disease is a life-threatening illness caused by the parasite Trypanosoma cruzi. The diagnosis of the acute form of the disease is performed by trained microscopists who detect parasites in blood smear samples. Since this method requires a dedicated high-resolution camera system attached to the microscope, the diagnostic method is more expensive and often prohibitive for low-income settings. Here, we present a machine learning approach based on a random forest (RF) algorithm for the detection and counting of T. cruzi trypomastigotes in mobile phone images. We analyzed micrographs of blood smear samples that were acquired using a mobile device camera capable of capturing images in a resolution of 12 megapixels. We extracted a set of features that describe morphometric parameters (geometry and curvature), as well as color, and texture measurements of 1,314 parasites. The features were divided into train and test sets (4:1) and classified using the RF algorithm. The values of precision, sensitivity, and area under the receiver operating characteristic (ROC) curve of the proposed method were 87.6%, 90.5%, and 0.942, respectively. Automating image analysis acquired with a mobile device is a viable alternative for reducing costs and gaining efficiency in the use of the optical microscope.
Assuntos
Telefone Celular , Doença de Chagas , Parasitos , Trypanosoma cruzi , Animais , Doença de Chagas/diagnóstico , Curva ROCRESUMO
To develop novel chemotherapeutic alternatives for the treatment of Chagas disease, in this study, a set of new amino naphthoquinone derivatives were synthesised and evaluated in vitro on the epimastigote and trypomastigote forms of Trypanosoma cruzi strains (NINOA and INC-5) and on J774 murine macrophages. The design of the new naphthoquinone derivatives considered the incorporation of nitrogenous fragments with different substitution patterns present in compounds with activity on T. cruzi, and, thus, 19 compounds were synthesised in a simple manner. Compounds 2e and 7j showed the lowest IC50 values (0.43 µM against both strains for 2e and 0.19 µM and 0.92 µM for 7j). Likewise, 7j was more potent than the reference drug, benznidazole, and was more selective on epimastigotes. To postulate a possible mechanism of action, molecular docking studies were performed on T. cruzi trypanothione reductase (TcTR), specifically at a site in the dimer interface, which is a binding site for this type of naphthoquinone. Interestingly, 7j was one of the compounds that showed the best interaction profile on the enzyme; therefore, 7j was evaluated on TR, which behaved as a non-competitive inhibitor. Finally, 7j was predicted to have a good pharmacokinetic profile for oral administration. Thus, the naphthoquinone nucleus should be considered in the search for new trypanocidal agents based on our hit 7j.
RESUMO
Background: Chagas is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. On the order of seven million people are infected worldwide and current therapies are limited, highlighting the urgent need for new interventions. T. cruzi trypomastigotes can infect a variety of mammalian cells, recognition and adhesion to the host cell being critical for parasite entry. This study focuses on trypomastigote surface ligands involved in cell invasion. Methods: Three selection rounds of a phage peptide display library for isolation of phages that bind to trypomastigotes, resulted in the identification of the N3 dodecapeptide. N3 peptide binding to T. cruzi developmental forms (trypomastigotes, amastigotes and epimastigotes) was evaluated by flow cytometry and immunofluorescence assays. Parasite invasion of Vero cells was assessed by flow cytometry and immunofluorescence assays. Results: Phage display screening identified the N3 peptide that binds preferentially to the surface of the trypomastigote and amastigote infective forms as opposed to non-infective epimastigotes. Importantly, the N3 peptide, but not a control scrambled peptide, inhibits trypomastigote invasion of Vero cells by 50%. Conclusion: The N3 peptide specifically binds to T. cruzi, and by doing so, inhibits Vero cell infection. Follow-up studies will identify the molecule on the parasite surface to which the N3 peptide binds. This putative T. cruzi ligand may advance chemotherapy design and vaccine development.
RESUMO
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.
