RESUMEN

The rhizosphere is a vital soil compartment providing key plant-beneficial functions. However, little is known about the mechanisms driving viral diversity in the rhizosphere. Viruses can establish lytic or lysogenic interactions with their bacterial hosts. In the latter, they assume a dormant state integrated in the host genome and can be awakened by different perturbations that impact host cell physiology, triggering a viral bloom, which is potentially a fundamental mechanism driving soil viral diversity, as 22%-68% of soil bacteria are predicted to harbour dormant viruses. Here we assessed the viral bloom response in rhizospheric viromes by exposing them to three contrasting soil perturbation agents: earthworms, herbicide and antibiotic pollutant. The viromes were next screened for rhizosphere-relevant genes and also used as inoculant on microcosms incubations to test their impacts on pristine microbiomes. Our results show that while post-perturbation viromes diverged from control conditions, viral communities exposed to both herbicide and antibiotic pollutant were more similar to each other than those influenced by earthworms. The latter also favoured an increase in viral populations harbouring genes involved in plant-beneficial functions. Post-perturbation viromes inoculated on soil microcosms changed the diversity of pristine microbiomes, suggesting that viromes are important components of the soil ecological memory driving eco-evolutionary processes that determine future microbiome trajectories according to past events. Our findings demonstrate that viromes are active players in the rhizosphere and need to be considered in efforts to understand and control the microbial processes towards sustainable crop production.

Asunto(s)

Herbicidas , Virus , Rizosfera , Viroma , Microbiología del Suelo , Bacterias/genética , Virus/genética , Suelo , Antibacterianos

RESUMEN

Blastocladiella emersonii is an early diverging fungus of the phylum Blastocladiomycota. During the life cycle of the fungus, mitochondrial morphology changes significantly, from a fragmented form in sessile vegetative cells to a fused network in motile zoospores. In this study, we visualize these morphological changes using a mitochondrial fluorescent probe and show that the respiratory capacity in zoospores is much higher than in vegetative cells, suggesting that mitochondrial morphology could be related to the differences in oxygen consumption. While studying the respiratory chain of the fungus, we observed an antimycin A and cyanide-insensitive, salicylhydroxamic (SHAM)-sensitive respiratory activity, indicative of a mitochondrial alternative oxidase (AOX) activity. The presence of AOX was confirmed by the finding of a B. emersonii cDNA encoding a putative AOX, and by detection of AOX protein in immunoblots. Inhibition of AOX activity by SHAM was found to significantly alter the capacity of the fungus to grow and sporulate, indicating that AOX participates in life cycle control in B. emersonii.

Asunto(s)

Blastocladiella/crecimiento & desarrollo , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Proteínas Mitocondriales/metabolismo , Oxidorreductasas/metabolismo , Proteínas de Plantas/metabolismo , Esporas Fúngicas/crecimiento & desarrollo , Transporte de Electrón , Oxidación-Reducción , Oxígeno/metabolismo

RESUMEN

Sensing light is the fundamental property of visual systems, with vision in animals being based almost exclusively on opsin photopigments [1]. Rhodopsin also acts as a photoreceptor linked to phototaxis in green algae [2, 3] and has been implicated by chemical means as a light sensor in the flagellated swimming zoospores of the fungus Allomyces reticulatus [4]; however, the signaling mechanism in these fungi remains unknown. Here we use a combination of genome sequencing and molecular inhibition experiments with light-sensing phenotype studies to examine the signaling pathway involved in visual perception in the closely related fungus Blastocladiella emersonii. Our data show that in these fungi, light perception is accomplished by the function of a novel gene fusion (BeGC1) of a type I (microbial) rhodopsin domain and guanylyl cyclase catalytic domain. Photobleaching of rhodopsin function prevents accumulation of cGMP levels and phototaxis of fungal zoospores exposed to green light, whereas inhibition of guanylyl cyclase activity negatively affects fungal phototaxis. Immunofluorescence microscopy localizes the BeGC1 protein to the external surface of the zoospore eyespot positioned close to the base of the swimming flagellum [4, 5], demonstrating this is a photoreceptive organelle composed of lipid droplets. Taken together, these data indicate that Blastocladiomycota fungi have a cGMP signaling pathway involved in phototaxis similar to the vertebrate vision-signaling cascade but composed of protein domain components arranged as a novel gene fusion architecture and of distant evolutionary ancestry to type II rhodopsins of animals.

