RESUMO
Adhesion of T. cruzi trypomastigotes to components of the extracellular matrix (ECM) is an important step in mammalian host cell invasion. We have recently described a significant increase in the tyrosine nitration levels of histones H2A and H4 when trypomastigotes are incubated with components of the ECM. In this work, we used chromatin immunoprecipitation (ChIP) with an anti-nitrotyrosine antibody followed by mass spectrometry to identify nitrated DNA binding proteins in T. cruzi and to detect alterations in nitration levels induced upon parasite incubation with the ECM. Histone H1, H2B, H2A and H3 were detected among the 9 most abundant nitrated DNA binding proteins using this proteomic approach. One nitrated tyrosine residue (Y29) was identified in Histone H2B in the MS/MS spectrum. In addition, we observed a significant increase in the nitration levels of histones H1, H2B, H2A and H4 upon parasite incubation with ECM. Finally, we used ChIP-Seq to map global changes in the DNA binding profile of nitrated proteins. We observed a significant change in the binding pattern of nitrated proteins to DNA after parasite incubation with ECM. This work provides the first global profile of nitrated DNA binding proteins in T. cruzi and additional evidence for modification in the nitration profile of histones upon parasite incubation with ECM. Our data also indicate that the parasite interaction with the ECM induces alterations in chromatin structure, possibly affecting nuclear functions.
Assuntos
Matriz Extracelular/parasitologia , Histonas/análise , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/análise , Trypanosoma cruzi/química , Trypanosoma cruzi/crescimento & desenvolvimento , Imunoprecipitação da Cromatina , Matriz Extracelular/metabolismo , Histonas/metabolismo , Espectrometria de Massas , Nitrosação , Proteômica , Proteínas de Protozoários/metabolismo , Tirosina/análogos & derivados , Tirosina/imunologiaRESUMO
OBJECTIVE: To analyze the remodeling dynamics of total collagen, type I and III, the expression of ICAM-1 and 5-HT in the jejunum of rats. METHODS: Twenty-eight Wistar rats were randomly assigned to two experimental groups: the control group (CG, n = 7) and the infected group (receiving 5,000 sporulated T. gondii oocysts - ME49 strain, genotype II, n = 21). Seven infected rats each at 6 (G6), 12 (G12), and 24 (G24) hours post infection were sacrificed and segments of jejunum were collected for standard histological, histochemical, and immunohistochemistry processing techniques. RESULTS: The infection promoted ICAM-1 and 5-HT expression, type III collagen, and total mast cell increases. However, it also caused a reduction in the area occupied by type I collagen fibers, and in submucosa thickness, and caused ganglion and peri-ganglion alterations. CONCLUSION: The structural damage caused by toxoplasmic infection is intense during the first 24 h post inoculation. At peak dissemination, from 12 to 24 h, there is an increase in ICAM-1 and 5-HT expression, with intense migration of mast cells to the site of infection. There was also a reduction in submucosa thickness, and an effective loss of extracellular matrix (ECM) organization, which included changes in the dynamics of type I and III total collagen deposition.
Assuntos
Molécula 1 de Adesão Intercelular/metabolismo , Jejuno/metabolismo , Jejuno/parasitologia , Serotonina/metabolismo , Toxoplasma/patogenicidade , Toxoplasmose/metabolismo , Toxoplasmose/parasitologia , Animais , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Matriz Extracelular/metabolismo , Matriz Extracelular/parasitologia , Cistos Glanglionares/metabolismo , Cistos Glanglionares/parasitologia , Masculino , Mastócitos/metabolismo , Mastócitos/parasitologia , Ratos , Ratos WistarRESUMO
Trypanosoma cruzi, the etiological agent of Chagas' disease, affects 8 million people predominantly living in socioeconomic underdeveloped areas. T. cruzi trypomastigotes (Ty), the classical infective stage, interact with the extracellular matrix (ECM), an obligatory step before invasion of almost all mammalian cells in different tissues. Here we have characterized the proteome and phosphoproteome of T. cruzi trypomastigotes upon interaction with ECM (MTy) and the data are available via ProteomeXchange with identifier PXD010970. Proteins involved with metabolic processes (such as the glycolytic pathway), kinases, flagellum and microtubule related proteins, transport-associated proteins and RNA/DNA binding elements are highly represented in the pool of proteins modified by phosphorylation. Further, important metabolic switches triggered by this interaction with ECM were indicated by decreases in the phosphorylation of hexokinase, phosphofructokinase, fructose-2,6-bisphosphatase, phosphoglucomutase, phosphoglycerate kinase in MTy. Concomitantly, a decrease in the pyruvate and lactate and an increase of glucose and succinate contents were detected by GC-MS. These observations led us to focus on the changes in the glycolytic pathway upon binding of the parasite to the ECM. Inhibition of hexokinase, pyruvate kinase and lactate dehydrogenase activities in MTy were observed and this correlated with the phosphorylation levels of the respective enzymes. Putative kinases involved in protein phosphorylation altered upon parasite incubation with ECM were suggested by in silico analysis. Taken together, our results show that in addition to cytoskeletal changes and protease activation, a reprogramming of the trypomastigote metabolism is triggered by the interaction of the parasite with the ECM prior to cell invasion and differentiation into amastigotes, the multiplicative intracellular stage of T. cruzi in the vertebrate host.
