RESUMEN
Chagas disease is a major public health issue, affecting â¼10 million people worldwide. Transmitted by a protozoan named Trypanosoma cruzi, this infection triggers a chronic inflammatory process that can lead to cardiomyopathy (Chagas disease). Resolvin D1 (RvD1) is a novel proresolution lipid mediator whose effects on inflammatory diseases dampens pathological inflammatory responses and can restore tissue homeostasis. Current therapies are not effective in altering the outcome of T. cruzi infection, and as RvD1 has been evaluated as a therapeutic agent in various inflammatory diseases, we examined if exogenous RvD1 could modulate the pathogenesis of Chagas disease in a murine model. CD-1 mice infected with the T. cruzi Brazil strain were treated with RvD1. Mice were administered 3 µg/kg of body weight RvD1 intraperitoneally on days 5, 10, and 15 to examine the effect of RvD1 on acute disease or administered the same dose on days 60, 65, and 70 to examine its effects on chronic infection. RvD1 therapy increased the survival rate and controlled parasite replication in mice with acute infection and reduced the levels of interferon gamma and transforming growth factor ß (TGF-ß) in mice with chronic infection. In addition, there was an increase in interleukin-10 levels with RvD1 therapy in both mice with acute infection and mice with chronic infection and a decrease in TGF-ß levels and collagen content in cardiac tissue. Together, these data indicate that RvD1 therapy can dampen the inflammatory response, promote the resolution of T. cruzi infection, and prevent cardiac fibrosis.
Asunto(s)
Antiinflamatorios/administración & dosificación , Enfermedad de Chagas/microbiología , Ácidos Docosahexaenoicos/administración & dosificación , Interacciones Huésped-Patógeno/efectos de los fármacos , Trypanosoma cruzi/efectos de los fármacos , Animales , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/mortalidad , Enfermedad de Chagas/patología , Modelos Animales de Enfermedad , Ecocardiografía , Fibrosis , Corazón , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Leucocitos/patología , Ratones , Miocardio/metabolismo , Miocardio/patología , Tamaño de los Órganos , Índice de Severidad de la EnfermedadRESUMEN
Chagas disease affects millions of people, and it is a major cause of death in Latin America. Prevention and development of an effective treatment for this infection can be favored by a more thorough understanding of T. cruzi interaction with the microbiome of vectors and hosts. Next-generation sequencing technology vastly broadened the knowledge about intestinal bacteria composition, showing that microbiota within each host (triatomines and mammals) is composed by high diversity of species, although few dominant phyla. This fact may represent an ecological balance that was acquired during the evolutionary process of the microbiome-host complex, and that serves to perpetuate this system. In this context, commensal microbiota is also essential to protect hosts, conferring them resistance to pathogens colonization. However, in some situations, the microbiota is not able to prevent infection but only modulate it. Here we will review the role of the microbiota on the parasite-vector-host triad with a focus on the kinetoplastida of medical importance Trypanosoma cruzi. Novel strategies to control Chagas disease based on intestinal microbiome will also be discussed.
Asunto(s)
Enfermedad de Chagas/microbiología , Microbioma Gastrointestinal/fisiología , Insectos Vectores/microbiología , Animales , Evolución Biológica , Disbiosis/microbiología , Disbiosis/parasitología , Ecología , Interacciones Microbiota-Huesped/inmunología , Interacciones Huésped-Patógeno/inmunología , Humanos , Trypanosoma cruziRESUMEN
The M17 leucyl-aminopeptidase of Trypanosoma cruzi (LAPTc) is a novel drug target for Chagas disease. The objective of this work was to obtain recombinant LAPTc (rLAPTc) in Escherichia coli. A LAPTc gene was designed, optimized for its expression in E. coli, synthesized and cloned into the vector pET-19b. Production of rLAPTc in E. coli BL21(DE3)pLysS, induced for 20 h at 25 °C with 1 mM IPTG, yielded soluble rLAPTC that was catalytically active. The rLAPTc enzyme was purified in a single step by IMAC. The recombinant protein was obtained with a purity of 90% and a volumetric yield of 90 mg per liter of culture. The enzymatic activity has an optimal pH of 9.0, and preference for Leu-p-nitroanilide (appKM = 74 µM, appkcat = 4.4 s-1). The optimal temperature is 50 °C, and the cations Mg2+, Cd2+, Ba2+, Ca2+ and Zn2+ at 4 mM inhibited the activity by 60% or more, while Mn2+ inhibited by only 15% and addition of Co2+ activated by 40%. The recombinant enzyme is insensitive toward the protease inhibitors PMSF, TLCK, E-64 and pepstatin A, but is inhibited by EDTA and bestatin. Bestatin is a non-competitive inhibitor of the enzyme with a Ki value of 881 nM. The enzyme is a good target for inhibitor identification.