Asunto(s)

Blastocladiella/fisiología , Proteínas Fúngicas/genética , Guanilato Ciclasa/genética , Luz , Rodopsina/genética , Transducción de Señal , Secuencia de Aminoácidos , Secuencia de Bases , Blastocladiella/genética , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Proteínas Fúngicas/metabolismo , Fusión Génica , Guanilato Ciclasa/metabolismo , Datos de Secuencia Molecular , Filogenia , Estructura Terciaria de Proteína , Rodopsina/metabolismo , Alineación de Secuencia , Percepción Visual

RESUMEN

BACKGROUND: The unicellular parasite Trypanosoma cruzi is the causative agent of Chagas disease in humans. Adherence of the infective stage to elements of the extracellular matrix (ECM), as laminin and fibronectin, is an essential step in host cell invasion. Although members of the gp85/TS, as Tc85, were identified as laminin and fibronectin ligands, the signaling events triggered on the parasite upon binding to these molecules are largely unexplored. METHODOLOGY/PRINCIPAL FINDINGS: Viable infective parasites were incubated with laminin, fibronectin or bovine serum albumin for different periods of time and the proteins were separated by bidimensional gels. The phosphoproteins were envisaged by specific staining and the spots showing phosphorylation levels significantly different from the control were excised and identified by MS/MS. The results of interest were confirmed by immunoblotting or immunoprecipitation and the localization of proteins in the parasite was determined by immunofluorescence. Using a host cell-free system, our data indicate that the phosphorylation contents of T. cruzi proteins encompassing different cellular functions are modified upon incubation of the parasite with fibronectin or laminin. CONCLUSIONS/SIGNIFICANCE: Herein it is shown, for the first time, that paraflagellar rod proteins and α-tubulin, major structural elements of the parasite cytoskeleton, are predominantly dephosphorylated during the process, probably involving the ERK1/2 pathway. It is well established that T. cruzi binds to ECM elements during the cell infection process. The fact that laminin and fibronectin induce predominantly dephosphorylation of the main cytoskeletal proteins of the parasite suggests a possible correlation between cytoskeletal modifications and the ability of the parasite to internalize into host cells.

Asunto(s)

Fibronectinas/metabolismo , Laminina/metabolismo , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/fisiología , Fosforilación , Proteínas Protozoarias/metabolismo , Transducción de Señal/fisiología , Tubulina (Proteína)/metabolismo

RESUMEN

Different types of shed vesicles as, for example, exosomes, plasma-membrane-derived vesicles or microparticles, are the focus of intense research in view of their potential role in cell-cell communication and under the perspective that they might be good tools for immunotherapy, vaccination or diagnostic purposes. This review discusses ways employed by pathogenic trypanosomatids to interact with the host by shedding vesicles that contain molecules important for the establishment of infection, as opposed to previous beliefs considering them as a waste of cellular metabolism. Trypanosomatids are compared with Apicomplexa, which circulate parasite antigens bound to vesicles shed by host cells. The knowledge of the origin and chemical composition of these different vesicles might lead to the understanding of the mechanisms that determine their biological function.

Asunto(s)

Exosomas/metabolismo , Infecciones por Protozoos/parasitología , Factores de Virulencia/metabolismo , Animales , Apicomplexa/metabolismo , Apicomplexa/patogenicidad , Exosomas/parasitología , Interacciones Huésped-Parásitos , Humanos , Trypanosomatina/metabolismo , Trypanosomatina/patogenicidad

RESUMEN

Mutations in the gene encoding cytosolic Cu,Zn-superoxide dismutase (SOD1) have been linked to familial amyotrophic lateral sclerosis (FALS). However the molecular mechanisms of motor neuron death are multi-factorial and remain unclear. Here we examined DNA damage, p53 activity and apoptosis in SH-SY5Y human neuroblastoma cells transfected to achieve low-level expression of either wild-type or mutant Gly(93)-->Ala (G93A) SOD1, typical of FALS. DNA damage was investigated by evaluating the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and DNA strand breaks. Significantly higher levels of DNA damage, increased p53 activity, and a greater percentage of apoptotic cells were observed in SH-SY5Y cells transfected with G93A SOD1 when compared to cells overexpressing wild-type SOD1 and untransfected cells. Western blot, FACS, and confocal microscopy analysis demonstrated that G93A SOD1 is present in the nucleus in association with DNA. Nuclear G93A SOD1 has identical superoxide dismutase activity but displays increased peroxidase activity when compared to wild-type SOD1. These results indicate that the G93A mutant SOD1 association with DNA might induce DNA damage and trigger the apoptotic response by activating p53. This toxic activity of mutant SOD1 in the nucleus may play an important role in the complex mechanisms associated with motor neuron death observed in ALS pathogenesis.

