RESUMO
Endocytosis of caveolae has previously been implicated in the repair of plasma membrane wounds. Here, we show that caveolin-1-deficient fibroblasts lacking caveolae upregulate a tubular endocytic pathway and have a reduced capacity to reseal after permeabilization with pore-forming toxins compared with wild-type cells. Silencing endophilin-A2 expression inhibited fission of endocytic tubules and further reduced plasma membrane repair in cells lacking caveolin-1, supporting a role for tubular endocytosis as an alternative pathway for the removal of membrane lesions. Endophilin-A2 was visualized in association with cholera toxin B-containing endosomes and was recruited to recently formed intracellular vacuoles containing Trypanosoma cruzi, a parasite that utilizes the plasma membrane wounding repair pathway to invade host cells. Endophilin-A2 deficiency inhibited T. cruzi invasion, and fibroblasts deficient in both caveolin-1 and endophilin-A2 did not survive prolonged exposure to the parasites. These findings reveal a novel crosstalk between caveolin-1 and endophilin-A2 in the regulation of clathrin-independent endocytosis and plasma membrane repair, a process that is subverted by T. cruzi parasites for cell invasion.
Assuntos
Aciltransferases/fisiologia , Membrana Celular , Endocitose , Trypanosoma cruzi , Células 3T3 , Animais , Cavéolas , Clatrina , CamundongosRESUMO
Chemokines and other chemoattractants direct leukocyte migration and are essential for the development and delivery of immune and inflammatory responses. To probe the molecular mechanisms that underlie chemoattractant-guided migration, we did an RNA-mediated interference screen that identified several members of the synaptotagmin family of calcium-sensing vesicle-fusion proteins as mediators of cell migration: SYT7 and SYTL5 were positive regulators of chemotaxis, whereas SYT2 was a negative regulator of chemotaxis. SYT7-deficient leukocytes showed less migration in vitro and in a gout model in vivo. Chemoattractant-induced calcium-dependent lysosomal fusion was impaired in SYT7-deficient neutrophils. In a chemokine gradient, SYT7-deficient lymphocytes accumulated lysosomes in their uropods and had impaired uropod release. Our data identify a molecular pathway required for chemotaxis that links chemoattractant-induced calcium flux to exocytosis and uropod release.
Assuntos
Movimento Celular/fisiologia , Sinaptotagminas/metabolismo , Animais , Quimiocina CXCL12/metabolismo , Quimiotaxia , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase , Receptores CXCR4/metabolismo , Sinaptotagmina II/metabolismo , Sinaptotagminas/genética , Linfócitos T/imunologiaRESUMO
Gaucher disease (GD) is caused by bi-allelic mutations in GBA1, the gene that encodes acid ß-glucocerebrosidase (GCase). Individuals affected by GD have hematologic, visceral and bone abnormalities, and in severe cases there is also neurodegeneration. To shed light on the mechanisms by which mutant GBA1 causes bone disease, we examined the ability of human induced pluripotent stem cells (iPSC) derived from patients with Types 1, 2 and 3 GD, to differentiate to osteoblasts and carry out bone deposition. Differentiation of GD iPSC to osteoblasts revealed that these cells had developmental defects and lysosomal abnormalities that interfered with bone matrix deposition. Compared with controls, GD iPSC-derived osteoblasts exhibited reduced expression of osteoblast differentiation markers, and bone matrix protein and mineral deposition were defective. Concomitantly, canonical Wnt/ß catenin signaling in the mutant osteoblasts was downregulated, whereas pharmacological Wnt activation with the GSK3ß inhibitor CHIR99021 rescued GD osteoblast differentiation and bone matrix deposition. Importantly, incubation with recombinant GCase (rGCase) rescued the differentiation and bone-forming ability of GD osteoblasts, demonstrating that the abnormal GD phenotype was caused by GCase deficiency. GD osteoblasts were also defective in their ability to carry out Ca2+-dependent exocytosis, a lysosomal function that is necessary for bone matrix deposition. We conclude that normal GCase enzymatic activity is required for the differentiation and bone-forming activity of osteoblasts. Furthermore, the rescue of bone matrix deposition by pharmacological activation of Wnt/ß catenin in GD osteoblasts uncovers a new therapeutic target for the treatment of bone abnormalities in GD.
