RESUMEN

Diguanylate cyclases (DGCs) synthesize the bacterial second messenger cyclic 3',5'-diguanosine monophosphate (c-di-GMP), which is degraded by specific phosphodiesterases. c-di-GMP levels control the transition of bacteria from a motile to a biofilm-forming lifestyle. These bacterial communities are highly resistant to antibiotic treatment and represent the predominant lifestyle in most chronic infections. Hence, DGCs serve as starting point for the development of novel therapeutics interfering with the second messenger-signaling network in bacteria. In previous studies, we showed that 2'(3')-O-(N-methylanthraniloyl) (MANT)- and 2',3'-O-(2,4,6-trinitrophenyl) (TNP)-substituted nucleotides are potent adenylyl and guanylyl cyclase inhibitors. The catalytic domain of DGCs is homologous to the mammalian adenylyl cyclase catalytic domain. Therefore, we investigated the interaction of various MANT purine and pyrimidine nucleotides with the model DGC YdeH from Escherichia coli. We observed strong fluorescence resonance energy transfer between tryptophan and tyrosine residues of YdeH and the MANT group of MANT-NTPs (MANT-ATP, -CTP, -GTP, -ITP, -UTP, and -XTP) and an enhanced direct MANT fluorescence upon interaction with YdeH. We assessed the affinity of MANT-NTPs to YdeH by performing competition assays with NTPs. We conducted an amino acid alignment of YdeH with the earlier crystallized Caulobacter crescentus DGC PleD and found high similarities in the nucleotide-binding site of PleD. In vitro mass-spectrometric activity assays with YdeH resulted in the identification of new MANT/TNP nucleotide-based inhibitors of DGC activity. Together, the analysis of interactions between MANT/TNP nucleotides and YdeH provides a new basis for the identification and development of DGC inhibitors and allows insights into nucleotide-protein interactions.

Asunto(s)

Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Liasas de Fósforo-Oxígeno/metabolismo , Nucleótidos de Purina/metabolismo , Nucleótidos de Pirimidina/metabolismo , Secuencia de Aminoácidos , Proteínas de Escherichia coli/genética , Datos de Secuencia Molecular , Unión Proteica/fisiología , Nucleótidos de Purina/genética , Nucleótidos de Pirimidina/genética

RESUMEN

Cyclic di-guanosine monophosphate (c-di-GMP) represents an important ubiquitous second messenger in bacteria. It controls the transition between a sessile and a motile lifestyle of bacteria and, hence, affects the formation of biofilms which are highly resistant to antimicrobial treatment. c-di-GMP is synthesized by di-guanylate cyclases (DGCs) and degraded by specific phosphodiesterases (PDEs), two highly abundant protein families in bacteria. We have established a robust and highly sensitive high performance liquid chromatography-coupled tandem mass spectrometry (HPLC-MS/MS) based method for the quantitation of c-di-GMP and investigated various method performance parameters such as limit of detection (LOD), lower limit of quantitation (LLOQ), linearity, accuracy, recovery and analyte stability. As a proof of principle we used this method to accurately measure the activity of the prototype DGC PleD* from Caulobacter crescentus in vitro. In addition the methodology was successfully applied to determine in vivo levels of c-di-GMP in bacterial extracts of E. coli at different stages of bacterial growth. This demonstrates that our method is suitable for the sensitive and specific quantitation of c-di-GMP in bacterial cell extracts.

Asunto(s)

GMP Cíclico/análogos & derivados , Espectrometría de Masas en Tándem/métodos , Caulobacter crescentus/enzimología , Cromatografía Liquida/métodos , GMP Cíclico/análisis , Escherichia coli/química , Proteínas de Escherichia coli , Liasas de Fósforo-Oxígeno/metabolismo , Sensibilidad y Especificidad

RESUMEN

Bacteria swim by means of rotating flagella that are powered by ion influx through membrane-spanning motor complexes. Escherichia coli and related species harness a chemosensory and signal transduction machinery that governs the direction of flagellar rotation and allows them to navigate in chemical gradients. Here, we show that Escherichia coli can also fine-tune its swimming speed with the help of a molecular brake (YcgR) that, upon binding of the nucleotide second messenger cyclic di-GMP, interacts with the motor protein MotA to curb flagellar motor output. Swimming velocity is controlled by the synergistic action of at least five signaling proteins that adjust the cellular concentration of cyclic di-GMP. Activation of this network and the resulting deceleration coincide with nutrient depletion and might represent an adaptation to starvation. These experiments demonstrate that bacteria can modulate flagellar motor output and thus swimming velocity in response to environmental cues.

