RESUMEN
Pathogenic bacteria's metabolic adaptation for survival and proliferation within hosts is a crucial aspect of bacterial pathogenesis. Here, we demonstrate that citrate, the first intermediate of the tricarboxylic acid (TCA) cycle, plays a key role as a regulator of gene expression in Staphylococcus aureus. We show that citrate activates the transcriptional regulator CcpE and thus modulates the expression of numerous genes involved in key cellular pathways such as central carbon metabolism, iron uptake and the synthesis and export of virulence factors. Citrate can also suppress the transcriptional regulatory activity of ferric uptake regulator. Moreover, we determined that accumulated intracellular citrate, partly through the activation of CcpE, decreases the pathogenic potential of S. aureus in animal infection models. Therefore, citrate plays a pivotal role in coordinating carbon metabolism, iron homeostasis, and bacterial pathogenicity at the transcriptional level in S. aureus, going beyond its established role as a TCA cycle intermediate.
Asunto(s)
Carbono , Ácido Cítrico , Regulación Bacteriana de la Expresión Génica , Homeostasis , Hierro , Infecciones Estafilocócicas , Staphylococcus aureus , Staphylococcus aureus/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Hierro/metabolismo , Carbono/metabolismo , Ácido Cítrico/metabolismo , Infecciones Estafilocócicas/metabolismo , Infecciones Estafilocócicas/microbiología , Animales , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Ciclo del Ácido Cítrico , Ratones , Transducción de SeñalRESUMEN
Epigenetics is brought to RNA, introducing a new dimension to gene expression regulation. Among numerous RNA modifications, N6-methyladenosine (m6A) is an abundant internal modification on eukaryote mRNA first identified in the 1970s. However, the significance of m6A modification in mRNA had been long neglected until the fat mass and obesity-associated (FTO) enzyme was identified as the first m6A demethylase almost 40 years later. The m6A modification influences nearly every step of RNA metabolism and thus broadly affects gene expression at multiple levels, playing a critical role in many biological processes, including cancer progression, metastasis, and immune evasion. The m6A level is dynamically regulated by RNA epigenetic machinery comprising methyltransferases such as methyltransferase-like protein 3 (METTL3), demethylases FTO and AlkB human homologue 5 (ALKBH5), and multiple reader proteins. The understanding of the biology of RNA epigenetics and its translational drug discovery is still in its infancy. It is essential to further develop chemical probes and lead compounds for an in-depth investigation into m6A biology and the translational discovery of anticancer drugs targeting m6A modifying oncogenic proteins.In this Account, we present our work on the development of chemical inhibitors to regulate m6A in mRNA by targeting the FTO demethylase, and the elucidation of their mode of action. We reported rhein to be the first substrate competitive FTO inhibitor. Due to rhein's poor selectivity, we identified meclofenamic acid (MA) that selectively inhibits FTO compared with ALKBH5. Based on the structural complex of MA bound with FTO, we designed MA analogs FB23-2 and Dac51, which exhibit significantly improved activities compared with MA. For example, FB23-2 is specific to FTO inhibition in vitro among over 400 other oncogenic proteins, including kinases, proteases, and DNA and histone epigenetic proteins. Mimicking FTO depletion, FB23-2 promotes the differentiation/apoptosis of human acute myeloid leukemia (AML) cells and inhibits the progression of primary cells in xenotransplanted mice. Dac51 treatment impairs the glycolytic activity of tumor cells and restores the function of CD8+ T cells, thereby inhibiting the growth of solid tumors in vivo. These FTO inhibitors were and will continue to be used as probes to promote biological studies of m6A modification and as lead compounds to target FTO in anticancer drug discovery.Toward the end, we also include a brief review of ALKBH5 demethylase inhibitors and METTL3 methyltransferase modulators. Collectively, these small-molecule modulators that selectively target RNA epigenetic proteins will promote in-depth studies on the regulation of gene expression and potentially accelerate anticancer target discovery.