Asunto(s)

Esclerosis Amiotrófica Lateral/metabolismo , Apoptosis , Cromatina/metabolismo , Daño del ADN , Neuroblastoma/metabolismo , Superóxido Dismutasa/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , 8-Hidroxi-2'-Desoxicoguanosina , Esclerosis Amiotrófica Lateral/patología , Núcleo Celular/enzimología , Núcleo Celular/patología , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Humanos , Técnicas para Inmunoenzimas , Peroxidación de Lípido , Superóxido Dismutasa-1 , Células Tumorales Cultivadas

RESUMEN

Trypanosoma cruzi, the agent of Chagas' disease, alternates between different morphogenetic stages that face distinct physiological conditions in their invertebrate and vertebrate hosts, likely in the availability of glucose. While the glucose transport is well characterized in epimastigotes of T. cruzi, nothing is known about how the mammalian stages acquire this molecule. Herein glucose transport activity and expression were analyzed in the three developmental stages present in the vertebrate cycle of T. cruzi. The infective trypomastigotes showed the highest transport activity (V(max)=5.34+/-0.54 nmol/min per mg of protein; K(m)=0.38+/-0.01 mM) when compared to intracellular epimastigotes (V(max)=2.18+/-0.20 nmol/min per mg of protein; K(m)=0.39+/-0.01 mM). Under the conditions employed no transport activity could be detected in amastigotes. The gene of the glucose transporter is expressed at the mRNA level in trypomastigotes and in intracellular epimastigotes but not in amastigotes, as revealed by real-time PCR. In both trypomastigotes and intracellular epimastigotes protein expression could be detected by Western blot with an antibody raised against the glucose transporter correlating well with the transport activity measured experimentally. Interestingly, anti-glucose transporter antibodies showed a strong reactivity with glycosome and reservosome organelles. A comparison between proline and glucose transport among the intracellular differentiation forms is presented. The data suggest that the regulation of glucose transporter reflects different energy and carbon requirements along the intracellular life cycle of T. cruzi.

Asunto(s)

Glucosa/metabolismo , Trypanosoma cruzi/metabolismo , Animales , Western Blotting , Perfilación de la Expresión Génica , Prolina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Vesículas Transportadoras

RESUMEN

Fluorescent proteins from the green fluorescent protein family strongly interact with CdSe/ZnS and ZnSe/ZnS nanocrystals at neutral pH. Green emitting CdSe/ZnS nanocrystals and red emitting fluorescent protein dTomato constitute a 72% efficiency FRET system with the largest alteration of the overall photoluminescence profile, following complex formation, observed so far. The substitution of ZnSe/ZnS for CdSe/ZnS nanocrystals as energy donors enabled the use of a green fluorescent protein, GFP5, as energy acceptor. Violet emitting ZnSe/ZnS nanocrystals and green GFP5 constitute a system with 43% FRET efficiency and an unusually strong sensitized emission. ZnSe/ZnS-GFP5 provides a cadmium-free, high-contrast FRET system that covers only the high-energy part of the visible spectrum, leaving room for simultaneous use of the yellow and red color channels. Anisotropic fluorescence measurements confirmed the depolarization of GFP5 sensitized emission.

Asunto(s)

Proteínas Luminiscentes/química , Nanopartículas/química , Compuestos de Selenio/química , Compuestos de Zinc/química , Sulfato de Zinc/química , Color , Polarización de Fluorescencia , Transferencia Resonante de Energía de Fluorescencia , Proteínas Luminiscentes/metabolismo , Compuestos de Selenio/metabolismo , Compuestos de Zinc/metabolismo , Sulfato de Zinc/metabolismo

