RESUMEN
Glioblastoma is the most common primary brain tumor and has a dismal prognosis. The development of central necrosis represents a tipping point in the evolution of these tumors that foreshadows aggressive expansion, swiftly leading to mortality. The onset of necrosis, severe hypoxia and associated radial glioma expansion correlates with dramatic tumor microenvironment (TME) alterations that accelerate tumor growth. In the past, most have concluded that hypoxia and necrosis must arise due to "cancer outgrowing its blood supply" when rapid tumor growth outpaces metabolic supply, leading to diffusion-limited hypoxia. However, growing evidence suggests that microscopic intravascular thrombosis driven by the neoplastic overexpression of pro-coagulants attenuates glioma blood supply (perfusion-limited hypoxia), leading to TME restructuring that includes breakdown of the blood-brain barrier, immunosuppressive immune cell accumulation, microvascular hyperproliferation, glioma stem cell enrichment and tumor cell migration outward. Cumulatively, these adaptations result in rapid tumor expansion, resistance to therapeutic interventions and clinical progression. To inform future translational investigations, the complex interplay among environmental cues and myriad cell types that contribute to this aggressive phenotype requires better understanding. This review focuses on contributions from intratumoral thrombosis, the effects of hypoxia and necrosis, the adaptive and innate immune responses, and the current state of targeted therapeutic interventions.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Glioma , Neoplasias Encefálicas/metabolismo , Progresión de la Enfermedad , Glioblastoma/patología , Glioma/patología , Humanos , Necrosis/complicaciones , Microambiente TumoralRESUMEN
Widespread tumor cell invasion is a fundamental property of diffuse gliomas and is ultimately responsible for their poor prognosis. A greater understanding of basic mechanisms underlying glioma invasion is needed to provide insights into therapies that could potentially counteract them. While none of the currently available in vitro models can fully recapitulate the complex interactions of glioma cells within the brain tumor microenvironment, if chosen and developed appropriately, these models can provide controlled experimental settings to study molecular and cellular phenomena that are challenging or impossible to model in vivo. Therefore, selecting the most appropriate in vitro model, together with its inherent advantages and limitations, for specific hypotheses and experimental questions achieves primary significance. In this review, we describe and discuss commonly used methods for modeling and studying glioma invasion in vitro, including platforms, matrices, cell culture, and visualization techniques, so that choices for experimental approach are informed and optimal.
Asunto(s)
Neoplasias Encefálicas , Glioma , Encéfalo , Humanos , Invasividad Neoplásica , Microambiente TumoralRESUMEN
The flagellar calcium-binding protein (FCaBP) of Trypanosoma cruzi is localized to the flagellar membrane in all life cycle stages of the parasite. Myristoylation and palmitoylation of the N terminus of FCaBP are necessary for flagellar membrane targeting. Not all dually acylated proteins in T. cruzi are flagellar, however. Other determinants of FCaBP therefore likely contribute to flagellar specificity. We generated T. cruzi transfectants expressing the N-terminal 24 or 12 amino acids of FCaBP fused to GFP. Analysis of these mutants revealed that although amino acids 1-12 are sufficient for dual acylation and membrane binding, amino acids 13-24 are required for flagellar specificity and lipid raft association. Mutagenesis of several conserved lysine residues in the latter peptide demonstrated that these residues are essential for flagellar targeting and lipid raft association. Finally, FCaBP was expressed in the protozoan Leishmania amazonensis, which lacks FCaBP. The flagellar localization and membrane association of FCaBP in L. amazonensis suggest that the mechanisms for flagellar targeting, including a specific palmitoyl acyltransferase, are conserved in this organism.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Cilios/metabolismo , Membranas Intracelulares/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/metabolismo , Acilación/efectos de los fármacos , Secuencia de Aminoácidos , Proteínas de Unión al Calcio/química , Secuencia Conservada , Detergentes/farmacología , Proteínas Fluorescentes Verdes/metabolismo , Membranas Intracelulares/efectos de los fármacos , Leishmania/efectos de los fármacos , Leishmania/metabolismo , Lisina/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Ácido Mirístico/metabolismo , Palmitatos/metabolismo , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Protozoarias/química , Proteínas Recombinantes de Fusión/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Trypanosoma cruzi/efectos de los fármacosRESUMEN