Assuntos
Doença de Gaucher/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Lisossomos/patologia , Osteoblastos/patologia , Matriz Óssea/patologia , Estudos de Casos e Controles , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Exocitose/genética , Doença de Gaucher/genética , Glucosilceramidase/genética , Glucosilceramidase/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Lisossomos/genética , Mutação , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , Via de Sinalização Wnt , beta Catenina/metabolismoRESUMO
Iron is essential for many cellular processes, but can generate highly toxic hydroxyl radicals in the presence of oxygen. Therefore, intracellular iron accumulation must be tightly regulated, by balancing uptake with storage or export. Iron uptake in Leishmania is mediated by the coordinated action of two plasma membrane proteins, the ferric iron reductase LFR1 and the ferrous iron transporter LIT1. However, how these parasites regulate their cytosolic iron concentration to prevent toxicity remains unknown. Here we characterize Leishmania Iron Regulator 1 (LIR1), an iron responsive protein with similarity to membrane transporters of the major facilitator superfamily (MFS) and plant nodulin-like proteins. LIR1 localizes on the plasma membrane of L. amazonensis promastigotes and intracellular amastigotes. After heterologous expression in Arabidopsis thaliana, LIR1 decreases the iron content of leaves and worsens the chlorotic phenotype of plants lacking the iron importer IRT1. Consistent with a role in iron efflux, LIR1 deficiency does not affect iron uptake by L. amazonensis but significantly increases the amount of iron retained intracellularly in the parasites. LIR1 null parasites are more sensitive to iron toxicity and have drastically impaired infectivity, phenotypes that are reversed by LIR1 complementation. We conclude that LIR1 functions as a plasma membrane iron exporter with a critical role in maintaining iron homeostasis and promoting infectivity in L. amazonensis.
Assuntos
Membrana Celular/metabolismo , Ferro/farmacologia , Leishmania/efeitos dos fármacos , Leishmaniose/prevenção & controle , Proteínas de Protozoários/metabolismo , Virulência/efeitos dos fármacos , Animais , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/parasitologia , Transporte Biológico , Células Cultivadas , Feminino , Homeostase , Ferro/toxicidade , Leishmaniose/metabolismo , Leishmaniose/parasitologia , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Protozoários/genéticaRESUMO
Acid sphingomyelinase (ASM) is a lysosomal enzyme that cleaves the phosphorylcholine head group of sphingomyelin, generating ceramide. Recessive mutations in SMPD1, the gene encoding ASM, cause Niemann-Pick Disease Types A and B. These disorders are attributed not only to lipid accumulation inside lysosomes but also to changes on the outer leaflet of the plasma membrane, highlighting an extracellular role for ASM. Secretion of ASM occurs under physiological conditions, and earlier studies proposed two forms of the enzyme, one resident in lysosomes and another form that would be diverted to the secretory pathway. Such differential intracellular trafficking has been difficult to explain because there is only one SMPD1 transcript that generates an active enzyme, found primarily inside lysosomes. Unexpectedly, studies of cell invasion by the protozoan parasite Trypanosoma cruzi revealed that conventional lysosomes can fuse with the plasma membrane in response to elevations in intracellular Ca2+ , releasing their contents extracellularly. ASM exocytosed from lysosomes remodels the outer leaflet of the plasma membrane, promoting parasite invasion and wound repair. Here, we discuss the possibility that ASM release during lysosomal exocytosis, in response to various forms of stress, may represent a major source of the secretory form of this enzyme.
