RESUMEN
The flow diverter is becoming a standard device for treating cerebral aneurysms. The aim of this in vitro study was to evaluate the impact of flow complexity on the effectiveness of flow diverter stents in a cerebral aneurysm model. The flow pattern of a carotid artery was decomposed into harmonics to generate four flow patterns with different pulsatility indexes ranging from 0.72 to 1.44. The effect of flow diverters on the aneurysm was investigated by injecting red dye or erythrocytes as markers. The recorded images were postprocessed to evaluate the maximum filling of the aneurysm cavity and the washout time. There were significant differences in the cut-off flows between the markers, linked to the flow complexity. Increasing the pulsatility index altered the performance of the flow diverter. The red dye was more sensitive to changes in flow than the red blood cell markers. The flow cut-off depended on the diverter design and the diverter deployment step was crucial for reproducibility of the results. These results strongly suggest that flow complexity should be considered when selecting a flow diverter.
Asunto(s)
Circulación Cerebrovascular/fisiología , Procedimientos Endovasculares/instrumentación , Aneurisma Intracraneal/cirugía , Modelos Cardiovasculares , Stents , Arterias Carótidas/diagnóstico por imagen , Arterias Carótidas/fisiopatología , Procedimientos Endovasculares/métodos , Humanos , Aneurisma Intracraneal/diagnóstico por imagen , Aneurisma Intracraneal/fisiopatología , Diseño de Prótesis , Reproducibilidad de los Resultados , Resultado del Tratamiento , Grabación en VideoRESUMEN
The C-terminal variants G1 and G2 of apolipoprotein L1 (APOL1) confer human resistance to the sleeping sickness parasite Trypanosoma rhodesiense, but they also increase the risk of kidney disease. APOL1 and APOL3 are death-promoting proteins that are partially associated with the endoplasmic reticulum and Golgi membranes. We report that in podocytes, either APOL1 C-terminal helix truncation (APOL1Δ) or APOL3 deletion (APOL3KO) induces similar actomyosin reorganization linked to the inhibition of phosphatidylinositol-4-phosphate [PI(4)P] synthesis by the Golgi PI(4)-kinase IIIB (PI4KB). Both APOL1 and APOL3 can form K+ channels, but only APOL3 exhibits Ca2+-dependent binding of high affinity to neuronal calcium sensor-1 (NCS-1), promoting NCS-1-PI4KB interaction and stimulating PI4KB activity. Alteration of the APOL1 C-terminal helix triggers APOL1 unfolding and increased binding to APOL3, affecting APOL3-NCS-1 interaction. Since the podocytes of G1 and G2 patients exhibit an APOL1Δ or APOL3KO-like phenotype, APOL1 C-terminal variants may induce kidney disease by preventing APOL3 from activating PI4KB, with consecutive actomyosin reorganization of podocytes.
Asunto(s)
Actomiosina/metabolismo , Apolipoproteína L1/química , Apolipoproteína L1/genética , Apolipoproteínas L/metabolismo , Enfermedades Renales/metabolismo , Mutación/genética , Secuencia de Aminoácidos , Apolipoproteína L1/orina , Calcio/metabolismo , Línea Celular , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Humanos , Enfermedades Renales/orina , Glomérulos Renales/metabolismo , Glomérulos Renales/patología , Antígenos de Histocompatibilidad Menor/metabolismo , Proteínas Sensoras del Calcio Neuronal/metabolismo , Neuropéptidos/metabolismo , Fenotipo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Podocitos/efectos de los fármacos , Podocitos/metabolismo , Podocitos/ultraestructura , Poli I-C/farmacología , Canales de Potasio/metabolismo , Unión Proteica/efectos de los fármacos , Estructura Secundaria de ProteínaRESUMEN
The primate-specific serum protein apolipoprotein L1 (APOL1) is the only secreted member of a family of cell death promoting proteins 1-4 . APOL1 kills the bloodstream parasite Trypanosoma brucei brucei, but not the human sleeping sickness agents T.b. rhodesiense and T.b. gambiense 3 . We considered the possibility that intracellular members of the APOL1 family, against which extracellular trypanosomes could not have evolved resistance, could kill pathogenic T. brucei subspecies. Here we show that recombinant APOL3 (rAPOL3) kills all African trypanosomes, including T.b. rhodesiense, T.b. gambiense and the animal pathogens Trypanosoma evansi, Trypanosoma congolense and Trypanosoma vivax. However, rAPOL3 did not kill more distant trypanosomes such as Trypanosoma theileri or Trypanosoma cruzi. This trypanolytic potential was partially shared by rAPOL1 from Papio papio (rPpAPOL1). The differential killing ability of rAPOL3 and rAPOL1 was associated with a distinct dependence on acidic pH for activity. Due both to its instability and toxicity when injected into mice, rAPOL3 cannot be used for the treatment of infection, but an experimental rPpAPOL1 mutant inspired by APOL3 exhibited enhanced trypanolytic activity in vitro and the ability to completely inhibit T.b. gambiense infection in mice. We conclude that pH dependence influences the trypanolytic potential of rAPOLs.