Asunto(s)
Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Linfocitos T CD8-positivos , Humanos , Ratones , Animales , Linfocitos T CD8-positivos/metabolismo , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/genética , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato/metabolismo , Proteínas/química , ARN , ARN Mensajero/metabolismo , Ácido Meclofenámico/farmacología , MetiltransferasasRESUMEN
Intracellular protein degradation is essential for the survival of all organisms, but its role in interspecies interaction is unknown. Here, we show that the ClpXP protease of Pseudomonas aeruginosa suppresses its antimicrobial activity against Staphylococcus aureus, a common pathogen co-isolated with P. aeruginosa from polymicrobial human infections. Using proteomic, biochemical, and molecular genetic approaches, we found that this effect is due to the inhibitory effects of ClpXP on the quorum sensing (QS) of P. aeruginosa, mainly by degrading proteins (e.g., PhnA, PhnB, PqsR, and RhlI) which are critical for the production of QS signal molecules PQS and C4-HSL. We provide evidence that co-culturing with S. aureus induces a decrease in the activity of ClpXP in P. aeruginosa, an effect which was also achieved by the treatment of P. aeruginosa with N-acetylglucosamine (GlcNAc), a widespread chemical present on the surface of diverse cell types from bacteria to humans. These findings extend the range of biological events governed by proteolytic machinery to microbial community structure, thus also suggesting that a chemical-induced alteration of protein homeostasis is a mechanism for interspecies interactions.
Asunto(s)
Acetilglucosamina/farmacología , Endopeptidasa Clp/metabolismo , Pseudomonas aeruginosa/fisiología , Percepción de Quorum/genética , Staphylococcus aureus/fisiología , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biopelículas/efectos de los fármacos , Endopeptidasa Clp/genética , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Humanos , Interacciones Microbianas , Mutación , Proteolisis/efectos de los fármacos , Proteómica , Proteostasis , Infecciones por Pseudomonas/microbiología , Percepción de Quorum/efectos de los fármacos , Infecciones Estafilocócicas/microbiologíaRESUMEN
Despite the fact that the introduction of a fluorine atom at the C-6 position has resulted in the evolution of fluoroquinolones, fluoroquinolone-induced cardiac toxicity has drawn considerable attention. In this context, desfluoroquinolone-based hybrids with involvement of C-7 aminopyrimidine functional group were designed and synthesized. The biological results showed majority of these hybrids still demonstrated potent anti-MRSA activity with MIC values between 0.38 and 1.5 µg/mL, despite the lack of the typical C-6 fluorine atom. Particularly, the most active B14 exhibited activities at submicromolar concentrations against a panel of MRSA strains including vancomycin-intermediate strains, levofloxacin-resistant isolates, and linezolid-resistant isolates, etc. As expected, it also displayed highly selective toxicity toward bacterial cells and low hERG inhibition. Further resistance development study indicated MRSA is unlikely to acquire resistance against B14. The docking study revealed that two hydrogen bonds were formed between the C-7 substituent and the surrounding DNA bases, which might contribute to overcome resistance by reducing the dependence on the magnesium-water bridge interactions with topoisomerase IV. These results indicate a promising strategy for developing new antibiotic quinolones to combat multidrug resistance and cardiotoxicity.
Asunto(s)
Fluoroquinolonas/síntesis química , Fluoroquinolonas/uso terapéutico , Pirimidinas/síntesis química , Pirimidinas/uso terapéutico , Fluoroquinolonas/farmacología , Humanos , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Estructura Molecular , Pirimidinas/farmacología , Relación Estructura-ActividadRESUMEN
The surge of antibiotic resistance in Staphylococcus aureus has created a dire need for innovative anti-infective agents that attack new targets, to overcome resistance. In S. aureus, carotenoid pigment is an important virulence factor because it shields the bacterium from host oxidant killing. Here we show that naftifine, a US Food and Drug Administration (FDA)-approved antifungal drug, blocks biosynthesis of carotenoid pigment at nanomolar concentrations. This effect is mediated by competitive inhibition of S. aureus diapophytoene desaturase (CrtN), an essential enzyme for carotenoid pigment synthesis. We found that naftifine attenuated the virulence of a variety of clinical S. aureus isolates, including methicillin-resistant S. aureus (MRSA) strains, in mouse infection models. Specifically, we determined that naftifine is a lead compound for potent CrtN inhibitors. In sum, these findings reveal that naftifine could serve as a chemical probe to manipulate CrtN activity, providing proof of concept that CrtN is a druggable target against S. aureus infections.
