RESUMEN
Two-component systems are crucial for signal perception and modulation of bacterial behavior. Nevertheless, to date, very few ligands have been identified that directly interact with histidine kinases. The histidine kinase/response regulator system YehU/YehT of Escherichia coli is part of a nutrient-sensing network. Here we demonstrate that this system senses the onset of nutrient limitation in amino acid rich media and responds to extracellular pyruvate. Binding of radiolabeled pyruvate was found for full-length YehU in right-side-out membrane vesicles as well as for a truncated, membrane-integrated variant, confirming that YehU is a high-affinity receptor for extracellular pyruvate. Therefore we propose to rename YehU/YehT as BtsS/BtsR, after "Brenztraubensäure", the name given to pyruvic acid when it was first synthesized. The function of BtsS/BtsR was also assessed in a clinically relevant uropathogenic E. coli strain. Quantitative transcriptional analysis revealed BtsS/BtsR importance during acute and chronic urinary-tract infections.
Asunto(s)
Proteínas de Escherichia coli/farmacología , Ácido Pirúvico/administración & dosificación , Escherichia coli , Transducción de Señal , Infecciones Urinarias/metabolismo , Escherichia coli Uropatógena/metabolismoRESUMEN
Extra-intestinal pathogenic E. coli (ExPEC) infections are common in mammals and birds. The predominant ExPEC types are avian pathogenic E. coli (APEC), neonatal meningitis causing E. coli/meningitis associated E. coli (NMEC/MAEC), and uropathogenic E. coli (UPEC). Many reviews have described current knowledge on ExPEC infection strategies and virulence factors, especially for UPEC. However, surprisingly little has been reported on the regulatory modules that have been identified as critical in ExPEC pathogenesis. Two-component systems (TCSs) comprise the predominant method by which bacteria respond to changing environments and play significant roles in modulating bacterial fitness in diverse niches. Recent studies have highlighted the potential of manipulating signal transduction systems as a means to chemically re-wire bacterial pathogens, thereby reducing selective pressure and avoiding the emergence of antibiotic resistance. This review begins by providing a brief introduction to characterized infection strategies and common virulence factors among APEC, NMEC, and UPEC and continues with a comprehensive overview of two-component signal transduction networks that have been shown to influence ExPEC pathogenesis.
Asunto(s)
Infecciones por Escherichia coli/microbiología , Escherichia coli/patogenicidad , Intestinos/microbiología , Transducción de Señal/fisiología , Enfermedades de los Animales/microbiología , Animales , Modelos Animales de Enfermedad , Farmacorresistencia Microbiana , Infecciones por Escherichia coli/tratamiento farmacológico , Proteínas de Escherichia coli/fisiología , Humanos , Lactante , Mortalidad Infantil , Ratones , Proteínas Quinasas/fisiología , Escherichia coli Uropatógena/patogenicidad , Factores de Virulencia , Zoonosis/microbiología , Zoonosis/transmisiónRESUMEN
Two-component systems are prototypically comprised of a histidine kinase (sensor) and a response regulator (responder). The sensor kinases autophosphorylate at a conserved histidine residue, acting as a phosphodonor for subsequent phosphotransfer to and activation of a cognate response regulator. In rare cases, the histidine residue is also essential for response regulator dephosphorylation via a reverse-phosphotransfer reaction. In this work, we present an example of a kinase that relies on reverse phosphotransfer to catalyze the dephosphorylation of its cognate partner. The QseC sensor kinase is conserved across several Gram-negative pathogens; its interaction with its cognate partner QseB is critical for maintaining pathogenic potential. Here, we demonstrate that QseC-mediated dephosphorylation of QseB occurs via reverse phosphotransfer. In previous studies, we demonstrated that, in uropathogenic Escherichia coli, exposure to high concentrations of ferric iron (Fe3+) stimulates the PmrB sensor kinase. This stimulation, in turn, activates the cognate partner, PmrA, and noncognate QseB to enhance tolerance to polymyxin B. We demonstrate that in the absence of signal, kinase-inactive QseC variants, in which the H246 residue was changed to alanine (A) aspartate (D) or leucine (L), rescued a ΔqseC deletion mutant, suggesting