RESUMEN
Trypanosoma brucei colonise and multiply in the blood vasculature, as well as in various organs of the host's body. Lymph nodes have been previously shown to harbour large numbers of parasites, and the lymphatic system has been proposed as a key site that allows T. brucei distribution through, and colonization of the mammalian body. However, visualization of host-pathogen interactions in the lymphatic system has never captured dynamic events with high spatial and temporal resolution throughout infection. In our work, we used a mixture of tools including intravital microscopy and ex vivo imaging to study T. brucei distribution in 20 sets of lymph nodes. We demonstrate that lymph node colonization by T. brucei is different across lymph node sets, with the most heavily colonised being the draining lymph nodes of main tissue reservoirs: the gonadal white adipose tissue and pancreas. Moreover, we show that the lymphatic vasculature is a pivotal site for parasite dispersal, and altering this colonization by blocking LYVE-1 is detrimental for parasite survival. Additionally, parasites within the lymphatic vasculature have unique morphological and behavioural characteristics, different to those found in the blood, demonstrating that across both types of vasculature, these environments are physically separated. Finally, we demonstrate that the lymph nodes and the lymphatic vasculature undergo significant alterations during T. brucei infection, resulting in oedema throughout the host's body.
Asunto(s)
Trypanosoma brucei brucei , Tripanosomiasis Africana , Animales , Tripanosomiasis Africana/parasitología , Tripanosomiasis Africana/patología , Sistema Linfático , MamíferosRESUMEN
The first stage of the drug discovery process involves the identification of small compounds with biological activity. Iboga alkaloids are monoterpene indole alkaloids (MIAs) containing a fused isoquinuclidine-tetrahydroazepine ring. Both the natural products and the iboga-inspired synthetic analogs have shown a wide variety of biological activities. Herein, we describe the chemoenzymatic preparation of a small library of novel N-indolylethyl-substituted isoquinuclidines as iboga-inspired compounds, using toluene as a starting material and an imine Diels-Alder reaction as the key step in the synthesis. The new iboga series was investigated for its potential to promote the release of glial cell line-derived neurotrophic factor (GDNF) by C6 glioma cells, and to inhibit the growth of infective trypanosomes. GDNF is a neurotrophic factor widely recognized by its crucial role in development, survival, maintenance, and protection of dopaminergic neuronal circuitries affected in several neurological and psychiatric pathologies. Four compounds of the series showed promising activity as GDNF releasers, and a leading structure (compound 11) was identified for further studies. The same four compounds impaired the growth of bloodstream Trypanosoma brucei brucei (EC50 1-8 µM) and two of them (compounds 6 and 14) showed a good selectivity index.
Asunto(s)
Alcaloides , Antiprotozoarios , Factor Neurotrófico Derivado de la Línea Celular Glial/biosíntesis , Tabernaemontana/química , Trypanosoma brucei brucei/crecimiento & desarrollo , Tripanosomiasis Africana/tratamiento farmacológico , Alcaloides/síntesis química , Alcaloides/química , Alcaloides/farmacología , Animales , Antiprotozoarios/síntesis química , Antiprotozoarios/química , Antiprotozoarios/farmacología , Línea Celular Tumoral , Ratones , Ratas , Tripanosomiasis Africana/metabolismo , Tripanosomiasis Africana/patologíaRESUMEN
BACKGROUND: Nifurtimox-eflornithine combination therapy (NECT) for the treatment of second stage gambiense human African trypanosomiasis (HAT) was added to the World Health Organization's Essential Medicines List in 2009 after demonstration of its non-inferior efficacy compared to eflornithine therapy. A study of NECT use in the field showed acceptable safety and high efficacy until hospital discharge in a wide population, including children, pregnant and breastfeeding women, and patients with a HAT treatment history. We present here the effectiveness results after the 24-month follow-up visit. METHODOLOGY/PRINCIPAL FINDINGS: In a multicenter, open label, single arm phase IIIb study, second stage gambiense HAT patients were treated with NECT in the Democratic Republic of Congo. Clinical cure was defined 24 months after treatment as survival without clinical and/or parasitological signs of HAT. Of the 629 included patients, 619 (98.4%) were discharged alive after treatment and were examined for the presence of trypanosomes, white blood cell count in cerebro-spinal fluid, and disease symptoms. The clinical cure rate of 94.1% was comparable for all subpopulations analyzed at the 24-month follow-up visit. Self-reported adverse events during follow-up were few and concerned mainly nervous system disorders, infections, and gastro-intestinal disorders. Overall, 28 patients (4.3%) died during the course of the trial. The death of 16 of the 18 patients who died during the follow-up period was assessed as unlikely or not related to NECT. Within 24 months, eight patients (1.3%) relapsed and received rescue treatment. Sixteen patients were completely lost to follow-up. CONCLUSIONS/SIGNIFICANCE: NECT treatment administered under field conditions was effective and sufficiently well tolerated, no major concern arose for children or pregnant or breastfeeding women. Patients with a previous HAT treatment history had the same response as those who were naïve. In conclusion, NECT was confirmed as effective and appropriate for use in a broad population, including vulnerable subpopulations. TRIAL REGISTRATION: The trial is registered at ClinicalTrials.gov, number NCT00906880.