Dynamic post-translational modifications allow the rapid, specific, and tunable regulation of protein functions in eukaryotic cells. S-acylation is the only reversible lipid modification of proteins, in which a fatty acid, usually palmitate, is covalently attached to a cysteine residue of a protein by a zDHHC palmitoyl acyltransferase enzyme. Depalmitoylation is required for acylation homeostasis and is catalyzed by an enzyme from the alpha/beta hydrolase family of proteins usually acyl-protein thioesterase (APT1). The enzyme responsible for depalmitoylation in Trypanosoma brucei parasites is currently unknown. We demonstrate depalmitoylation activity in live bloodstream and procyclic form trypanosomes sensitive to dose-dependent inhibition with the depalmitoylation inhibitor, palmostatin B. We identified a homologue of human APT1 in Trypanosoma brucei which we named TbAPT-like (TbAPT-L). Epitope-tagging of TbAPT-L at N- and C- termini indicated a cytoplasmic localization. Knockdown or over-expression of TbAPT-L in bloodstream forms led to robust changes in TbAPT-L mRNA and protein expression but had no effect on parasite growth in vitro, or cellular depalmitoylation activity. Esterase activity in cell lysates was also unchanged when TbAPT-L was modulated. Unexpectedly, recombinant TbAPT-L possesses esterase activity with specificity for short- and medium-chain fatty acid substrates, leading to the conclusion, TbAPT-L is a lipase, not a depalmitoylase.
RESUMEN
Chagas disease is caused by infection with the protozoan parasite Trypanosoma cruzi (T. cruzi), an intracellular pathogen that causes significant morbidity and death among millions in the Americas from Canada to Argentina. Current therapy involves oral administration of the nitroimidazole benznidazole (BNZ), which has serious side effects that often necessitate cessation of treatment. To both avoid off-target side effects and reduce the necessary dosage of BNZ, we packaged the drug within poly(ethylene glycol)-block-poly(propylene sulfide) polymersomes (BNZ-PSs). We show that these vesicular nanocarriers enhanced intracellular delivery to phagocytic cells and tested this formulation in a mouse model of T. cruzi infection. BNZ-PS is not only nontoxic but also significantly more potent than free BNZ, effectively reducing parasitemia, intracellular infection, and tissue parasitosis at a 466-fold lower dose of BNZ. We conclude that BNZ-PS was superior to BNZ for treatment of T. cruzi infection in mice and that further modifications of this nanocarrier formulation could lead to a wide range of custom controlled delivery applications for improved treatment of Chagas disease in humans.
Asunto(s)
Enfermedad de Chagas/tratamiento farmacológico , Sistema de Administración de Fármacos con Nanopartículas , Nitroimidazoles/administración & dosificación , Fagocitos/parasitología , Tripanocidas/administración & dosificación , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Portadores de Fármacos , Ratones , Nitroimidazoles/farmacología , Fagocitos/efectos de los fármacos , Polietilenglicoles , Sulfuros , Tripanocidas/farmacología , Trypanosoma cruzi/efectos de los fármacosRESUMEN
The protozoan Trypanosoma cruzi expresses multiple isoforms of the GP63 family of metalloproteases. Polyclonal antiserum against recombinant GP63 of T. cruzi (TcGP63) was used to study TcGP63 expression and localization in this organism. Western blot analysis revealed that TcGP63 is 61 kDa in epimastigotes, amastigotes, and tissue culture-derived trypomastigotes but 55 kDa in metacyclic trypomastigotes. Antiserum specific for Leishmania amazonensis GP63 specifically reacted with a 55-kDa TcGP63 form in metacyclic trypomastigotes, suggesting stage-specific expression of different isoforms. Surface biotinylation and endoglycosidase digestion experiments showed that TcGP63 is an ecto-glycoprotein in epimastigotes but is intracellular and lacking in N-linked glycans in metacyclic trypomastigotes. Immunofluorescence microscopy showed that TcGP63 is localized on the surfaces of epimastigotes but distributed intracellularly in metacyclic trypomastigotes. TcGP63 is soluble in cold Triton X-100, in contrast to Leishmania GP63, which is detergent resistant in this medium, suggesting that GP63 is not raft associated in T. cruzi. Western blot comparison of our antiserum to a previously described anti-peptide TcGP63 antiserum indicates that each antiserum recognizes distinct TcGP63 proteins. Preincubation of trypomastigotes with either TcGP63 antiserum or a purified TcGP63 C-terminal subfragment reduced infection of host myoblasts. These results show that TcGP63 is expressed at all life stages and that individual isoforms play a role in host cell infection.