Asunto(s)
Alilamina/análogos & derivados , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Oxidorreductasas/antagonistas & inhibidores , Staphylococcus aureus/efectos de los fármacos , Alilamina/farmacología , Animales , Unión Competitiva/efectos de los fármacos , Carotenoides/metabolismo , Diseño de Fármacos , Farmacorresistencia Bacteriana , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Ratones , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/enzimología , Staphylococcus aureus/patogenicidad , Factores de Virulencia , Xantófilas/antagonistas & inhibidores , Xantófilas/biosíntesisRESUMEN
Sortase A (SrtA) anchors surface proteins to the cell wall and aids biofilm formation during infection, which functions as a key virulence factor of important Gram-positive pathogens, such as Staphylococcus aureus. At present researchers need a way in which to validate whether or not SrtA is a druggable target alternative to the conventional antibiotic targets in the mechanism. In this study, we performed a high-throughput screening and identified a new class of potential inhibitors of S. aureus SrtA, which are derived from natural products and contain the quinone skeleton. Compound 283 functions as an irreversible inhibitor that covalently alkylates the active site Cys184 of SrtA. NMR analysis confirms the direct interaction of the small-molecule inhibitor towards SrtA protein. The anchoring of protein A (SpA) to the cell wall and the biofilm formation are significantly attenuated when the S. aureus Newman strain is cultured in the presence of inhibitor. Our study indicates that compound 283 could be a potential hit for the development of new anti-virulence agents against S. aureus infections by covalently targeting SrtA.
Asunto(s)
Aminoaciltransferasas/antagonistas & inhibidores , Proteínas Bacterianas/antagonistas & inhibidores , Benzoquinonas/química , Inhibidores de Cisteína Proteinasa/química , Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Pared Celular/metabolismo , Cisteína Endopeptidasas/metabolismo , Inhibidores de Cisteína Proteinasa/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Transferencia Resonante de Energía de Fluorescencia , Concentración 50 Inhibidora , Espectroscopía de Resonancia Magnética , Péptidos/análisis , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/enzimologíaRESUMEN
A series of N-sulfonaminoethyloxime derivatives of dehydroabietic acid were synthesized and investigated for their antibacterial activity against Staphylococcus aureus Newman strain and multidrug-resistant strains (NRS-1, NRS-70, NRS-100, NRS-108 and NRS-271). Most of the target compounds having chloro, bromo, trifluoromethyl phenyl moiety exhibited potent in vitro antistaphylococcal activity. The meta-CF3 phenyl derivative T23 showed the highest activity with MIC of 0.39-0.78⯵g/mL against S. aureus Newman, while several analogues showed similar potent antibacterial activity with MIC values between 0.78 and 1.56⯵g/mL against five multidrug-resistant S. aureus. The stability of T35 in plasma of SD rat and the cellular cytotoxicity were also evaluated.
