Participation of heparin binding proteins from the surface of Leishmania (Viannia) braziliensis promastigotes in the adhesion of parasites to Lutzomyia longipalpis cells (Lulo) in vitro

Background: Leishmania (V.) braziliensis is a causative agent of cutaneous leishmaniasis in Brazil. During the parasite life cycle, the promastigotes adhere to the gut of sandflies, to avoid being eliminated with the dejection. the Lulo cell line, derived from Lutzomyia longipalpis (Diptera: Psychodidae), is a suitable in vitro study model to understand the features of parasite adhesion. Here, we analyze the role of glycosaminoglycans (GAGs) from Lulo cells and proteins from the parasites in this event.Methods: Flagellar (F-f) and membrane (M-f) fractions from promastigotes were obtained by differential centrifugation and the purity of fractions confirmed by western blot assays, using specific antibodies for cellular compartments. Heparin-binding proteins (HBP) were isolated from both fractions using a HiTrap-Heparin column. in addition, binding of promastigotes to Lulo cells or to a heparin-coated surface was assessed by inhibition assays or surface plasmon resonance (SPR) analysis.Results: the success of promastigotes subcellular fractionation led to the obtainment of F-f and M-f proteins, both of which presented two main protein bands (65.0 and 55.0kDa) with affinity to heparin. the contribution of HBPs in the adherence of promastigotes to Lulo cells was assessed through competition assays, using HS or the purified HBPs fractions. All tested samples presented a measurable inhibition rate when compared to control adhesion rate (17 +/- 2.0% of culture cells with adhered parasites): 30% (for HS 20 mu g/ml) and 16% (for HS 10 mu g/ml); HBP M-f (35.2% for 10 mu g/ml and 25.4% for 20 mu g/ml) and HBP F-f (10.0% for 10 mu g/ml and 31.4% for 20 mu g/ml). Additionally, to verify the presence of sulfated GAGs in Lulo cells surface and intracellular compartment, metabolic labeling with radioactive sulfate was performed, indicating the presence of an HS and chondroitin sulfate in both cell sections. the SPR analysis performed further confirmed the presence of GAGs ligands on L. (V.) braziliensis promastigote surfaces.Conclusions: the data presented here point to evidences that HBPs present on the surface of L. (V.) braziliensis promastigotes participate in adhesion of these parasites to Lulo cells through HS participation.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ)Lab Biol Mol & Doencas Endem, BR-21040360 Rio de Janeiro, BrazilLab Ultraestrutura Celular, BR-21040360 Rio de Janeiro, BrazilFiocruz MS, IOC, Lab Bioquim & Fisiol Insetos, BR-21040360 Rio de Janeiro, BrazilFiocruz MS, IPEC, Lab Vigilancia Leishmanioses, BR-21040360 Rio de Janeiro, BrazilUniversidade Federal de São Paulo, UNIFESP, Dept Bioquim, São Paulo, BrazilUniv Rosario, Escuela Med, Bogota, DC, ColombiaUniversidade Federal de São Paulo, UNIFESP, Dept Bioquim, São Paulo, BrazilCNPq: 300731/2010-8CNPq: 509737/2010-2CAPES: EDITAL - 11/2009FAPERJ: E-26/103.060/2008FAPERJ: E-26/110.257/2010Web of Scienc

Atuação das proteínas que ligam à heparina no ciclo biológico do Trypanosoma cruzi