Asunto(s)
Metaloproteasas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/fisiología , Factores de Virulencia/metabolismo , Animales , Membrana Celular/química , Citosol/química , Metaloproteasas/química , Microscopía Fluorescente , Peso Molecular , Proteínas Protozoarias/química , Trypanosoma cruzi/química , Factores de Virulencia/químicaRESUMEN
Lipid rafts are liquid-ordered membrane microdomains that form by preferential association of 3-ß-hydroxysterols, sphingolipids and raft-associated proteins often having acyl modifications. We isolated lipid rafts of the protozoan parasite Trypanosoma brucei and determined the protein composition of lipid rafts in the cell. This analysis revealed a striking enrichment of flagellar proteins and several putative signaling proteins in the lipid raft proteome. Calpains and intraflagellar transport proteins, in particular, were found to be abundant in the lipid raft proteome. These findings provide additional evidence supporting the notion that the eukaryotic cilium/flagellum is a lipid raft-enriched specialized structure with high concentrations of sterols, sphingolipids and palmitoylated proteins involved in environmental sensing and cell signaling.
RESUMEN
Cilia play important roles in cell signaling, facilitated by the unique lipid environment of a ciliary membrane containing high concentrations of sterol-rich lipid rafts. The African trypanosome Trypanosoma brucei is a single-celled eukaryote with a single cilium/flagellum. We tested whether flagellar sterol enrichment results from selective flagellar partitioning of specific sterol species or from general enrichment of all sterols. While all sterols are enriched in the flagellum, cholesterol is especially enriched. T. brucei cycles between its mammalian host (bloodstream cell), in which it scavenges cholesterol, and its tsetse fly host (procyclic cell), in which it both scavenges cholesterol and synthesizes ergosterol. We wondered whether the insect and mammalian life cycle stages possess chemically different lipid rafts due to different sterol utilization. Treatment of bloodstream parasites with cholesterol-specific methyl-ß-cyclodextrin disrupts both membrane liquid order and localization of a raft-associated ciliary membrane calcium sensor. Treatment with ergosterol-specific amphotericin B does not. The opposite results were observed with ergosterol-rich procyclic cells. Further, these agents have opposite effects on flagellar sterol enrichment and cell metabolism in the two life cycle stages. These findings illuminate differences in the lipid rafts of an organism employing life cycle-specific sterols and have implications for treatment.