RESUMEN

Aminoacetone (AA), triose phosphates, and acetone are putative endogenous sources of potentially cytotoxic and genotoxic methylglyoxal (MG), which has been reported to be augmented in the plasma of diabetic patients. In these patients, accumulation of MG derived from aminoacetone, a threonine and glycine catabolite, is inferred from the observed concomitant endothelial overexpression of circulating semicarbazide-sensitive amine oxidases. These copper-dependent enzymes catalyze the oxidation of primary amines, such as AA and methylamine, by molecular oxygen, to the corresponding aldehydes, NH4(+) ion and H2O2. We recently reported that AA aerobic oxidation to MG also takes place immediately upon addition of catalytic amounts of copper and iron ions. Taking into account that (i) MG and H2O2 are reportedly cytotoxic to insulin-producing cell lineages such as RINm5f and that (ii) the metal-catalyzed oxidation of AA is propagated by O2(*-) radical anion, we decided to investigate the possible pro-oxidant action of AA on these cells taken here as a reliable model system for pancreatic beta-cells. Indeed, we show that AA (0.10-5.0 mM) administration to RINm5f cultures induces cell death. Ferrous (50-300 microM) and Fe(3+) ion (100 microM) addition to the cell cultures had no effect, whereas Cu(2+) (5.0-100 microM) significantly increased cell death. Supplementation of the AA- and Cu(2+)-containing culture medium with antioxidants, such as catalase (5.0 microM), superoxide dismutase (SOD, 50 U/mL), and N-acetylcysteine (NAC, 5.0 mM) led to partial protection. mRNA expression of MnSOD, CuZnSOD, glutathione peroxidase, and glutathione reductase, but not of catalase, is higher in cells treated with AA (0.50-1.0 mM) plus Cu(2+) ions (10-50 microM) relative to control cultures. This may imply higher activity of antioxidant enzymes in RINm5f AA-treated cells. In addition, we have found that AA (0.50-1.0 mM) plus Cu(2+) (100 microM) (i) increase RINm5f cytosolic calcium; (ii) promote DNA fragmentation; and (iii) increase the pro-apoptotic (Bax)/antiapoptotic (Bcl-2) ratio at the level of mRNA expression. In conclusion, although both normal and pathological concentrations of AA are probably much lower than those used here, it is tempting to propose that excess AA in diabetic patients may drive oxidative damage and eventually the death of pancreatic beta-cells.

Asunto(s)

Acetona/análogos & derivados , Insulina/metabolismo , Estrés Oxidativo/efectos de los fármacos , Acetona/química , Acetona/farmacología , Acetilcisteína/farmacología , Animales , Antioxidantes/farmacología , Catalasa/farmacología , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Cobre/farmacología , Relación Dosis-Respuesta a Droga , Secreción de Insulina , Ratones , Estructura Molecular , Células 3T3 NIH , ARN Mensajero/metabolismo , Superóxido Dismutasa/farmacología , Triosas/farmacología

RESUMEN

Fluorescent proteins from the green fluorescent protein (GFP) family interact strongly with CdSe/ZnS quantum dots. Photoluminescence of GFP5 is suppressed by red-emitting CdSe/ZnS quantum dots with high efficiency in a pH-dependent manner. The elevated degree of quenching, around 90%, makes it difficult to analyze the remaining signal, and it is not clear yet whether FRET is the reason behind the quenching. When the donor is a green-emitting CdSe/ZnS quantum dot and the acceptor is the HcRed1 protein, it is possible to detect quenching of the donor and sensitized emission from the acceptor. It was verified that the sensitized emission has the low anisotropy characteristic of FRET. The present characterization identifies donor-acceptor pairs formed by fluorescent proteins and CdSe/ZnS quantum dots that are suitable for the exploration of cellular events. These donor-acceptor pairs take advantage of the exceptional photochemical properties of quantum dots allied with the unique ability of fluorescent proteins to act as gene-based fluorescent probes.

Asunto(s)

Compuestos de Cadmio/química , Proteínas Fluorescentes Verdes/química , Puntos Cuánticos , Compuestos de Selenio/química , Dicroismo Circular , Transferencia Resonante de Energía de Fluorescencia , Concentración de Iones de Hidrógeno , Fotoquímica

RESUMEN

Chagas' disease is a chronic, debilitating and incapacitating illness, caused by the protozoan parasite Trypanosoma cruzi when infective trypomastigotes invade host cells. Although the mechanism of trypomastigotes interaction with mammalian cells has been intensively studied, a final and integrated picture of the signal transduction mechanisms involved still remains to be elucidated. Our group has previously shown that the conserved FLY domain (VTVXNVFLYNR), present in all members of the gp85/trans-sialidase glycoprotein family coating the surface of trypomastigotes, binds to cytokeratin 18 (CK18) on the surface of LLC-MK(2) epithelial cells, and significantly increases parasite entry into mammalian cells. Now it is reported that FLY, present on the surface of trypomastigotes or on latex beads binds to CK18, promotes dephosphorylation and reorganization of CK18 and activation of the ERK1/2 signaling cascade culminating in an increase of approximately 9-fold in the number of parasites/cell. Inhibition of ERK1/2 phosphorylation completely blocks the adhesion of FLY to cells and blocks by 57% the host cell infection by T. cruzi. Taken together our results indicate that the conserved FLY domain is an important tool that trypomastigotes have evolved to specific exploit the host cell machinery and guarantee a successful infection.