Asunto(s)
Clonación Molecular/métodos , Escherichia coli/genética , Leucil Aminopeptidasa/biosíntesis , Proteínas Protozoarias/biosíntesis , Trypanosoma cruzi/enzimología , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/microbiología , Concentración de Iones de Hidrógeno , Cinética , Leucina/análogos & derivados , Leucina/química , Leucil Aminopeptidasa/antagonistas & inhibidores , Leucil Aminopeptidasa/química , Leucil Aminopeptidasa/aislamiento & purificación , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Especificidad por Sustrato , TemperaturaRESUMEN
BACKGROUND: Chagas disease is still prevalent in rural areas of South America. In endemic areas of Bolivia, school children are screened for the program of Chagas disease eradication of the Ministry of Health, and positive children are treated. Here, we compared the fecal, oral and skin microbiomes of children with or without Chagas disease, and before and after benznidazol treatment of infected children. METHODS: A total of 543 Bolivian children (5-14 years old) were tested for Chagas disease, and 20 positive children were treated with Benznidazole. Fecal samples and oral and skin swabs were obtained before and after treatment, together with samples from a group of 35 uninfected controls. The 16S rRNA genes were sequenced and analyzed using QIIME to determine Alpha diversity differences and community distances, and linear discriminant analyses to determine marker taxa by infection status or treatment. RESULTS: Twenty out of 543 children screened were seropositive for Chagas disease (3.7%) and were included in the study, together with 35 control children that were seronegative for the disease. Fecal samples, oral and skin swabs were taken at the beginning of the study and after the anti-protozoa therapy with Benznidazole to the chagasic children. Infected children had higher fecal Firmicutes (Streptococcus, Roseburia, Butyrivibrio, and Blautia), and lower Bacteroides and also showed some skin -but not oral- microbiota differences. Treatment eliminated the fecal microbiota differences from control children, increasing Dialister (class Clostridia) and members of the Enterobacteriaceae, and decreasing Prevotella and Coprococcus, with minor effects on the oral and skin bacterial diversity. CONCLUSIONS: The results of this study show differences in the fecal microbiota associated with Chagas disease in children, and also evidence that treatment normalizes fecal microbiota (makes it more similar to that in controls), but is associated with oral and skin microbiota differences from control children. Since microbiota impacts in children, it is important to determine the effect of drugs on the children microbiota, since dysbiosis could lead to physiological effects which might be avoidable with microbiota restoration interventions.