that QseC can control QseB activation via a mechanism that is independent of reverse phosphotransfer. However, in the presence of Fe3+, the same QseC variants were unable to mediate a wild-type stimulus response, indicating that QseC-mediated dephosphorylation is required for maintaining proper QseB-PmrB-PmrA interactions.IMPORTANCE Two-component signaling networks constitute one of the predominant methods by which bacteria sense and respond to their changing environments. Two-component systems allow bacteria to thrive and survive in a number of different environments, including within a human host. Uropathogenic Escherichia coli, the causative agent of urinary tract infections, rely on two interacting two-component systems, QseBC and PmrAB, to induce intrinsic resistance to the colistin antibiotic polymyxin B, which is a last line of defense drug. The presence of one sensor kinase, QseC, is required to regulate the interaction between the other sensor kinase, PmrB and the response regulators from both systems, QseB and PmrA, effectively creating a "four-component" system required for virulence. Understanding the important role of the sensor kinase QseC will provide insight into additional ways to therapeutically target uropathogens that harbor these signaling systems.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Histidina/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Escherichia coli Uropatógena/fisiología , Proteínas de Escherichia coli/genética , Eliminación de Gen , Histidina/genética , Hierro/metabolismo , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Escherichia coli Uropatógena/genéticaRESUMEN
Bacteria use two-component systems (TCSs) to react appropriately to environmental stimuli. Typical TCSs comprise a sensor histidine kinase that acts as a receptor coupled to a partner response regulator that coordinates changes in bacterial behavior, often through its activity as a transcriptional regulator. TCS interactions are typically confined to cognate pairs of histidine kinases and response regulators. We describe two distinct TCSs in uropathogenic Escherichia coli (UPEC) that interact to mediate a response to ferric iron. The PmrAB and QseBC TCSs were both required for proper transcriptional response to ferric iron. Ferric iron induced the histidine kinase PmrB to phosphotransfer to both its cognate response regulator PmrA and the noncognate response regulator QseB, leading to transcriptional responses coordinated by both regulators. Pretreatment of the UPEC strain UTI89 with ferric iron led to increased resistance to polymyxin B that required both PmrA and QseB. Similarly, pretreatment of several UPEC isolates with ferric iron increased tolerance to polymyxin B. This study defines physiologically relevant cross talk between TCSs in a bacterial pathogen and provides a potential mechanism for antibiotic resistance of some strains of UPEC.
Asunto(s)
Tolerancia a Medicamentos/genética , Proteínas de Escherichia coli/genética , Polimixina B/farmacología , Transducción de Señal/genética , Escherichia coli Uropatógena/efectos de los fármacos , Escherichia coli Uropatógena/genética , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Compuestos Férricos/farmacología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Iones/farmacología , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The ability to de novo synthesize purines has been associated with the intracellular survival of multiple bacterial pathogens. Uropathogenic Escherichia coli (UPEC), the predominant cause of urinary tract infections, undergoes a transient intracellular lifestyle during which bacteria clonally expand into multicellular bacterial communities within the cytoplasm of bladder epithelial cells. Here, we characterized the contribution of the conserved de novo purine biosynthesis-associated locus cvpA-purF to UPEC pathogenesis. Deletion of cvpA-purF, or of purF alone, abolished de novo purine biosynthesis but did not impact bacterial adherence properties in vitro or in the bladder lumen. However, upon internalization by bladder epithelial cells, UPEC deficient in de novo purine biosynthesis was unable to expand into intracytoplasmic bacterial communities over time, unless it was extrachromosomally complemented. These findings indicate that UPEC is deprived of purine nucleotides within the intracellular niche and relies on de novo purine synthesis to meet this metabolic requirement.