Assuntos
Matriz Extracelular/parasitologia , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Cromatografia Gasosa-Espectrometria de Massas , Regulação da Expressão Gênica/fisiologia , Interações Hospedeiro-Parasita , Humanos , Proteínas de Protozoários/genéticaRESUMO
The spleen is one of the main affected organs in canine visceral leishmaniasis (CVL). Disorganization of the splenic white pulp (SWP) has been associated with immunosuppression and disease progression. This study aims to assess structural and cellular changes in the splenic extracellular matrix of dogs with CVL, correlating these changes with the parasite load and clinical signs. Splenic fragments were collected from 41 naturally infected animals for parasite load quantification by quantitative PCR, histopathological analysis and immunohistochemistry for CD3+, CD4+, and CD8+ T cells; CD21+ B cells; Ki-67+, IFN-γ+, and IL-10+ cells; and the MMP-9 and ADAM-10 enzymes. Laminin, collagen and fibronectin deposition were also evaluated. The animals were grouped according to the level of SWP organization. SWP disorganization was accompanied by a reduction in the quantity of lymphoid follicles/mm2 (p > 0.0001). Animals with moderate to intense SWP disorganization showed more clinical signs (p = 0.021), higher laminin (p = 0.045) and collagen deposition (p = 0.036), higher MMP-9 expression (p = 0.035) and lower numbers of CD4+ T cells (p = 0.027) in the spleen than the animals with organized SWP. These data suggest that splenic structure and function are drastically altered and compromised during CVL.
Assuntos
Doenças do Cão/patologia , Matriz Extracelular/patologia , Leishmania infantum/fisiologia , Leishmaniose Visceral/veterinária , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Doenças do Cão/imunologia , Doenças do Cão/parasitologia , Cães , Matriz Extracelular/imunologia , Matriz Extracelular/parasitologia , Imuno-Histoquímica/veterinária , Leishmania infantum/imunologia , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/parasitologia , Leishmaniose Visceral/patologia , Linfócitos/imunologia , Linfócitos/patologia , Carga Parasitária/veterinária , Baço/imunologia , Baço/parasitologia , Baço/patologiaRESUMO
Experimental cerebral malaria (ECM) is characterized by a strong immune response, with leukocyte recruitment, blood-brain barrier breakdown and hemorrhage in the central nervous system. Phosphatidylinositol 3-kinase γ (PI3Kγ) is central in signaling diverse cellular functions. Using PI3Kγ-deficient mice (PI3Kγ-/-) and a specific PI3Kγ inhibitor, we investigated the relevance of PI3Kγ for the outcome and the neuroinflammatory process triggered by Plasmodium berghei ANKA (PbA) infection. Infected PI3Kγ-/- mice had greater survival despite similar parasitemia levels in comparison with infected wild type mice. Histopathological analysis demonstrated reduced hemorrhage, leukocyte accumulation and vascular obstruction in the brain of infected PI3Kγ-/- mice. PI3Kγ deficiency also presented lower microglial activation (Iba-1+ reactive microglia) and T cell cytotoxicity (Granzyme B expression) in the brain. Additionally, on day 6 post-infection, CD3+CD8+ T cells were significantly reduced in the brain of infected PI3Kγ-/- mice when compared to infected wild type mice. Furthermore, expression of CD44 in CD8+ T cell population in the brain tissue and levels of phospho-IkB-α in the whole brain were also markedly lower in infected PI3Kγ-/- mice when compared with infected wild type mice. Finally, AS605240, a specific PI3Kγ inhibitor, significantly delayed lethality in infected wild type mice. In brief, our results indicate a pivotal role for PI3Kγ in the pathogenesis of ECM.