Asunto(s)
Antiprotozoarios/administración & dosificación , Eflornitina/administración & dosificación , Nifurtimox/administración & dosificación , Tripanocidas/administración & dosificación , Tripanosomiasis Africana/tratamiento farmacológico , Adolescente , Adulto , Anciano , Antiprotozoarios/efectos adversos , Niño , Preescolar , República Democrática del Congo , Quimioterapia Combinada , Eflornitina/efectos adversos , Femenino , Estudios de Seguimiento , Humanos , Lactante , Masculino , Persona de Mediana Edad , Nifurtimox/efectos adversos , Embarazo , Resultado del Tratamiento , Trypanosoma brucei gambiense/efectos de los fármacos , Trypanosoma brucei gambiense/genética , Trypanosoma brucei gambiense/fisiología , Tripanosomiasis Africana/parasitología , Tripanosomiasis Africana/patología , Adulto JovenRESUMEN
The infection by Trypanosoma brucei brucei (T.b.b.), a protozoan parasite, is characterized by an early-systemic stage followed by a late stage in which parasites invade the brain parenchyma in a T cell-dependent manner. Here we found that early after infection effector-memory T cells were predominant among brain T cells, whereas, during the encephalitic stage T cells acquired a tissue resident memory phenotype (TRM) and expressed PD1. Both CD4 and CD8 T cells were independently redundant for the penetration of T.b.b. and other leukocytes into the brain parenchyma. The role of lymphoid cells during the T.b.b. infection was studied by comparing T- and B-cell deficient rag1-/- and WT mice. Early after infection, parasites located in circumventricular organs, brain structures with increased vascular permeability, particularly in the median eminence (ME), paced closed to the sleep-wake regulatory arcuate nucleus of the hypothalamus (Arc). Whereas parasite levels in the ME were higher in rag1-/- than in WT mice, leukocytes were instead reduced. Rag1-/- infected mice showed increased levels of meca32 mRNA coding for a blood /hypothalamus endothelial molecule absent in the blood-brain-barrier (BBB). Both immune and metabolic transcripts were elevated in the ME/Arc of WT and rag1-/- mice early after infection, except for ifng mRNA, which levels were only increased in WT mice. Finally, using a non-invasive sleep-wake cycle assessment method we proposed a putative role of lymphocytes in mediating sleep alterations during the infection with T.b.b. Thus, the majority of T cells in the brain during the early stage of T.b.b. infection expressed an effector-memory phenotype while TRM cells developed in the late stage of infection. T cells and parasites invade the ME/Arc altering the metabolic and inflammatory responses during the early stage of infection and modulating sleep disturbances.
Asunto(s)
Enfermedades del Sistema Nervioso Central/inmunología , Enfermedades del Sistema Nervioso Central/parasitología , Subgrupos de Linfocitos T/fisiología , Trypanosoma brucei brucei , Tripanosomiasis Africana/inmunología , Tripanosomiasis Africana/patología , Animales , Encéfalo/parasitología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Memoria Inmunológica , Leucocitos , Ratones , Ratones Noqueados , SueñoRESUMEN
Trypanosoma brucei is a protozoan parasite that causes important human and livestock diseases in sub-Saharan Africa. By overexpressing a single RNA-binding protein, RBP6, in non-infectious procyclics trypanosomes, we previously recapitulated in vitro the events occurring in the tsetse fly vector, namely the development of epimastigotes and infectious, quiescent metacyclic parasites. To identify genes involved in this developmental progression, we individually targeted 86 transcripts by RNAi in the RBP6 overexpression cell line and assessed the loss-of-function phenotypes on repositioning the kinetoplast, an organelle that contains the mitochondrial genome, the expression of BARP or brucei alanine rich protein, a marker for epimastigotes, and metacyclic variant surface glycoprotein. This screen identified 22 genes that positively or negatively regulate the stepwise progression towards infectivity at different stages. Two previously uncharacterized putative nucleic acid binding proteins emerged as potent regulators, namely the cold shock domain-containing proteins CSD1 and CSD2. RNA-Seq data from a selected group of cell lines further revealed that the components of gene expression regulatory networks identified in this study affected the abundance of a subset of transcripts in very similar fashion. Finally, our data suggest a considerable overlap between the genes that regulate the formation of stumpy bloodstream form trypanosomes and the genes that govern the development of metacyclic form parasites.