Asunto(s)
Abietanos/química , Antibacterianos/síntesis química , Oximas/química , Animales , Antibacterianos/metabolismo , Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Estabilidad de Medicamentos , Pruebas de Sensibilidad Microbiana , Oximas/metabolismo , Oximas/farmacología , Ratas , Ratas Sprague-Dawley , Staphylococcus aureus/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
The AlkB repair enzymes, including Escherichia coli AlkB and two human homologues, ALKBH2 and ALKBH3, are iron(II)- and 2-oxoglutarate-dependent dioxygenases that efficiently repair N(1)-methyladenine and N(3)-methylcytosine methylated DNA damages. The development of small molecule inhibitors of these enzymes has seen less success. Here we have characterized a previously discovered natural product rhein and tested its ability to inhibit AlkB repair enzymes in vitro and to sensitize cells to methyl methane sulfonate that mainly produces N(1)-methyladenine and N(3)-methylcytosine lesions. Our investigation of the mechanism of rhein inhibition reveals that rhein binds to AlkB repair enzymes in vitro and promotes thermal stability in vivo In addition, we have determined a new structural complex of rhein bound to AlkB, which shows that rhein binds to a different part of the active site in AlkB than it binds to in fat mass and obesity-associated protein (FTO). With the support of these observations, we put forth the hypothesis that AlkB repair enzymes would be effective pharmacological targets for cancer treatment.
Asunto(s)
Antraquinonas/farmacología , Enzimas Reparadoras del ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Oxigenasas de Función Mixta/antagonistas & inhibidores , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/antagonistas & inhibidores , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/genética , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/antagonistas & inhibidores , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/genética , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB/metabolismo , Antraquinonas/química , Dominio Catalítico , Línea Celular , Cristalografía por Rayos X , Daño del ADN , Metilación de ADN , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Inhibidores Enzimáticos/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Metilmetanosulfonato/farmacología , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Modelos Moleculares , Interferencia de ARN , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
As a master redox-sensing MarR-family transcriptional regulator, AbfR participates in oxidative stress responses and virulence regulations in Staphylococcus epidermidis. Here, we present structural insights into the DNA-binding mechanism of AbfR in different oxidation states by determining the X-ray crystal structures of a reduced-AbfR/DNA complex, an overoxidized (Cys13-SO2H and Cys13-SO3H) AbfR/DNA, and 2-disulfide cross-linked AbfR dimer. Together with biochemical analyses, our results suggest that the redox regulation of AbfR-sensing displays two novel features: (i) the reversible disulfide modification, but not the irreversible overoxidation, significantly abolishes the DNA-binding ability of the AbfR repressor; (ii) either 1-disulfide cross-linked or 2-disulfide cross-linked AbfR dimer is biologically significant. The overoxidized species of AbfR, resembling the reduced AbfR in conformation and retaining the DNA-binding ability, does not exist in biologically significant concentrations, however. The 1-disulfide cross-linked modification endows AbfR with significantly weakened capability for DNA-binding. The 2-disulfide cross-linked AbfR adopts a very "open" conformation that is incompatible with DNA-binding. Overall, the concise oxidation chemistry of the redox-active cysteine allows AbfR to sense and respond to oxidative stress correctly and efficiently.
Asunto(s)
ADN/metabolismo , Staphylococcus epidermidis/metabolismo , Factores de Transcripción/metabolismo , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/metabolismo , Cristalografía por Rayos X , ADN/química , Disulfuros/química , Disulfuros/metabolismo , Modelos Moleculares , Oxidación-Reducción , Conformación Proteica , Factores de Transcripción/químicaRESUMEN