Asunto(s)
Microdominios de Membrana/química , Esteroles/análisis , Trypanosoma brucei brucei/crecimiento & desarrollo , Moscas Tse-Tse/parasitología , Anfotericina B/farmacología , Animales , Colesterol/análisis , Ergosterol/análisis , Flagelos/química , Estadios del Ciclo de Vida/efectos de los fármacos , Microdominios de Membrana/efectos de los fármacos , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/efectos de los fármacos , beta-Ciclodextrinas/farmacologíaRESUMEN
African trypanosomiasis is caused by infection with the protozoan parasite Trypanosoma brucei During infection, this pathogen divides rapidly to high density in the bloodstream of its mammalian host in a manner similar to that of leukemia. Like all eukaryotes, T. brucei has a cell cycle involving the de novo synthesis of DNA regulated by ribonucleotide reductase (RNR), which catalyzes the conversion of ribonucleotides into their deoxy form. As an essential enzyme for the cell cycle, RNR is a common target for cancer chemotherapy. We hypothesized that inhibition of RNR by genetic or pharmacological means would impair parasite growth in vitro and prolong the survival of infected animals. Our results demonstrate that RNR inhibition is highly effective in suppressing parasite growth both in vitro and in vivo These results support drug discovery efforts targeting the cell cycle, not only for African trypanosomiasis but possibly also for other infections by eukaryotic pathogens.IMPORTANCE The development of drugs to treat infections with eukaryotic pathogens is challenging because many key virulence factors have closely related homologues in humans. Drug toxicity greatly limits these development efforts. For pathogens that replicate at a high rate, especially in the blood, an alternative approach is to target the cell cycle directly, much as is done to treat some hematologic malignancies. The results presented here indicate that targeting the cell cycle via inhibition of ribonucleotide reductase is effective at killing trypanosomes and prolonging the survival of infected animals.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Inhibidores Enzimáticos/uso terapéutico , Hidroxiurea/uso terapéutico , Ribonucleótido Reductasas/antagonistas & inhibidores , Tripanocidas/uso terapéutico , Trypanosoma brucei brucei/efectos de los fármacos , Tripanosomiasis Africana/tratamiento farmacológico , Animales , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Humanos , Hidroxiurea/administración & dosificación , Hidroxiurea/farmacología , Ratones , Ribonucleótido Reductasas/metabolismo , Tripanocidas/farmacología , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/fisiología , Tripanosomiasis Africana/parasitologíaRESUMEN
Flagellar calcium-binding protein (FCaBP) is a dually acylated Ca(2+) sensor in the Trypanosoma cruzi flagellar membrane that undergoes a massive conformational change upon Ca(2+) binding. It is similar to neuronal Ca(2+) sensors, like recoverin, which regulate their binding partners through a calcium acyl switch mechanism. FCaBP is washed out of permeabilized cells with buffers containing EDTA, indicating Ca(2+)-dependent flagellar membrane association. We hypothesized that, like recoverin, FCaBP projects its acyl groups in the presence of Ca(2+), permitting flagellar membrane and binding partner association and that it sequesters the acyl groups in low Ca(2+), disassociating from the membrane and releasing its binding partner to perform a presumed enzymatic function. The X-ray crystal structure of FCaBP suggests that the acyl groups are always exposed, so we set out to test our hypothesis directly. We generated T. cruzi transfectants expressing FCaBP or Ca(2+)-binding mutant FCaBP(E151Q/E188Q) and recombinant wildtype and mutant proteins as well. Both FCaBP and FCaBP(E151Q/E188Q) were found to associate with lipid rafts, indicating the Ca(2+)-independence of this association. To our initial surprise, FCaBP(E151Q/E188Q), like wildtype FCaBP, exhibited Ca(2+)-dependent flagellar membrane association, even though this protein does not bind Ca(2+) itself [16]. One possible explanation for this is that FCaBP(E151Q/E188Q), like some other Ca(2+) sensors, may form dimers and that dimerization of FCaBP(E151Q/E188Q) with endogenous wildtype FCaBP might explain its Ca(2+)-dependent localization. Indeed both proteins are able to form dimers in the presence and absence of Ca(2+). These results suggest that FCaBP possesses two distinct Ca(2+)-dependent interactions-one involving a Ca(2+)-induced change in conformation and another perhaps involving binding partner association.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Calcio/metabolismo , Membrana Celular/metabolismo , Flagelos/efectos de los fármacos , Proteínas Protozoarias/metabolismo , Trypanosoma cruzi/efectos de los fármacos , Proteínas de Unión al Calcio/genética , Microdominios de Membrana/química , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión Proteica , Multimerización de Proteína , Proteínas Protozoarias/genética , Trypanosoma cruzi/genéticaRESUMEN