Asunto(s)
Progresión de la Enfermedad , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/patogenicidad , Tripanosomiasis Africana/patología , Tripanosomiasis Africana/parasitología , Línea Celular , Regulación hacia Abajo/genética , Perfilación de la Expresión Génica , Polirribosomas/metabolismo , Biosíntesis de Proteínas , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , RNA-Seq , Tripanosomiasis Africana/genética , Regulación hacia Arriba/genéticaRESUMEN
Bovine African Trypanosomosis is an infectious parasitic disease affecting livestock productivity and thereby impairing the economic development of Sub-Saharan Africa. The most important trypanosome species implicated is T. congolense, causing anemia as most important pathological feature. Using murine models, it was shown that due to the parasite's efficient immune evasion mechanisms, including (i) antigenic variation of the variable surface glycoprotein (VSG) coat, (ii) induction of polyclonal B cell activation, (iii) loss of B cell memory and (iv) T cell mediated immunosuppression, disease prevention through vaccination has so far been impossible. In trypanotolerant models a strong, early pro-inflammatory immune response involving IFN-γ, TNF and NO, combined with a strong humoral anti-VSG response, ensures early parasitemia control. This potent protective inflammatory response is counterbalanced by the production of the anti-inflammatory cytokine IL-10, which in turn prevents early death of the host from uncontrolled hyper-inflammation-mediated immunopathologies. Though at this stage different hematopoietic cells, such as NK cells, T cells and B cells as well as myeloid cells (i.e. alternatively activated myeloid cells (M2) or Ly6c- monocytes), were found to produce IL-10, the contribution of non-hematopoietic cells as potential IL-10 source during experimental T. congolense infection has not been addressed. Here, we report for the first time that during the chronic stage of T. congolense infection non-hematopoietic cells constitute an important source of IL-10. Our data shows that hepatocyte-derived IL-10 is mandatory for host survival and is crucial for the control of trypanosomosis-induced inflammation and associated immunopathologies such as anemia, hepatosplenomegaly and excessive tissue injury.
Asunto(s)
Hepatocitos , Evasión Inmune , Interleucina-10/inmunología , Trypanosoma congolense , Tripanosomiasis Africana , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Enfermedad Crónica , Modelos Animales de Enfermedad , Femenino , Hepatocitos/inmunología , Hepatocitos/parasitología , Hepatocitos/patología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/patología , Activación de Linfocitos , Ratones , Monocitos/inmunología , Monocitos/patología , Linfocitos T/inmunología , Linfocitos T/patología , Trypanosoma congolense/inmunología , Trypanosoma congolense/patogenicidad , Tripanosomiasis Africana/inmunología , Tripanosomiasis Africana/patologíaAsunto(s)
Núcleos Cerebelosos/patología , Hipotálamo/patología , Imagen por Resonancia Magnética , Trypanosoma brucei gambiense , Tripanosomiasis Africana/patología , Núcleos Cerebelosos/parasitología , Quimioterapia Combinada , Eflornitina/administración & dosificación , Eflornitina/uso terapéutico , Humanos , Hipotálamo/parasitología , Masculino , Persona de Mediana Edad , Nifurtimox/administración & dosificación , Nifurtimox/uso terapéutico , Tripanocidas/administración & dosificación , Tripanocidas/uso terapéutico , Tripanosomiasis Africana/tratamiento farmacológico , Tripanosomiasis Africana/parasitologíaRESUMEN
Trypanosomosis has been described as the single largest disease entity limiting livestock development in sub-Saharan Africa. The effects on ovarian weight, follicle count and retrieved oocyte characteristics in ten West African dwarf goat does (control=5, infected=5) experimentally infected with Trypanosoma brucei were investigated. The does were fed with elephant grass and supplement (15.23% CP) daily. Infected does received 4.8x105 T. brucei intravenously and thereafter, all does were synchronized using Lutalyse®. The results showed that the differences between control and infected does for ovarian weight (0.68±0.56 g and 0.40±0.09 g) and follicle count (10.50±1.25 and 2.50±1.22), respectively were significant (P<0.05). The difference in retrieved-oocytes-count between control (30, 57.7%) and infected (22, 42.3%) does was not significant (P>0.05). The differences in proportion between control and infected does for well-formed-oocytes (90.5% and 9.5%), completely-denuded-oocytes (30.8% and 69.2%) and proportion per group of oocytes with substantial-investment-of-cumulus (63.3% and 9.1%), respectively were significant (P<0.05). The difference in extensively-denuded-oocytes between control (38.9%) and infected (61.1%) does was not significant (P>0.05). These findings suggest that experimental Trypanosoma brucei infection caused reduction in ovarian weight and follicle count, number of oocytes as well as proportion of well-formed oocytes that are capable of supporting embryonic development.