Asunto(s)
Apolipoproteína L1/farmacología , Apolipoproteínas L/farmacología , Trypanosoma/efectos de los fármacos , Animales , Apolipoproteína L1/genética , Apolipoproteínas L/genética , Concentración de Iones de Hidrógeno , Ratones , Papio papio , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/farmacología , Trypanosoma/fisiología , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei gambiense/efectos de los fármacos , Trypanosoma brucei rhodesiense/efectos de los fármacos , Trypanosoma congolense/efectos de los fármacos , Trypanosoma vivax/efectos de los fármacos , Tripanosomiasis Africana/parasitologíaRESUMEN
Phosphodiesterases (PDEs) may modulate inflammatory pathways, but PDE expression is poorly documented in humans with sepsis. Using quantitative PCR on whole blood leukocytes, we characterized PDE mRNA expression in healthy volunteers (n = 20), healthy volunteers given lipopolysaccharide (LPS; n = 18), and critically ill patients with (n = 20) and without (n = 20) sepsis. PDE4B protein expression was also studied in magnetic-activated cell sorting (MACS)-isolated CD15+ neutrophils (from 7 healthy volunteers, 5 patients without and 5 with sepsis). We studied relationships between PDE expression, HLA-DR (mRNA and expression on CD14+ monocytes), tumor necrosis factor (TNF)-α, and interleukin (IL)-10 levels. LPS administration in volunteers was associated with increases in PDE4B and PDE4D and decreases in PDE4A and PDE7A mRNAs. The observed global down-regulation of the HLA-DR complex was correlated with PDE7A. Critically ill patients had lower TNF-α/IL-10 mRNA ratios than the volunteers had and global down-regulation of the HLA-DR complex. Septic patients had persistently lower mRNA levels of PDE7A, PDE4A, and 4B (also at a protein level) and decreasing levels of PDE4D over time. Low PDE4D mRNA levels correlated negatively with HLA-DMA and HLA-DMB. LPS administration and sepsis are, therefore, associated with different PDE mRNA expression patterns. The effect of PDE changes on immune dysfunction and HLA-DR expression requires further investigation.
Asunto(s)
Antígenos HLA-DR/metabolismo , Leucocitos/enzimología , Lipopolisacáridos/farmacología , Neutrófilos/enzimología , Hidrolasas Diéster Fosfóricas/metabolismo , Sepsis/fisiopatología , Estudios de Casos y Controles , Humanos , Leucocitos/efectos de los fármacos , Masculino , Neutrófilos/efectos de los fármacos , Hidrolasas Diéster Fosfóricas/genética , Estudios ProspectivosRESUMEN
Delayed neutrophil apoptosis has been demonstrated in sepsis and may contribute to organ damage. It has recently been proposed that apolipoprotein L (ApoL) may be involved in programmed cell death, but the expression and functions of ApoLs in leukocytes (especially neutrophils) during sepsis and other inflammatory conditions are currently unknown. In this prospective observational study in a 36-bed university hospital medicosurgical intensive care unit (ICU), we included 78 adult ICU patients with (nâ=â41) or without (nâ=â37) sepsis and 47 healthy volunteers. We analyzed ApoL mRNA expression using quantitative polymerase chain reaction in whole blood leukocytes and protein expression in CD15 isolated neutrophils using Western blotting. Neutrophil apoptosis was assessed using the APO-BRDU method. Apolipoprotein L mRNA was downregulated in whole blood leukocytes and neutrophils in ICU patients compared with in healthy volunteers, and this effect translated at the protein level as indicated by Western blot analysis of neutrophils. There was a negative correlation between ApoL expression in neutrophils and C-reactive protein levels and a positive correlation between the number of apoptotic neutrophils and mRNA levels of ApoL1 and ApoL2. The degree of neutrophil apoptosis in critically ill patients is therefore correlated with modified expression profiles of ApoLs.