Made available in DSpace on 2014-05-29T12:32:35Z (GMT). No. of bitstreams: 4 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 69316.pdf: 16389099 bytes, checksum: 74eb72078f131d9d9be7efc37391f1e6 (MD5) 69316.pdf.txt: 484412 bytes, checksum: 46c33af71747059817ce7b4a20b609af (MD5) 69316.pdf.jpg: 1433 bytes, checksum: 084dde94865696f7af0cab1566875cf5 (MD5) Previous issue date: 2014-05-06Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil.A doença de Chagas, causada pelo protozoário Trypanosoma cruzi, é uma doença tropical negligenciada que representa um grave problema de saúde pública. Assim, compreender a biologia da interação T. cruzi-hospedeiros constitui um grande desafio, uma vez que o ciclo de vida deste parasito exige um repertório de adaptações para garantir sua dispersão em hospedeiros vertebrados e invertebrados. O presente estudo demonstra o potencial das proteínas com propriedade de ligação à heparina (PLHs) em atuar no ciclo biológico do T. cruzi. Durante este trabalho foi oportuno isolar uma fração de proteínas hidrofóbicas com propriedade de ligação à heparina, com massas moleculares entre 70 kDa e 59 kDa em formas epimastigotas e tripomastigotas de T. cruzi por cromatografia de afinidade à heparina. A presença destas proteínas na superfície celular destes parasitos foi confirmada por ressonância plasmônica de superfície. Tais ensaios também foram decisivos na determinação da especificidade e estabilidade da ligação das PLHs a heparina, heparam sulfato (HS) e condroitim sulfato (CS). Os ensaios de competição realizados indicaram que a interação entre PLHs e GAGs pode influenciar a adesão dos epimastigotas à superfície de células epiteliais do trato intestinal de Rhodnius prolixus O envolvimento de GAGs na invasão de amastigotas em cardiomiócitos, célula alvo da infecção pelo T. cruzi, também foi demonstrado através de ensaios de competição com 20 \03BCg/ml de GAGs solúveis, incluindo heparina, HS, CS, dermatam sulfato (DS) e queratam sulfato (KS). Uma drástica redução no nível de infecção foi evidenciada apenas com heparina e HS, atingindo 82% e 65% de redução da invasão, respectivamente. Ensaios com células deficientes em GAGs (CHO-745) corroboraram o importante papel destes componentes de matriz extracelular no processo de reconhecimento e invasão de amastigotas. Na continuidade deste estudo, avançamos na caracterização bioquímica de PLHs, na determinação da expressão e distribuição espacial destas proteínas em tripomastigotas. As análises por citometria de fluxo revelaram que PLHs são abundantes na superfície de tripomastigotas, clone Dm28c, e a detecção destas proteínas por imunofluorescência indireta revelou uma localização predominante na membrana flagelar do parasito. Com os ensaios de zimografia realizados neste trabalho, revelamos que as PLHs de tripomastigotas tem atividade de protease sobre gelatina em uma ampla faixa de pH (5,5 - 8,0). A sensibilidade destas enzimas a presença de inibidores de serino protease indicam que as PLHs de tripomastigotas têm propriedades similares à tripsina. O conjunto de resultados deste trabalho aponta para o importante papel das PLHs em todas as etapas do ciclo biológico do T. cruzi a partir de eventos de adesão e invasão celular, através do reconhecimento de glicosaminoglicanos sulfatadosChagas disease, caused by the protozoan Trypanosoma cruzi, is a neglected tropical disease that causes a serious public health problem. Thus, understanding the biology of the T. cruzi-hosts interaction is a major challenge, since the life cycle of this parasite requires a repertoire of adaptations to ensure their dispersion in vertebrate and invertebrate hosts. The present study demonstrates the potential of proteins with heparin-binding property (HBPs) to act in the biological cycle of T. cruzi. During this study, it was opportune to isolate a hydrophobic protein fraction with property to bind to heparin, with molecular weights ranging from 70 kDa to 59 kDa, in epimastigotes and trypomastigotes of T. cruzi by heparin affinity chromatography. The presence of these proteins on the cell surface of these parasites was confirmed by surface plasmon resonance. These assays were also decisive in determining the specificity and stability of the binding of HBPs to heparin, heparan sulfate (HS) and chondroitin sulfate (CS). The competition assays indicated that the interaction between HBPs and GAGs can influence the adhesion of the epimastigote to the surface of epithelial cells of the intestinal tract of Rhodnius prolixus. The involvement of GAGs in the invasion of amastigotes in cardiomyocytes, target cell of T. cruzi infection, was also demonstrated by competition assays with 20 μg/ml of soluble GAGs, including heparin, HS, CS, dermatan sulfate (DS) and keratan sulfate (KS). A drastic reduction in the level of infection was observed only with heparin and HS, reaching 82% and 65% reduction of the invasion, respectively. Experimental assays using cells deficient in GAGs (CHO-745) corroborated the important role of extracellular matrix components in the recognition and invasion of amastigotes. In continuation of this study, we advanced in the biochemical characterization of HBPs and also determined the expression and spatial distribution of these proteins in trypomastigotes. The flow cytometric analysis revealed that HBPs are abundant at the surface of trypomastigotes, Dm28c clone, and the detection of these proteins by indirect immunofluorescence revealed a predominant location in the flagellar membrane of the parasite. With the zymography assays conducted in this work, we revealed that HBPs of trypomastigotes have proteinase activity on gelatin in a wide pH range (5.5 - 8.0). The sensitivity of these enzymes in the presence of serine proteinase inhibitors indicates that HBPs of trypomastigotes have properties similar to trypsin. All together, the results of this study points out to the important role of HBPs at all stages of the life cycle of T. cruzi from events of adhesion and cell invasion, through the recognition of sulfated glycosaminoglycan

