RESUMEN
The discovery of mitochondrion-type genes in organisms thought to lack mitochondria led to the demonstration that hydrogenosomes share a common ancestry with mitochondria, as well as the discovery of mitosomes in multiple eukaryotic lineages. No examples of examined eukaryotes lacking a mitochondrion-related organelle exist, implying that the endosymbiont that gave rise to the mitochondrion was present in the first eukaryote. These organelles, known as hydrogenosomes, mitosomes, or mitochondrion-like organelles, are typically reduced, both structurally and biochemically, relative to classical mitochondria. However, despite their diversification and adaptation to different niches, all appear to play a role in Fe-S cluster assembly, as observed for mitochondria. Although evidence supports the use of common protein targeting mechanisms in the biogenesis of these diverse organelles, divergent features are also apparent. This review examines the metabolism and biogenesis of these organelles in divergent unicellular microbes, with a focus on parasitic protists.
Asunto(s)
Eucariontes/genética , Eucariontes/metabolismo , Genes Mitocondriales , Mitocondrias/genética , Mitocondrias/metabolismo , Eucariontes/ultraestructura , Proteínas Hierro-Azufre/metabolismo , Redes y Vías Metabólicas , Mitocondrias/ultraestructura , Filogenia , Homología de SecuenciaRESUMEN
RNAi knockdown was employed to study the function of p67, a lysosome-associated membrane protein (LAMP)-like type I transmembrane lysosomal glycoprotein in African trypanosomes. Conditional induction of p67 dsRNA resulted in specific approximately 90% reductions in de novo p67 synthesis in both mammalian bloodstream and procyclic insect-stage parasites. Bloodstream cell growth was severely retarded with extensive death after > 24 h of induction. Biosynthetic trafficking of residual p67, and of the soluble lysosomal protease trypanopain, were unimpaired. Endocytosis of tomato lectin, a surrogate receptor-mediated cargo, was only mildly impaired (approximately 20%), but proper lysosomal targeting was unaffected. p67 ablation had dramatic effects on lysosomal morphology with gross enlargement (four- to fivefold) and internal membrane profiles reminiscent of autophagic vacuoles. Ablation of p67 expression rendered bloodstream trypanosomes refractory to lysis by human trypanolytic factor (TLF), a lysosomally activated host innate immune mediator. Similar effects on lysosomal morphology and TLF sensitivity were also obtained by two pharmacological agents that neutralize lysosomal pH--chloroquine and bafilomycin A1. Surprisingly, however, lysosomal pH was not affected in ablated cells suggesting that other physiological alterations must account for increased resistance to TLF. These results indicate p67 plays an essential role in maintenance of normal lysosomal structure and physiology in bloodstream-stage African trypanosomes.
Asunto(s)
Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/metabolismo , Tripanosomiasis Africana/parasitología , Secuencia de Aminoácidos , Animales , Endocitosis , Orden Génico , Genoma de Protozoos , Humanos , Concentración de Iones de Hidrógeno , Lipoproteínas HDL/inmunología , Proteínas de Membrana de los Lisosomas/genética , Lisosomas/ultraestructura , Datos de Secuencia Molecular , Péptido Hidrolasas/metabolismo , Proteínas Protozoarias/genética , Interferencia de ARN , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/ultraestructuraRESUMEN
Trypanosome lytic factor (TLF) is a high-density lipoprotein (HDL) subclass providing innate protection to humans against infection by the protozoan parasite Trypanosoma brucei brucei. Two primate-specific plasma proteins, haptoglobin-related protein (Hpr) and apolipoprotein L-1 (ApoL-1), have been proposed to kill T. b. brucei both singularly or when co-assembled into the same HDL. To better understand the mechanism of T. b. brucei killing by TLF, the protein composition of TLF was investigated using a gentle immunoaffinity purification technique that avoids the loss of weakly associated proteins. HDL particles recovered by immunoaffinity absorption, with either anti-Hpr or anti-ApoL-1, were identical in protein composition and specific activity for T. b. brucei killing. Here, we show that TLF-bound Hpr strongly binds Hb and that addition of Hb stimulates TLF killing of T. b. brucei by increasing the affinity of TLF for its receptor, and by inducing Fenton chemistry within the trypanosome lysosome. These findings suggest that TLF in uninfected humans may be inactive against T. b. brucei prior to initiation of infection. We propose that infection of humans by T. b. brucei causes hemolysis that triggers the activation of TLF by the formation of Hpr-Hb complexes, leading to enhanced binding, trypanolytic activity, and clearance of parasites.