Asunto(s)
Bacterias , Enfermedad de Chagas , Heces/microbiología , Microbiota , Mucosa Bucal/microbiología , Nitroimidazoles/administración & dosificación , Piel/microbiología , Adolescente , Bacterias/clasificación , Bacterias/genética , Bolivia , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/microbiología , Niño , Preescolar , Femenino , Humanos , Masculino , Población RuralRESUMEN
The IL-17 family contributes to host defense against many intracellular pathogens by mechanisms that are not fully understood. CD8+ T lymphocytes are key elements against intracellular microbes, and their survival and ability to mount cytotoxic responses are orchestrated by several cytokines. Here, we demonstrated that IL-17RA-signaling cytokines sustain pathogen-specific CD8+ T cell immunity. The absence of IL-17RA and IL-17A/F during Trypanosoma cruzi infection resulted in increased tissue parasitism and reduced frequency of parasite-specific CD8+ T cells. Impaired IL-17RA-signaling in vivo increased apoptosis of parasite-specific CD8+ T cells, while in vitro recombinant IL-17 down-regulated the pro-apoptotic protein BAD and promoted the survival of activated CD8+ T cells. Phenotypic, functional, and transcriptomic profiling showed that T. cruzi-specific CD8+ T cells derived from IL-17RA-deficient mice presented features of cell dysfunction. PD-L1 blockade partially restored the magnitude of CD8+ T cell responses and parasite control in these mice. Adoptive transfer experiments established that IL-17RA-signaling is intrinsically required for the proper maintenance of functional effector CD8+ T cells. Altogether, our results identify IL-17RA and IL-17A as critical factors for sustaining CD8+ T cell immunity to T. cruzi.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Enfermedad de Chagas/inmunología , Enfermedad de Chagas/metabolismo , Receptores de Interleucina-17/metabolismo , Transducción de Señal , Trypanosoma cruzi/inmunología , Traslado Adoptivo , Animales , Apoptosis , Supervivencia Celular , Enfermedad de Chagas/microbiología , Citocinas/metabolismo , Femenino , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Inmunomodulación/genética , Interleucina-17/metabolismo , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Recuento de Linfocitos , Masculino , Ratones , Ratones Noqueados , Receptores de Interleucina-17/deficiencia , Transcripción GenéticaRESUMEN
The activation of macrophage respiratory burst in response to infection with Trypanosoma cruzi inflicts oxidative damage to the host's tissues. For decades, the role of reactive oxygen species (ROS) in the elimination of T. cruzi was taken for granted, but recent evidence suggests parasite growth is stimulated in oxidative environments. It is still a matter of debate whether indeed oxidative environments provide ideal conditions (e.g., iron availability in macrophages) for T. cruzi growth and whether indeed ROS signals directly to stimulate growth. Nitric oxide (NO) and ROS combine to form peroxynitrite, participating in the killing of phagocytosed parasites by activated macrophages. In response to infection, mitochondrial ROS are produced by cardiomyocytes. They contribute to oxidative damage that persists at the chronic stage of infection and is involved in functional impairment of the heart. In this review, we discuss how oxidative stress helps parasite growth during the acute stage and how it participates in the development of cardiomyopathy at the chronic stage.
Asunto(s)
Enfermedad de Chagas/complicaciones , Cardiopatías/etiología , Macrófagos/microbiología , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Trypanosoma cruzi/patogenicidad , Animales , Enfermedad de Chagas/microbiología , HumanosRESUMEN
Trypanosoma cruzi parasites are the causative agents of Chagas disease. These parasites infect cardiac and gastrointestinal tissues, leading to local inflammation and tissue damage. Digestive Chagas disease is associated with perturbations in food absorption, intestinal traffic and defecation. However, the impact of T. cruzi infection on the gut microbiota and metabolome have yet to be characterized. In this study, we applied mass spectrometry-based metabolomics and 16S rRNA sequencing to profile infection-associated alterations in fecal bacterial composition and fecal metabolome through the acute-stage and into the chronic stage of infection, in a murine model of Chagas disease. We observed joint microbial and chemical perturbations associated with T. cruzi infection. These included alterations in conjugated linoleic acid (CLA) derivatives and in specific members of families Ruminococcaceae and Lachnospiraceae, as well as alterations in secondary bile acids and members of order Clostridiales. These results highlight the importance of multi-'omics' and poly-microbial studies in understanding parasitic diseases in general, and Chagas disease in particular.
