RESUMEN
Fidaxomicin (Fdx) is a natural product antibiotic with potent activity against Clostridioides difficile and other Gram-positive bacteria such as Mycobacterium tuberculosis. Only a few Fdx derivatives have been synthesized and examined for their biological activity in the 50â years since its discovery. Fdx has a well-studied mechanism of action, namely inhibition of the bacterial RNA polymerase. Yet, the targeted organisms harbor different target protein sequences, which poses a challenge for the rational development of new semisynthetic Fdx derivatives. We introduced substituents on the two phenolic hydroxy groups of Fdx and evaluated the resulting trends in antibiotic activity against M.â tuberculosis, C.â difficile, and the Gram-negative model organism Caulobacter crescentus. As suggested by the target protein structures, we identified the preferable derivatisation site for each organism. The derivative ortho-methyl Fdx also exhibited activity against the Gram-negative C.â crescentus wild type, a first for fidaxomicin antibiotics. These insights will guide the synthesis of next-generation fidaxomicin antibiotics.
Asunto(s)
Clostridioides difficile , Mycobacterium tuberculosis , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Fidaxomicina , Aminoglicósidos/farmacología , ARN Polimerasas Dirigidas por ADNRESUMEN
Mitochondria are dynamic organelles, the morphology of which results from an equilibrium between two opposing processes, fusion and fission. Mitochondrial fusion relies on dynamin-related GTPases, the mitofusins (MFN1 and 2) in the outer mitochondrial membrane and OPA1 (optic atrophy 1) in the inner mitochondrial membrane. Apart from a role in the maintenance of mitochondrial DNA, little is known about the physiological role of mitochondrial fusion. Here we report that mitochondria hyperfuse and form a highly interconnected network in cells exposed to selective stresses. This process precedes mitochondrial fission when it is triggered by apoptotic stimuli such as UV irradiation or actinomycin D. Stress-induced mitochondrial hyperfusion (SIMH) is independent of MFN2, BAX/BAK, and prohibitins, but requires L-OPA1, MFN1, and the mitochondrial inner membrane protein SLP-2. In the absence of SLP-2, L-OPA1 is lost and SIMH is prevented. SIMH is accompanied by increased mitochondrial ATP production and represents a novel adaptive pro-survival response against stress.
Asunto(s)
Fibroblastos/fisiología , Proteínas de la Membrana/fisiología , Mitocondrias/fisiología , Estrés Fisiológico , Adenosina Trifosfato/metabolismo , Animales , Células Cultivadas , Dactinomicina/toxicidad , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , GTP Fosfohidrolasas/fisiología , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/efectos de la radiación , Rayos UltravioletaRESUMEN
Phage T4, the archetype of lytic bacterial viruses, needs only 62 genes to propagate under standard laboratory conditions. Interestingly, the T4 genome contains more than 100 putative genes of unknown function, with few detectable homologues in cellular genomes. To characterize this uncharted territory of genetic information, we have identified several T4 genes that prevent bacterial growth when expressed from plasmids under inducible conditions. Here, we report on the various phenotypes and molecular characterization of 55.1, one of the genes of unknown function. High-level expression from the arabinose-inducible P(BAD) promoter is toxic to the bacteria and delays the intracellular accumulation of phage without affecting the final burst size. Low-level expression from T4 promoter(s) renders bacteria highly sensitive to UV irradiation and hypersensitive to trimethoprim, an inhibitor of dihydrofolate reductase. The delay in intracellular phage accumulation requires UvsW, a T4 helicase that is also a suppressor of 55.1-induced toxicity and UV sensitivity. Genetic and biochemical experiments demonstrate that gp55.1 binds to FolD, a key enzyme of the folate metabolism and suppressor of 55.1. Finally, we show that gp55.1 prevents the repair of UV-induced DNA photoproducts by the nucleotide excision repair (NER) pathway through interaction with the UvrA and UvrB proteins.