Asunto(s)
Hemoglobinas/metabolismo , Lipoproteínas HDL/metabolismo , Tripanosomiasis Africana/inmunología , Tripanosomiasis Africana/metabolismo , Animales , Antígenos de Neoplasias/metabolismo , Proteínas Sanguíneas/metabolismo , Eritrocitos/metabolismo , Haptoglobinas/metabolismo , Hemólisis , Humanos , Inmunidad Innata/fisiología , Lisosomas/fisiología , Trypanosoma brucei brucei/inmunología , Trypanosoma brucei brucei/patogenicidadRESUMEN
The human pathogen Trichomonas vaginalis lacks conventional mitochondria and instead contains divergent mitochondrial-related organelles. These double-membrane bound organelles, called hydrogenosomes, produce molecular hydrogen. Phylogenetic and biochemical analyses of hydrogenosomes indicate a common origin with mitochondria; however identification of hydrogenosomal proteins and studies on its metabolism have been limited. Here we provide a detailed proteomic analysis of the T. vaginalis hydrogenosome. The proteome of purified hydrogenosomes consists of 569 proteins, a number substantially lower than the 1,000-1,500 proteins reported for fungal and animal mitochondrial proteomes, yet considerably higher than proteins assigned to mitosomes. Pathways common to and distinct from both mitochondria and mitosomes were revealed by the hydrogenosome proteome. Proteins known to function in amino acid and energy metabolism, Fe-S cluster assembly, flavin-mediated catalysis, oxygen stress response, membrane translocation, chaperonin functions, proteolytic processing and ATP hydrolysis account for â¼30% of the hydrogenosome proteome. Of the 569 proteins in the hydrogenosome proteome, many appear to be associated with the external surface of hydrogenosomes, including large numbers of GTPases and ribosomal proteins. Glycolytic proteins were also found to be associated with the hydrogenosome proteome, similar to that previously observed for mitochondrial proteomes. Approximately 18% of the hydrogenosomal proteome is composed of hypothetical proteins of unknown function, predictive of multiple activities and properties yet to be uncovered for these highly adapted organelles.
Asunto(s)
Mitocondrias/metabolismo , Orgánulos/metabolismo , Proteoma/metabolismo , Proteínas Protozoarias/metabolismo , Trichomonas vaginalis/metabolismo , Humanos , Espectrometría de Masas , Mitocondrias/química , Mitocondrias/genética , Orgánulos/química , Orgánulos/genética , Filogenia , Proteoma/química , Proteoma/genética , Proteómica , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Trichomonas vaginalis/química , Trichomonas vaginalis/clasificación , Trichomonas vaginalis/genéticaRESUMEN
Trypanosoma brucei brucei is the causative agent of Nagana in cattle and can infect a wide range of mammals but is unable to infect humans because it is susceptible to the innate cytotoxic activity of normal human serum. A minor subfraction of human high-density lipoprotein (HDL), containing apolipoprotein A-I (APOA1), apolipoprotein L-I (APOL1) and haptoglobin-related protein (HPR) provides this innate protection against T. b. brucei infection. Both HPR and APOL1 are cytotoxic to T. b. brucei but their specific activities for killing increase several hundred-fold when assembled in the same HDL. This HDL is called trypanosome lytic factor (TLF) and kills T. b. brucei following receptor binding, endocytosis, and lysosomal localization. Trypanosome lytic factor is activated in the acidic lysosome and facilitates lysosomal membrane disruption. Lysosomal localization is necessary for T. b. brucei killing by TLF. Trypanosoma brucei rhodesiense, which is indistinguishable from T. b. brucei, is resistant to TLF killing and causes human African sleeping sickness. Human infectivity by T. b. rhodesiense correlates with the evolution of a human serum resistance associated protein (SRA) that is able to ablate TLF killing. When T. b. brucei is transfected with the SRA gene it becomes highly resistant to TLF and human serum. In the SRA transfected cells, intracellular trafficking of TLF is altered and TLF mainly localizes to a subset of SRA containing cytoplasmic vesicles but not to the lysosome. These findings indicate that the cellular distribution of TLF is influenced by SRA expression and may directly determine susceptibility.
Asunto(s)
Endocitosis , Lipoproteínas HDL/inmunología , Glicoproteínas de Membrana/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/inmunología , Trypanosoma brucei rhodesiense/inmunología , Tripanosomiasis Africana/inmunología , Animales , Antígenos de Neoplasias/inmunología , Apolipoproteína L1 , Apolipoproteínas/inmunología , Proteínas Sanguíneas/inmunología , Haptoglobinas/inmunología , Humanos , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Lisosomas/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/inmunología , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei rhodesiense/metabolismoRESUMEN
Trypanosoma brucei brucei is the causative agent of nagana in cattle and can infect a wide range of mammals but is unable to infect humans because it is susceptible to the innate cytotoxic activity of normal human serum. A minor subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I (apoA-I), apolipoprotein L-I (apoL-I), and haptoglobin-related protein (Hpr) provides this innate protection against T. b. brucei infection. This HDL subfraction, called trypanosome lytic factor (TLF), kills T. b. brucei following receptor binding, endocytosis, and lysosomal localization. Trypanosoma brucei rhodesiense, which is morphologically and physiologically indistinguishable from T. b. brucei, is resistant to TLF-mediated killing and causes human African sleeping sickness. Human infectivity by T. b. rhodesiense correlates with the evolution of a resistance-associated protein (SRA) that is able to ablate TLF killing. To examine the mechanism of TLF resistance, we transfected T. b. brucei with an epitope-tagged SRA gene. Transfected T. b. brucei expressed SRA mRNA at levels comparable to those in T. b. rhodesiense and was highly resistant to TLF. In the SRA-transfected cells, intracellular trafficking of TLF was altered, with TLF being mainly localized to a subset of SRA-containing cytoplasmic vesicles but not to the lysosome. These results indicate that the cellular distribution of TLF is influenced by SRA expression and may directly determine the organism's susceptibility to TLF.