UNLABELLED: Sphingolipids are important constituents of cell membranes and also serve as mediators of cell signaling and cell recognition. Sphingolipid metabolites such as sphingosine-1-phosphate and ceramide regulate signaling cascades involved in cell proliferation and differentiation, autophagy, inflammation, and apoptosis. Little is known about how sphingolipids and their metabolites function in single-celled eukaryotes. In the present study, we investigated the role of sphingosine kinase (SPHK) in the biology of the protozoan parasite Trypanosoma brucei, the agent of African sleeping sickness. T. brucei SPHK (TbSPHK) is constitutively but differentially expressed during the life cycle of T. brucei. Depletion of TbSPHK in procyclic-form T. brucei causes impaired growth and attenuation in the G1/S phase of the cell cycle. TbSPHK-depleted cells also develop organelle positioning defects and an accumulation of tyrosinated α-tubulin at the elongated posterior end of the cell, known as the "nozzle" phenotype, caused by other molecular perturbations in this organism. Our studies indicate that TbSPHK is involved in G1-to-S cell cycle progression, organelle positioning, and maintenance of cell morphology. Cytotoxicity assays using TbSPHK inhibitors revealed a favorable therapeutic index between T. brucei and human cells, suggesting TbSPHK to be a novel drug target. IMPORTANCE: Trypanosoma brucei is a single-celled parasite that is transmitted between humans and other animals by the tsetse fly. T. brucei is endemic in sub-Saharan Africa, where over 70 million people and countless livestock are at risk of developing T. brucei infection, called African sleeping sickness, resulting in economic losses of ~$35 million from the loss of cattle alone. New drugs for this infection are sorely needed and scientists are trying to identify essential enzymes in the parasite that can be targets for new therapies. One possible enzyme target is sphingosine kinase, an enzyme involved in the synthesis of lipids important for cell surface integrity and regulation of cell functions. In this study, we found that sphingosine kinase is essential for normal growth and structure of the parasite, raising the possibility that it could be a good target for new chemotherapy for sleeping sickness.
Asunto(s)
Ciclo Celular , Proliferación Celular , Microtúbulos/metabolismo , Biogénesis de Organelos , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Trypanosoma brucei brucei/fisiología , Secuencia de Aminoácidos , Transporte Biológico , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei brucei/metabolismoRESUMEN
Flagellar calcium binding proteins are expressed in a variety of trypanosomes and are potential drug targets for Chagas disease and African sleeping sickness. We report complete NMR chemical shift assignments of the flagellar calcium binding protein calflagin Tb24 of Trypanosoma brucei. (BMRB no. 18011).
Asunto(s)
Proteínas de Unión al Calcio/química , Flagelos , Resonancia Magnética Nuclear Biomolecular , Proteínas Protozoarias/química , Trypanosoma brucei brucei/citología , Secuencia de Aminoácidos , Datos de Secuencia MolecularRESUMEN
Chagas heart disease, the leading cause of heart failure in Latin America, results from infection with the parasite Trypanosoma cruzi. Although T. cruzi disseminates intravascularly, how the parasite contends with the endothelial barrier to escape the bloodstream and infect tissues has not been described. Understanding the interaction between T. cruzi and the vascular endothelium, likely a key step in parasite dissemination, could inform future therapies to interrupt disease pathogenesis. We adapted systems useful in the study of leukocyte transmigration to investigate both the occurrence of parasite transmigration and its determinants in vitro. Here we provide the first evidence that T. cruzi can rapidly migrate across endothelial cells by a mechanism that is distinct from productive infection and does not disrupt monolayer integrity or alter permeability. Our results show that this process is facilitated by a known modulator of cellular infection and vascular permeability, bradykinin, and can be augmented by the chemokine CCL2. These represent novel findings in our understanding of parasite dissemination, and may help identify new therapeutic strategies to limit the dissemination of the parasite.