Asunto(s)
Enfermedad de Chagas/metabolismo , Enfermedad de Chagas/microbiología , Heces/microbiología , Microbioma Gastrointestinal , Tracto Gastrointestinal/microbiología , Parasitosis Intestinales/metabolismo , Parasitosis Intestinales/microbiología , Metaboloma , Animales , Ácidos y Sales Biliares/análisis , Enfermedad de Chagas/parasitología , Enfermedad de Chagas/fisiopatología , Modelos Animales de Enfermedad , Tracto Gastrointestinal/parasitología , Secuenciación de Nucleótidos de Alto Rendimiento , Parasitosis Intestinales/parasitología , Ácidos Linoleicos Conjugados/análisis , Espectrometría de Masas , Metabolómica , Ratones , Interacciones Microbianas , ARN Ribosómico 16S/genética , Trypanosoma cruzi/patogenicidadRESUMEN
Some 1,3-diarylureas and 1-((1,4-trans)-4-aryloxycyclohexyl)-3-arylureas (cHAUs) activate heme-regulated kinase causing protein synthesis inhibition via phosphorylation of the eukaryotic translation initiation factor 2 (eIF2) in mammalian cancer cells. To evaluate if these agents have potential to inhibit trypanosome multiplication by also affecting the phosphorylation of eIF2 alpha subunit (eIF2α), we tested 25 analogs of 1,3-diarylureas and cHAUs against Trypanosoma cruzi, the agent of Chagas disease. One of them (I-17) presented selectivity close to 10-fold against the insect replicative forms and also inhibited the multiplication of T. cruzi inside mammalian cells with an EC50 of 1-3 µM and a selectivity of 17-fold. I-17 also prevented replication of African trypanosomes (Trypanosoma brucei bloodstream and procyclic forms) at similar doses. It caused changes in the T. cruzi morphology, arrested parasite cell cycle in G1 phase, and promoted phosphorylation of eIF2α with a robust decrease in ribosome association with mRNA. The activity against T. brucei also implicates eIF2α phosphorylation, as replacement of WT-eIF2α with a non-phosphorylatable eIF2α, or knocking down eIF2 protein kinase-3 by RNAi increased resistance to I-17. Therefore, we demonstrate that eIF2α phosphorylation can be engaged to develop trypanosome-static agents in general, and particularly by interfering with activity of eIF2 kinases.
Asunto(s)
Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/metabolismo , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/metabolismo , Urea/metabolismo , Urea/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Enfermedad de Chagas/microbiología , Factor 2 Eucariótico de Iniciación/metabolismo , Fase G1/efectos de los fármacos , Hemo/metabolismo , Humanos , Mioblastos/efectos de los fármacos , Mioblastos/parasitología , Pruebas de Sensibilidad Parasitaria , Fosforilación , Ratas , Urea/análogos & derivados , eIF-2 Quinasa/metabolismoRESUMEN
Chagas disease (CD) is a parasitic zoonosis endemic in most mainland countries of Central and South America affecting nearly 10 million people, with 100 million people at high risk of contracting the disease. Treatment is only effective if received at the early stages of the disease. Only two drugs (benznidazole and nifurtimox) have so far been marketed, and both share various limitations such as variable efficacy, many side effects, and long duration of treatment, thus reducing compliance. The in vitro and in vivo efficacy of poly-aggregated amphotericin B (AmB), encapsulated poly-aggregated AmB in albumin microspheres (AmB-AME), and dimeric AmB-sodium deoxycholate micelles (AmB-NaDC) was evaluated. Dimeric AmB-NaDC exhibited a promising selectivity index (SI = 3164) against amastigotes, which was much higher than those obtained for licensed drugs (benznidazole and nifurtimox). AmB-AME, but not AmB-NaDC, significantly reduced the parasitemia levels (3.6-fold) in comparison to the control group after parenteral administration at day 7 postinfection. However, the oral administration of AmB-NaDC (10-15 mg/kg/day for 10 days) resulted in a 75% reduction of parasitemia levels and prolonged the survival rate in 100% of the tested animals. Thus, the results presented here illustrate for the first time the oral efficacy of AmB in the treatment of trypanosomiasis. AmB-NaDC is an easily scalable, affordable formulation prepared from GRAS excipients, enabling treatment access worldwide, and therefore it can be regarded as a promising therapy for trypanosomiasis.
