RESUMEN
Forty percent of the US population and 1 in 6 individuals worldwide are obese, and the incidence of this disease is surging globally1,2. Various dietary interventions, including carbohydrate and fat restriction, and more recently amino acid restriction, have been explored to combat this epidemic3-6. We sought to investigate the impact of removing individual amino acids on the weight profiles of mice. Compared to essential amino acid restriction, induction of conditional cysteine restriction resulted in the most dramatic weight loss, amounting to 20% within 3 days and 30% within one week, which was readily reversed. This weight loss occurred despite the presence of substantial cysteine reserves stored in glutathione (GSH) across various tissues7. Further analysis demonstrated that the weight reduction primarily stemmed from an increase in the utilization of fat mass, while locomotion, circadian rhythm and histological appearance of multiple other tissues remained largely unaffected. Cysteine deficiency activated the integrated stress response (ISR) and NRF2-mediated oxidative stress response (OSR), which amplify each other, leading to the induction of GDF15 and FGF21, hormones associated with increased lipolysis, energy homeostasis and food aversion8-10. We additionally observed rapid tissue coenzyme A (CoA) depletion, resulting in energetically inefficient anaerobic glycolysis and TCA cycle, with sustained urinary excretion of pyruvate, orotate, citrate, α-ketoglutarate, nitrogen rich compounds and amino acids. In summary, our investigation highlights that cysteine restriction, by depleting GSH and CoA, exerts a maximal impact on weight loss, metabolism, and stress signaling compared to other amino acid restrictions. These findings may pave the way for innovative strategies for addressing a range of metabolic diseases and the growing obesity crisis.
RESUMEN
Glutathione (GSH) is the most abundant cellular antioxidant. As reactive oxygen species (ROS) are widely believed to promote aging and age-related diseases, and antioxidants can neutralize ROS, it follows that GSH and its precursor, N-acetyl cysteine (NAC), are among the most popular dietary supplements. However, the long- term effects of GSH or NAC on healthy animals have not been thoroughly investigated. We employed C. elegans to demonstrate that chronic administration of GSH or NAC to young or aged animals perturbs global gene expression, inhibits skn-1-mediated transcription, and accelerates aging. In contrast, limiting the consumption of dietary thiols, including those naturally derived from the microbiota, extended lifespan. Pharmacological GSH restriction activates the unfolded protein response and increases proteotoxic stress resistance in worms and human cells. It is thus advantageous for healthy individuals to avoid excessive dietary antioxidants and, instead, rely on intrinsic GSH biosynthesis, which is fine-tuned to match the cellular redox status and to promote homeostatic ROS signaling.
Asunto(s)
Acetilcisteína/farmacología , Envejecimiento/efectos de los fármacos , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/fisiología , Glutatión/farmacología , Envejecimiento/genética , Envejecimiento/fisiología , Animales , Animales Modificados Genéticamente , Proteínas de Caenorhabditis elegans/genética , Proteínas de Unión al ADN/genética , Suplementos Dietéticos , Escherichia coli , Femenino , Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glutatión/metabolismo , Humanos , Masculino , Paraquat/farmacología , Especies Reactivas de Oxígeno/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Factores de Transcripción/genética , Respuesta de Proteína Desplegada/fisiologíaRESUMEN
Glycogen is synthesized and stored to maintain postprandial blood glucose homeostasis and to ensure an uninterrupted energy supply between meals. Although the regulation of glycogen turnover has been well studied, the effects of glycogen on aging and disease development have been largely unexplored. In Caenorhabditis elegans fed a high sugar diet, glycogen potentiates resistance to oxidants, but paradoxically, shortens lifespan. Depletion of glycogen by oxidants or inhibition of glycogen synthesis extends the lifespan of worms by an AMPK-dependent mechanism. Thus, glycogen is not merely an inert storage molecule, but also an active regulator of energy balance and aging. Its depletion by oxidants may be beneficial in the treatment of hyperglycemia and glycogen-related diseases.
Asunto(s)
Envejecimiento/metabolismo , Envejecimiento/fisiología , Metabolismo Energético/fisiología , Glucógeno/metabolismo , Estrés Fisiológico/fisiología , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Homeostasis/fisiología , Humanos , Longevidad/fisiología , Oxidantes/metabolismoRESUMEN
Reports by Seth et al. (2018) and Wolhuter et al. (2018) in this issue of Molecular Cell highlight the enzymatic synthesis, functionality, and propagation of S-nitrosylation-based signaling and address its low stability due to the elevated reactivity toward other cellular thiols.