Asunto(s)
Permeabilidad Capilar , Cardiomiopatía Chagásica/metabolismo , Endotelio Vascular , Células Endoteliales de la Vena Umbilical Humana , Trypanosoma cruzi/metabolismo , Animales , Bradiquinina/metabolismo , Quimiocina CCL2/metabolismo , Endotelio Vascular/metabolismo , Endotelio Vascular/parasitología , Femenino , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/parasitología , Humanos , Masculino , RatonesRESUMEN
Flagellar calcium binding proteins are expressed in a variety of trypanosomes and are potential drug targets for Chagas disease and African sleeping sickness. The flagellar calcium binding protein calflagin of Trypanosoma brucei (called Tb24) is a myristoylated and palmitoylated EF-hand protein that is targeted to the inner leaflet of the flagellar membrane. The Tb24 protein may also interact with proteins on the membrane surface that may be different from those bound to flagellar calcium binding proteins (FCaBPs) in T. cruzi. We report here the NMR structure of Tb24 that contains four EF-hand motifs bundled in a compact arrangement, similar to the overall fold of T. cruzi FCaBP (RMSD = 1.0 Å). A cluster of basic residues (K22, K25, K31, R36, and R38) located on a surface near the N-terminal myristoyl group may be important for membrane binding. Non-conserved residues on the surface of a hydrophobic groove formed by EF2 (P91, Q95, D103, and V108) and EF4 (C194, T198, K199, Q202, and V203) may serve as a target protein binding site and could have implications for membrane target recognition.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas Protozoarias/química , Trypanosoma brucei brucei/química , Secuencia de Aminoácidos , Enfermedad de Chagas/parasitología , Motivos EF Hand , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Alineación de Secuencia , Trypanosoma cruzi/química , Tripanosomiasis Africana/parasitologíaRESUMEN
Protein palmitoylation has diverse effects in regulating protein membrane affinity, localization, binding partner interactions, turnover and function. Here, we show that palmitoylation also contributes to the sorting of proteins to the eukaryotic flagellum. African trypanosomes are protozoan pathogens that express a family of unique Ca(2+)-binding proteins, the calflagins, which undergo N-terminal myristoylation and palmitoylation. The localization of calflagins depends on their acylation status. Myristoylation alone is sufficient for membrane association, but, in the absence of palmitoylation, the calflagins localize to the pellicular (cell body) membrane. Palmitoylation, which is mediated by a specific palmitoyl acyltransferase, is then required for subsequent trafficking of calflagin to the flagellar membrane. Coincident with the redistribution of calflagin from the pellicular to the flagellar membrane is their association with lipid rafts, which are highly enriched in the flagellar membrane. Screening of candidate palmitoyl acyltranferases identified a single enzyme, TbPAT7, that is necessary for calflagin palmitoylation and flagellar membrane targeting. Our results implicate protein palmitoylation in flagellar trafficking, and demonstrate the conservation and specificity of palmitoyl acyltransferase activity by DHHC-CRD proteins across kingdoms.
Asunto(s)
Aciltransferasas/metabolismo , Membrana Celular/enzimología , Flagelos/enzimología , Lipoilación , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/enzimología , Acilación , Secuencia de Aminoácidos , Animales , Biotina/metabolismo , Proteínas de Unión al Calcio/metabolismo , Genes Protozoarios , Cinética , Datos de Secuencia Molecular , Mutagénesis , Mutación/genética , Ácido Mirístico/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Protozoarias/química , Trypanosoma brucei brucei/genéticaRESUMEN
The eukaryotic flagellar membrane has a distinct composition from other domains of the plasmalemma. Our work shows that the specialized composition of the trypanosome flagellar membrane reflects increased concentrations of sterols and saturated fatty acids, correlating with direct observation of high liquid order by laurdan fluorescence microscopy. These findings indicate that the trypanosome flagellar membrane possesses high concentrations of lipid rafts: discrete regions of lateral heterogeneity in plasma membranes that serve to sequester and organize specialized protein complexes. Consistent with this, a dually acylated Ca(2+) sensor that is concentrated in the flagellum is found in detergent-resistant membranes and mislocalizes if the lipid rafts are disrupted. Detergent-extracted cells have discrete membrane patches localized on the surface of the flagellar axoneme, suggestive of intraflagellar transport particles. Together, these results provide biophysical and biochemical evidence to indicate that lipid rafts are enriched in the trypanosome flagellar membrane, providing a unique mechanism for flagellar protein localization and illustrating a novel means by which specialized cellular functions may be partitioned to discrete membrane domains.