Assuntos
Trypanosoma cruzi , Citoesqueleto de Actina/metabolismo , Lisossomos/metabolismo , Lisossomos/parasitologia , Trypanosoma cruzi/metabolismoRESUMO
Metacyclic trypomastigote (MT) forms of Trypanosoma cruzi have been shown to release into medium gp82 and gp90, the stage-specific surface molecules that regulate host cell invasion, either in vesicles or in soluble form. Here, we found that during interaction of poorly invasive G strain with the host cell, gp82 and gp90 were released in vesicle-like forms, whereas no such release by highly invasive CL strain was observed. Shedding of vesicles of varying sizes by CL and G strains was visualized by scanning electron microscopy, and the protein profile of conditioned medium (CM) of the two strains was similar, but the content of gp82 and gp90 differed, with both molecules being detected in G strain as bands of high intensity in Western blotting, whereas in CL strain, they were barely detectable. Confocal images revealed a distinct distribution of gp82 and gp90 on MT surface of CL and G strains. In cell invasion assays, addition of G strain CM resulted in decreased CL strain internalization. Depletion of gp82 in G strain CM, by treatment with specific mAb-coupled magnetic beads, increased its inhibitory effect on CL strain invasion, in contrast to CM depleted in gp90. The effect of cholesterol-depleting drug methyl-ß-cyclodextrin (MßCD) on gp82 and gp90 release by MTs was also examined. G strain MTs, untreated or treated with MßCD, were incubated in serum-containing medium or in nutrient-depleted PBS++, and the CM generated under these conditions was analyzed by Western blotting. In PBS++, gp82 and gp90 were released at lower levels by untreated MTs, as compared with MßCD-treated parasites. CM from untreated and MßCD-treated G strain, generated in PBS++, inhibited CL strain internalization. Treatment of CL strain MTs with MßCD resulted in increased gp82 and gp90 shedding and in decreased host cell invasion. The involvement of phospholipase C (PLC) on gp82 and gp90 shedding was also investigated. The CM from G strain MTs pretreated with specific PLC inhibitor contained lower levels of gp82 and gp90, as compared with untreated parasites. Our results contribute to shed light on the mechanism by which T. cruzi releases surface molecules implicated in host cell invasion.
Assuntos
Trypanosoma cruzi , Células HeLa , Humanos , Proteínas de Protozoários , Esteróis , Fosfolipases Tipo C , Glicoproteínas Variantes de Superfície de TrypanosomaRESUMO
Chagas' disease arises as a direct consequence of the lytic cycle of Trypanosoma cruzi in the mammalian host. While invasion is well studied for this pathogen, study of egress has been largely neglected. Here, we provide the first description of T. cruzi egress documenting a coordinated mechanism by which T. cruzi engineers its escape from host cells in which it has proliferated and which is essential for maintenance of infection and pathogenesis. Our results indicate that this parasite egress is a sudden event involving coordinated remodeling of host cell cytoskeleton and subsequent rupture of host cell plasma membrane. We document that host cells maintain plasma membrane integrity until immediately prior to parasite release and report the sequential transformation of the host cell's actin cytoskeleton from normal meshwork in noninfected cells to spheroidal cages-a process initiated shortly after amastigogenesis. Quantification revealed gradual reduction in F-actin over the course of infection, and using cytoskeletal preparations and electron microscopy, we were able to observe disruption of the F-actin proximal to intracellular trypomastigotes. Finally, Western blotting experiments suggest actin degradation driven by parasite proteases, suggesting that degradation of cytoskeleton is a principal component controlling the initiation of egress. Our results provide the first description of the cellular mechanism that regulates the lytic component of the T. cruzi lytic cycle. We show graphically how it is possible to preserve the envelope of host cell plasma membrane during intracellular proliferation of the parasite and how, in cells packed with amastigotes, differentiation into trypomastigotes may trigger sudden egress. IMPORTANCE Understanding how Trypanosoma cruzi interacts with host cells has been transformed by high-quality studies that have examined in detail the mechanisms of T. cruzi host cell invasion. In contrast, little is known about the latter stages of the parasite's lytic cycle: how parasites egress and thereby sustain round after round of infection. Our results show that once in the host cell cytosol and having undergone amastigogenesis, T. cruzi begins to alter the host cell cytoskeleton, remodeling normal F-actin meshworks into encapsulating spheroidal cages. Filamentous actin diminishes over the course of the lytic cycle, and just prior to egress, the filaments comprising the cages are severely degraded where adjacent to the parasites. We conclude that sudden egress follows breach of the containment afforded by the actin cytoskeleton and subsequent plasma membrane rupture-a process that when understood in molecular detail may serve as a target for future novel therapeutic interventions.