Asunto(s)
Recuperación del Oocito/métodos , Oocitos/patología , Folículo Ovárico/patología , Trypanosoma brucei brucei , Tripanosomiasis Africana/patología , Animales , Femenino , Cabras , Nigeria , Tamaño de los Órganos , Ovario/patología , EmbarazoRESUMEN
OBJECTIVE: Where human African trypanosomiasis (HAT) patients are seen, failure to microscopically diagnose infections by Trypanosoma brucei gambiense in blood smears and/or cerebrospinal fluid (CSF) in the critical early stages of the disease is the single most important factor in treatment failure, a result of delayed treatment onset or its absence. We hypothesized that the enhanced sensitivity of detergent-enhanced loop-mediated isothermal amplification (LAMP) will allow for point of care (POC) detection of African trypanosomes in the CSF of HAT patients where the probability for detecting a single parasite or parasite DNA molecule in 1 µL of CSF sample is negligible by current methods. METHODOLOGY: We used LAMP targeting the multicopy pan-T. brucei repetitive insertion mobile element (RIME LAMP) and the Trypanosoma brucei gambiense 5.8S rRNA-internal transcribed spacer 2 gene (TBG1 LAMP). We tested 1 µL out of 20 µL sham or Triton X-100 treated CSFs from 73 stage-1 and 77 stage-2 HAT patients from the Central African Republic and 100 CSF negative controls. RESULTS: Under sham conditions, parasite DNA was detected by RIME and TBG1 LAMP in 1.4% of the stage-1 and stage-2 gambiense HAT CSF samples tested. After sample incubation with detergent, the number of LAMP parasite positive stage-2 CSF's increased to 26%, a value which included the 2 of the 4 CSF samples where trypanosomes were identified microscopically. Unexpected was the 41% increase in parasite positive stage-1 CSF's detected by LAMP. Cohen's kappa coefficients for RIME versus TBG1 LAMP of 0.92 (95%CI: 0.82-1.00) for stage-1 and 0.90 (95%CI: 0.80-1.00) for stage-2 reflected a high level of agreement between the data sets indicating that the results were not due to amplicon contamination, data confirmed in χ2 tests (p<0.001) and Fisher's exact probability test (p = 4.7e-13). CONCLUSION: This study detected genomic trypanosome DNA in the CSF independent of the HAT stage and may be consistent with early CNS entry and other scenarios that identify critical knowledge gaps for future studies. Detergent-enhanced LAMP could be applicable for non-invasive African trypanosome detection in human skin and saliva or as an epidemiologic tool for the determination of human (or animal) African trypanosome prevalence in areas where chronically low parasitemias are present.
Asunto(s)
Líquido Cefalorraquídeo/parasitología , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificación de Ácido Nucleico/métodos , Índice de Severidad de la Enfermedad , Trypanosoma/aislamiento & purificación , Tripanosomiasis Africana/diagnóstico , Tripanosomiasis Africana/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , República Centroafricana , Niño , Preescolar , ADN Protozoario/genética , ADN Ribosómico/química , ADN Ribosómico/genética , ADN Espaciador Ribosómico/genética , Detergentes/metabolismo , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , ARN Ribosómico 5.8S/genética , Sensibilidad y Especificidad , Trypanosoma/genética , Adulto JovenRESUMEN
The objective of the present study was to evaluate the clinical signs, electrocardiographic signs and evolution of histopathological lesions in the heart of sheep experimentally infected by Trypanosoma vivax during the acute and chronic phases of infection as well as to investigate the presence of parasitic DNA in the heart using polymerase chain reaction (PCR). Twenty-two male sheep were divided into the following four groups: G1, which consisted of six sheep infected by T. vivax that were evaluated until 20 days post-infection (dpi; acute phase); G2, which consisted of six sheep infected by T. vivax that were evaluated until 90 dpi (chronic phase); and G3 and G4 groups, which each consisted of five uninfected sheep. At the end of the experimental period, electrocardiographic evaluations and necroscopic examinations were performed. Fragments of the heart were collected and stained by Hematoxylin-Eosin and Masson's trichrome, and the fragments were also evaluated by PCR for T. vivax. G2 animals presented clinical signs suggestive of heart failure and electrocardiogram alterations characterized by prolonged P, T and QRS complex durations as well as by a cardiac electrical axis shift to the left and increased heart rate. In these animals, mononuclear multifocal myocarditis and interstitial fibrosis were also observed. PCR revealed positivity for T. vivax in two G1 animals and in all G2 animals. Thus, these findings suggested that T. vivax is responsible for the occurrence of cardiac lesions, which are related to heart failure, electrocardiographic alterations and mortality of the infected animals.