Assuntos
Membrana Celular/parasitologia , Lisossomos/enzimologia , Esfingomielina Fosfodiesterase/metabolismo , Trypanosoma cruzi/patogenicidade , Animais , Secreções Corporais/efeitos da radiação , Cálcio/metabolismo , Membrana Celular/metabolismo , Membrana Celular/patologia , Ceramidas/metabolismo , Exocitose , Humanos , Lisossomos/metabolismo , Doença de Niemann-Pick Tipo A/enzimologia , Doença de Niemann-Pick Tipo B/enzimologia , Transporte Proteico , Esfingomielina Fosfodiesterase/deficiência , Esfingomielina Fosfodiesterase/genética , Esfingomielinas/metabolismo , Trypanosoma cruzi/metabolismoRESUMO
The molecular mechanisms underlying biological differences between two Leishmania species that cause cutaneous disease, L. major and L. amazonensis, are poorly understood. In L. amazonensis, reactive oxygen species (ROS) signaling drives differentiation of nonvirulent promastigotes into forms capable of infecting host macrophages. Tight spatial and temporal regulation of H2O2 is key to this signaling mechanism, suggesting a role for ascorbate-dependent peroxidase (APX), which degrades mitochondrial H2O2 Earlier studies showed that APX-null L. major parasites are viable, accumulate higher levels of H2O2, generate a greater yield of infective metacyclic promastigotes, and have increased virulence. In contrast, we found that in L. amazonensis, the ROS-inducible APX is essential for survival of all life cycle stages. APX-null promastigotes could not be generated, and parasites carrying a single APX allele were impaired in their ability to infect macrophages and induce cutaneous lesions in mice. Similar to what was reported for L. major, APX depletion in L. amazonensis enhanced differentiation of metacyclic promastigotes and amastigotes, but the parasites failed to replicate after infecting macrophages. APX expression restored APX single-knockout infectivity, while expression of catalytically inactive APX drastically reduced virulence. APX overexpression in wild-type promastigotes reduced metacyclogenesis, but enhanced intracellular survival following macrophage infection or inoculation into mice. Collectively, our data support a role for APX-regulated mitochondrial H2O2 in promoting differentiation of virulent forms in both L. major and L. amazonensis Our results also uncover a unique requirement for APX-mediated control of ROS levels for survival and successful intracellular replication of L. amazonensis.
Assuntos
Ascorbato Peroxidases/metabolismo , Leishmania major/patogenicidade , Leishmania mexicana/patogenicidade , Leishmaniose Cutânea/patologia , Macrófagos/parasitologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Ascorbato Peroxidases/genética , Células Cultivadas , Leishmania major/genética , Leishmania major/metabolismo , Leishmania mexicana/genética , Leishmania mexicana/metabolismo , Leishmaniose Cutânea/parasitologia , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/fisiologia , VirulênciaRESUMO
Leishmaniasis is one of the leading globally neglected diseases, affecting millions of people worldwide. Leishmania infection depends on the ability of insect-transmitted metacyclic promastigotes to invade mammalian hosts, differentiate into amastigotes, and replicate inside macrophages. To counter the hostile oxidative environment inside macrophages, these protozoans contain anti-oxidant systems that include iron-dependent superoxide dismutases (SODs) in mitochondria and glycosomes. Increasing evidence suggests that in addition to this protective role, Leishmania mitochondrial SOD may also initiate H2O2-mediated redox signaling that regulates gene expression and metabolic changes associated with differentiation into virulent forms. To investigate this hypothesis, we examined the specific role of SODA, the mitochondrial SOD isoform in Leishmania amazonensis Our inability to generate L. amazonensis SODA null mutants and the lethal phenotype observed following RNAi-mediated silencing of the Trypanosoma brucei SODA ortholog suggests that SODA is essential for trypanosomatid survival. L. amazonensis metacyclic promastigotes lacking one SODA allele failed to replicate in macrophages and were severely attenuated in their ability to generate cutaneous lesions in mice. Reduced expression of SODA also resulted in mitochondrial oxidative damage and failure of SODA/ΔsodA promastigotes to differentiate into axenic amastigotes. SODA expression above a critical threshold was also required for the development of metacyclic promastigotes, as SODA/ΔsodA cultures were strongly depleted in this infective form and more susceptible to reactive oxygen species (ROS)-induced stress. Collectively, our data suggest that SODA promotes Leishmania virulence by protecting the parasites against mitochondrion-generated oxidative stress and by initiating ROS-mediated signaling mechanisms required for the differentiation of infective forms.