Asunto(s)
Flagelos/metabolismo , Microdominios de Membrana/metabolismo , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/metabolismo , Animales , Axonema/efectos de los fármacos , Axonema/ultraestructura , Proteínas de Unión al Calcio/metabolismo , Detergentes/farmacología , Flagelos/efectos de los fármacos , Flagelos/ultraestructura , Microdominios de Membrana/efectos de los fármacos , Microdominios de Membrana/ultraestructura , Transporte de Proteínas/efectos de los fármacos , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/ultraestructuraRESUMEN
Sphingolipids and their metabolites have been thought crucial for cell growth and cell cycle progression, membrane and protein trafficking, signal transduction, and formation of lipid rafts; however, recent studies in trypanosomes point to the dispensability of sphingolipids in some of these processes. In this study, we explore the requirements for de novo sphingolipid biosynthesis in the insect life cycle stage of the African trypanosome Trypanosoma brucei by inhibiting the enzyme serine palmitoyltransferase (SPT2) by using RNA interference or treatment with a potent SPT2 inhibitor myriocin. Mass spectrometry revealed that upon SPT2 inhibition, the parasites contained substantially reduced levels of inositolphosphorylceramide. Although phosphatidylcholine and cholesterol levels were increased to compensate for this loss, the cells were ultimately not viable. The most striking result of sphingolipid reduction in procyclic T. brucei was aberrant cytokinesis, characterized by incomplete cleavage-furrow formation, delayed kinetoplast segregation and emergence of cells with abnormal DNA content. Organelle replication continued despite sphingolipid depletion, indicating that sphingolipids act as second messengers regulating cellular proliferation and completion of cytokinesis. Distention of the mitochondrial membrane, formation of multilamellar structures within the mitochondrion and near the nucleus, accumulation of lipid bodies and, less commonly, disruption of the Golgi complex were observed after prolonged sphingolipid depletion. These findings suggest that some aspects of vesicular trafficking may be compromised. However, flagellar membrane targeting and the association of the flagellar membrane protein calflagin with detergent-resistant membranes were not affected, indicating that the vesicular trafficking defects were mild. Our studies indicate that sphingolipid biosynthesis is vital for cell cycle progression and cell survival, but not essential for the normal trafficking of flagellar membrane-associated proteins or lipid raft formation in procyclic T. brucei.
Asunto(s)
Citocinesis/fisiología , Esfingolípidos/biosíntesis , Trypanosoma brucei brucei/metabolismo , Animales , Ciclo Celular/genética , Ciclo Celular/fisiología , Colesterol/metabolismo , Cromatografía en Capa Delgada , Citocinesis/genética , Ácidos Grasos Monoinsaturados/farmacología , Cromatografía de Gases y Espectrometría de Masas , Microscopía Electrónica de Transmisión , Microscopía Fluorescente , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Interferencia de ARN , Serina C-Palmitoiltransferasa/genética , Serina C-Palmitoiltransferasa/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Esteroles/metabolismo , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/ultraestructuraRESUMEN
Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is a major public health problem in Central and South America. The pathogenesis of Chagas disease is complex and the natural course of infection is not completely understood. The recent development of bioluminescence imaging technology has facilitated studies of a number of infectious and non-infectious diseases. We developed luminescent T. cruzi to facilitate similar studies of Chagas disease pathogenesis. Luminescent T. cruzi trypomastigotes and amastigotes were imaged in infections of rat myoblast cultures, which demonstrated a clear correlation of photon emission signal strength to the number of parasites used. This was also observed in mice infected with different numbers of luminescent parasites, where a stringent correlation of photon emission to parasite number was observed early at the site of inoculation, followed by dissemination of parasites to different sites over the course of a 25-day infection. Whole animal imaging from ventral, dorsal and lateral perspectives provided clear evidence of parasite dissemination. The tissue distribution of T. cruzi was further determined by imaging heart, spleen, skeletal muscle, lungs, kidneys, liver and intestines ex vivo. These results illustrate the natural dissemination of T. cruzi during infection and unveil a new tool for studying a number of aspects of Chagas disease, including rapid in vitro screening of potential therapeutical agents, roles of parasite and host factors in the outcome of infection, and analysis of differential tissue tropism in various parasite-host strain combinations.