Asunto(s)
ADN Protozoario/aislamiento & purificación , Insuficiencia Cardíaca/veterinaria , Corazón/parasitología , Enfermedades de las Ovejas/parasitología , Trypanosoma vivax/patogenicidad , Tripanosomiasis Africana/veterinaria , Enfermedad Aguda , Animales , Anticuerpos Antiprotozoarios/sangre , Enfermedad Crónica/veterinaria , Electrocardiografía/veterinaria , Técnica del Anticuerpo Fluorescente Indirecta/veterinaria , Insuficiencia Cardíaca/mortalidad , Insuficiencia Cardíaca/parasitología , Inmunoglobulina G/sangre , Masculino , Miocardio/patología , Parasitemia/veterinaria , Pericarditis/parasitología , Pericarditis/patología , Pericarditis/veterinaria , Reacción en Cadena de la Polimerasa/veterinaria , Distribución Aleatoria , Ovinos , Enfermedades de las Ovejas/mortalidad , Enfermedades de las Ovejas/patología , Trypanosoma vivax/genética , Trypanosoma vivax/inmunología , Trypanosoma vivax/aislamiento & purificación , Tripanosomiasis Africana/complicaciones , Tripanosomiasis Africana/mortalidad , Tripanosomiasis Africana/patologíaRESUMEN
Trypanosomiasis is caused by a pathogenic protozoan of the genus Trypanosoma, being Trypanosoma vivax the most important agent for cattle. The aim of the present study was to demonstrate the expansion of T. vivax infection in different mesoregions of Minas Gerais, Brazil, and describe the clinicopathological findings of trypanosomiasis in cattle. The diagnosis was based on visualization of the parasite in blood smears and DNA detection of T. vivax in the blood of live cows and tissues of necropsied animals by the polymerase chain reaction (PCR). Thirty suspected herds were tested, of which 11 were positive for T. vivax. The most frequent clinical signs were anemia, apathy, drop in milk production, weight loss, reproductive disorders, and nervous signs. Concomitant diseases, such as malignant edema, pneumonia and increased cases of mastitis were associated with T. vivax infection. Three cows were necropsied and the most significant findings were low body condition score, pale mucous and spleen with white pulp hyperplasia. The results demonstrated the expansion of T. vivax infection in Minas Gerais, that PCR-associated blood smears are promising for diagnosis, and that other diseases often occur concomitantly to T. vivax infection in regions with trypanosomiasis in cattle.
Asunto(s)
Enfermedades de los Bovinos/epidemiología , Enfermedades de los Bovinos/patología , Trypanosoma vivax/aislamiento & purificación , Tripanosomiasis Africana/veterinaria , Estructuras Animales/parasitología , Estructuras Animales/patología , Animales , Sangre/parasitología , Brasil/epidemiología , Bovinos , Enfermedades de los Bovinos/parasitología , Microscopía , Reacción en Cadena de la Polimerasa , Tripanosomiasis Africana/epidemiología , Tripanosomiasis Africana/parasitología , Tripanosomiasis Africana/patologíaRESUMEN
Infection by Trypanosoma brucei gambiense is characterized by a wide array of clinical outcomes, ranging from asymptomatic to acute disease and even spontaneous cure. In this study, we investigated the association between macrophage migrating inhibitory factor (MIF), an important pro-inflammatory cytokine that plays a central role in both innate and acquired immunity, and disease outcome during T. b. gambiense infection. A comparative expression analysis of patients, individuals with latent infection and controls found that MIF had significantly higher expression in patients (nâ¯=â¯141; 1.25⯱â¯0.07; pâ¯<â¯.0001) and latent infections (nâ¯=â¯25; 1.23⯱â¯0.13; pâ¯=â¯.0005) relative to controls (nâ¯=â¯46; 0.94⯱â¯0.11). Furthermore, expression decreased significantly after treatment (patients before treatment nâ¯=â¯33; 1.40⯱â¯0.18 versus patients after treatment nâ¯=â¯33; 0.99⯱â¯0.10, pâ¯=â¯.0001). We conducted a genome wide eQTL analysis on 29 controls, 128 cases and 15 latently infected individuals for whom expression and genotype data were both available. Four loci, including one containing the chemokine CXCL13, were found to associate with MIF expression. Genes at these loci are candidate regulators of increased expression of MIF after infection. Our study is the first data demonstrating that MIF expression is elevated in T. b. gambiense-infected human hosts but does not appear to contribute to pathology.