Asunto(s)
Purinas/biosíntesis , Purinas/metabolismo , Escherichia coli Uropatógena/metabolismo , Animales , Citoplasma/metabolismo , Citoplasma/microbiología , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Femenino , Humanos , Ratones , Ratones Endogámicos C3H , Vejiga Urinaria/metabolismo , Vejiga Urinaria/microbiología , Infecciones Urinarias/metabolismo , Infecciones Urinarias/microbiología , Virulencia/genéticaRESUMEN
The rise in quinolone resistance is threatening the clinical use of this important class of broad-spectrum antibacterials. Quinolones kill bacteria by increasing the level of DNA strand breaks generated by the type II topoisomerases gyrase and topoisomerase IV. Most commonly, resistance is caused by mutations in the serine and acidic amino acid residues that anchor a water-metal ion bridge that facilitates quinolone-enzyme interactions. Although other mutations in gyrase and topoisomerase IV have been reported in quinolone-resistant strains, little is known regarding their contributions to cellular quinolone resistance. To address this issue, we characterized the effects of the V96A mutation in the A subunit of Bacillus anthracis topoisomerase IV on quinolone activity. The results indicate that this mutation causes an â¼ 3-fold decrease in quinolone potency and reduces the stability of covalent topoisomerase IV-cleaved DNA complexes. However, based on metal ion usage, the V96A mutation does not disrupt the function of the water-metal ion bridge. A similar level of resistance to quinazolinediones (which do not use the bridge) was seen. V96A is the first topoisomerase IV mutation distal to the water-metal ion bridge demonstrated to decrease quinolone activity. It also represents the first A subunit mutation reported to cause resistance to quinazolinediones. This cross-resistance suggests that the V96A change has a global effect on the structure of the drug-binding pocket of topoisomerase IV.
Asunto(s)
Bacillus anthracis/química , Topoisomerasa de ADN IV/química , Manganeso/química , Mutación , Níquel/química , Subunidades de Proteína/química , Agua/química , Alanina/química , Alanina/genética , Antibacterianos/química , Bacillus anthracis/enzimología , Cationes Bivalentes , Ciprofloxacina/química , Topoisomerasa de ADN IV/antagonistas & inhibidores , Topoisomerasa de ADN IV/genética , ADN Bacteriano/química , Farmacorresistencia Bacteriana/genética , Fluoroquinolonas/química , Modelos Moleculares , Moxifloxacino , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Inhibidores de Topoisomerasa/química , Valina/química , Valina/genéticaRESUMEN
Although quinolones have been in clinical use for decades, the mechanism underlying drug activity and resistance has remained elusive. However, recent studies indicate that clinically relevant quinolones interact with Bacillus anthracis (Gram-positive) topoisomerase IV through a critical water-metal ion bridge and that the most common quinolone resistance mutations decrease drug activity by disrupting this bridge. As a first step toward determining whether the water-metal ion bridge is a general mechanism of quinolone-topoisomerase interaction, we characterized drug interactions with wild-type Escherichia coli (Gram-negative) topoisomerase IV and a series of ParC enzymes with mutations (S80L, S80I, S80F, and E84K) in the predicted bridge-anchoring residues. Results strongly suggest that the water-metal ion bridge is essential for quinolone activity against E. coli topoisomerase IV. Although the bridge represents a common and critical mechanism that underlies broad-spectrum quinolone function, it appears to play different roles in B. anthracis and E. coli topoisomerase IV. The water-metal ion bridge is the most important binding contact of clinically relevant quinolones with the Gram-positive enzyme. However, it primarily acts to properly align clinically relevant quinolones with E. coli topoisomerase IV. Finally, even though ciprofloxacin is unable to increase levels of DNA cleavage mediated by several of the Ser80 and Glu84 mutant E. coli enzymes, the drug still retains the ability to inhibit the overall catalytic activity of these topoisomerase IV proteins. Inhibition parallels drug binding, suggesting that the presence of the drug in the active site is sufficient to diminish DNA relaxation rates.