NalD was reported to be the secondary repressor of the MexAB-OprM multidrug efflux pump, the major system contributing to intrinsic multidrug resistance in Pseudomonas aeruginosa. Here, we show that novobiocin binds directly to NalD, which leads NalD to dissociate from the DNA promoter, and thus de-represses the expression of the MexAB-OprM pump. In addition, we have solved the crystal structure of NalD at a resolution of 2.90 Å. The structural alignment of NalD to its homologue TtgR reveals that the residues N129 and H167 in NalD are involved in its novobiocin-binding ability. We have confirmed the function of these two amino acids by EMSA and plate assay. The results presented here highlight the importance and diversity of regulatory mechanism in bacterial antibiotic resistance, and provide further insight for novel antimicrobial development.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana Múltiple , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/genética , Novobiocina/metabolismo , Pseudomonas aeruginosa/genética , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Proteínas Bacterianas/química , Cristalización , Cristalografía por Rayos X , Proteínas de Transporte de Membrana/metabolismo , Pruebas de Sensibilidad Microbiana , Novobiocina/química , Operón , Regiones Promotoras Genéticas , Unión Proteica , Pseudomonas aeruginosa/metabolismo , Proteínas Represoras/genéticaRESUMEN
Two human demethylases, the fat mass and obesity-associated (FTO) enzyme and ALKBH5, oxidatively demethylate abundant N(6)-methyladenosine (m(6)A) residues in mRNA. Achieving a method for selective inhibition of FTO over ALKBH5 remains a challenge, however. Here, we have identified meclofenamic acid (MA) as a highly selective inhibitor of FTO. MA is a non-steroidal, anti-inflammatory drug that mechanistic studies indicate competes with FTO binding for the m(6)A-containing nucleic acid. The structure of FTO/MA has revealed much about the inhibitory function of FTO. Our newfound understanding, revealed herein, of the part of the nucleotide recognition lid (NRL) in FTO, for example, has helped elucidate the principles behind the selectivity of FTO over ALKBH5. Treatment of HeLa cells with the ethyl ester form of MA (MA2) has led to elevated levels of m(6)A modification in mRNA. Our collective results highlight the development of functional probes of the FTO enzyme that will (i) enable future biological studies and (ii) pave the way for the rational design of potent and specific inhibitors of FTO for use in medicine.
Asunto(s)
Adenosina/análogos & derivados , Antiinflamatorios no Esteroideos/farmacología , Inhibidores Enzimáticos/farmacología , Ácido Meclofenámico/farmacología , Proteínas/antagonistas & inhibidores , Adenosina/metabolismo , Desmetilasa de ARN, Homólogo 5 de AlkB , Dioxigenasa FTO Dependiente de Alfa-Cetoglutarato , Antiinflamatorios no Esteroideos/química , Unión Competitiva , ADN de Cadena Simple/metabolismo , Dioxigenasas/antagonistas & inhibidores , Dioxigenasas/química , Inhibidores Enzimáticos/química , Células HeLa , Humanos , Ácido Meclofenámico/química , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/química , Proteínas/química , Proteínas/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Relación Estructura-ActividadRESUMEN
An effective metabolism is essential to all living organisms, including the important human pathogen Staphylococcus aureus. To establish successful infection, S. aureus must scavenge nutrients and coordinate its metabolism for proliferation. Meanwhile, it also must produce an array of virulence factors to interfere with host defenses. However, the ways in which S. aureus ties its metabolic state to its virulence regulation remain largely unknown. Here we show that citrate, the first intermediate of the tricarboxylic acid (TCA) cycle, binds to and activates the catabolite control protein E (CcpE) of S. aureus. Using structural and site-directed mutagenesis studies, we demonstrate that two arginine residues (Arg145 and Arg256) within the putative inducer-binding cavity of CcpE are important for its allosteric activation by citrate. Microarray analysis reveals that CcpE tunes the expression of 126 genes that comprise about 4.7% of the S. aureus genome. Intriguingly, although CcpE is a major positive regulator of the TCA-cycle activity, its regulon consists predominantly of genes involved in the pathogenesis of S. aureus. Moreover, inactivation of CcpE results in increased staphyloxanthin production, improved ability to acquire iron, increased resistance to whole-blood-mediated killing, and enhanced bacterial virulence in a mouse model of systemic infection. This study reveals CcpE as an important metabolic sensor that allows S. aureus to sense and adjust its metabolic state and subsequently to coordinate the expression of virulence factors and bacterial virulence.