Asunto(s)
Enfermedad de Chagas/parasitología , Luciferasas , Mediciones Luminiscentes/métodos , Trypanosoma cruzi , Animales , Luciferasas/genética , Ratones , Fotones , RatasRESUMEN
The flagellar calcium-binding protein (FCaBP) of the flagellated protozoan Trypanosoma cruzi associates with the flagellar membrane via its N-terminal myristate and palmitate moieties in a calcium-modulated, conformation-dependent manner. This mechanism of localization is similar to that described for neuronal calcium sensors, which undergo calcium-dependent changes in conformation, which modulate the availability of the acyl groups for membrane interaction and partner association. To test whether FCaBP undergoes a calcium-dependent conformational change and to explore the role of such a change in flagellar targeting, we first introduced point mutations into each of the two EF-hand calcium-binding sites of FCaBP to define their affinities. Analysis of recombinant EF-3 mutant (E151Q), EF-4 mutant (E188Q), and double mutant proteins showed EF-3 to be the high affinity site (Kd approximately 9 microM) and EF-4 the low affinity site (Kd approximately 120 microM). These assignments also correlated with partial (E188Q), nearly complete (E151Q), and complete (E151Q,E188Q) disruption of calcium-induced conformational changes determined by NMR spectrometry. We next expressed the FCaBP E151Q mutant and the double mutant in T. cruzi epimastigotes. These transproteins localized to the flagellum, suggesting the existence of a calcium-dependent interaction of FCaBP that is independent of its intrinsic calcium binding capacity. Several proteins were identified by FCaBP affinity chromatography that interact with FCaBP in a calcium-dependent manner, but with differential dependence on calcium-binding by FCaBP. These findings may have broader implications for the calcium acyl switch mechanism of protein regulation.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Calcio/metabolismo , Flagelos/metabolismo , Proteínas Protozoarias/fisiología , Animales , Sitios de Unión , Calcio/química , Proteínas de Unión al Calcio/metabolismo , Calorimetría , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Inmunoprecipitación , Cinética , Espectroscopía de Resonancia Magnética , Microscopía Fluorescente , Modelos Biológicos , Mutación , Mutación Puntual , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Protozoarias/metabolismo , Proteínas Recombinantes/química , Espectrometría de Fluorescencia , Transfección , Trypanosoma cruziRESUMEN
Antimicrobial peptides are components of the innate immune systems of a wide variety of eukaryotic organisms and are being developed as antibiotics in the fight against bacterial and fungal infections. We explored the potential activities of antimicrobial peptides against the African trypanosome Trypanosoma brucei, a vector-borne protozoan parasite that is responsible for significant morbidity and mortality in both humans and animals. Three classes of mammalian antimicrobial peptides were tested: alpha-defensins, beta-defensins, and cathelicidins. Although members of all 3 classes of antimicrobial peptides showed activity, those derived from the cathelicidin class were most effective, killing both insect and bloodstream forms of the parasite. The mechanism of action of the cathelicidins against T. brucei involves disruption of surface membrane integrity. Administration of cathelicidin antimicrobial peptides to mice with late-stage T. brucei infection acutely decreased parasitemia and prolonged survival. These results highlight the potential use of antimicrobial peptides for the treatment of African trypanosomiasis.