Asunto(s)
Anfotericina B/química , Anfotericina B/farmacología , Antifúngicos/química , Antifúngicos/farmacología , Enfermedad de Chagas/tratamiento farmacológico , Ácido Desoxicólico/química , Ácido Desoxicólico/farmacología , Trypanosoma cruzi/efectos de los fármacos , Albúminas/química , Animales , Enfermedad de Chagas/microbiología , Química Farmacéutica/métodos , Combinación de Medicamentos , Excipientes/química , Femenino , Ratones Endogámicos BALB C , Micelas , Microesferas , Tamaño de la PartículaRESUMEN
Since the immune response mounted by the host to a particular microorganism might be influenced by the acquired immunological experience due to previous contact with other microorganisms, we performed a cross-sectional study to explore the pattern of Trypanosoma cruzi infection-related antibodies in T. cruzi-seropositive individuals presenting concomitant tuberculosis, or the antecedent of BCG vaccination. Sampled individuals were grouped as follows: patients with Chagas disease, not vaccinated with BCG, who further developed pulmonary tuberculosis; individuals with Chagas disease, BCG-vaccinated; and subjects with Chagas disease, presenting neither BCG scar nor tuberculosis disease. Non-vaccinated individuals or without tuberculosis, presented the highest values of anti-PH (P < 0.001), anti-FRA (P < 0.001), anti-p2ß (P = 0.0023) and anti-B13 (P < 0.001) antibodies. The present findings constitute the first demonstration of the potential influence of concomitant tuberculosis on Chagas disease.
Asunto(s)
Anticuerpos Antiprotozoarios/inmunología , Enfermedad de Chagas/inmunología , Coinfección , Infecciones por Mycobacterium/inmunología , Mycobacterium/inmunología , Trypanosoma cruzi/inmunología , Anticuerpos Antiprotozoarios/sangre , Enfermedad de Chagas/microbiología , Enfermedad de Chagas/parasitología , Interacciones Huésped-Parásitos/inmunología , Interacciones Huésped-Patógeno/inmunología , Humanos , Infecciones por Mycobacterium/microbiologíaRESUMEN
This work describes the antitrypanocidal activity of two hydroxamic acid derivatives containing o-ethoxy (HAD1) and p-ethoxy (HAD2) as substituent in the aromatic ring linked to the isoxazoline ring. HAD1 and HAD2 induced a significant reduction in the number of intracellular parasites and consequently showed activity on the multiplication of the parasite. Treatment of cardiomyocytes and macrophages with the compounds revealed no significant loss in cell viability. Ultrastructural alterations after treatment of cardiomyocytes or macrophages infected by Trypanosoma cruzi with the IC50 value of HAD1 revealed alterations to amastigotes, showing initial damage seen as swelling of the kinetoplast. This gave a good indication of the ability of the drug to permeate through the host cell membrane as well as its selectivity to the parasite target. Both compounds HAD1 and 2 were able to reduce the cysteine peptidases and decrease the activity of metallopeptidases.