Assuntos
Classe Ib de Fosfatidilinositol 3-Quinase/genética , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Malária Cerebral/imunologia , Malária Cerebral/patologia , Plasmodium berghei/imunologia , Animais , Encéfalo/imunologia , Modelos Animais de Doenças , Matriz Extracelular/imunologia , Matriz Extracelular/parasitologia , Feminino , Humanos , Pulmão/enzimologia , Pulmão/parasitologia , Malária Cerebral/enzimologia , Malária Cerebral/parasitologia , Camundongos , Inibidores de Fosfoinositídeo-3 Quinase , Quinoxalinas/farmacologia , Análise de Sobrevida , Tiazolidinedionas/farmacologiaRESUMO
BACKGROUND: Chagas' disease is caused by the haemophlagelated protozoan Trypanosoma cruzi (T. cruzi). During congenital transmission the parasite breaks down the placental barrier. In the present study we analyzed the participation of matrix metalloproteases (MMPs) in the extracellular matrix (ECM) remodeling during T. cruzi ex vivo infection of human placental chorionic villi explants. METHODS: Chorionic villi from healthy woman placentas were incubated in the presence or absence of 105 or 106 T. cruzi trypomastigotes (Y strain) with or without the MMPs inhibitor doxycycline. Effective infection was tested measuring parasite DNA by real time PCR (qPCR). MMP-2 and MMP-9 expression were determined by western blotting and immunohistochemistry and their activities were measured by zymography. The effect of MMPs on ECM structure was analyzed histochemically. RESULTS: T. cruzi induces the expression and activity of MMP-2 and MMP-9 in chorionic villi. Inhibition of the MMPs prevents the tissue damage induced by T. cruzi and partially decreases the ex vivo infection of the chorionic villi. CONCLUSION: MMPs are partially responsible for the ECM changes observed in human chorionic villi during T. cruzi infection and participate in tissue invasion. On the other hand, MMPs may be part of a local placental antiparasitic mechanism.
Assuntos
Doença de Chagas/imunologia , Vilosidades Coriônicas/enzimologia , Resistência à Doença , Indução Enzimática , Metaloproteinase 2 da Matriz/biossíntese , Metaloproteinase 9 da Matriz/biossíntese , Trypanosoma cruzi/imunologia , Western Blotting , Doença de Chagas/patologia , Doença de Chagas/prevenção & controle , Doença de Chagas/transmissão , Vilosidades Coriônicas/imunologia , Vilosidades Coriônicas/parasitologia , Vilosidades Coriônicas/patologia , DNA de Protozoário/metabolismo , Doxiciclina/farmacologia , Matriz Extracelular/imunologia , Matriz Extracelular/metabolismo , Matriz Extracelular/parasitologia , Matriz Extracelular/patologia , Proteínas da Matriz Extracelular/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Metaloproteinase 2 da Matriz/química , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/química , Metaloproteinase 9 da Matriz/metabolismo , Gravidez , Inibidores de Proteases/farmacologia , Proteólise/efeitos dos fármacos , Técnicas de Cultura de Tecidos , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/isolamento & purificação , Trypanosoma cruzi/patogenicidadeRESUMO
Leishmania is inoculated, by the bite of an infected sandfly, into the skin of the host, where the promastigotes are phagocyted by dermal macrophages. The dermal region comprises cells and abundant extracellular matrix. Studies show that matrix metalloproteinases play an important role in host defense responses against pathogens in mammals and that their activities lead to the production of antimicrobial peptides. The aim of this study is to evaluate the changes in the distribution of fibronectin and laminin as well as in the elastic system fibres during the course of infection caused by Leishmania amazonensis in mice with distinct genetic backgrounds of susceptibility to this parasite. The results showed that BALB/c presented an enhancement of fibronectin during the course of infection when compared to their control group while the infected or non-infected C3H.He showed a decrease of this protein at end of the experiment. Laminin, on the other hand, remained unaltered in both strains. Also in both BALB/c and C3H.He mice the elastic and elaunin fibres remained unchanged while the oxytalan fibres decreased along the experiment. Ninety days after the infection C3H.He mice had recovered their capacity to produce oxytalan fibres.