Asunto(s)

Escherichia coli/fisiología , Flagelos/metabolismo , Sistemas de Mensajero Secundario , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/metabolismo , Datos de Secuencia Molecular , Movimiento , Liasas de Fósforo-Oxígeno/metabolismo , Alineación de Secuencia

RESUMEN

During long-term cystic fibrosis lung infections, Pseudomonas aeruginosa undergoes genetic adaptation resulting in progressively increased persistence and the generation of adaptive colony morphotypes. This includes small colony variants (SCVs), auto-aggregative, hyper-adherent cells whose appearance correlates with poor lung function and persistence of infection. The SCV morphotype is strongly linked to elevated levels of cyclic-di-GMP, a ubiquitous bacterial second messenger that regulates the transition between motile and sessile, cooperative lifestyles. A genetic screen in PA01 for SCV-related loci identified the yfiBNR operon, encoding a tripartite signaling module that regulates c-di-GMP levels in P. aeruginosa. Subsequent analysis determined that YfiN is a membrane-integral diguanylate cyclase whose activity is tightly controlled by YfiR, a small periplasmic protein, and the OmpA/Pal-like outer-membrane lipoprotein YfiB. Exopolysaccharide synthesis was identified as the principal downstream target for YfiBNR, with increased production of Pel and Psl exopolysaccharides responsible for many characteristic SCV behaviors. An yfi-dependent SCV was isolated from the sputum of a CF patient. Consequently, the effect of the SCV morphology on persistence of infection was analyzed in vitro and in vivo using the YfiN-mediated SCV as a representative strain. The SCV strain exhibited strong, exopolysaccharide-dependent resistance to nematode scavenging and macrophage phagocytosis. Furthermore, the SCV strain effectively persisted over many weeks in mouse infection models, despite exhibiting a marked fitness disadvantage in vitro. Exposure to sub-inhibitory concentrations of antibiotics significantly decreased both the number of suppressors arising, and the relative fitness disadvantage of the SCV mutant in vitro, suggesting that the SCV persistence phenotype may play a more important role during antimicrobial chemotherapy. This study establishes YfiBNR as an important player in P. aeruginosa persistence, and implicates a central role for c-di-GMP, and by extension the SCV phenotype in chronic infections.

Asunto(s)

Proteínas de la Membrana Bacteriana Externa/genética , GMP Cíclico/análogos & derivados , Proteínas Periplasmáticas/genética , Liasas de Fósforo-Oxígeno/genética , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Animales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Caenorhabditis elegans , Células Cultivadas , GMP Cíclico/metabolismo , Elementos Transponibles de ADN/genética , Proteínas de Escherichia coli , Macrófagos/citología , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Ratones Endogámicos C57BL , Mutagénesis/fisiología , Operón/genética , Proteínas Periplasmáticas/metabolismo , Fagocitosis/fisiología , Fenotipo , Liasas de Fósforo-Oxígeno/metabolismo , Neumonía Bacteriana/enzimología , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/microbiología , Infecciones por Pseudomonas/enzimología , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/metabolismo , Sistemas de Mensajero Secundario/fisiología

RESUMEN

Guanine nucleotide binding proteins, such as Ras proteins, play a pivotal role in maintaining the regular life cycle of cells. The involvement of Ras mutants in the progress of cancer has attracted many efforts to find detection methods for Ras activity. In this study we present a luminescent microwell plate assay for monitoring GTPase activity of Ras proteins. The luminescence intensity of the Tb-norfloxacin complex is influenced by nucleoside phosphates as well as by inorganic phosphates. Real-time kinetics of the GTPase activity of wild-type Ras and Ras mutants can be monitored online. The effect of a GTPase activating protein as well as of a downstream effector (Ras-binding domain of human Raf-1) on the GTPase activity of different Ras mutants is examined. In contrast to other methods, this assay does not require the use of radioactively labeled substrates or chromatographic separation steps. Moreover, the application of fluorescently labeled GTP substrates which often interfere with enzymatic activity can be avoided. This in vitro assay can serve as a model system for the screening of regulators affecting the GTPase activity of Ras proteins.