Asunto(s)
Enfermedad de Chagas/tratamiento farmacológico , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacología , Tripanocidas/química , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacos , Animales , Células Cultivadas , Enfermedad de Chagas/microbiología , Relación Dosis-Respuesta a Droga , Ácidos Hidroxámicos/síntesis química , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Ratones , Estructura Molecular , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/microbiología , Relación Estructura-Actividad , Tripanocidas/síntesis químicaRESUMEN
INTRODUÇÃO: A cardiomiopatia chagásica crônica (CCC), doença de elevada morbimortalidade, associada à grave disfunção ventricular e a arritmias cardíacas, caracteriza-se histologicamente por intensa reação inflamatória multifocal, com pronunciada fibrose miocárdica. Diante da ausência de uma terapia eficaz para os pacientes com as formas mais graves da doença, torna-se crucial a descoberta de biomarcadores que possam identificar pacientes em estágios mais precoces, sob risco mais elevado para a progressão da doença. Neste contexto, surge a syndecan-4, uma glicoproteína transmembrana associada à inflamação e fibrose, cujos níveis estão aumentados em indivíduos com insuficiência cardíaca. OBJETIVO: Neste trabalho, avaliamos o padrão de expressão da syndecan-4 no tecido cardíaco de camundongos e de indivíduos com cardiomiopatia chagásica e a possível correlação entre a concentração sérica de syndecan-4 com grau de fibrose miocárdica e com fração de ejeção do ventrículo esquerdo em indivíduos com doença de Chagas...
INTRODUCTION: The hallmark of chronic Chagas cardiomyopathy (CCC) is the presence of a multifocal inflammatory reaction, which leads to myocardial fibrosis, often followed by ventricular dysfunction and arrhythmias. Syndecan-4 is a transmembrane glycoprotein associated with inflammation and fibrosis. Syndecan-4 levels are increased in subjects with heart failure and it has been proposed as a biomarker to predict cardiovascular events. The expression of syndecan-4 is increased in the hearts of mice chronically infected with Trypanosoma cruzi, suggesting a role of this protein in the pathogenesis of CCC. OBJETIVE: Here we aimed to evaluate the pattern of expression of syndecan-4 in heart tissue of mice and subjects with Chagas cardiomyopathy, and to correlate with the degree of inflammation and fibrosis, as well as to determine the correlation of syndecan-4 serum concentration with the degree of myocardial fibrosis and with left ventricular ejection fraction in subjects with Chagas disease...
Asunto(s)
Enfermedad de Chagas/complicaciones , Enfermedad de Chagas/diagnóstico , Enfermedad de Chagas/inmunología , Enfermedad de Chagas/microbiología , Enfermedad de Chagas/patología , Enfermedad de Chagas/transmisiónRESUMEN
American trypanosomiasis, commonly known as Chagas disease, is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. The chronic form of the infection often causes debilitating morbidity and mortality. However, the current treatment for the disease is typically inadequate owing to drug toxicity and poor efficacy, necessitating a continual effort to discover and develop new antiparasitic therapeutic agents. The structure of T. cruzi histidyl-tRNA synthetase (HisRS), a validated drug target, has previously been reported. Based on this structure and those of human cytosolic HisRS, opportunities for the development of specific inhibitors were identified. Here, efforts are reported to identify small molecules that bind to T. cruzi HisRS through fragment-based crystallographic screening in order to arrive at chemical starting points for the development of specific inhibitors. T. cruzi HisRS was soaked into 68 different cocktails from the Medical Structural Genomics of Pathogenic Protozoa (MSGPP) fragment library and diffraction data were collected to identify bound fragments after soaking. A total of 15 fragments were identified, all bound to the same site on the protein, revealing a fragment-binding hotspot adjacent to the ATP-binding pocket. On the basis of the initial hits, the design of reactive fragments targeting the hotspot which would be simultaneously covalently linked to a cysteine residue present only in trypanosomatid HisRS was initiated. Inhibition of T. cruzi HisRS was observed with the resultant reactive fragments and the anticipated binding mode was confirmed crystallographically. These results form a platform for the development of future generations of selective inhibitors for trypanosomatid HisRS.
Asunto(s)
Inhibidores Enzimáticos/química , Histidina-ARNt Ligasa/antagonistas & inhibidores , Histidina-ARNt Ligasa/química , Bibliotecas de Moléculas Pequeñas/química , Trypanosoma cruzi/enzimología , Sitios de Unión , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/microbiología , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Histidina-ARNt Ligasa/metabolismo , Humanos , Modelos Moleculares , Bibliotecas de Moléculas Pequeñas/farmacología , Trypanosoma cruzi/química , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/metabolismoRESUMEN
Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1) in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50) of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.