Asunto(s)
Óxido Nítrico , Proteína S , Procesamiento Proteico-Postraduccional , Proteolisis , Transducción de SeñalRESUMEN
Bacteria naturally form communities of cells known as biofilms. However the physiological roles of biofilms produced by non-pathogenic microbiota remain largely unknown. To assess the impact of a biofilm on host physiology we explored the effect of several non-pathogenic biofilm-forming bacteria on Caenorhabditis elegans. We show that biofilm formation by Bacillus subtilis, Lactobacillus rhamnosus and Pseudomonas fluorescens induces C. elegans stress resistance. Biofilm also protects against pathogenic infection and prolongs lifespan. Total mRNA analysis identified a set of host genes that are upregulated in response to biofilm formation by B. subtilis. We further demonstrate that mtl-1 is responsible for the biofilm-mediated increase in oxidative stress resistance and lifespan extension. Induction of mtl-1 and hsp-70 promotes biofilm-mediated thermotolerance. ilys-2 activity accounts for biofilm-mediated resistance to Pseudomonas aeruginosa killing. These results reveal the importance of non-pathogenic biofilms for host physiology and provide a framework to study commensal biofilms in higher organisms.
Asunto(s)
Biopelículas , Caenorhabditis elegans/microbiología , Caenorhabditis elegans/fisiología , Longevidad , Estrés Fisiológico , Adaptación Biológica/genética , Alimentación Animal , Animales , Biomarcadores , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Interacciones Huésped-Patógeno , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , SimbiosisRESUMEN
A high-sugar diet has been associated with reduced lifespan in organisms ranging from worms to mammals. However, the mechanisms underlying the harmful effects of glucose are poorly understood. Here we establish a causative relationship between endogenous glucose storage in the form of glycogen, resistance to oxidative stress and organismal aging in Caenorhabditis elegans. We find that glycogen accumulated on high dietary glucose limits C. elegans longevity. Glucose released from glycogen and used for NADPH/glutathione reduction renders nematodes and human hepatocytes more resistant against oxidative stress. Exposure to low levels of oxidants or genetic inhibition of glycogen synthase depletes glycogen stores and extends the lifespan of animals fed a high glucose diet in an AMPK-dependent manner. Moreover, glycogen interferes with low insulin signalling and accelerates aging of long-lived daf-2 worms fed a high glucose diet. Considering its extensive evolutionary conservation, our results suggest that glycogen metabolism might also have a role in mammalian aging.
Asunto(s)
Caenorhabditis elegans/fisiología , Glucosa/metabolismo , Glucógeno/metabolismo , Estrés Oxidativo/fisiología , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Animales Modificados Genéticamente , Antioxidantes/metabolismo , Caenorhabditis elegans/efectos de los fármacos , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Diamida/farmacología , Glucosa/farmacología , Glutatión/metabolismo , Glucógeno Sintasa/genética , Glucógeno Sintasa/metabolismo , Células Hep G2 , Humanos , Longevidad/fisiología , NADP/metabolismo , Oxidantes/farmacología , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
Nitric oxide (NO) is an important signaling molecule in multicellular organisms. Most animals produce NO from L-arginine via a family of dedicated enzymes known as NO synthases (NOSes). A rare exception is the roundworm Caenorhabditis elegans, which lacks its own NOS. However, in its natural environment, C. elegans feeds on Bacilli that possess functional NOS. Here, we demonstrate that bacterially derived NO enhances C. elegans longevity and stress resistance via a defined group of genes that function under the dual control of HSF-1 and DAF-16 transcription factors. Our work provides an example of interspecies signaling by a small molecule and illustrates the lifelong value of commensal bacteria to their host.