Asunto(s)

GTP Fosfohidrolasas/metabolismo , Luminiscencia , Compuestos Organometálicos/química , Terbio/química , Proteínas ras/metabolismo , Calibración , GTP Fosfohidrolasas/química , Guanosina/química , Cinética , Mediciones Luminiscentes , Norfloxacino/química , Fosfatos/química , Reproducibilidad de los Resultados , Terbio/análisis , Factores de Tiempo , Proteínas ras/química

RESUMEN

Adenylyl cyclases catalyze the production of the second messenger cyclic AMP from ATP. Until now, there has been no fluorescent adenylyl cyclase assay known that is applicable to high-throughput screening and kinetic determinations that can directly monitor the turnover of the unmodified substrate ATP. In this study, a fluorescence-based assay is described using the Ca(II)- and calmodulin-dependent adenylyl cyclase edema factor (EF) from Bacillus anthracis and Tb(III)-norfloxacin as probe for the enzyme activity. This assay can be used to study enzyme regulators, allows real-time monitoring of adenylyl cyclase activity, and does not substitute ATP by fluorescent derivatives. These derivatives must be judged critically due to their interference on the activity of enzymes. Furthermore, the new assay makes redundant the application of radioactively labeled substrates such as [alpha-(32)P]ATP or fluorescently labeled antibodies such as anti-cyclic AMP. We determined the Michaelis-Menten constant (K(M)), the v(0)(max) value of ATP turnover, and the IC(50) values for three inhibitors of EF by this newly developed fluorescent method.

Asunto(s)

Adenilil Ciclasas/metabolismo , Fluorometría/métodos , Norfloxacino/metabolismo , Terbio/metabolismo , Venenos de Víboras/metabolismo , Adenosina Trifosfato/metabolismo , Toxina de Adenilato Ciclasa/metabolismo , Bacillus anthracis/enzimología , Calibración , Calmodulina/farmacología , AMP Cíclico/metabolismo , Cinética , Radiometría , Reproducibilidad de los Resultados , Venenos de Víboras/farmacología

RESUMEN

The determination of enzyme activities and the screening of enzyme regulators is a major task in clinical chemistry and the development of new drugs. A broad variety of enzymatic reactions is associated with the consumption or formation of small molecules like H(2)O(2), ATP, pyrophosphate, or phosphate. Luminescent lanthanide complexes can be applied to monitor these enzymatic conversions and therefore can serve as probes for the determination of enzyme activities. The utility of this concept will be demonstrated by means of some selected examples including europium and terbium complexes. Accordingly, this new approach could be already implemented for the determination of glucose oxidase, catalase, and peroxidase activity. In particular, enzymes that catalyze phosphorylation or dephosphorylation reactions came to the fore of interest because of their high relevance as drug targets. These include (protein) kinases, adenylyl cyclases, phosphodiesterases, phosphatases, and ATPases. The development and design of fluorescent lanthanide complexes should lead to probes with optimized selectivity and response times that can be applied for high-throughput screening of enzyme inhibitors and for real-time monitoring of enzyme kinetics. In contrast to other assays for enzyme activity determination, this method does not require the use of radioactively labelled substrates or the accomplishment of rather complex and expensive immunoassays.

Asunto(s)

Enzimas/química , Peróxido de Hidrógeno/química , Elementos de la Serie de los Lantanoides/química , Luminiscencia , Metales/química , Adenosina Trifosfatasas/química , Europio/química , Transferencia Resonante de Energía de Fluorescencia , Glucosa Oxidasa/química , Concentración de Iones de Hidrógeno , Cinética , Ligandos , Modelos Químicos , Nucleótidos/química , Peroxidasa/química , Terbio/química