Assuntos
Ferro/metabolismo , Leishmania mexicana/enzimologia , Mitocôndrias/enzimologia , Proteínas de Protozoários/metabolismo , Superóxido Dismutase/metabolismo , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Células da Medula Óssea/parasitologia , Células da Medula Óssea/patologia , Linhagem Celular , Células Cultivadas , Células Clonais , Feminino , Técnicas de Inativação de Genes , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Leishmania mexicana/crescimento & desenvolvimento , Leishmania mexicana/patogenicidade , Leishmania mexicana/ultraestrutura , Leishmaniose Cutânea/imunologia , Leishmaniose Cutânea/metabolismo , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/parasitologia , Macrófagos/patologia , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Carga Parasitária , Transporte Proteico , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/genética , Interferência de RNA , Superóxido Dismutase/antagonistas & inibidores , Superóxido Dismutase/genética , VirulênciaRESUMO
Iron, an essential co-factor of respiratory chain proteins, is critical for mitochondrial function and maintenance of its redox balance. We previously reported a role for iron uptake in differentiation of Leishmania amazonensis into virulent amastigotes, by a mechanism that involves reactive oxygen species (ROS) production and is independent of the classical pH and temperature cues. Iron import into mitochondria was proposed to be essential for this process, but evidence supporting this hypothesis was lacking because the Leishmania mitochondrial iron transporter was unknown. Here we describe MIT1, a homolog of the mitochondrial iron importer genes mrs3 (yeast) and mitoferrin-1 (human) that is highly conserved among trypanosomatids. MIT1 expression was essential for the survival of Trypanosoma brucei procyclic but not bloodstream forms, which lack functional respiratory complexes. L. amazonensis LMIT1 null mutants could not be generated, suggesting that this mitochondrial iron importer is essential for promastigote viability. Promastigotes lacking one LMIT1 allele (LMIT1/Δlmit1) showed growth defects and were more susceptible to ROS toxicity, consistent with the role of iron as the essential co-factor of trypanosomatid mitochondrial superoxide dismutases. LMIT1/Δlmit1 metacyclic promastigotes were unable to replicate as intracellular amastigotes after infecting macrophages or cause cutaneous lesions in mice. When induced to differentiate axenically into amastigotes, LMIT1/Δlmit1 showed strong defects in iron content and function of mitochondria, were unable to upregulate the ROS-regulatory enzyme FeSOD, and showed mitochondrial changes suggestive of redox imbalance. Our results demonstrate the importance of mitochondrial iron uptake in trypanosomatid parasites, and highlight the role of LMIT1 in the iron-regulated process that orchestrates differentiation of L. amazonensis into infective amastigotes.
Assuntos
Interações Hospedeiro-Parasita/fisiologia , Ferro/metabolismo , Leishmania/patogenicidade , Mitocôndrias/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Leishmania/crescimento & desenvolvimento , Leishmania/metabolismo , Leishmaniose , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteínas de Protozoários/genética , VirulênciaRESUMO
Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self-healing skin lesions to fatal visceralizing disease. As the host cells of choice for all species of Leishmania, macrophages are critical for the establishment of infections. How macrophages contribute to parasite homing to specific tissues and how parasites modulate macrophage function are still poorly understood. In this study, we show that Leishmania amazonensis infection inhibits macrophage roaming motility. The reduction in macrophage speed is not dependent on particle load or on factors released by infected macrophages. L. amazonensis-infected macrophages also show reduced directional migration in response to the chemokine MCP-1. We found that infected macrophages have lower levels of total paxillin, phosphorylated paxillin, and phosphorylated focal adhesion kinase when compared to noninfected macrophages, indicating abnormalities in the formation of signaling adhesion complexes that regulate motility. Analysis of the dynamics of actin polymerization at peripheral sites also revealed a markedly enhanced F-actin turnover frequency in L. amazonensis-infected macrophages. Thus, Leishmania infection inhibits macrophage motility by altering actin dynamics and impairing the expression of proteins that function in plasma membrane-extracellular matrix interactions.