Asunto(s)
Bacillus subtilis , Caenorhabditis elegans/fisiología , Longevidad , Óxido Nítrico/metabolismo , Animales , Proteínas de Caenorhabditis elegans/metabolismo , Dieta , Factores de Transcripción Forkhead , Tracto Gastrointestinal/microbiología , Temperatura , Factores de Transcripción/metabolismoRESUMEN
Staphylococcus aureus infections present an enormous global health concern complicated by an alarming increase in antibiotic resistance. S. aureus is among the few bacterial species that express nitric-oxide synthase (bNOS) and thus can catalyze NO production from L-arginine. Here we generate an isogenic bNOS-deficient mutant in the epidemic community-acquired methicillin-resistant S. aureus (MRSA) USA300 clone to study its contribution to virulence and antibiotic susceptibility. Loss of bNOS increased MRSA susceptibility to reactive oxygen species and host cathelicidin antimicrobial peptides, which correlated with increased MRSA killing by human neutrophils and within neutrophil extracellular traps. bNOS also promoted resistance to the pharmaceutical antibiotics that act on the cell envelope such as vancomycin and daptomycin. Surprisingly, bNOS-deficient strains gained resistance to aminoglycosides, suggesting that the role of bNOS in antibiotic susceptibility is more complex than previously observed in Bacillus species. Finally, the MRSA bNOS mutant showed reduced virulence with decreased survival and smaller abscess generation in a mouse subcutaneous infection model. Together, these data indicate that bNOS contributes to MRSA innate immune and antibiotic resistance phenotypes. Future development of specific bNOS inhibitors could be an attractive option to simultaneously reduce MRSA pathology and enhance its susceptibility to commonly used antibiotics.
Asunto(s)
Proteínas Bacterianas/metabolismo , Resistencia a la Meticilina , Staphylococcus aureus Resistente a Meticilina/enzimología , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Óxido Nítrico Sintasa/metabolismo , Infecciones Cutáneas Estafilocócicas/enzimología , Absceso/genética , Absceso/microbiología , Absceso/patología , Animales , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas Bacterianas/genética , Daptomicina/farmacología , Humanos , Staphylococcus aureus Resistente a Meticilina/genética , Ratones , Mutación , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/genética , Especies Reactivas de Oxígeno/metabolismo , Infecciones Cutáneas Estafilocócicas/genética , Infecciones Cutáneas Estafilocócicas/microbiología , Infecciones Cutáneas Estafilocócicas/patología , Vancomicina/farmacología , CatelicidinasRESUMEN
Most bacteria generate nitric oxide (NO) either aerobically by NO synthases or anaerobically from nitrite. Far from being a mere by-product of nitrate respiration, bacterial NO has diverse physiological roles. Many proteins undergo NO-mediated posttranslational modification (S-nitrosylation) in anaerobically grown Escherichia coli. The regulation of one such protein, OxyR, represents a redox signaling paradigm in which the same transcription factor controls different protective genes depending on its S-nitrosylation versus S-oxidation status. We discuss a structural model that may explain the remarkable stability and specificity of OxyR S-nitrosylation.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/fisiología , Modelos Moleculares , Óxido Nítrico/metabolismo , Procesamiento Proteico-Postraduccional/fisiología , Proteínas Represoras/metabolismo , S-Nitrosotioles/metabolismo , Escherichia coli/metabolismoRESUMEN
Bacterial nitric oxide synthases (bNOS) are present in many Gram-positive species and have been demonstrated to synthesize NO from arginine in vitro and in vivo. However, the physiological role of bNOS remains largely unknown. We show that NO generated by bNOS increases the resistance of bacteria to a broad spectrum of antibiotics, enabling the bacteria to survive and share habitats with antibiotic-producing microorganisms. NO-mediated resistance is achieved through both the chemical modification of toxic compounds and the alleviation of the oxidative stress imposed by many antibiotics. Our results suggest that the inhibition of NOS activity may increase the effectiveness of antimicrobial therapy.
Asunto(s)
Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/metabolismo , Acriflavina/metabolismo , Acriflavina/farmacología , Antibacterianos/metabolismo , Antibiosis , Bacillus anthracis/efectos de los fármacos , Bacillus anthracis/genética , Bacillus anthracis/crecimiento & desarrollo , Bacillus anthracis/metabolismo , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Bacillus subtilis/metabolismo , Bacterias/genética , Bacterias/crecimiento & desarrollo , Cefuroxima/farmacología , Mutación , Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/genética , Estrés Oxidativo , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/metabolismo , Piocianina/metabolismo , Piocianina/farmacología , Especies Reactivas de Oxígeno/metabolismo , Microbiología del Suelo , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
Bacterial nitric-oxide (NO) synthases (bNOSs) are smaller than their mammalian counterparts. They lack an essential reductase domain that supplies electrons during NO biosynthesis. This and other structural peculiarities have raised doubts about whether bNOSs were capable of producing NO in vivo. Here we demonstrate that bNOS enzymes from Bacillus subtilis and Bacillus anthracis do indeed produce NO in living cells and accomplish this task by hijacking available cellular redox partners that are not normally committed to NO production. These "promiscuous" bacterial reductases also support NO synthesis by the oxygenase domain of mammalian NOS expressed in Escherichia coli. Our results suggest that bNOS is an early precursor of eukaryotic NOS and that it acquired its dedicated reductase domain later in evolution. This work also suggests that alternatively spliced forms of mammalian NOSs lacking their reductase domains could still be functional in vivo. On a practical side, bNOS-containing probiotic bacteria offer a unique advantage over conventional chemical NO donors in generating continuous, readily controllable physiological levels of NO, suggesting a possibility of utilizing such live NO donors for research and clinical needs.