Asunto(s)
Apolipoproteína L1/metabolismo , Muerte Celular/fisiología , Neutrófilos/citología , Neutrófilos/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis/genética , Apoptosis/fisiología , Proteína C-Reactiva/metabolismo , Muerte Celular/genética , Enfermedad Crítica , Femenino , Voluntarios Sanos , Humanos , Unidades de Cuidados Intensivos/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Sepsis/metabolismoRESUMEN
BACKGROUND: Metabolic syndrome (MetS) is characterized by systemic disturbances that increase cardiovascular risk. Adiponectin (Ad) exhibits a cardioprotective function because of its anti-inflammatory and anti-atherosclerotic properties. In the bloodstream, this adipocytokine circulates on multimers (Admer), among which high molecular weight (HMW) are the most active forms. Because alterations of Ad plasmatic levels, Admer distribution and receptor (AdipoR) expression have been described in murine models and obese patients, strategies that aim to enhance Ad production or its effect on target tissues are the subject of intense investigations. While exercise training is well known to be beneficial for reducing cardiovascular risk, the contribution of Ad is still controversial. Our aim was to evaluate the effect of exercise training on Ad production, Admer distribution and AdipoR muscle expression in a murine model of MetS. METHODS: At 6 weeks of age, mice were submitted to a standard (SF) or high-fat high-sugar (HF) diet for 10 weeks. After 2 weeks, the SF- and HF-fed animals were randomly assigned to a training program (SFT, HFT) or not (SFC, HFC). The trained groups were submitted to sessions of running on a treadmill 5 days a week. RESULTS AND CONCLUSIONS: The HF mice presented the key problems associated with MetS (increased caloric intake, body weight, glycemia and fat mass), a change in Admer distribution in favor of the less-active forms and increased AdipoR2 expression in muscle. In contrast, exercise training reversed some of the adverse effects of a HF diet (increased glucose tolerance, better caloric intake control) without any modifications in Ad production and Admer distribution. However, increased AdipoR1 muscle expression was observed in trained mice, but this effect was hampered by HF diet. These data corroborate a recent hypothesis suggesting a functional divergence between AdipoR1 and AdipoR2, with AdipoR1 having the predominant protective action on metabolic function.
RESUMEN
Glutathione (GSH) is the keystone of the cellular response toward oxidative stress. Elevated GSH content correlates with increased resistance to chemotherapy and radiotherapy of head and neck (HN) tumors. The purpose of the present cross-sectional study was to evaluate whether the expression of glutamate-cysteine ligase (GCL) accounts for the increased GSH availability observed in HN squamous cell carcinoma (SCC). For that purpose, the messenger (m)RNA levels of the modifier (M) and catalytic (C) subunits of GCL and its putative regulators (namely, nuclear factor erythroid 2-related factor 2, heme oxygenase-1 and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) were monitored in 35 surgical resections of untreated HNSCC. The localization of GCLM was evaluated using in situ hybridization and immunohistochemistry. GCLM expression was significantly increased in tumor samples, compared with normal mucosa, both at the mRNA and protein level (P=0.029), but the pathway of GCLM activation remains to be elucidated. Protein expression of GCLM was detected in the cytoplasm and nucleus. GCLM and the proliferation marker Ki-67 displayed a similar distribution, being both mainly expressed at the periphery of tumor lobules. The present study reported increased expression of GCL and the rate-limiting enzyme of GSH synthesis, within HNSCC. The nuclear localization of GCLM and the concomitant expression of Ki-67 suggested that the localization of GSH synthesis contributes to the protection against oxidative stress within hotspots of cell proliferation.