Assuntos
Citoesqueleto de Actina/fisiologia , Membrana Celular/patologia , Citoesqueleto/metabolismo , Citoesqueleto/parasitologia , Interações Hospedeiro-Parasita , Trypanosoma cruzi/fisiologia , Actinas/metabolismo , Animais , Membrana Celular/parasitologia , Doença de Chagas/parasitologia , Chlorocebus aethiops , Células VeroRESUMO
The surface molecule gp82 of metacyclic trypomastigote (MT) forms of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, mediates the host cell invasion, a process critical for the establishment of infection. Gp82 is known to bind to the target cell in a receptor-dependent manner, triggering Ca2+ signal, actin cytoskeleton rearrangement and lysosome spreading. The host cell receptor for gp82 was recently identified as LAMP2, the major lysosome membrane-associated protein. To further clarify the mechanisms of MT invasion, we aimed in this study at identifying the LAMP2 domain that interacts with gp82 and investigated whether target cell PKC and ERK1/2, previously suggested to be implicated in MT invasion, are activated by gp82. Interaction of MT, or the recombinant gp82 (r-gp82), with human epithelial HeLa cells induced the activation of Ca2+-dependent PKC and ERK1/2. The LAMP2 sequence predicted to bind gp82 was mapped and the synthetic peptide based on that sequence inhibited MT invasion, impaired the binding of r-gp82 to HeLa cells, and blocked the PKC and ERK1/2 activation induced by r-gp82. Treatment of HeLa cells with specific inhibitor of focal adhesion kinase resulted in inhibition of r-gp82-induced PKC and ERK1/2 activation, as well as in alteration of the actin cytoskeleton architecture. PKC activation by r-gp82 was also impaired by treatment of HeLa cells with inhibitor of phospholipase C, which mediates the production of diacylglycerol, which activates PKC, and inositol 1,4,5-triphosphate that releases Ca2+ from intracellular stores. Taken together, our results indicate that recognition of MT gp82 by LAMP2 induces in the host cell the activation of phosholipase C, with generation of products that contribute for PKC activation and the downstream ERK1/2. This chain of events leads to the actin cytoskeleton disruption and lysosome spreading, promoting MT internalization.
Assuntos
Doença de Chagas , Trypanosoma cruzi , Ativação Enzimática , Células HeLa , Humanos , Proteína 2 de Membrana Associada ao Lisossomo , Proteína Quinase C , Proteínas de ProtozoáriosRESUMO
Trypanosoma cruzi, the protozoan agent of Chagas' disease, displays a complex population structure made up of multiple strains showing a diverse ecoepidemiological distribution. Parasite genetic variability may be associated with disease outcome, hence stressing the need to develop methods for T. cruzi typing in vivo. Serological typing methods that exploit the presence of host antibodies raised against polymorphic parasite antigens emerge as an appealing approach to address this issue. These techniques are robust, simple, cost-effective, and are not curtailed by methodological/biological limitations intrinsic to available genotyping methods. Here, we critically assess the progress towards T. cruzi serotyping and discuss the opportunity provided by high-throughput immunomics to improve this field.
Assuntos
Parasitologia/métodos , Testes Sorológicos/normas , Trypanosoma cruzi/classificação , Animais , Doença de Chagas/parasitologia , Humanos , Testes Sorológicos/economia , Testes Sorológicos/tendências , Especificidade da Espécie , Trypanosoma cruzi/imunologiaRESUMO
Ribose 5-phosphate isomerase type B (RPI-B) is a key enzyme of the pentose phosphate pathway that catalyzes the isomerization of ribose 5-phosphate (R5P) and ribulose 5-phosphate (Ru5P). Trypanosoma cruzi RPI-B (TcRPI-B) appears to be a suitable drug-target mainly due to: (i) its essentiality (as previously shown in other trypanosomatids), (ii) it does not present a homologue in mammalian genomes sequenced thus far, and (iii) it participates in the production of NADPH and nucleotide/nucleic acid synthesis that are critical for parasite cell survival. In this survey, we report on the competitive inhibition of TcRPI-B by a substrate - analogue inhibitor, Compound B (Ki = 5.5 ± 0.1 µM), by the Dixon method. This compound has an iodoacetamide moiety that is susceptible to nucleophilic attack, particularly by the cysteine thiol group. Compound B was conceived to specifically target Cys-69, an important active site residue. By incubating TcRPI-B with Compound B, a trypsin digestion LC-MS/MS analysis revealed the identification of Compound B covalently bound to Cys-69. This inhibitor also exhibited notable in vitro trypanocidal activity against T. cruzi infective life-stages co-cultured in NIH-3T3 murine host cells (IC50 = 17.40 ± 1.055 µM). The study of Compound B served as a proof-of-concept so that next generation inhibitors can potentially be developed with a focus on using a prodrug group in replacement of the iodoacetamide moiety, thus representing an attractive starting point for the future treatment of Chagas' disease.