Asunto(s)
Bacteriófago T4/genética , Reparación del ADN , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Fólico/metabolismo , Expresión Génica , Proteínas Virales/metabolismo , Bacteriófago T4/metabolismo , Daño del ADN/efectos de la radiación , Reparación del ADN/efectos de la radiación , Escherichia coli/efectos de la radiación , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Rayos Ultravioleta , Proteínas Virales/genéticaRESUMEN
Apoptosis, induced by a number of death stimuli, is associated with a fragmentation of the mitochondrial network. These morphological changes in mitochondria have been shown to require proteins, such as Drp1 or hFis1, which are involved in regulating the fission of mitochondria. However, the precise role of mitochondrial fission during apoptosis remains elusive. Here we report that inhibiting the fission machinery in Bax/Bak-mediated apoptosis, by down-regulating of Drp1 or hFis1, prevents the fragmentation of the mitochondrial network and partially inhibits the release of cytochrome c from the mitochondria but fails to block the efflux of Smac/DIABLO. In addition, preventing mitochondrial fragmentation does not inhibit cell death induced by Bax/Bak-dependent death stimuli, in contrast to the effects of Bcl-xL or caspase inhibition. Therefore, the fission of mitochondria is a dispensable event in Bax/Bak-dependent apoptosis.
Asunto(s)
Apoptosis , Mitocondrias/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Línea Celular , Chlorocebus aethiops , Citocromos c/metabolismo , Dinaminas , GTP Fosfohidrolasas/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/ultraestructura , Membranas Mitocondriales/metabolismo , Membranas Mitocondriales/ultraestructura , Proteínas Mitocondriales/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína X Asociada a bcl-2/genéticaRESUMEN
Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by fibrosis and vasculopathy. CXCL4 represents an early serum biomarker of severe SSc and likely contributes to inflammation via chemokine signaling pathways, but the exact role of CXCL4 in SSc pathogenesis is unclear. Here, we elucidate an unanticipated mechanism for CXCL4-mediated immune amplification in SSc, in which CXCL4 organizes "self" and microbial DNA into liquid crystalline immune complexes that amplify TLR9-mediated plasmacytoid dendritic cell (pDC)-hyperactivation and interferon-α production. Surprisingly, this activity does not require CXCR3, the CXCL4 receptor. Importantly, we find that CXCL4-DNA complexes are present in vivo and correlate with type I interferon (IFN-I) in SSc blood, and that CXCL4-positive skin pDCs coexpress IFN-I-related genes. Thus, we establish a direct link between CXCL4 overexpression and the IFN-I-gene signature in SSc and outline a paradigm in which chemokines can drastically modulate innate immune receptors without being direct agonists.
Asunto(s)
ADN Bacteriano/metabolismo , Interferón-alfa/metabolismo , Factor Plaquetario 4/metabolismo , Esclerodermia Sistémica/inmunología , Receptor Toll-Like 9/metabolismo , Adulto , Anciano , Biopsia , Estudios de Casos y Controles , ADN Bacteriano/inmunología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Voluntarios Sanos , Humanos , Interferón-alfa/inmunología , Cristales Líquidos , Masculino , Persona de Mediana Edad , Factor Plaquetario 4/inmunología , Receptores CXCR3/inmunología , Receptores CXCR3/metabolismo , Esclerodermia Sistémica/microbiología , Esclerodermia Sistémica/patología , Piel/citología , Piel/inmunología , Piel/microbiología , Piel/patología , Receptor Toll-Like 9/inmunologíaRESUMEN
Topoisomerases are enzymes that alter the topological properties of DNA. Phage T4 encodes its own topoisomerase but it can also utilize host-encoded topoisomerases. Here we characterized 55.2, a phage T4 predicted ORF of unknown function. High levels of expression of the cloned 55.2 gene are toxic in E. coli. This toxicity is suppressed either by increased topoisomerase I expression or by partial inactivation of the ATPase subunit of the DNA gyrase. Interestingly, very low-level expression of 55.2, which is non-lethal to wild type E. coli, prevents the growth of a deletion mutant of the topoisomerase I (topA) gene. In vitro, gp55.2 binds DNA and blocks specifically the relaxation of negatively supercoiled DNA by topoisomerase I. In vivo, expression of gp55.2 at low non-toxic levels alters the steady state DNA supercoiling of a reporter plasmid. Although 55.2 is not an essential gene, competition experiments indicate that it is required for optimal phage growth. We propose that the role of gp55.2 is to subtly modulate host topoisomerase I activity during infection to insure optimal T4 phage yield.