Asunto(s)
Bacillus subtilis/enzimología , Escherichia coli/enzimología , Óxido Nítrico Sintasa/metabolismo , Animales , Bacillus subtilis/genética , Escherichia coli/genética , Humanos , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa/genética , Oxidación-Reducción , Oxigenasas/genética , Oxigenasas/metabolismo , Filogenia , Especificidad por SustratoRESUMEN
Phagocytes generate nitric oxide (NO) and other reactive oxygen and nitrogen species in large quantities to combat infecting bacteria. Here, we report the surprising observation that in vivo survival of a notorious pathogen-Bacillus anthracis-critically depends on its own NO-synthase (bNOS) activity. Anthrax spores (Sterne strain) deficient in bNOS lose their virulence in an A/J mouse model of systemic infection and exhibit severely compromised survival when germinating within macrophages. The mechanism underlying bNOS-dependent resistance to macrophage killing relies on NO-mediated activation of bacterial catalase and suppression of the damaging Fenton reaction. Our results demonstrate that pathogenic bacteria use their own NO as a key defense against the immune oxidative burst, thereby establishing bNOS as an essential virulence factor. Thus, bNOS represents an attractive antimicrobial target for treatment of anthrax and other infectious diseases.
Asunto(s)
Bacillus anthracis/patogenicidad , Macrófagos/citología , Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Animales , Carbunco/metabolismo , Carbunco/patología , Línea Celular , Supervivencia Celular , Ratones , Estrés Oxidativo , Tasa de Supervivencia , Factores de Tiempo , VirulenciaRESUMEN
Numerous sophisticated systems have been described that protect bacteria from increased levels of reactive oxygen species. Although indispensable during prolonged oxidative stress, these response systems depend on newly synthesized proteins, and are hence both time and energy consuming. Here, we describe an "express" cytoprotective system in Bacillus subtilis which depends on nitric oxide (NO). We show that NO immediately protects bacterial cells from reactive oxygen species by two independent mechanisms. NO transiently suppresses the enzymatic reduction of free cysteine that fuels the damaging Fenton reaction. In addition, NO directly reactivates catalase, a major antioxidant enzyme that has been inhibited in vivo by endogenous cysteine. Our data also reveal a critical role for bacterial NO-synthase in adaptation to oxidative stress associated with fast metabolic changes, and suggest a possible role for NO in defending pathogens against immune oxidative attack.
Asunto(s)
Bacillus subtilis/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico/farmacología , Estrés Oxidativo , Adaptación Fisiológica , Antioxidantes/metabolismo , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/genética , Catalasa/metabolismo , Citoprotección , Activación Enzimática , Eliminación de Gen , Peróxido de Hidrógeno/farmacología , Hierro/farmacología , Óxido Nítrico Sintasa/genética , Especies Reactivas de Oxígeno/metabolismo , Agua/farmacologíaAsunto(s)
Bioquímica/métodos , Transcripción Genética , Secuencia de Bases , Relación Dosis-Respuesta a Droga , Escherichia coli/enzimología , Proteínas de Escherichia coli , Modelos Biológicos , Datos de Secuencia Molecular , Terminación de la Cadena Péptídica Traduccional , Factores de Elongación de Péptidos/química , Regiones Promotoras Genéticas , Factores de Tiempo , Factores de Transcripción/química , Factores de Elongación TranscripcionalRESUMEN
Thiamin and riboflavin are precursors of essential coenzymes-thiamin pyrophosphate (TPP) and flavin mononucleotide (FMN)/flavin adenine dinucleotide (FAD), respectively. In Bacillus spp, genes responsible for thiamin and riboflavin biosynthesis are organized in tightly controllable operons. Here, we demonstrate that the feedback regulation of riboflavin and thiamin genes relies on a novel transcription attenuation mechanism. A unique feature of this mechanism is the formation of specific complexes between a conserved leader region of the cognate RNA and FMN or TPP. In each case, the complex allows the termination hairpin to form and interrupt transcription prematurely. Thus, sensing small molecules by nascent RNA controls transcription elongation of riboflavin and thiamin operons and possibly other bacterial operons as well.