Assuntos
Actinas/metabolismo , Movimento Celular , Leishmania mexicana/patogenicidade , Macrófagos/fisiologia , Macrófagos/parasitologia , Proteína-Tirosina Quinases de Adesão Focal/análise , Macrófagos/química , Paxilina/análiseRESUMO
The process of host cell invasion by Trypanosoma cruzi shares mechanistic elements with plasma membrane injury and repair. Both processes require Ca(2+)-triggered exocytosis of lysosomes, exocytosis of acid sphingomyelinase and formation of ceramide-enriched endocytic compartments. T. cruzi invades at peripheral sites, suggesting a need for spatial regulation of membrane traffic. Here, we show that Exo70 and Sec8 (also known as EXOC7 and EXOC4, respectively), components of the exocyst complex, accumulate in nascent T. cruzi vacuoles and at sites of mechanical wounding. Exo70 or Sec8 depletion inhibits T. cruzi invasion and Ca(2+)-dependent resealing of mechanical wounds, but does not affect the repair of smaller lesions caused by pore-forming toxins. Thus, T. cruzi invasion and mechanical lesion repair share a unique requirement for the exocyst, consistent with a dependence on targeted membrane delivery.
Assuntos
Membrana Celular/metabolismo , Doença de Chagas/metabolismo , Endocitose , Trypanosoma cruzi/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Cálcio/metabolismo , Membrana Celular/patologia , Doença de Chagas/patologia , Células HeLa , HumanosRESUMO
Leishmania parasites infect macrophages, cells that play an important role in organismal iron homeostasis. By expressing ferroportin, a membrane protein specialized in iron export, macrophages release iron stored intracellularly into the circulation. Iron is essential for the intracellular replication of Leishmania, but how the parasites compete with the iron export function of their host cell is unknown. Here, we show that infection with Leishmania amazonensis inhibits ferroportin expression in macrophages. In a TLR4-dependent manner, infected macrophages upregulated transcription of hepcidin, a peptide hormone that triggers ferroportin degradation. Parasite replication was inhibited in hepcidin-deficient macrophages and in wild type macrophages overexpressing mutant ferroportin that is resistant to hepcidin-induced degradation. Conversely, intracellular growth was enhanced by exogenously added hepcidin, or by expression of dominant-negative ferroportin. Importantly, dominant-negative ferroportin and macrophages from flatiron mice, a mouse model for human type IV hereditary hemochromatosis, restored the infectivity of mutant parasite strains defective in iron acquisition. Thus, inhibition of ferroportin expression is a specific strategy used by L. amazonensis to inhibit iron export and promote their own intracellular growth.
Assuntos
Ferro/metabolismo , Leishmania/metabolismo , Leishmaniose/metabolismo , Macrófagos/metabolismo , Animais , Transporte Biológico Ativo/genética , Proteínas de Transporte de Cátions/biossíntese , Proteínas de Transporte de Cátions/deficiência , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Hemocromatose/genética , Hemocromatose/metabolismo , Hemocromatose/parasitologia , Hemocromatose/patologia , Hepcidinas/biossíntese , Hepcidinas/genética , Humanos , Leishmaniose/genética , Leishmaniose/patologia , Macrófagos/parasitologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , MutaçãoRESUMO
Cells permeabilized by the bacterial pore-forming toxin streptolysin O (SLO) reseal their plasma membrane in a Ca(2+) -dependent manner. Resealing involves Ca(2+) -dependent exocytosis of lysosomes, release of acid sphingomyelinase and rapid formation of endosomes that carry the transmembrane pores into the cell. The intracellular fate of the toxin-carrying endocytic vesicles, however, is still unknown. Here, we show that SLO pores removed from the plasma membrane by endocytosis are sorted into the lumen of lysosomes, where they are degraded. SLO-permeabilized cells contain elevated numbers of total endosomes, which increase gradually in size while transitioning from endosomes with flat clathrin coats to large multivesicular bodies (MVBs). Under conditions that allow endocytosis and plasma membrane repair, SLO is rapidly ubiquitinated and gradually degraded, in a process sensitive to inhibitors of lysosomal hydrolysis but not of proteasomes. The endosomes induced by SLO permeabilization become increasingly acidified and promote SLO degradation under normal conditions, but not in cells silenced for expression of Vps24, an ESCRT-III complex component required for the release of intraluminal vesicles into MVBs. Thus, cells dispose of SLO transmembrane pores by ubiquitination/ESCRT-dependent sorting into the lumen of late endosomes/lysosomes.