Asunto(s)
Quimiocina CXCL13/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Sitios de Carácter Cuantitativo/inmunología , Trypanosoma brucei gambiense/patogenicidad , Tripanosomiasis Africana/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Quimiocina CXCL13/genética , Niño , Preescolar , Femenino , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Guinea , Humanos , Factores Inhibidores de la Migración de Macrófagos/genética , Masculino , Persona de Mediana Edad , Tripanosomiasis Africana/inmunología , Tripanosomiasis Africana/patología , Adulto JovenRESUMEN
The flagellated parasite Trypanosoma brucei is the causative agent of Human African Trypanosomiasis (HAT). By a mechanism not well understood yet, trypanosomes enter the central nervous system (CNS), invade the brain parenchyma, and cause a fatal encephalopathy if is not treated. Trypanosomes are fast dividing organisms that, without any immune response, would kill the host in a short time. However, infected individuals survive either 6-12 months or more than 3 years for the acute and chronic forms, respectively. Thus, only when the brain defense collapses a lethal encephalopathy will occur. Here, we evaluated interactions between trypanosomes and microglial cells, which are the primary immune effector cells within the CNS. Using co-cultures of primary microglia and parasites, we found clear evidences of trypanosome phagocytosis by microglial cells. Microglia activation was also evident; analysis of its ultrastructure showed changes that have been reported in activated microglia undergoing oxidative stress caused by infections or degenerative diseases. Accordingly, an increase of the nitric oxide production was detected in supernatants of microglia/parasite co-cultures. Altogether, our results demonstrate that microglial cells respond to the presence of the parasite, leading to parasite's engulfment and elimination.
Asunto(s)
Encefalopatías/metabolismo , Microglía/metabolismo , Trypanosoma brucei brucei/metabolismo , Tripanosomiasis Africana/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/parasitología , Encéfalo/patología , Encefalopatías/complicaciones , Encefalopatías/parasitología , Encefalopatías/patología , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/parasitología , Sistema Nervioso Central/patología , Técnicas de Cocultivo , Humanos , Activación de Macrófagos/fisiología , Macrófagos/metabolismo , Macrófagos/parasitología , Microglía/parasitología , Microglía/patología , Óxido Nítrico/biosíntesis , Óxido Nítrico/metabolismo , Estrés Oxidativo , Fagocitosis/genética , Trypanosoma brucei brucei/patogenicidad , Tripanosomiasis Africana/parasitología , Tripanosomiasis Africana/patologíaRESUMEN
Messenger RNA is modified by the addition of a 5' methylated cap structure, which protects the transcript and recruits protein complexes that mediate RNA processing and/or the initiation of translation. Two genes encoding mRNA cap methyltransferases have been identified in T. brucei: TbCMT1 and TbCGM1. Here we analysed the impact of TbCMT1 gene deletion on bloodstream form T. brucei cells. TbCMT1 was dispensable for parasite proliferation in in vitro culture. However, significantly decreased parasitemia was observed in mice inoculated with TbCMT1 null and conditional null cell lines. Using RNA-Seq, we observed that several cysteine peptidase mRNAs were downregulated in TbCMT1 null cells lines. The cysteine peptidase Cathepsin-L was also shown to be reduced at the protein level in TbCMT1 null cell lines. Our data suggest that TbCMT1 is not essential to bloodstream form T. brucei growth in vitro or in vivo but that it contributes significantly to parasite virulence in vivo.
Asunto(s)
Metiltransferasas/genética , Proteínas Protozoarias/genética , Caperuzas de ARN/genética , ARN Protozoario/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/patogenicidad , Tripanosomiasis Africana/parasitología , Animales , Femenino , Eliminación de Gen , Regulación de la Expresión Génica , Ratones Endogámicos BALB C , Trypanosoma brucei brucei/crecimiento & desarrollo , Tripanosomiasis Africana/patología , Tripanosomiasis Africana/veterinaria , Virulencia , Factores de Virulencia/genéticaRESUMEN
African trypanosomes, Trypanosoma brucei spp., are lethal pathogens that cause substantial human suffering and limit economic development in some of the world's most impoverished regions. The name Trypanosoma ("auger cell") derives from the parasite's distinctive motility, which is driven by a single flagellum. However, despite decades of study, a requirement for trypanosome motility in mammalian host infection has not been established. LC1 is a conserved dynein subunit required for flagellar motility. Prior studies with a conditional RNAi-based LC1 mutant, RNAi-K/R, revealed that parasites with defective motility could infect mice. However, RNAi-K/R retained residual expression of wild-type LC1 and residual motility, thus precluding definitive interpretation. To overcome these limitations, here we generate constitutive mutants in which both LC1 alleles are replaced with mutant versions. These double knock-in mutants show reduced motility compared to RNAi-K/R and are viable in culture, but are unable to maintain bloodstream infection in mice. The virulence defect is independent of infection route but dependent on an intact host immune system. By comparing different mutants, we also reveal a critical dependence on the LC1 N-terminus for motility and virulence. Our findings demonstrate that trypanosome motility is critical for establishment and maintenance of bloodstream infection, implicating dynein-dependent flagellar motility as a potential drug target.