Asunto(s)
Lipoproteínas HDL/metabolismo , Lisosomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/metabolismo , Animales , Núcleo Celular/metabolismo , Genotipo , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Mitocondrias/metabolismo , Fenotipo , Unión Proteica , Transporte de Proteínas , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , TransfecciónRESUMEN
The host range of African trypanosomes is influenced by innate protective molecules in the blood of primates. A subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I, apolipoprotein L-I, and haptoglobin-related protein is toxic to Trypanosoma brucei brucei but not the human sleeping sickness parasite Trypanosoma brucei rhodesiense. It is thought that T. b. rhodesiense evolved from a T. b. brucei-like ancestor and expresses a defense protein that ablates the antitrypanosomal activity of human HDL. To directly investigate this possibility, we developed an in vitro selection to generate human HDL-resistant T. b. brucei. Here we show that conversion of T. b. brucei from human HDL sensitive to resistant correlates with changes in the expression of the variant surface glycoprotein (VSG) and abolished uptake of the cytotoxic human HDLs. Complete transcriptome analysis of the HDL-susceptible and -resistant trypanosomes confirmed that VSG switching had occurred but failed to reveal the expression of other genes specifically associated with human HDL resistance, including the serum resistance-associated gene (SRA) of T. b. rhodesiense. In addition, we found that while the original active expression site was still utilized, expression of three expression site-associated genes (ESAG) was altered in the HDL-resistant trypanosomes. These findings demonstrate that resistance to human HDLs can be acquired by T. b. brucei.
Asunto(s)
Resistencia a Medicamentos/genética , Lipoproteínas HDL/fisiología , Glicoproteínas de Membrana/genética , Proteínas Protozoarias/genética , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei brucei/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Humanos , Lipoproteínas HDL/genética , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL/farmacología , Datos de Secuencia Molecular , Alineación de Secuencia , Trypanosoma brucei brucei/efectos de los fármacos , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismoRESUMEN
Human innate immunity to non-pathogenic species of African trypanosomes is provided by human high density lipoprotein (HDL) particles. Here we show that native human HDLs containing haptoglobin-related protein (Hpr), apolipoprotein L-I (apoL-I) and apolipoprotein A-I (apoA-I) are the principle antimicrobial molecules providing protection from trypanosome infection. Other HDL subclasses containing either apoA-I and apoL-I or apoA-I and Hpr have reduced trypanolytic activity, whereas HDL subclasses lacking apoL-I and Hpr are non-toxic to trypanosomes. Highly purified, lipid-free Hpr and apoL-I were both toxic to Trypanosoma brucei brucei but with specific activities at least 500-fold less than those of native HDLs, suggesting that association of these apolipoproteins within the HDL particle was necessary for optimal cytotoxicity. These studies show that HDLs can serve as platforms for the assembly of multiple synergistic proteins and that these assemblies may play a critical role in the evolution of primate-specific innate immunity to trypanosome infection.
Asunto(s)
Sistema Inmunológico/parasitología , Lipoproteínas HDL/metabolismo , Animales , Apolipoproteína A-I/metabolismo , Apolipoproteína L1 , Apolipoproteínas/metabolismo , Western Blotting , Cromatografía de Afinidad , Dimerización , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Evolución Molecular , Humanos , Lipoproteínas HDL/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Trypanosoma brucei brucei , Tripanosomiasis , Tripsina/farmacologíaRESUMEN
In the plant pathogenic ascomycete Nectria haematococca mating population (MP) VI, the conditionally dispensable chromosomes are unstable during sexual reproduction. During mapping of such a chromosome, three dispersed repeats were identified. Nht2, one of these repeated elements, is a long terminal repeat (LTR) retrotransposon that is 5.9 kb in length. Its deduced amino acid sequence is homologous to the four enzymatic domains characteristic of copia retrotransposons, but it contains multiple stop codons and probably is no longer able to transpose autonomously. Nht2's LTRs differ at ten positions and the characteristics of these differences resemble the changes induced by repeat-induced point mutation (RIP) in Neurospora crassa. The likelihood that Nectria haematococca MP VI has a RIP-like process, however, is reduced by the fact that a multi-copy transposon cloned from the same ascospore isolate as Nht2 encodes an intact open reading frame. Nht2 is broadly distributed among isolates collected from a variety of host plants. A limited survey of three field isolates suggests that Nht2 is on only one or a few chromosomes in every genome. Nht2's degeneracy and its widespread distribution within the species both suggest that it is an ancient element within N. haematococca MP VI.