Assuntos
Tecido Elástico/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Matriz Extracelular/imunologia , Leishmaniose/imunologia , Animais , Suscetibilidade a Doenças , Matriz Extracelular/parasitologia , Feminino , Colágenos Fibrilares/metabolismo , Fibronectinas/metabolismo , Laminina/metabolismo , Leishmania/fisiologia , Leishmaniose/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Microscopia ConfocalRESUMO
Cardiac damages caused by in vivo infection with Trypanosoma cruzi are still not fully clarified. Here we describe for the first time an in vitro model of fibrosis, hypertrophy, and remodeling induced by T. cruzi in cardiomyocyte spheroids (cardiac microtissues). In this new 3-dimensional system, cardiac spheroids showed spontaneous contractility, with typical cardiac morphology and production of extracellular matrix components. There were 4- and 6-fold increases, respectively, in the area and the volume of T. cruzi-infected cardiomyocytes and whole microtissues, together with a 50% reduction of the cell population. Immunofluorescence showed increased expression of fibronectin, collagen IV, and laminin in the microtissues 144 h after infection. T. cruzi infection induced an increase in both the cellular area and the extracellular matrix components in cardiac spheroids, which contributed to an increase in total microtissue volume, making this a powerful 3-dimensional in vitro model for the study of cardiac-tissue hypertrophy, fibrosis, and remodeling.
Assuntos
Cardiomiopatia Chagásica/patologia , Cardiomiopatia Chagásica/parasitologia , Miócitos Cardíacos/patologia , Miócitos Cardíacos/parasitologia , Trypanosoma cruzi/fisiologia , Animais , Chlorocebus aethiops , Matriz Extracelular/parasitologia , Matriz Extracelular/fisiologia , Fibrose/parasitologia , Camundongos , Células VeroRESUMO
We previously showed migration disturbances in the thymus during experimental infection with Trypanosoma cruzi, the causative agent of Chagas disease. These changes were related to the enhanced expression of extracellular matrix ligands and receptors, leading to the escape of immature cells to the periphery. Here, we analyzed the expression and role of selected chemokines (CXCL12 and CCL4) and their receptors (CXCR4 and CCR5) in regulating thymocyte migration in conjunction with extracellular matrix during acute T. cruzi infection. We found increased chemokine deposition in the thymus of infected mice when compared to controls, accompanied by enhanced co-localization with fibronectin as well as up-regulated surface expression of CXCR4 and CCR5 in thymocytes. We also noticed altered thymocyte migration towards the chemokines analyzed. Such an enhancement was even more prominent when fibronectin was added as a haptotatic stimulus in combination with a given chemokine. Our findings suggest that thymocyte migration results from a combined action of chemokines and extracellular matrix (ECM), which can be altered during pathological conditions such as T. cruzi infection, and may be at the origin of the changes in the T cell repertoire seen in this pathological process.
Assuntos
Movimento Celular/imunologia , Doença de Chagas/imunologia , Quimiocinas CC/imunologia , Quimiocinas CXC/imunologia , Fibronectinas/imunologia , Proteínas Inflamatórias de Macrófagos/imunologia , Linfócitos T/imunologia , Trypanosoma cruzi/imunologia , Animais , Doença de Chagas/parasitologia , Quimiocina CCL4 , Quimiocina CXCL12 , Quimiocinas CC/biossíntese , Quimiocinas CC/genética , Quimiocinas CXC/biossíntese , Quimiocinas CXC/genética , Matriz Extracelular/imunologia , Matriz Extracelular/parasitologia , Imuno-Histoquímica , Proteínas Inflamatórias de Macrófagos/biossíntese , Proteínas Inflamatórias de Macrófagos/genética , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores CCR5/biossíntese , Receptores CCR5/genética , Receptores CCR5/imunologia , Receptores CXCR4/biossíntese , Receptores CXCR4/genética , Receptores CXCR4/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/citologia , Linfócitos T/parasitologia , Timo/citologia , Timo/imunologia , Timo/parasitologiaRESUMO
Here we describe extracellular matrix alterations in footpad lesions and draining lymph nodes caused by Leishmania (L.) amazonensis in mouse strains with distinct susceptibilities to this parasite: BALB/c (susceptible), C57BL/6 (intermediate), and DBA/2 (resistant). Changes in ECM were observed mainly in BALB/c mice that, in general, presented tissue damage associated with high parasite burden. Under polarized light, Sirius Red revealed type I collagen that was predominant in the primary lesion in all strains studied at the early phase of infection, but gradually decreased and was replaced by abundant type III collagen fibres in chronic phase lesions. The presence of type III collagen seemed to provide support to inflammatory cells, mainly vacuolated and parasitized macrophages. Laminin expression was not altered during infection by L. (L.) amazonensis in any of the mouse strains studied. Furthermore, the decreased fibronectin expression, in all strains, in areas where amastigotes have been found, indicated that this decline was also not related to the genetic background.