Assuntos
Aldose-Cetose Isomerases/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/química , Proteínas de Protozoários/antagonistas & inibidores , Tripanossomicidas/síntese química , Trypanosoma cruzi/enzimologia , Células 3T3 , Aldose-Cetose Isomerases/metabolismo , Animais , Sítios de Ligação , Domínio Catalítico , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Cinética , Camundongos , Simulação de Dinâmica Molecular , Proteínas de Protozoários/metabolismo , Especificidade por Substrato , Tripanossomicidas/metabolismo , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Focal adhesion kinase (FAK), a cytoplasmic protein tyrosine kinase (PTK), is implicated in diverse cellular processes, including the regulation of F-actin dynamics. Host cell F-actin rearrangement is critical for invasion of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. It is unknown whether FAK is involved in the internalization process of metacyclic trypomastigote (MT), the parasite form that is important for vectorial transmission. MT can enter the mammalian host through the ocular mucosa, lesion in the skin, or by the oral route. Oral infection by MT is currently a mode of transmission responsible for outbreaks of acute Chagas disease. Here we addressed the question by generating HeLa cell lines deficient in FAK. Host cell invasion assays showed that, as compared to control wild type (WT) cells, FAK-deficient cells were significantly more susceptible to parasite invasion. Lysosome spreading and a disarranged actin cytoskeleton, two features associated with susceptibility to MT invasion, were detected in FAK-deficient cells, as opposed to WT cells that exhibited a more organized F-actin arrangement, and lysosomes concentrated in the perinuclear area. As compared to WT cells, the capacity of FAK-deficient cells to bind a recombinant protein based on gp82, the MT surface molecule that mediates invasion, was higher. On the other hand, when treated with FAK-specific inhibitor PF573228, WT cells exhibited a dense meshwork of actin filaments, lysosome accumulation around the nucleus, and had increased resistance to MT invasion. In cells treated with PF573228, the phosphorylation levels of FAK were reduced and, as a consequence of FAK inactivation, diminished phosphorylation of extracellular signal-regulated protein kinases (ERK1/2) was observed. Fibronectin, known to impair MT invasion, induced the formation of thick bundles of F-actin and ERK1/2 dephosphorylation.
Assuntos
Suscetibilidade a Doenças/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo , Actinas/metabolismo , Doença de Chagas/metabolismo , Doença de Chagas/parasitologia , Suscetibilidade a Doenças/parasitologia , Quinase 1 de Adesão Focal/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/genética , Células HeLa , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Lisossomos/metabolismo , Sistema de Sinalização das MAP Quinases , Fosforilação , Proteínas de Protozoários/genética , Quinolonas/metabolismo , Proteínas Recombinantes/metabolismo , Sulfonas/metabolismo , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidade , Glicoproteínas Variantes de Superfície de Trypanosoma/genéticaRESUMO
Oral infection by Trypanosoma cruzi has been responsible for frequent outbreaks of acute Chagas disease in the north of South America and in the Amazon region, where T. cruzi genetic group TcI predominates. TcI strains from different geographical regions have been used in oral infection in mice, but there is no information about strains from Mexico where TcI is prevalent. Here, we analyzed four Mexican strains as concerns the course of oral infection, the ability to invade host cells in vitro, and the profile of metacyclic trypomastigote surface molecules gp82 and gp90 that are implicated in parasite internalization. Oral infection of mice with metacyclic forms of all strains resulted in reduced blood and tissue parasitism, and mild to moderate inflammatory process in the heart/skeletal muscle. They expressed pepsin-resistant gp82 and gp90 molecules at high levels and invaded host cells poorly in full nutrient medium and efficiently under nutrient-deprived condition. The properties exhibited by Mexican strains were similar to those displayed by TcI strains from other geographical regions, reinforcing the notion that these features are common to the genetic group TcI as a whole.