Caracterização de ligantes de heparina em Trypanossoma cruzi e determinação do domínio de heparam sulfato envolvido no processo de invasão T. cruzi-cardiomiócito in vitro

Made available in DSpace on 2016-02-26T13:34:40Z (GMT). No. of bitstreams: 2 francisco_junior_ioc_mest_2007.pdf: 5291215 bytes, checksum: 88ec236330fd3ac74ad110315d9ceb1b (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2016-01-13Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, BrasilA capacidade do Trypanosoma cruzi de reconhecer moléculas na superfície de células fagociticas e não-fagociticas profissionais é essencial para sua sobrevivência no hospedeiro vertebrado. O papel de proteoglicanos sulfatados no processo de reconhecimento celular tem sido relatado em muitos patógenos humanos, incluindo o T. cruzi. Dados do nosso grupo demonstraram a participação de proteoglicanos de heparam sulfato (PGHS) de cardiomiócitos na invasão por formas tripomastigotas. Entretanto, a estrutura da molécula de PGHS envolvida na interação receptor-ligante e o papel de outros glicosaminoglicanos (GAGs) sulfatados no processo de invasão ainda não foram elucidados. Para avaliar a participação dos GAGs na invasão do T. cruzi, tripomastigotas, clone Dm28c, foram pré-tratados com 20µg/ml de heparina, queratam sulfato (KS) ou três fragmentos distintos de heparam sulfato (HS), os quais foram obtidos por tratamento enzimático (heparitinase I e II) e ácido nitroso. Nos ensaios de competição, os parasitas controles ou pré-tratados com GAGs solúveis foram incubados por 2hs a 37°C com culturas de cardiomiócitos e o percentual de infecção foi determinado após coloração pelo Giemsa. Nossos resultados revelaram uma inibição significante no índice de infecção de 84,8% e 45% após tratamento dos parasitas com heparina e com o fragmento N-acetilado/N-sulfatado (NA/NS), respectivamente, sugerindo o importante papel do domínio ([IdoUA-GlcNAc]-[GlcUA-GlcNS]3-[GlcUA-GlcNAc]4[GlcNAc]), da cadeia de HS no reconhecimento T. cruzi-cardiomiócito. Em contraste, o tratamento dos parasitas com KS, fragmento N-acetilado ou N-sulfatado não apresentou efeito no processo de invasão. O papel da sulfatação no processo de reconhecimento e invasão foi avaliado pelo tratamento de culturas de cardiomiócitos por 16hs a 37°C com diferentes concentrações de clorato de sódio e posteriormente, infectados com tripomastigotas (2hs)... (...) O declínio da sulfatação resultou na redução (dose dependente) do índice de infecção, alcançando níveis de inibição de 26%, 48,5% e 73,6% após o tratamento de cardiomiócitos com 25 mM, 50 mM e 75 mM de clorato de sódio, respectivamente, sugerindo a participação da carga negativa como moduladora do reconhecimento específico com o domínio NA/NS da cadeia de HS. Adicionalmente, ensaios bioquímicos foram realizados para caracterizar a proteína de ligação a heparina presente na superfície do T. cruzi. Duas bandas majoritárias de 65,8 kDa e 59 kDa foram identificadas no extrato protéico total das 3 formas evolutivas do T. cruzi por Western blotting, utilizando heparina, condroitim sulfato (CS) e HS conjugados a biotina. O ligante de heparina de T. cruzi foi isolado pela associação do método do Triton X-114 e cromatografia de afinidade a heparina-Sepharose. Após marcação metabólica (35S-Metionina), as proteínas hidrofóbicas foram isoladas em coluna de afinidade e separadas por SDS-PAGE, revelando um perfil protéico, similar ao extrato total, com duas bandas majoritárias (65,8 kDa e 59 kDa) eluídas com 0,5 M e 1,0 M de NaCl em tripomastigotas e epimastigotas, respectivamente. A análise isotópica também revelou uma expressão superior deste ligante (1,3-2 vezes) em tripomastigotas quando comparado com epimastigotas. As proteínas de ligação a heparina (65,8 kDa e 59 kDa) foram detectadas na fração de membrana de epimastigotas obtida pelo método de fracionamento subcelular associado a purificação em coluna de afinidade. A detecção das proteínas eluídas da coluna de afinidade a heparina por Western blotting com heparina-, HS- e CS-biotinilados revelou intensa marcação principalmente na proteína de 59 kDa. Além disso, a análise