Asunto(s)
Bacteriófago T4/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Escherichia coli/enzimología , Sistemas de Lectura Abierta/genética , Bacteriófago T4/crecimiento & desarrollo , Bacteriófago T4/fisiología , Clonación Molecular , ADN-Topoisomerasas de Tipo I/química , ADN-Topoisomerasas de Tipo I/genética , ADN Superhelicoidal/química , ADN Superhelicoidal/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/virología , Plásmidos/genética , Plásmidos/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Inhibidores de Topoisomerasa I/química , Inhibidores de Topoisomerasa I/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Mitochondrial fusion is a poorly characterized process which has mainly been studied in yeast and Drosophila but is thought to occur in all eukaryotes. Until now, there was only indirect evidence to support such a process in mammalian cells. In this study, using a cell fusion system, we found that mitochondrial fusion occurs rapidly in mammalian cells and is completed in less than 24 h. We report that the fusion of mitochondria requires an intact mitochondrial inner membrane potential but is independent of a functional cytoskeleton.
Asunto(s)
Membranas Intracelulares/fisiología , Fusión de Membrana , Potenciales de la Membrana , Mitocondrias/fisiología , Actinas/fisiología , Animales , Células Cultivadas , Perros , Células HeLa , Humanos , Tubulina (Proteína)/fisiologíaRESUMEN
BACKGROUND: Staphylococcus aureus is a major human pathogen responsible for a variety of nosocomial and community-acquired infections. Recent reports show that the prevalence of Methicillin-Resistant S. aureus (MRSA) infections in cystic fibrosis (CF) patients is increasing. In 2006 in Marseille, France, we have detected an atypical MRSA strain with a specific antibiotic susceptibility profile and a unique growth phenotype. Because of the clinical importance of the spread of such strain among CF patients we decided to sequence the genome of one representative isolate (strain CF-Marseille) to compare this to the published genome sequences. We also conducted a retrospective epidemiological analysis on all S. aureus isolated from 2002 to 2007 in CF patients from our institution. RESULTS: CF-Marseille is multidrug resistant, has a hetero-Glycopeptide-Intermediate resistance S. aureus phenotype, grows on Cepacia agar with intense orange pigmentation and has a thickened cell wall. Phylogenetic analyses using Complete Genome Hybridization and Multi Locus VNTR Assay showed that CF-Marseille was closely related to strain Mu50, representing vancomycin-resistant S. aureus. Analysis of CF-Marseille shows a similar core genome to that of previously sequenced MRSA strains but with a different genomic organization due to the presence of specific mobile genetic elements i.e. a new SCCmec type IV mosaic cassette that has integrated the pUB110 plasmid, and a new phage closely related to phiETA3. Moreover this phage could be seen by electron microscopy when mobilized with several antibiotics commonly used in CF patients including, tobramycin, ciprofloxacin, cotrimoxazole, or imipenem. Phylogenetic analysis of phenotypically similar h-GISA in our study also suggests that CF patients are colonized by polyclonal populations of MRSA that represents an incredible reservoir for lateral gene transfer. CONCLUSION: In conclusion, we demonstrated the emergence and spreading of a new isolate of MRSA in CF patients in Marseille, France, that has probably been selected in the airways by antibiotic pressure. Antibiotic-mediated phage induction may result in high-frequency transfer and the unintended consequence of promoting the spread of virulence and/or antibiotic resistance determinants. The emergence of well-adapted MRSA is worrying in such population chronically colonized and receiving many antibiotics and represents a model for emergence of uncontrollable super bugs in a specific niche. REVIEWERS: This article was reviewed by Eric Bapteste, Pierre Pontarotti, and Igor Zhulin. For the full reviews, please go to the Reviewers' comments section.