Assuntos
Toxinas Bacterianas/metabolismo , Permeabilidade da Membrana Celular , Membrana Celular/metabolismo , Corpos Multivesiculares/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Western Blotting , Células Cultivadas , Endocitose , Modelos Biológicos , Estreptolisinas/metabolismoRESUMO
Trypanosomatid protozoan parasites lack a functional heme biosynthetic pathway, so must acquire heme from the environment to survive. However, the molecular pathway responsible for heme acquisition by these organisms is unknown. Here we show that L. amazonensis LHR1, a homolog of the C. elegans plasma membrane heme transporter HRG-4, functions in heme transport. Tagged LHR1 localized to the plasma membrane and to endocytic compartments, in both L. amazonensis and mammalian cells. Heme deprivation in L. amazonensis increased LHR1 transcript levels, promoted uptake of the fluorescent heme analog ZnMP, and increased the total intracellular heme content of promastigotes. Conversely, deletion of one LHR1 allele reduced ZnMP uptake and the intracellular heme pool by approximately 50%, indicating that LHR1 is a major heme importer in L. amazonensis. Viable parasites with correct replacement of both LHR1 alleles could not be obtained despite extensive attempts, suggesting that this gene is essential for the survival of promastigotes. Notably, LHR1 expression allowed Saccharomyces cerevisiae to import heme from the environment, and rescued growth of a strain deficient in heme biosynthesis. Syntenic genes with high sequence identity to LHR1 are present in the genomes of several species of Leishmania and also Trypanosoma cruzi and Trypanosoma brucei, indicating that therapeutic agents targeting this transporter could be effective against a broad group of trypanosomatid parasites that cause serious human disease.
Assuntos
Heme/metabolismo , Leishmania mexicana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Células HeLa , Heme/deficiência , Humanos , Leishmania mexicana/patogenicidade , Macrófagos/metabolismo , Macrófagos/parasitologia , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Metaloporfirinas/metabolismo , Camundongos , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
The protozoan parasite Trypanosoma cruzi, the aetiological agent of Chagas' disease, has two infective life cycle stages, trypomastigotes and amastigotes. While trypomastigotes actively enter mammalian cells, highly infective extracellular amastigotes (type Iâ T. cruzi) rely on actin-mediated uptake, which is generally inefficient in non-professional phagocytes. We found that extracellular amastigotes (EAs) of T. cruziâ G strain (type I), but not Y strain (type II), were taken up 100-fold more efficiently than inert particles. Mammalian cell lines showed levels of parasite uptake comparable to macrophages, and extensive actin recruitment and polymerization was observed at the site of entry. EA uptake was not dependent on parasite-secreted molecules and required the same molecular machinery utilized by professional phagocytes during large particle phagocytosis. Transcriptional silencing of synaptotagmin VII and CD63 significantly inhibited EA internalization, demonstrating that delivery of supplemental lysosomal membrane to form the phagosome is involved in parasite uptake. Importantly, time-lapse live imaging using fluorescent reporters revealed phagosome-associated modulation of phosphoinositide metabolism during EA uptake that closely resembles what occurs during phagocytosis by macrophages. Collectively, our results demonstrate that T. cruziâ EAs are potent inducers of phagocytosis in non-professional phagocytes, a process that may facilitate parasite persistence in infected hosts.