das proteínas por eletroforese não desnaturante revelou a presença de duas bandas nas formas tripomastigotas e epimastigotas de T. cruzi... (...) Ensaios bioquímicos complementares serão realizados a fim de obter informações detalhadas sobre a proteína de ligação a heparina de T. cruzi.The ability of Trypanosoma cruzi to recognize molecules at the surface of both the phagocytic and non-phagocytic cells is essential to its survival in the vertebrate host. The role of the sulfated proteoglycans in the cell reco gnition process has been reported in several human pathogens, including T. cruzi . Data from our group have demonstrated the participation of heparan sulfated proteoglycan (HSP G) of cardiomyocytes in the invasion for forms trypomastigotes. However, the structure of th e HSPG molecule involved in the receptor-ligand interaction and the role of other s ulfated glycosaminoglycans (GAGs) in the invasion process have not been elucidated yet. To evaluate the participation of GAGs in T. cruzi invasion, trypomastigotes, clone Dm28c, were pre-treated with 20μg/ml of heparin, ke ratan sulfate (KS) or three distinct fragments of heparan sulfate (HS) obtained by enzym atic (heparitinase I and II) and nitrous acid treatments. For competition assays, the untrea ted or soluble GAGs pre-treated parasites were incubated for 2h at 37°C with the ca rdiomyocyte cultures and the percentage of infection was determined after Giemsa staining. Our results revealed a significant inhibition of the infection index of 84.8% and 45% after treatment of the parasites with heparin and the N-acetylated/ N-sulfated fragment, respectively, suggesting the important role of the ([IdoUA-GlcNAc]-[GlcUA-GlcNS] 3 -[GlcUA-GlcNAc] 4 [GlcNAc]) domain of the HS chain in the T. cruzi -cardiomyocyte recognition. In contrast, the treatm ent of the parasites with KS, N- acetylated or N-sulfated fragments did not display any effect in the invasion process. The role of sulfation in the recognition and invasion p rocess was evaluated by treating the cardiomyocytes cultures for 16h at 37°C with differ ent concentrations of sodium chlorate, followed by trypomastigotes infection (2h). The dec line of sulfation resulted in the reduction (dose dependent) of the infection index, achieving inhibition levels of 26%, 48.5% and 73.6% after treatment of cardiomyocyte cultures with 25mM , 50mM and 75mM of sodium chlorate, respectively, suggesting the participation of negat ive charge as modulator of the specific recognition with the NA/NS domain of HS chain. Additionately, biochemical assays were performed to characterize the heparin binding protein present at the surface of T. cruzi . Two major protein bands of 65.8 kDa and 59 kDa were identified in the total protein extract of all evolutive forms of T. cruzi by Western blotting , using biotin conjugated-heparin, -chondroitin sulfa te (CS) and -HS. The T. cruzi heparin ligand was isolated by association of Triton X-114 extraction method and heparin-Sepharose affinity chromatography. After metabolic labeling ( 35 S-Methionine), the hydrophobic proteins were isolated by affinity column and separated by S DS-PAGE, revealing a protein profile, similar to total protein extract, with two major ba nds (65.8 kDa and 59 kDa) eluted with 0.5M and 1M NaCl in trypomastigotes and epimastigotes, r espectively. The isotopic analysis also revealed a higher expression of heparin ligand (1.3 -2X) in trypomastigotes when compared to epimastigotes. The heparin binding proteins were detected at membrane fraction of epimastigotes obtained by cell fractionation method ology associated to the affinity column purification. The detection of proteins eluted from heparin affinity column with biotinilated heparin, CS and HS by Western blotting revealed intense labeling mainly at the 59 kDa protein. In addition, the analysis of the proteins by denaturant electrophoresis revealed the presence of two protein band in both trypomastigote s and epimastigotes forms of T. cruzi . Complementary biochemical assays will be carried ou t to get more detailed information about the T. cruzi heparin binding protein