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas Represoras/fisiología , Staphylococcus aureus/patogenicidad , Absceso/microbiología , Aconitato Hidratasa/genética , Aconitato Hidratasa/metabolismo , Regulación Alostérica , Animales , Arginina/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Ácido Cítrico/metabolismo , Cristalografía por Rayos X , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Hierro/metabolismo , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Pigmentación/genética , Unión Proteica , Conformación Proteica , Proteínas Represoras/química , Proteínas Represoras/genética , Eliminación de Secuencia , Infecciones Estafilocócicas/microbiología , Transcripción Genética/fisiología , Virulencia/fisiologíaRESUMEN
Methicillin-resistant Staphylococcus aureus (MRSA) is the most frequent cause of hospital-acquired infection, which manifests as surgical site infections, bacteremia, and sepsis. Due to drug-resistance, prophylaxis of MRSA infection with antibiotics frequently fails or incites nosocomial diseases such as Clostridium difficile infection. Sortase A is a transpeptidase that anchors surface proteins in the envelope of S. aureus, and sortase mutants are unable to cause bacteremia or sepsis in mice. Here we used virtual screening and optimization of inhibitor structure to identify 3-(4-pyridinyl)-6-(2-sodiumsulfonatephenyl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole and related compounds, which block sortase activity in vitro and in vivo. Sortase inhibitors do not affect in vitro staphylococcal growth yet protect mice against lethal S. aureus bacteremia. Thus, sortase inhibitors may be useful as antiinfective therapy to prevent hospital-acquired S. aureus infection in high-risk patients without the side effects of antibiotics.
Asunto(s)
Aminoaciltransferasas/antagonistas & inhibidores , Antiinfecciosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores de Proteasas/farmacología , Bibliotecas de Moléculas Pequeñas/farmacología , Staphylococcus aureus/enzimología , Animales , Antiinfecciosos/química , Biocatálisis/efectos de los fármacos , Cisteína Endopeptidasas , Femenino , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Péptidos/metabolismo , Inhibidores de Proteasas/química , Bibliotecas de Moléculas Pequeñas/química , Staphylococcus aureus/efectos de los fármacos , Streptococcus pyogenes/efectos de los fármacos , Streptococcus pyogenes/enzimología , Tiadiazoles/química , Tiadiazoles/farmacologíaRESUMEN
Rhamnolipid acts as a virulence factor during Pseudomonas aeruginosa infection. Here, we show that deletion of the catabolite repression control (crc) gene in P. aeruginosa leads to a rhamnolipid-negative phenotype. This effect is mediated by the down-regulation of rhl quorum sensing (QS). We discover that a disruption of the gene encoding the Lon protease entirely offsets the effect of crc deletion on the production of both rhamnolipid and rhlâ QS signal C4-HSL. Crc is unable to bind lon mRNAâ in vitro in the absence of the RNA chaperon Hfq, while Crc contributes to Hfq-mediated repression of the lon gene expression at a posttranscriptional level. Deletion of crc, which results in up-regulation of lon, significantly reduces the in vivo stability and abundance of the RhlI protein that synthesizes C4-HSL, causing the attenuation of rhlâ QS. Lon is also capable of degrading the RhlI protein in vitro. In addition, constitutive expression of rhlI suppresses the defects of the crc deletion mutant in rhamnolipid, C4-HSL and virulence on lettuce leaves. This study therefore uncovers a novel posttranscriptional regulatory cascade, Crc-Hfq/Lon/RhlI, for the regulation of rhamnolipid production and rhlâ QS in P. aeruginosa.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Glucolípidos/metabolismo , Proteasa La/metabolismo , Pseudomonas aeruginosa/fisiología , Percepción de Quorum , Proteínas Represoras/metabolismo , Proteínas Bacterianas/genética , Eliminación de Gen , Proteína de Factor 1 del Huésped/metabolismo , Ligasas/metabolismo , Unión Proteica , Proteolisis , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , ARN Mensajero/metabolismo , Proteínas Represoras/genética , Factores de Transcripción/metabolismoRESUMEN