Asunto(s)
Antifúngicos/farmacología , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/microbiología , Citocromos b/metabolismo , Trypanosoma cruzi/efectos de los fármacos , Animales , Antimicina A/metabolismo , Enfermedad de Chagas/genética , Citocromos b/genética , Transporte de Electrón/efectos de los fármacos , Transporte de Electrón/inmunología , Genómica , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mutación , Consumo de Oxígeno/efectos de los fármacos , Trypanosoma cruzi/aislamiento & purificación , Trypanosoma cruzi/metabolismoRESUMEN
Cruzain is a primary cysteine protease expressed by the protozoan parasite Trypanosoma cruzi during Chagas disease infection, and thus, the development of inhibitors of this protein is a promising target for designing an effective therapy against the disease. In this paper, the mechanism of inhibition of cruzain by two different irreversible peptidyl halomethyl ketones (PHK) inhibitors has been studied by means of hybrid quantum mechanics/molecular mechanics-molecular dynamics (MD) simulations to obtain a complete representation of the possible free energy reaction paths. These have been traced on free energy surfaces in terms of the potential of mean force computed at AM1d/MM and DFT/MM levels of theory. An analysis of the possible reaction mechanisms of the inhibition process has been performed showing that the nucleophilic attack of an active site cysteine, Cys25, on a carbon atom of the inhibitor and the cleavage of the halogen-carbon bond take place in a single step. PClK appears to be much more favorable than PFK from a kinetic point of view. This result would be in agreement with experimental studies in other papain-like enzymes. A deeper analysis of the results suggests that the origin of the differences between PClK and PFK can be the different stabilizing interactions established between the inhibitors and the residues of the active site of the protein. Any attempt to explore the viability of the inhibition process through a stepwise mechanism involving the formation of a thiohemiketal intermediate and a three-membered sulfonium intermediate has been unsuccessful. Nevertheless, a mechanism through a protonated thiohemiketal, with participation of His159 as a proton donor, appears to be feasible despite showing higher free energy barriers. Our results suggest that PClK can be used as a starting point to develop a proper inhibitor of cruzain.
Asunto(s)
Enfermedad de Chagas/microbiología , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/química , Inhibidores de Cisteína Proteinasa/farmacología , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/enzimología , Dominio Catalítico/efectos de los fármacos , Cisteína Endopeptidasas/química , Humanos , Cetonas/química , Cetonas/farmacología , Simulación de Dinámica Molecular , Péptidos/química , Péptidos/farmacología , Proteínas Protozoarias/química , Trypanosoma cruzi/efectos de los fármacosRESUMEN
Many infectious diseases arise from co-infections or re-infections with more than one genotype of the same pathogen. These mixed infections could alter host fitness, the severity of symptoms, success in pathogen transmission and the epidemiology of the disease. Trypanosoma cruzi, the etiological agent of Chagas disease, exhibits a high biological variability often correlated with its genetic diversity. Here, we developed an experimental approach in order to evaluate biological interaction between three T. cruzi isolates belonging to different Discrete Typing Units (DTUs TcIII, TcV and TcVI). These isolates were obtained from a restricted geographical area in the Chaco Region. Different mixed infections involving combinations of two isolates (TcIII + TcV, TcIII + TcVI and TcV + TcVI) were studied in a mouse model. The parameters evaluated were number of parasites circulating in peripheral blood, histopathology and genetic characterization of each DTU in different tissues by DNA hybridization probes. We found a predominance of TcVI isolate in blood and tissues respect to TcIII and TcV; and a decrease of the inflammatory response in heart when the damage of mice infected with TcVI and TcIII + TcVI mixture were compared. In addition, simultaneous presence of two isolates in the same tissue was not detected. Our results show that biological interactions between isolates with different biological behaviors lead to changes in their biological properties. The occurrence of interactions among different genotypes of T. cruzi observed in our mouse model suggests that these phenomena could also occur in natural cycles in the Chaco Region.