Assuntos
Doença de Chagas/transmissão , Proteínas de Protozoários/biossíntese , Trypanosoma cruzi/genética , Trypanosoma cruzi/patogenicidade , Glicoproteínas Variantes de Superfície de Trypanosoma/biossíntese , Animais , Linhagem Celular Tumoral , Doença de Chagas/parasitologia , Células HeLa , Humanos , México , Camundongos , Proteínas de Protozoários/genética , América do Sul , Trypanosoma cruzi/classificação , Glicoproteínas Variantes de Superfície de Trypanosoma/genéticaRESUMO
Trypanosoma cruzi, the causative agent of Chagas' disease survives to DNA damage generated by ROS/RNS inside to their different hosts. In recent eukaryotes, oxidative DNA damage is repaired mainly by the Base Excision Repair (BER) pathway, being essential the apurinic/apyrimidinic endonuclease activity. Using a pTREX-gfp vector, the nucleotide sequence that encodes T. cruzi AP endonuclease TcAP1 (orthologue of human APE1) and a putative TcAP1 dominant negative (TcAP1DN), were transfectedand expressed in T. cruzi epimastigotes. TcAP1-GFP and TcAP1DN-GFP were expressed in those modified epimastigotes and found in the parasite nucleus. The endonucleases were purified under native conditions and the AP endonuclease activity was evaluated. While TcAP1 presents the expected AP endonuclease activity TcAP1DN does not. Moreover, TcAP1DN partially inhibits in vitro TcAP1 enzymatic activity. Transfected epimastigotes expressing TcAP1-GFP and TcAP1DN-GFP were differentiated to infective trypomastigotes. The infective parasites maintained both proteins (TcAP1-GFP and TcAP1DN-GFP) in the nucleus. The overexpression of TcAP1-GFP in epimastigotes and trypomastigotes increases the viability of both parasite forms when exposed to oxidative stress while the expression of TcAP1DN-GFP did not show any in vivo inhibitory effect, suggesting that endogenous TcAP1 constitutive expression overcomes the TcAP1DN inhibitory activity. Our results show that TcAP1 is important for trypomastigote survival under oxidative conditions similar to those found in infected mammalian cells, then increasing its permanence in the infected cells and the possibility of development of Chagas disease.
Assuntos
Doença de Chagas/patologia , Dano ao DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Estresse Oxidativo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismo , Sequência de Aminoácidos , Doença de Chagas/genética , Doença de Chagas/parasitologia , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Humanos , Estágios do Ciclo de Vida , Mutação , Oxirredução , Proteínas de Protozoários/genética , Homologia de Sequência , Trypanosoma cruzi/genéticaRESUMO
In this study, Moringa oleifera flower extract and a trypsin inhibitor (MoFTI) isolated from it were evaluated for anti-protozoal activity against Trypanosoma cruzi and cytotoxicity to mammalian cells. The presence of flavonoids was remarkable in the HPLC fingerprints of the extract at 254 and 360 nm. Amino acid sequences of peptides derived from in-gel digestion of MoFTI were determined. Both the extract and MoFTI caused lysis of T. cruzi trypomastigotes with LC50/24 h of 54.18 ± 6.62 and 41.20 ± 4.28 µg/mL, respectively. High selectivity indices (7.9 to >12) for T. cruzi cells over murine peritoneal macrophages and Vero cells were found for the extract and MoFTI. The results show that MoFTI is a trypanocidal principle of the flower extract.
Assuntos
Flavonoides , Flores/química , Moringa oleifera/química , Extratos Vegetais/química , Tripanossomicidas , Trypanosoma cruzi/efeitos dos fármacos , Inibidores da Tripsina/isolamento & purificação , Animais , Linhagem Celular , Chlorocebus aethiops , Flavonoides/análise , Macrófagos Peritoneais/efeitos dos fármacos , Mamíferos , Camundongos , Extratos Vegetais/farmacologia , Tripanossomicidas/farmacologia , Inibidores da Tripsina/farmacologia , Células Vero/efeitos dos fármacosRESUMO
OBJECTIVES: The impact of Chagas disease (CD) in HIV-infected patients is relevant throughout the world. In fact, the characterization of the adaptive immune response in the context of co-infection is important for predicting the need for interventions in areas in which HIV and Chagas disease co-exist. METHODS: We described and compared the frequency of cytokine-producing T cells stimulated with soluble antigen of Trypanosoma cruzi (T. cruzi) using a cytometric assay for the following groups: individuals with chronic Chagas disease (CHR, n=10), those with Chagas disease and HIV infection (CO, n=11), those with only HIV (HIV, n=14) and healthy individuals (C, n=15). RESULTS: We found 1) a constitutively lower frequency of IL-2+ and IFN-γ+ T cells in the CHR group compared with the HIV, CO and healthy groups; 2) a suppressive activity of soluble T. cruzi antigen, which down-regulated IL-2+CD4+ and IFN-γ+CD4+ phenotypes, notably in the healthy group; 3) a down-regulation of inflammatory cytokines on CD8+ T cells in the indeterminate form of Chagas disease; and 4) a significant increase in IL-10+CD8+ cells distinguishing the indeterminate form from the cardiac/digestive form of Chagas disease, even in the presence of HIV infection. CONCLUSIONS: Taken together, our data suggest the presence of an immunoregulatory response in chronic Chagas disease, which seems to be driven by T. cruzi antigens. Our findings provide new insights into immunotherapeutic strategies for people living with HIV/AIDS and Chagas disease.