Asunto(s)

Glicoproteínas/química , Glicoproteínas/fisiología , Neuraminidasa/química , Neuraminidasa/fisiología , Proteínas Protozoarias/química , Proteínas Protozoarias/fisiología , Trypanosoma cruzi/fisiología , Trypanosoma cruzi/patogenicidad , Secuencia de Aminoácidos , Animales , Línea Celular , Secuencia Conservada , Glicoproteínas/genética , Humanos , Queratina-18/química , Queratina-18/metabolismo , Sistema de Señalización de MAP Quinasas , Neuraminidasa/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Proteínas Protozoarias/genética , Trypanosoma cruzi/genética , Virulencia

RESUMEN

5-Aminolevulinic acid (ALA) is a heme precursor accumulated in plasma and in organs in acute intermittent porphyria (AIP), a disease associated with neuromuscular dysfunction and increased incidence of hepatocellular carcinoma (HCC). Liver biopsies of AIP patients showed odd-shaped mitochondria and autophagic vacuoles containing well-preserved mitochondria. ALA yields reactive oxygen species upon metal-catalyzed oxidation and causes in vivo and in vitro impairment of rat liver mitochondria and DNA damage. Using a quantitative polymerase chain reaction assay, we demonstrated that ALA induces a dose-dependent damage in nuclear and mitochondrial DNA in human SVNF fibroblasts and rat PC12 cells. CHO cells treated with ALA also show nuclear DNA damage and human HepG2 cells entered in apoptosis and necrosis induced by ALA and its dimerization product, DHPY. The present data provide additional information on the genotoxicity of ALA, reinforcing the hypothesis that it may be involved in the development of HCC in AIP patients.

Asunto(s)

Ácido Aminolevulínico/farmacología , Daño del ADN , ADN/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Animales , Apoptosis/efectos de los fármacos , Células CHO , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/ultraestructura , Supervivencia Celular/efectos de los fármacos , Cricetinae , ADN/ultraestructura , Relación Dosis-Respuesta a Droga , Fibroblastos/ultraestructura , Hepatocitos/ultraestructura , Humanos , Mitocondrias/ultraestructura , Células PC12 , Ratas

RESUMEN

Here we report on the mitochondrial permeability transition (MPT), which refers to the morphology of mitochondria whose inner membrane has lost its selective permeability. In all types of apoptotic cells so far examined, we found outer mitochondrial membranes that had been ruptured. These mitochondria present a swollen matrix covered by an inner membrane herniating into the cytoplasm through the breached outer membrane. Similarly ruptured outer mitochondrial membranes have been reported in studies on mitochondrial fractions induced to undergo MPT, carried out by others. Our observations were made on five types of rat tissue cells and six different cultured cell lines in the early stages of apoptosis. Samples from the cell lines HL-60, HeLa, WEHI-164, and a special batch of PC-12 cells were subjected to various apoptogenic agents and analyzed morphometrically. Nonapoptotic companion cells with unaltered nuclear structure (CUNS) were also analyzed. The mitochondrial volume in microm(3) and the volume fraction of the cytoplasm occupied by mitochondria in cells with typical nuclear signs of apoptosis and also in CUNS were evaluated. The volume of the mitochondria with ruptured membrane represents at least 69% (47-89%) of the total mitochondrial volume of the apoptotic cells. Thus, a considerable fraction of the cellular mitochondrial mass is or was in the state of permeability transition and probably involved in enhancement of the apoptotic program. In all samples, a fraction of the cells with normal nuclei possessed mitochondria with breached outer membranes as described above. In these cells, MPT occurred before the appearance of the typical nuclear phenotype of the apoptotic cells.

Asunto(s)

Apoptosis , Membranas Intracelulares/metabolismo , Mitocondrias/química , Mitocondrias/metabolismo , Animales , Femenino , Humanos , Macrófagos/metabolismo , Macrófagos/ultraestructura , Masculino , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/ultraestructura , Microscopía Electrónica de Transmisión , Permeabilidad , Células Plasmáticas/metabolismo , Células Plasmáticas/ultraestructura , Próstata/metabolismo , Próstata/ultraestructura , Ratas , Células Tumorales Cultivadas/ultraestructura