Ziziphus joazeiro Stem Bark Extract as a Green Corrosion Inhibitor for Mild Steel in Acid Medium

The aqueous extract of Joazeiro stem bark (EJSB) and its high molecular weight fraction (HMWF) were examined as potential corrosion inhibitors of mild steel in 1 mol L−1 hydrochloric acid media, using weight-loss measurements, potentiodynamic polarization curves and an electrochemical impedance spectroscopy (EIS).Varying the concentration of the inhibitors from 100 to 800 mg L−1, the results show an increase in anticorrosive efficiency from 85.4 to 89.8 and 89.8 to 93.0% for EJSB and its HMWF, respectively, using the data of the gravimetric essay, and from 84.5 to 94.5 and 89.9 to 94.7% for EJSB and its HMWF, respectively, from the impedance data. The composition of the crude extract was chemically characterized by liquid chromatography-high resolution mass spectrometry. Additionally, scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS) were used, respectively, to morphologically and chemically characterize the surface. Considering that the saponin molecules, the main constituent from juá, are responsible for its inhibitory action, quantum chemical calculations showed that the C67, C69 and O144 atoms likely have an important role in the process of electron-donation of saponin to metal, due to the higher values of ƒk+ and %HOMO observed on these atoms

Morphologic study of the effect of iron on pseudocyst formation in Trichomonas vaginalis and its interaction with human epithelial cells

BACKGROUND Trichomonas vaginalis is the aetiological agent of human trichomoniasis, which is one of the most prevalent sexually transmitted diseases in humans. Iron is an important element for the survival of this parasite and the colonisation of the host urogenital tract. OBJECTIVES In this study, we investigated the effects of iron on parasite proliferation in the dynamics of pseudocyst formation and morphologically characterised iron depletion-induced pseudocysts. METHODS We performed structural and ultrastructural analyses using light microscopy, scanning electron microscopy and transmission electron microscopy. FINDINGS It was observed that iron depletion (i) interrupts the proliferation of T. vaginalis, (ii) induces morphological changes in typical multiplicative trophozoites to spherical non-proliferative, non-motile pseudocysts, and (iii) induces the arrest of cell division at different stages of the cell cycle; (iv) iron is the fundamental element for the maintenance of typical trophozoite morphology; (v) pseudocysts induced by iron depletion are viable and reversible forms; and, finally, (vi) we demonstrated that pseudocysts induced by iron depletion are able to interact with human epithelial cells maintaining their spherical forms. MAIN CONCLUSIONS Together, these data suggest that pseudocysts could be induced as a response to iron nutritional stress and could have a potential role in the transmission and infection of T. vaginalis