Asunto(s)
Flagelos , Mutación , Trypanosoma brucei brucei , Tripanosomiasis Africana , Animales , Bovinos , Femenino , Flagelos/genética , Flagelos/metabolismo , Ratones , Ratones Endogámicos BALB C , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/patogenicidad , Tripanosomiasis Africana/genética , Tripanosomiasis Africana/metabolismo , Tripanosomiasis Africana/patologíaRESUMEN
Human African trypanosomiasis imported to nonendemic countries is rare. It is very difficult to establish the correct diagnosis of human African trypanosomiasis in nonendemic areas. We present a case of human African trypanosomiasis with MRI and FDG PET/CT findings. Head MRI showed hyperintense areas in bilateral internal capsules. Abdominal and pelvic MRI showed hepatosplenomegaly and multiple enlarged lymph nodes. FDG PET/CT showed generalized hypermetabolic lymph nodes, diffuse FDG uptake of the spleen, and hepatosplenomegaly mimicking lymphoma. In addition, FDG PET/CT revealed decreased FDG uptake in the medial occipital cortex and cardiomegaly with pericardial effusion.
Asunto(s)
Fluorodesoxiglucosa F18 , Tomografía Computarizada por Tomografía de Emisión de Positrones , Tripanosomiasis Africana/diagnóstico por imagen , Diagnóstico Diferencial , Humanos , Masculino , Persona de Mediana Edad , Tripanosomiasis Africana/patologíaRESUMEN
Trypanosoma brucei gambiense (T. b. gambiense) is the major causative agent of human African trypanosomiasis (HAT). A great variety of clinical outcomes have been observed in West African foci, probably due to complex host-parasite interactions. In order to separate the roles of parasite genetic diversity and host variability, we have chosen to precisely characterize the pathogenicity and virulence of T. b. gambiense field isolates in a mouse model. Thirteen T. b. gambiense strains were studied in experimental infections, with 20 Balb/C infected mice per isolate. Mice were monitored for 30â¯days, in which mortality, parasitemia, anemia, and weight were recorded. Mortality rate, prepatent period, and maximum parasitemia were estimated, and a survival analysis was performed to compare strain pathogenicity. Mixed models were used to assess parasitemia dynamics, weight, and changes in Packed Cell Volume (PCV). Finally, a multivariate analysis was performed to infer relationships between all variables. A large phenotypic diversity was observed. Pathogenicity was highly variable, ranging from strains that kill their host within 9â¯days to a non-pathogenic strain (no deaths during the experiment). Virulence was also variable, with maximum parasitemia values ranging from 42 million to 1 billion trypanosomes/ml. Reduced PCV and weight occurred in the first two weeks of the infection, with the exception of two strains. Finally, the global analysis highlighted three groups of strains: a first group with highly pathogenic strains showing an early mortality associated with a short prepatent period; a second group of highly virulent strains with intermediate pathogenicity; and a third group of isolates characterized by low pathogenicity and virulence patterns. Such biological differences could be related to the observed clinical diversity in HAT. A better understanding of the biological pathways underlying the observed phenotypic diversity could thus help to clarify the complex nature of the host-parasite interactions that determine the resistance/susceptibility status to T. brucei gambiense.