Asunto(s)
Ciprofloxacina/metabolismo , Topoisomerasa de ADN IV/metabolismo , Escherichia coli/enzimología , Metales/química , Agua/química , Biocatálisis , ADN/químicaRESUMEN
Bacterial two-component systems (TCSs) mediate specific responses to distinct conditions and/or stresses. TCS interactions are highly specific between cognate partners to avoid unintended cross-talk. Although cross-talk between a sensor kinase and a noncognate response regulator has been previously demonstrated, the majority of reported interactions have not been robust. Here, we report that in the case of the quorum-sensing Escherichia coli (Qse)BC TCS, absence of the cognate sensor QseC leads to robust, constitutive activation of the QseB response regulator by the noncognate polymyxin resistance (Pmr) sensor kinase PmrB. Remarkably, the noncognate PmrB exhibits a kinetic preference for QseB that is similar to QseC. However, although PmrB readily phosphorylates QseB in vitro, it is significantly less efficient at dephosphorylating QseB, compared with QseC, thereby explaining the increased levels of active QseB in the qseC mutant. In addition to PmrB activating QseB on the protein level, we found that the PmrA response regulator contributes to qseB transcription in the absence of QseC and PmrA specifically binds the qseBC promoter, indicative of a direct regulation of qseBC gene transcription by PmrAB under physiological conditions. Addition of ferric iron in the growth medium of wild-type uropathogenic E. coli induced the expression of qseBC in a PmrB-dependent manner. Taken together, our findings suggest that (i) robust cross-talk between noncognate partners is possible and (ii) this interaction can be manipulated for the development of antivirulence strategies aimed at targeting uropathogenic Escherichia coli and potentially other QseBC-PmrAB-bearing pathogens.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Percepción de Quorum/fisiología , Receptor Cross-Talk/fisiología , Transducción de Señal/fisiología , Proteínas Bacterianas/metabolismo , Cartilla de ADN/genética , Elementos Transponibles de ADN/genética , Ensayo de Cambio de Movilidad Electroforética , Escherichia coli/patogenicidad , Proteínas de Escherichia coli/fisiología , Immunoblotting , Mutagénesis , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND: The R6K replicon is one of the best studied bacterial plasmid replicons. Replication of the R6K plasmid and derivatives harboring its γ origin of replication (ori(R6Kγ)) is dependent on the pir gene-encoded π protein. Originally encoded by R6K, this protein is usually provided in trans in hosts engineered to support replication of plasmids harboring ori(R6Kγ). In Escherichia coli this is commonly achieved by chromosomal integration of pir either via lysogenization with a λpir phage or homologous recombination at a pre-determined locus. FINDINGS: Current methods for construction of host strains for ori(R6Kγ)-containing plasmids involve procedures that do not allow selection for presence of the pir gene and require cumbersome and time-consuming screening steps. In this study, we established a mini-Tn7-based method for rapid and reliable construction of pir+ host strains. Using a curable mini-Tn7 delivery plasmid, pir expressing derivatives of several commonly used E. coli cloning and mobilizer strains were isolated using both the wild-type pir+ gene as well as the copy-up pir-116 allele. In addition, we isolated pir+ and pir-116 expressing derivatives of a clinical isolate of Salmonella enterica serovar Typhimurium. In both E. coli and S. enterica serovar Typhimurium, the presence of the pir+ wild-type or pir-116 alleles allowed the replication of ori(R6Kγ)-containing plasmids. CONCLUSIONS: A mini-Tn7 system was employed for rapid and reliable engineering of E. coli and S. enterica serovar Typhimurium host strains for plasmids containing ori(R6Kγ). Since mini-Tn7 elements transpose in most, if not all, Gram negative bacteria, we anticipate that with relatively minor modifications this newly established method will for the first time allow engineering of other bacterial species to enable replication of plasmids with ori(R6Kγ).