RESUMEN
Apolipoproteins L (ApoLs) are Bcl-2-like proteins expressed under inflammatory conditions in myeloid and endothelial cells. We found that Toll-like receptor (TLR) stimuli, particularly the viral mimetic polyinosinic:polycytidylic acid (poly(I:C)), specifically induce ApoLs7/11 subfamilies in murine CD8α(+) dendritic cells (DCs). This induction requires the TLR3/TRIF (where TRIF is TIR domain containing adapter-inducing interferon ß) signaling pathway and is dependent on IFN-ß in all ApoLs subfamilies except for ApoL7c. Poly(I:C) treatment of DCs is also associated with induction of both cell death and autophagy. ApoLs expression is related to promotion of DC death by poly(I:C), as ApoLs7/11 knockdown increases DC survival and ApoLs7 are associated with the anti-apoptotic protein Bcl-xL (where Bcl-xL is B-cell lymphoma extra large). Similarly, in human monocyte-derived DCs poly(I:C) induces both cell death and the expression of ApoLs, principally ApoL3. Finally, the BH3-like peptide of ApoLs appears to be involved in the DC death-promoting activity. We would like to propose that ApoLs are involved in cell death linked to activation of DCs by viral stimuli.
Asunto(s)
Apolipoproteínas/inmunología , Apoptosis , Células Dendríticas/citología , Transducción de Señal , Receptor Toll-Like 3/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Animales , Antígenos CD8/metabolismo , Línea Celular , Células Cultivadas , Células Dendríticas/metabolismo , Humanos , Interferón beta/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Poli I-C/farmacología , Isoformas de Proteínas/inmunología , Proteína bcl-X/metabolismoRESUMEN
Platelet spreading and retraction play a pivotal role in the platelet plugging and the thrombus formation. In routine laboratory, platelet function tests include exhaustive information about the role of the different receptors present at the platelet surface without information on the 3D structure of platelet aggregates. In this work, we develop, a method in Digital Holographic Microscopy (DHM) to characterize the platelet and aggregate 3D shapes using the quantitative phase contrast imaging. This novel method is suited to the study of platelets physiology in clinical practice as well as the development of new drugs.
RESUMEN
Human-infectious trypanosomes such as Trypanosoma cruzi, T. brucei rhodesiense, and T. b. gambiense can be discriminated from those only infecting animals by their resistance to normal human serum (NHS). These parasites are naturally resistant to trypanolysis induced by the human-specific pore-forming serum protein apolipoprotein L1 (ApoL-1). T. lewisi, a worldwide distributed parasite, has been considered as rat-specific and non-pathogenic to the natural hosts. Here we provide evidence that 19 tested T. lewisi isolates from Thailand and China share resistance to NHS. Further investigation on one selected isolate CPO02 showed that it could resist at least 90% NHS or 30 µg/ml recombinant human ApoL-1 (rhApoL-1) in vitro, in contrast to T. b. brucei which could not survive in 0.0001% NHS and 0.1 µg/ml rhApoL-1. In vivo tests in rats also demonstrated that this parasite is fully resistant to lysis by NHS. Together with recent reports of atypical human infection by T. lewisi, these data allow the conclusion that T. lewisi is potentially an underestimated and thus a neglected human pathogen.
Asunto(s)
Apolipoproteínas/metabolismo , Lipoproteínas HDL/metabolismo , Suero/inmunología , Suero/parasitología , Trypanosoma lewisi/inmunología , Trypanosoma lewisi/fisiología , Animales , Apolipoproteína L1 , Supervivencia Celular/efectos de los fármacos , China , Humanos , Ratas , Tailandia , Trypanosoma lewisi/efectos de los fármacos , Trypanosoma lewisi/aislamiento & purificaciónRESUMEN
Human apolipoprotein L1 (APOL1) kills African trypanosomes except Trypanosoma rhodesiense and Trypanosoma gambiense, the parasites causing sleeping sickness. APOL1 uptake into trypanosomes is favoured by its association with the haptoglobin-related protein-haemoglobin complex, which binds to the parasite surface receptor for haptoglobin-haemoglobin. As haptoglobin-haemoglobin can saturate the receptor, APOL1 uptake is increased in haptoglobin-poor (hypohaptoglobinaemic) serum (HyHS). While T. rhodesiense resists APOL1 by RNA polymerase I (pol-I)-mediated expression of the serum resistance-associated (SRA) protein, T. gambiense resists by pol-II-mediated expression of the T. gambiense-specific glycoprotein (TgsGP). Moreover, in T. gambiense resistance to HyHS is linked to haptoglobin-haemoglobin receptor inactivation by mutation. We report that unlike T. gambiense, T. rhodesiense possesses a functional haptoglobin-haemoglobin receptor, and that like T. gambiense experimentally provided with active receptor, this parasite is killed in HyHS because of receptor-mediated APOL1 uptake. However, T. rhodesiense could adapt to low haptoglobin by increasing transcription of SRA. When assayed in Trypanosoma brucei, resistance to HyHS occurred with pol-I-, but not with pol-II-mediated SRA expression. Similarly, T. gambiense provided with active receptor acquired resistance to HyHS only when TgsGP was moved to a pol-I locus. Thus, transcription by pol-I favours adaptive gene regulation, explaining the presence of SRA in a pol-I locus.