Asunto(s)
Interacciones Huésped-Parásitos , Parasitemia/patología , Fenotipo , Trypanosoma brucei gambiense/patogenicidad , Tripanosomiasis Africana/patología , África Occidental , Animales , Peso Corporal , Modelos Animales de Enfermedad , Índices de Eritrocitos , Eritrocitos/parasitología , Eritrocitos/patología , Humanos , Ratones , Ratones Endogámicos BALB C , Análisis Multivariante , Parasitemia/mortalidad , Parasitemia/parasitología , Análisis de Componente Principal , Análisis de Supervivencia , Trypanosoma brucei gambiense/clasificación , Trypanosoma brucei gambiense/aislamiento & purificación , Tripanosomiasis Africana/mortalidad , Tripanosomiasis Africana/parasitología , VirulenciaRESUMEN
Trypanosoma brucei glutaredoxin 2 (Grx2) is a dithiol glutaredoxin that is specifically located in the mitochondrial intermembrane space. Bloodstream form parasites lacking Grx2 or both, Grx2 and the cytosolic Grx1, are viable in vitro and infectious to mice suggesting that neither oxidoreductase is needed for survival or infectivity to mammals. A 37⯰C to 39⯰C shift changes the cellular redox milieu of bloodstream cells to more oxidizing conditions and induces a significantly stronger growth arrest in wildtype parasites compared to the mutant cells. Grx2-deficient cells ectopically expressing the wildtype form of Grx2 with its C31QFC34 active site, but not the C34S mutant, regain the sensitivity of the parental strain, indicating that the physiological role of Grx2 requires both active site cysteines. In the procyclic insect stage of the parasite, Grx2 is essential. Both alleles can be replaced if procyclic cells ectopically express authentic or C34S, but not C31S/C34S Grx2, pointing to a redox role that relies on a monothiol mechanism. RNA-interference against Grx2 causes a virtually irreversible proliferation defect. The cells adopt an elongated morphology but do not show any significant alteration in the cell cycle. The growth retardation is attenuated by high glucose concentrations. Under these conditions, procyclic cells obtain ATP by substrate level phosphorylation suggesting that Grx2 might regulate a respiratory chain component.
Asunto(s)
Adaptación Fisiológica/genética , Glutarredoxinas/genética , Trypanosoma brucei brucei/metabolismo , Tripanosomiasis Africana/metabolismo , Adenosina Trifosfato/metabolismo , Alelos , Animales , Dominio Catalítico , Proliferación Celular/genética , Citosol/metabolismo , Glutarredoxinas/química , Glutarredoxinas/metabolismo , Calor , Humanos , Ratones , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/parasitología , Membranas Mitocondriales/metabolismo , Mutación , Oxidación-Reducción , Trypanosoma brucei brucei/patogenicidad , Tripanosomiasis Africana/parasitología , Tripanosomiasis Africana/patologíaRESUMEN
The trypanosomatids, Trypanosoma brucei, Trypanosoma cruzi and Leishmania spp, are causative agents of important human diseases such as African sleeping sickness, Chagas' disease and Leishmaniasis, respectively. The high impact of these diseases on human health and economy worldwide, the unsatisfactory available chemotherapeutic options and the absence of human effective vaccines, strongly justifies the search for new drugs. The pentose phosphate pathway has been proposed to be a viable strategy to defeat several infectious diseases, including those from trypanosomatids, as it includes an oxidative branch, important in the maintenance of cell redox homeostasis, and a non-oxidative branch in which ribose 5-phosphate and erythrose 4-phosphate, precursors of nucleic acids and aromatic amino acids, are produced. This review provides an overview of the available chemotherapeutic options against these diseases and discusses the potential of genetically validated enzymes from the pentose phosphate pathway of trypanosomatids to be explored as potential drug targets.
Asunto(s)
Vía de Pentosa Fosfato/genética , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/metabolismo , Antiprotozoarios/uso terapéutico , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/metabolismo , Enfermedad de Chagas/patología , Glucosafosfato Deshidrogenasa/antagonistas & inhibidores , Glucosafosfato Deshidrogenasa/metabolismo , Humanos , Leishmaniasis/tratamiento farmacológico , Leishmaniasis/patología , Proteínas Protozoarias/antagonistas & inhibidores , Trypanosoma cruzi/metabolismo , Tripanosomiasis Africana/tratamiento farmacológico , Tripanosomiasis Africana/patologíaRESUMEN
Inositol polyphosphates are a diverse and multifaceted class of intracellular messengers omnipresent in eukaryotic cells. These water-soluble molecules regulate many aspects of fundamental cell physiology. Removing this metabolic pathway is deleterious: inositol phosphate kinase null mutations can result in lethality or substantial growth phenotypes. Inositol polyphosphate synthesis occurs through the actions of a set of kinases that phosphorylate phospholipase-generated IP3 to higher phosphorylated forms, such as the fully phosphorylated IP6 and the inositol pyrophosphates IP7 and IP8. Unicellular organisms have a reduced array of the kinases for synthesis of higher phosphorylated inositol polyphosphates, while human cells possess two metabolic routes to IP6. The enzymes responsible for inositol polyphosphate synthesis have been identified in all eukaryote genomes, although their amino acid sequence homology is often barely detectable by common search algorithms. Homology between human and microbial inositol phosphate kinases is restricted to a few catalytically important residues. Recent studies of the inositol phosphate metabolic pathways in pathogenic fungi (Cryptococcus neoformans) and protozoa (Trypanosome brucei) have revealed the importance of the highly phosphorylated inositol polyphosphates to the fitness and thus virulence of these pathogens. Given this, identification of inositol kinase inhibitors specifically targeting the kinases of pathogenic microorganisms is desirable and achievable.