Asunto(s)
Enfermedad de Chagas/genética , Inflamación/genética , Trypanosoma cruzi/genética , Animales , Enfermedad de Chagas/microbiología , Enfermedad de Chagas/fisiopatología , Variación Genética , Genotipo , Corazón/microbiología , Corazón/fisiopatología , Humanos , Inflamación/microbiología , Inflamación/patología , Ratones , Trypanosoma cruzi/patogenicidadRESUMEN
BACKGROUND: Clinical and experimental conflicting data have questioned the relationship between infectious agents, inflammation and dilated cardiomyopathy (DCM). OBJECTIVES: The aim of this study was to determine the frequency of infectious agents and inflammation in endomyocardial biopsy (EMB) specimens from patients with idiopathic DCM, explanted hearts from different etiologies, including Chagas' disease, compared to donated hearts. METHODS: From 2008 to 2011, myocardial samples from 29 heart donors and 55 patients with DCMs from different etiologies were studied (32 idiopathic, 9 chagasic, 6 ischemic and 8 other specific etiologies). Inflammation was investigated by immunohistochemistry and infectious agents by immunohistochemistry, molecular biology, in situ hybridization and electron microscopy. RESULTS: There were no differences regarding the presence of macrophages, expression of HLA class II and ICAM-I in donors and DCM. Inflammation in Chagas' disease was predominant. By immunohistochemistry, in donors, there was a higher expression of antigens of enterovirus and Borrelia, hepatitis B and C in DCMs. By molecular biology, in all groups, the positivity was elevated to microorganisms, including co-infections, with a higher positivity to adenovirus and HHV6 in donors towards DCMs. This study was the first to demonstrate the presence of virus in the heart tissue of chagasic DCM. CONCLUSIONS: The presence of inflammation and infectious agents is frequent in donated hearts, in the myocardium of patients with idiopathic DCM, myocardial dysfunction related to cardiovascular diseases, and primary and secondary cardiomyopathies, including Chagas' disease. The role of co-infection in Chagas' heart disease physiopathology deserves to be investigated in future studies.
Asunto(s)
Cardiomiopatía Dilatada/diagnóstico , Cardiomiopatía Dilatada/microbiología , Enfermedad de Chagas/diagnóstico , Enfermedad de Chagas/microbiología , Corazón/microbiología , Donantes de Tejidos , Adulto , Cardiomiopatías/diagnóstico , Cardiomiopatías/microbiología , Femenino , Trasplante de Corazón/normas , Humanos , Inflamación/diagnóstico , Inflamación/microbiología , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Adulto JovenRESUMEN
Introduction Most studies that have evaluated the stomachs of patients with Chagas disease were performed before the discovery of Helicobacter pylori and used no control groups. This study compared the gastric features of chagasic and non-chagasic patients and assessed whether gastritis could be associated with Chagas disease. Methods Gastric biopsy samples were taken from patients who underwent endoscopy for histological analysis according to the Updated Sydney System. H. pylori infection was assessed by histology, 16S ribosomal ribonucleic acid (rRNA) polymerase chain reaction (PCR), serology and the 13C-urea breath test. Patients were considered H. pylori-negative when all of these diagnostic tests were negative. Clinical and socio-demographic data were obtained by reviewing medical records and using a questionnaire. Results The prevalence of H. pylori infection (70.3% versus 71.7%) and chronic gastritis (92.2% versus 85%) was similar in the chagasic and non-chagasic groups, respectively; such as peptic ulcer, atrophy and intestinal metaplasia. Gastritis was associated with H. pylori infection independent of Chagas disease in a log-binomial regression model. However, the chagasic H. pylori-negative patients showed a significantly higher grade of mononuclear (in the corpus) and polymorphonuclear ...