Asunto(s)
Antibacterianos/farmacología , Bacteriófagos/efectos de los fármacos , Fibrosis Quística/microbiología , Farmacorresistencia Bacteriana Múltiple/genética , Genoma Bacteriano/genética , Staphylococcus aureus/citología , Staphylococcus aureus/genética , Secuencia de Bases , Proliferación Celular/efectos de los fármacos , Pared Celular/ultraestructura , Fibrosis Quística/epidemiología , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Francia/epidemiología , Perfilación de la Expresión Génica , Genes Bacterianos , Humanos , Resistencia a la Meticilina/efectos de los fármacos , Resistencia a la Meticilina/genética , Repeticiones de Minisatélite/genética , Datos de Secuencia Molecular , Fenotipo , Filogenia , Staphylococcus aureus/ultraestructura , Transcripción Genética/efectos de los fármacosRESUMEN
Mitochondria form a highly dynamic tubular network, the morphology of which is regulated by frequent fission and fusion events. However, the role of mitochondrial fission in homeostasis of the organelle is still unknown. Here we report that preventing mitochondrial fission, by down-regulating expression of Drp1 in mammalian cells leads to a loss of mitochondrial DNA and a decrease of mitochondrial respiration coupled to an increase in the levels of cellular reactive oxygen species (ROS). At the cellular level, mitochondrial dysfunction resulting from the lack of fission leads to a drop in the levels of cellular ATP, an inhibition of cell proliferation and an increase in autophagy. In conclusion, we propose that mitochondrial fission is required for preservation of mitochondrial function and thereby for maintenance of cellular homeostasis.
Asunto(s)
ADN Mitocondrial/metabolismo , Regulación de la Expresión Génica , Mitocondrias/fisiología , Adenosina Trifosfato/metabolismo , Autofagia , Proliferación Celular , Dinaminas , GTP Fosfohidrolasas/metabolismo , Células HeLa , Homeostasis , Humanos , Potencial de la Membrana Mitocondrial , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Modelos Biológicos , Consumo de Oxígeno , Interferencia de ARN , Especies Reactivas de OxígenoRESUMEN
Mitochondria form filamentous networks that undergo continuous fission/fusion. In the pancreatic beta-cells, mitochondria are essential for the transduction of signals linking nutrient metabolism to insulin granule exocytosis. Here we have studied mitochondrial networks in the insulinoma cell line INS-1E, primary rat and human beta-cells. We have further investigated the impact of mitochondrial fission/fusion on metabolism-secretion coupling in INS-1E cells. Overexpression of hFis1 caused dramatic mitochondrial fragmentation, whereas Mfn1 evoked hyperfusion and the aggregation of mitochondria. Cells overexpressing hFis1 or Mfn1 showed reduced mitochondrial volume, lowered cellular ATP levels, and as a consequence, impaired glucose-stimulated insulin secretion. Decreased mitochondrial ATP generation was partially compensated for by enhanced glycolysis as indicated by increased lactate production in these cells. Dominant-negative Mfn1 elicited mitochondrial shortening and fragmentation of INS-1E cell mitochondria, similar to hFis1. However, the mitochondrial volume, cytosolic ATP levels, and glucose-stimulated insulin secretion were little affected. We conclude that mitochondrial fragmentation per se does not impair metabolism-secretion coupling. Through their impact on mitochondrial bioenergetics and distribution, hFis1 and Mfn1 activities influence mitochondrial signal generation thereby insulin exocytosis.
Asunto(s)
GTP Fosfohidrolasas/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Fusión de Membrana/fisiología , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Animales , Línea Celular Tumoral , Exocitosis/fisiología , GTP Fosfohidrolasas/genética , Glucosa/genética , Glucosa/metabolismo , Humanos , Insulina/genética , Secreción de Insulina , Células Secretoras de Insulina/citología , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial , Proteínas Mitocondriales/genética , Ratas , Ratas Wistar , Vesículas Secretoras/genética , Vesículas Secretoras/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The balance between the fission and fusion mechanisms regulate the morphology of mitochondria. In this study we have identified a mammalian protein that we call hFis1, which is the orthologue of the yeast Fis1p known to participate in yeast mitochondrial division. hFis1, when overexpressed in various cell types, localized to the outer mitochondrial membrane and induced mitochondrial fission. This event was inhibited by a dominant negative mutant of Drp1 (Drp1(K38A)), a major component of the fission apparatus. Fragmentation of the mitochondrial network by hFis1 was followed by the release of cytochrome c and ultimately apoptosis. Bcl-xL was able to block cytochrome c release and apoptosis but failed to prevent mitochondrial fragmentation. Our studies show that hFis1 is part of the mammalian fission machinery and suggest that regulation of the fission processes might be involved in apoptotic mechanisms.