Assuntos
Doença de Chagas/fisiopatologia , Células HeLa/parasitologia , Estágios do Ciclo de Vida/fisiologia , Fagocitose/fisiologia , Trypanosoma cruzi/crescimento & desenvolvimento , Actinas/metabolismo , Animais , Doença de Chagas/metabolismo , Humanos , Metabolismo dos Lipídeos/fisiologia , Fosfatidilinositóis/metabolismo , Sinaptotagminas/metabolismo , Tetraspanina 30/metabolismo , Trypanosoma cruzi/patogenicidadeRESUMO
The protozoan parasite Leishmania amazonensis is a heme auxotroph and must acquire this essential factor from the environment. Previous studies showed that L. amazonensis incorporates heme through the transmembrane protein LHR1 (Leishmania Heme Response 1). LHR1-null promastigotes were not viable, suggesting that the transporter is essential for survival. Here, we compared the growth, differentiation, and infectivity for macrophages and mice of wild-type, LHR1-single-knockout (LHR1/Δlhr1), and LHR1-complemented (LHR1/Δlhr1 plus LHR1) L. amazonensis strains. LHR1/Δlhr1 promastigotes replicated poorly in heme-deficient media and had lower intracellular heme content than wild-type parasites. LHR1/Δlhr1 promastigotes were also less effective in reducing ferric iron to ferrous iron, a reaction mediated by the heme-containing parasite enzyme LFR1 (Leishmania Ferric Reductase 1). LHR1/Δlhr1 parasites differentiated normally into aflagellated forms expressing amastigote-specific markers but were not able to replicate intracellularly after infecting macrophages. Importantly, the intracellular growth of LHR1/Δlhr1 amastigotes was fully restored when macrophages were allowed to phagocytose red blood cells prior to infection. LHR1/Δlhr1 parasites were also severely defective in the development of cutaneous lesions in mice. All phenotypes observed in LHR1/Δlhr1 L. amazonensis were rescued by expression of episomal LHR1. Our results reveal the importance of efficient heme uptake for L. amazonensis replication and vertebrate host infectivity, reinforcing the potential usefulness of LHR1 as a target for new antileishmanial drugs.
Assuntos
Heme/metabolismo , Leishmania/patogenicidade , Macrófagos/parasitologia , Proteínas de Protozoários/metabolismo , Animais , Deleção de Genes , Leishmania/classificação , Camundongos , Proteínas de Protozoários/genética , VirulênciaRESUMO
The protozoan parasite Leishmania is the causative agent of serious human infections worldwide. The parasites alternate between insect and vertebrate hosts and cause disease by invading macrophages, where they replicate. Parasites lacking the ferrous iron transporter LIT1 cannot grow intracellularly, indicating that a plasma membrane-associated mechanism for iron uptake is essential for the establishment of infections. Here, we identify and functionally characterize a second member of the Leishmania iron acquisition pathway, the ferric iron reductase LFR1. The LFR1 gene is up-regulated under iron deprivation and accounts for all the detectable ferric reductase activity exposed on the surface of Leishmania amazonensis. LFR1 null mutants grow normally as promastigote insect stages but are defective in differentiation into the vertebrate infective forms, metacyclic promastigotes and amastigotes. LFR1 overexpression partially restores the abnormal morphology of infective stages but markedly reduces parasite viability, precluding its ability to rescue LFR1 null replication in macrophages. However, LFR1 overexpression is not toxic for amastigotes lacking the ferrous iron transporter LIT1 and rescues their growth defect. In addition, the intracellular growth of both LFR1 and LIT1 null parasites is rescued in macrophages loaded with exogenous iron. This indicates that the Fe(3+) reductase LFR1 functions upstream of LIT1 and suggests that LFR1 overexpression results in excessive Fe(2+) production, which impairs parasite viability after intracellular transport by LIT1.
Assuntos
FMN Redutase/biossíntese , Regulação Enzimológica da Expressão Gênica/fisiologia , Ferro/metabolismo , Leishmania/enzimologia , Leishmania/patogenicidade , Leishmaniose/enzimologia , Proteínas de Protozoários/biossíntese , Sequência de Aminoácidos , Animais , Células Cultivadas , FMN Redutase/genética , Humanos , Leishmania/genética , Leishmaniose/genética , Macrófagos/metabolismo , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas de Protozoários/genéticaRESUMO
Infection of mammalian hosts with Leishmania amazonensis depends on the remarkable ability of these parasites to replicate within macrophage phagolysosomes. A critical adaptation for survival in this harsh environment is an efficient mechanism for gaining access to iron. In this study, we identify and characterize LIT1, a novel L. amazonensis membrane protein with extensive similarity to IRT1, a ZIP family ferrous iron transporter from Arabidopsis thaliana. The ability of LIT1 to promote iron transport was demonstrated after expression in yeast and in L. amazonensis LIT1-null amastigotes. Endogenous LIT1 was only detectable in amastigotes replicating intracellularly, and its intracellular expression was accelerated under conditions predicted to result in iron deprivation. Although L. amazonensis lacking LIT1 grew normally in axenic culture and had no defects differentiating into infective forms, replication within macrophages was abolished. Consistent with an essential role for LIT1 in intracellular growth as amastigotes, Deltalit1 parasites were avirulent. After inoculation into highly susceptible mice, no lesions were detected, even after extensive periods of time. Despite the absence of pathology, viable Deltalit1 parasites were recovered from the original sites of inoculation, indicating that L. amazonensis can persist in vivo independently of the ability to grow in macrophages. Our findings highlight the essential role played by intracellular iron acquisition in Leishmania virulence and identify this pathway as a promising target for therapeutic intervention.