Asunto(s)
Apolipoproteínas/toxicidad , Regulación de la Expresión Génica , Lipoproteínas HDL/toxicidad , ARN Polimerasa I/metabolismo , Transcripción Genética , Trypanosoma brucei rhodesiense/efectos de los fármacos , Trypanosoma brucei rhodesiense/fisiología , Adaptación Fisiológica , Apolipoproteína L1 , Haptoglobinas/análisis , Humanos , Glicoproteínas de Membrana/biosíntesis , Receptores de Superficie Celular/metabolismo , Suero/química , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei gambiense/efectos de los fármacos , Trypanosoma brucei gambiense/genética , Trypanosoma brucei gambiense/crecimiento & desarrollo , Trypanosoma brucei rhodesiense/genética , Trypanosoma brucei rhodesiense/crecimiento & desarrolloRESUMEN
Normal human serum (NHS) confers human resistance to infection by the parasite Trypanosoma brucei owing to the trypanolytic activity of apolipoprotein L1 (APOL1), present in two serum complexes termed Trypanolytic Factors (TLF-1 and -2). In order to identify parasite components involved in the intracellular trafficking and activity of TLFs, an inducible RNA interference (RNAi) genomic DNA library constructed in bloodstream form T. brucei was subjected to RNAi induction and selection for resistant parasites under NHS conditions favouring either TLF-1 or TLF-2 uptake. While TLF-1 conditions readily selected the haptoglobin-haemoglobin (HP-HB) surface receptor TbHpHbR as expected, given its known ability to bind TLF-1, under TLF-2 conditions no specific receptor for TLF-2 was identified. Instead, the screen allowed the identification of five distinct factors expected to be involved in the assembly of the vacuolar proton pump V-ATPase and consecutive endosomal acidification. These data confirm that lowering the pH during endocytosis is required for APOL1 toxic activity.
Asunto(s)
Apolipoproteínas/metabolismo , Citotoxinas/metabolismo , Lipoproteínas HDL/metabolismo , Suero/metabolismo , Trypanosoma brucei brucei/efectos de los fármacos , Apolipoproteína L1 , Endocitosis , Concentración de Iones de Hidrógeno , Proteínas Protozoarias/análisis , Proteínas Protozoarias/genética , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/genética , Trypanosoma brucei brucei/genéticaRESUMEN
Humans can survive bloodstream infection by African trypanosomes, owing to the activity of serum complexes that have efficient trypanosome-killing ability. The two trypanosome subspecies that are responsible for human sleeping sickness--Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense--can evade this defence mechanism by expressing distinct resistance proteins. In turn, sequence variation in the gene that encodes the trypanosome-killing component in human serum has enabled populations in western Africa to restore resistance to T. b. rhodesiense, at the expense of the high probability of developing kidney sclerosis. These findings highlight the importance of resistance to trypanosomes in human evolution.