The rhl quorum-sensing (QS) system plays critical roles in the pathogenesis of P. aeruginosa. However, the regulatory effects that occur directly upstream of the rhl QS system are poorly understood. Here, we show that deletion of gene encoding for the two-component sensor BfmS leads to the activation of its cognate response regulator BfmR, which in turn directly binds to the promoter and decreases the expression of the rhlR gene that encodes the QS regulator RhlR, causing the inhibition of the rhl QS system. In the absence of bfmS, the Acka-Pta pathway can modulate the regulatory activity of BfmR. In addition, BfmS tunes the expression of 202 genes that comprise 3.6% of the P. aeruginosa genome. We further demonstrate that deletion of bfmS causes substantially reduced virulence in lettuce leaf, reduced cytotoxicity, enhanced invasion, and reduced bacterial survival during acute mouse lung infection. Intriguingly, specific missense mutations, which occur naturally in the bfmS gene in P. aeruginosa cystic fibrosis (CF) isolates such as DK2 strains and RP73 strain, can produce BfmS variants (BfmSL181P, BfmSL181P/E376Q, and BfmSR393H) that no longer repress, but instead activate BfmR. As a result, BfmS variants, but not the wild-type BfmS, inhibit the rhl QS system. This study thus uncovers a previously unexplored signal transduction pathway, BfmS/BfmR/RhlR, for the regulation of rhl QS in P. aeruginosa. We propose that BfmRS TCS may have an important role in the regulation and evolution of P. aeruginosa virulence during chronic infection in CF lungs.
Asunto(s)
Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa/patogenicidad , Percepción de Quorum/fisiología , Transducción de Señal/fisiología , Animales , Secuencia de Bases , Western Blotting , Ensayo de Cambio de Movilidad Electroforética , Femenino , Regulación Bacteriana de la Expresión Génica , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Infecciones por Pseudomonas/genética , Pseudomonas aeruginosa/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , VirulenciaRESUMEN
A series of 12-oxime and O-oxime ether derivatives of dehydroabietic acid were synthesized and investigated for the antibacterial activity against Staphylococcus aureus Newman strain and five multidrug-resistant strains (NRS-1, NRS-70, NRS-100, NRS-108, and NRS-271). The aromatic oximate derivative 11a showed the highest activity with MIC of 0.39-0.78µg/mL against S. aureus Newman. Of note, compounds 10b, 11 and 14 showed the most potent antibacterial activity against five multidrug-resistant S. aureus with MIC values of 1.25-3.13µg/mL. These results offered useful information for further strategic optimization in search of the antibacterial candidates against infection of multidrug-resistant Gram-positive bacteria.
Asunto(s)
Abietanos/química , Abietanos/farmacología , Antibacterianos/química , Antibacterianos/farmacología , Oximas/química , Oximas/farmacología , Staphylococcus aureus/efectos de los fármacos , Abietanos/síntesis química , Antibacterianos/síntesis química , Farmacorresistencia Bacteriana Múltiple , Humanos , Pruebas de Sensibilidad Microbiana , Oximas/síntesis química , Infecciones Estafilocócicas/tratamiento farmacológico , Relación Estructura-ActividadRESUMEN
Mononuclear iron-containing oxygenases conduct a diverse variety of oxidation functions in biology, including the oxidative demethylation of methylated nucleic acids and histones. Escherichia coli AlkB is the first such enzyme that was discovered to repair methylated nucleic acids, which are otherwise cytotoxic and/or mutagenic. AlkB human homologues are known to play pivotal roles in various processes. Here we present structural characterization of oxidation intermediates for these demethylases. Using a chemical cross-linking strategy, complexes of AlkB-double stranded DNA (dsDNA) containing 1,N(6)-etheno adenine (εA), N(3)-methyl thymine (3-meT) and N(3)-methyl cytosine (3-meC) are stabilized and crystallized, respectively. Exposing these crystals, grown under anaerobic conditions containing iron(II) and α-ketoglutarate (αKG), to dioxygen initiates oxidation in crystallo. Glycol (from εA) and hemiaminal (from 3-meT) intermediates are captured; a zwitterionic intermediate (from 3-meC) is also proposed, based on crystallographic observations and computational analysis. The observation of these unprecedented intermediates provides direct support for the oxidative demethylation mechanism for these demethylases. This study also depicts a general mechanistic view of how a methyl group is oxidatively removed from different biological substrates.