Assuntos
Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Leishmania/metabolismo , Leishmania/patogenicidade , Lisossomos/parasitologia , Macrófagos/parasitologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Primers do DNA , Ferro/metabolismo , Leishmania/fisiologia , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Reprodução/fisiologia , Alinhamento de Sequência , Análise de Sequência de DNA , Virulência , LevedurasRESUMO
Removing membrane pores may help cancer cells survive T cell assault.
RESUMO
Although macrophages have long been considered key players in the course of Leishmania infections, other non-professional phagocytes have lately been shown to maintain low levels of the parasite in safe intracellular niches. Recently, it was demonstrated that the adipose tissue is capable of harboring Old World L. (L.) infantum in mice. However, there is no evidence of experimental adipocyte infection with New World Leishmania species so far. In addition, it was not known whether adipocytes would be permissive for formation of the unique, large and communal parasitophorous vacuoles that are typical of L. (L.) amazonensis in macrophages. Here we evaluated the ability of L. (L.) amazonensis and L. (V.) braziliensis promastigotes and amastigotes to infect 3T3-L1 fibroblast-derived adipocytes (3T3-Ad) using light and transmission electron microscopy. Our results indicate that amastigotes and promastigotes of both species were capable of infecting and surviving inside pre- and fully differentiated 3T3-Ad for up to 144 h. Importantly, L. (L.) amazonensis amastigotes resided in large communal parasitophorous vacuoles in pre-adipocytes, which appeared to be compressed between large lipid droplets in mature adipocytes. In parallel, individual L. (V.) braziliensis amastigotes were detected in single vacuoles 144 h post-infection. We conclude that 3T3-Ad may constitute an environment that supports low loads of viable parasites perhaps contributing to parasite maintenance, since amastigotes of both species recovered from these cells differentiated into replicative promastigotes. Our findings shed light on the potential of a new host cell model that can be relevant to the persistence of New World Leishmania species.
Assuntos
Leishmania , Leishmaniose Cutânea , Leishmaniose , Células 3T3-L1 , Adipócitos , Animais , Leishmaniose/parasitologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Synaptotagmin (Syt) VII is a ubiquitously expressed member of the Syt family of Ca2+ sensors. It is present on lysosomes in several cell types, where it regulates Ca2+-dependent exocytosis. Because [Ca2+]i and exocytosis have been associated with phagocytosis, we investigated the phagocytic ability of macrophages from Syt VII-/- mice. Syt VII-/- macrophages phagocytose normally at low particle/cell ratios but show a progressive inhibition in particle uptake under high load conditions. Complementation with Syt VII rescues this phenotype, but only when functional Ca2+-binding sites are retained. Reinforcing a role for Syt VII in Ca2+-dependent phagocytosis, particle uptake in Syt VII-/- macrophages is significantly less dependent on [Ca2+]i. Syt VII is concentrated on peripheral domains of lysosomal compartments, from where it is recruited to nascent phagosomes. Syt VII recruitment is rapidly followed by the delivery of Lamp1 to phagosomes, a process that is inhibited in Syt VII-/- macrophages. Thus, Syt VII regulates the Ca2+-dependent mobilization of lysosomes as a supplemental source of membrane during phagocytosis.