Asunto(s)
Trypanosoma brucei gambiense/inmunología , Trypanosoma brucei rhodesiense/inmunología , Tripanosomiasis Africana/inmunología , Animales , Apolipoproteína L1 , Apolipoproteínas/genética , Apolipoproteínas/metabolismo , Evolución Biológica , Resistencia a la Enfermedad/inmunología , Humanos , Lipoproteínas HDL/genética , Lipoproteínas HDL/metabolismo , Trypanosoma brucei gambiense/metabolismo , Trypanosoma brucei rhodesiense/metabolismo , Tripanosomiasis Africana/genética , Tripanosomiasis Africana/metabolismoRESUMEN
BACKGROUND: In order to promote infection, the blood-borne parasite Trypanosoma brucei releases factors that upregulate arginase expression and activity in myeloid cells. METHODOLOGY/PRINCIPAL FINDINGS: By screening a cDNA library of T. brucei with an antibody neutralizing the arginase-inducing activity of parasite released factors, we identified a Kinesin Heavy Chain isoform, termed TbKHC1, as responsible for this effect. Following interaction with mouse myeloid cells, natural or recombinant TbKHC1 triggered SIGN-R1 receptor-dependent induction of IL-10 production, resulting in arginase-1 activation concomitant with reduction of nitric oxide (NO) synthase activity. This TbKHC1 activity was IL-4Rα-independent and did not mirror M2 activation of myeloid cells. As compared to wild-type T. brucei, infection by TbKHC1 KO parasites was characterized by strongly reduced parasitaemia and prolonged host survival time. By treating infected mice with ornithine or with NO synthase inhibitor, we observed that during the first wave of parasitaemia the parasite growth-promoting effect of TbKHC1-mediated arginase activation resulted more from increased polyamine production than from reduction of NO synthesis. In late stage infection, TbKHC1-mediated reduction of NO synthesis appeared to contribute to liver damage linked to shortening of host survival time. CONCLUSION: A kinesin heavy chain released by T. brucei induces IL-10 and arginase-1 through SIGN-R1 signaling in myeloid cells, which promotes early trypanosome growth and favors parasite settlement in the host. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 contributes to liver pathogenicity.
Asunto(s)
Arginasa/inmunología , Cinesinas/inmunología , Proteínas Protozoarias/inmunología , Trypanosoma brucei brucei/inmunología , Tripanosomiasis Africana/inmunología , Animales , Arginasa/genética , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/inmunología , Activación Enzimática/genética , Activación Enzimática/inmunología , Interleucina-10/genética , Interleucina-10/inmunología , Cinesinas/genética , Lectinas Tipo C/genética , Lectinas Tipo C/inmunología , Ratones , Ratones Noqueados , Óxido Nítrico/genética , Óxido Nítrico/inmunología , Proteínas Protozoarias/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/inmunología , Trypanosoma brucei brucei/genética , Tripanosomiasis Africana/genética , Tripanosomiasis Africana/patologíaRESUMEN
The African parasite Trypanosoma brucei gambiense accounts for 97% of human sleeping sickness cases. T. b. gambiense resists the specific human innate immunity acting against several other tsetse-fly-transmitted trypanosome species such as T. b. brucei, the causative agent of nagana disease in cattle. Human immunity to some African trypanosomes is due to two serum complexes designated trypanolytic factors (TLF-1 and -2), which both contain haptoglobin-related protein (HPR) and apolipoprotein LI (APOL1). Whereas HPR association with haemoglobin (Hb) allows TLF-1 binding and uptake via the trypanosome receptor TbHpHbR (ref. 5), TLF-2 enters trypanosomes independently of TbHpHbR (refs 4, 5). APOL1 kills trypanosomes after insertion into endosomal/lysosomal membranes. Here we report that T. b. gambiense resists TLFs via a hydrophobic ß-sheet of the T. b. gambiense-specific glycoprotein (TgsGP), which prevents APOL1 toxicity and induces stiffening of membranes upon interaction with lipids. Two additional features contribute to resistance to TLFs: reduction of sensitivity to APOL1 requiring cysteine protease activity, and TbHpHbR inactivation due to a L210S substitution. According to such a multifactorial defence mechanism, transgenic expression of T. b. brucei TbHpHbR in T. b. gambiense did not cause parasite lysis in normal human serum. However, these transgenic parasites were killed in hypohaptoglobinaemic serum, after high TLF-1 uptake in the absence of haptoglobin (Hp) that competes for Hb and receptor binding. TbHpHbR inactivation preventing high APOL1 loading in hypohaptoglobinaemic serum may have evolved because of the overlapping endemic area of T. b. gambiense infection and malaria, the main cause of haemolysis-induced hypohaptoglobinaemia in western and central Africa.