Asunto(s)
Reparación del ADN , Dioxigenasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Hierro/metabolismo , Oxigenasas de Función Mixta/metabolismo , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 2 de AlkB , Dioxigenasa Dependiente de Alfa-Cetoglutarato, Homólogo 3 de AlkB , Catálisis , Reactivos de Enlaces Cruzados/química , Cristalización , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Dioxigenasas/química , Proteínas de Escherichia coli/química , Humanos , Ácidos Cetoglutáricos/metabolismo , Metilación , Oxigenasas de Función Mixta/química , Modelos Moleculares , Oxidación-Reducción , Electricidad Estática , Especificidad por SustratoAsunto(s)
Inhibidores Enzimáticos/farmacología , Metiltransferasas/antagonistas & inhibidores , Oxidorreductasas N-Desmetilantes/antagonistas & inhibidores , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Adenosina/análogos & derivados , Adenosina/química , Animales , Línea Celular Tumoral , Epigénesis Genética , Humanos , Metiltransferasas/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , ARN/química , ARN/metabolismo , TranscriptomaRESUMEN
Ribonucleotide reductase (RR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides for DNA synthesis. Human RR small subunit M2 exists in a homodimer form. However, the importance of the dimer form to the enzyme and the related mechanism remain unclear. In this study, we tried to identify the interfacial residues that may mediate the assembly of M2 homodimer by computational alanine scanning based on the x-ray crystal structure. Co-immunoprecipitation, size exclusion chromatography, and RR activity assays showed that the K95E mutation in M2 resulted in dimer disassembly and enzyme activity inhibition. In comparison, the charge-exchanging double mutation of K95E and E98K recovered the dimerization and activity. Structural comparisons suggested that a conserved cluster of charged residues, including Lys-95, Glu-98, Glu-105, and Glu-174, at the interface may function as an ionic lock for M2 homodimer. Although the measurements of the radical and iron contents showed that the monomer (the K95E mutant) was capable of generating the diiron and tyrosyl radical cofactor, co-immunoprecipitation and competitive enzyme inhibition assays indicated that the disassembly of M2 dimer reduced its interaction with the large subunit M1. In addition, the immunofluorescent and fusion protein-fluorescent imaging analyses showed that the dissociation of M2 dimer altered its subcellular localization. Finally, the transfection of the wild-type M2 but not the K95E mutant rescued the G1/S phase cell cycle arrest and cell growth inhibition caused by the siRNA knockdown of M2. Thus, the conserved Lys-95 charged residue cluster is critical for human RR M2 homodimerization, which is indispensable to constitute an active holoenzyme and function in cells.
Asunto(s)
Ácido Glutámico/metabolismo , Lisina/metabolismo , Multimerización de Proteína , Ribonucleósido Difosfato Reductasa/metabolismo , Sustitución de Aminoácidos , Biocatálisis , Proliferación Celular , Cristalografía por Rayos X , Espectroscopía de Resonancia por Spin del Electrón , Puntos de Control de la Fase G1 del Ciclo Celular/genética , Ácido Glutámico/genética , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Lisina/genética , Microscopía Confocal , Modelos Moleculares , Mutación , Interferencia de ARN , Ribonucleósido Difosfato Reductasa/química , Ribonucleósido Difosfato Reductasa/genéticaRESUMEN
The FTO protein is unequivocally reported to play a critical role in human obesity and in the regulation of cellular levels of m(6)A modification, which makes FTO a significant and worthy subject of study. Here, we identified that fluorescein derivatives can selectively inhibit FTO demethylation, and the mechanisms behind these activities were elucidated after we determined the X-ray crystal structures of FTO/fluorescein and FTO/5-aminofluorescein. Furthermore, these inhibitors can also be applied to the direct labeling and enrichment of FTO protein combined with photoaffinity labeling assay.