Asunto(s)
Apolipoproteínas/sangre , Apolipoproteínas/metabolismo , Lipoproteínas HDL/sangre , Lipoproteínas HDL/metabolismo , Trypanosoma brucei gambiense/fisiología , África , Animales , Animales Modificados Genéticamente , Apolipoproteína L1 , Apolipoproteínas/antagonistas & inhibidores , Apolipoproteínas/toxicidad , Membrana Celular/química , Membrana Celular/metabolismo , Proteasas de Cisteína/metabolismo , Haptoglobinas/metabolismo , Hemoglobinas/metabolismo , Hemólisis , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Metabolismo de los Lípidos , Lipoproteínas HDL/antagonistas & inhibidores , Lipoproteínas HDL/química , Lipoproteínas HDL/toxicidad , Parásitos/patogenicidad , Parásitos/fisiología , Estructura Secundaria de Proteína , Suero/química , Suero/parasitología , Trypanosoma brucei gambiense/efectos de los fármacos , Trypanosoma brucei gambiense/patogenicidad , Tripanosomiasis Africana/parasitología , Glicoproteínas Variantes de Superficie de Trypanosoma/química , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismoRESUMEN
The parasite Trypanosoma brucei possesses a large family of transmembrane receptor-like adenylate cyclases. Activation of these enzymes requires the dimerization of the catalytic domain and typically occurs under stress. Using a dominant-negative strategy, we found that reducing adenylate cyclase activity by about 50% allowed trypanosome growth but reduced the parasite's ability to control the early innate immune defense of the host. Specifically, activation of trypanosome adenylate cyclase resulting from parasite phagocytosis by liver myeloid cells inhibited the synthesis of the trypanosome-controlling cytokine tumor necrosis factor-α through activation of protein kinase A in these cells. Thus, adenylate cyclase activity of lyzed trypanosomes favors early host colonization by live parasites. The role of adenylate cyclases at the host-parasite interface could explain the expansion and polymorphism of this gene family.
Asunto(s)
Adenilil Ciclasas/metabolismo , Inmunidad Innata , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/inmunología , Tripanosomiasis Africana/inmunología , Adenilil Ciclasas/química , Adenilil Ciclasas/genética , Animales , Dominio Catalítico , Línea Celular , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática , Interacciones Huésped-Parásitos , Hígado/citología , Ratones , Ratones Endogámicos C57BL , Mutagénesis Sitio-Dirigida , Células Mieloides/inmunología , Parasitemia , Fagocitosis , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/metabolismo , Trypanosoma brucei brucei/crecimiento & desarrollo , Tripanosomiasis Africana/metabolismo , Tripanosomiasis Africana/parasitología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/sangreRESUMEN
Antigenic variation of the parasite Trypanosoma brucei operates by monoallelic expression of a variant surface glycoprotein (VSG) from a collection of multiple telomeric expression sites (ESs). Each of these ESs harbours a long polycistronic transcription unit containing several expression site-associated genes (ESAGs). ESAG4 copies encode bloodstream stage-specific adenylyl cyclases (AC) and belong to a larger gene family of around 80 members, the majority of which, termed genes related to ESAG4 (GRESAG4s), are not encoded in ESs and are expressed constitutively in the life cycle. Here we report that ablation of ESAG4 from the active ES did not affect parasite growth, neither in culture nor upon rodent infection, and did not significantly change total AC activity. In contrast, inducible RNAi-mediated knock-down of an AC subfamily that includes ESAG4 and two ESAG4-like GRESAG4 (ESAG4L) genes, decreased total AC activity and induced a lethal phenotype linked to impaired cytokinesis. In the Δesag4 line compensatory upregulation of apparently functionally redundant ESAG4L genes was observed, suggesting that the ESAG4/ESAG4L-subfamily ACs are involved in the control of cell division. How deregulated adenylyl cyclases or cAMP might impair cytokinesis is discussed.
Asunto(s)
Adenilil Ciclasas/metabolismo , Citocinesis , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/fisiología , Adenilil Ciclasas/genética , Análisis por Conglomerados , Eliminación de Gen , Expresión Génica , Prueba de Complementación Genética , Filogenia , Proteínas Protozoarias/genética , Homología de Secuencia de Aminoácido , Trypanosoma brucei brucei/genéticaRESUMEN
African Americans have higher rates of kidney disease than European Americans. Here, we show that, in African Americans, focal segmental glomerulosclerosis (FSGS) and hypertension-attributed end-stage kidney disease (H-ESKD) are associated with two independent sequence variants in the APOL1 gene on chromosome 22 {FSGS odds ratio = 10.5 [95% confidence interval (CI) 6.0 to 18.4]; H-ESKD odds ratio = 7.3 (95% CI 5.6 to 9.5)}. The two APOL1 variants are common in African chromosomes but absent from European chromosomes, and both reside within haplotypes that harbor signatures of positive selection. ApoL1 (apolipoprotein L-1) is a serum factor that lyses trypanosomes. In vitro assays revealed that only the kidney disease-associated ApoL1 variants lysed Trypanosoma brucei rhodesiense. We speculate that evolution of a critical survival factor in Africa may have contributed to the high rates of renal disease in African Americans.
Asunto(s)
Apolipoproteínas/genética , Negro o Afroamericano/genética , Glomeruloesclerosis Focal y Segmentaria/genética , Fallo Renal Crónico/genética , Lipoproteínas HDL/genética , Polimorfismo de Nucleótido Simple , Trypanosoma brucei rhodesiense/metabolismo , África , Alelos , Apolipoproteína L1 , Apolipoproteínas/sangre , Apolipoproteínas/metabolismo , Estudios de Casos y Controles , Estudios de Cohortes , Frecuencia de los Genes , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Glomeruloesclerosis Focal y Segmentaria/etnología , Haplotipos , Humanos , Hipertensión/complicaciones , Inmunidad Innata , Fallo Renal Crónico/etnología , Fallo Renal Crónico/etiología , Desequilibrio de Ligamiento , Lipoproteínas HDL/sangre , Lipoproteínas HDL/metabolismo , Modelos Logísticos , Proteínas Motoras Moleculares/genética , Cadenas Pesadas de Miosina/genética , Proteínas Recombinantes/metabolismo , Selección Genética , Eliminación de Secuencia , Tripanosomiasis Africana/genética , Tripanosomiasis Africana/parasitologíaRESUMEN
During the course of mammalian infection, African trypanosomes undergo extensive cellular differentiation, as actively dividing long slender (SL) forms progressively transform into intermediate (I) forms and finally quiescent G(1)/G(0)-locked short stumpy (ST) forms. ST forms maintain adaptations compatible with their survival in the mammalian bloodstream, such as high endocytic activity, but they already show preadaptations to the insect midgut conditions. The nutritional requirements of ST forms must differ from those of SL forms because the ST forms stop multiplying. We report that the uptake of several ligands was reduced in ST forms compared with that in SL forms. In particular, the haptoglobin-hemoglobin (Hp-Hb) complex was no longer taken up due to dramatic downregulation of its cognate receptor, TbHpHbR. As this receptor also allows uptake of trypanolytic particles from human serum, ST forms were resistant to trypanolysis by human serum lipoproteins. These observations allowed both flow cytometry analysis of SL-to-ST differentiation and the generation of homogeneous ST populations after positive selection upon exposure to trypanolytic particles. In addition, we observed that in ST forms the lysosome relocates anterior to the nucleus. Altogether, we identified novel morphological and molecular features that characterize SL-to-ST differentiation.
Asunto(s)
Diferenciación Celular , Endocitosis , Estadios del Ciclo de Vida , Transducción de Señal , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/crecimiento & desarrollo , Animales , División Celular , Frío , Regulación hacia Abajo/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Espacio Intracelular/metabolismo , Ligandos , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Ratones , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Fracciones Subcelulares/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/ultraestructuraRESUMEN
Nuclear transcription of Trypanosoma brucei displays unusual features. Most protein-coding genes are organized in large directional gene clusters, which are transcribed polycistronically by RNA polymerase II (pol II) with subsequent processing to generate mature mRNA. Here, we describe the identification and characterization of two trypanosome homologues of transcription elongation factor TFIIS (TbTFIIS1 and TbTFIIS2-1). TFIIS has been shown to aid transcription elongation by relieving arrested pol II. Our phylogenetic analysis demonstrated the existence of four independent TFIIS expansions across eukaryotes. While TbTFIIS1 contains only the canonical domains II and III, the N-terminus of TbTFIIS2-1 contains a PWWP domain and a domain I. TbTFIIS1 and TbTFIIS2-1 are expressed in procyclic and bloodstream form cells and localize to the nucleus in similar, but distinct, punctate patterns throughout the cell cycle. Neither TFIIS protein was enriched in the major pol II sites of spliced-leader RNA transcription. Single RNA interference (RNAi)-mediated knock-down and knockout showed that neither protein is essential. Double knock-down, however, impaired growth. Repetitive failure to generate a double knockout of TbTFIIS1 and TbTFIIS2-1 strongly suggests synthetical lethality and thus an essential function shared by the two proteins in trypanosome growth.