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Anthony Maxwell - One of the best experts on this subject based on the ideXlab platform.
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A Common Origin for the Bacterial Toxin-AntiToxin Systems parD and ccd, Suggested by Analyses of Toxin/ Target and Toxin/AntiToxin Interactions
2016Co-Authors: Andrew Smith. B. ¤a, Lesley A Mitchenall, Elizabeth Diago-navarro, Arjan Barendregt, Albert J. Heck, Anthony MaxwellAbstract:Bacterial Toxin-antiToxin (TA) systems encode two proteins, a potent inhibitor of cell proliferation (Toxin) and its specific antidote (antiToxin). Structural data has revealed striking similarities between the two model TA Toxins CcdB, a DNA gyrase inhibitor encoded by the ccd system of plasmid F, and Kid, a site-specific endoribonuclease encoded by the parD system of plasmid R1. While a common structural fold seemed at odds with the two clearly different modes of action of these Toxins, the possibility of functional crosstalk between the parD and ccd systems, which would further point to their common evolutionary origin, has not been documented. Here, we show that the cleavage of RNA and the inhibition of protein synthesis by the Kid Toxin, two activities that are specifically counteracted by its cognate Kis antiToxin, are altered, but not inhibited, by the CcdA antiToxin. In addition, Kis was able to inhibit the stimulation of DNA gyrase-mediated cleavage of DNA by CcdB, albeit less efficiently than CcdA. We further show that physical interactions between the Toxins and antiToxins of the different systems do occur and define the stoichiometry of the complexes formed. We found that CcdB did not degrade RNA nor did Kid have any reproducible effect on the tested DNA gyrase activities, suggesting that these Toxin
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fragments of the Bacterial Toxin microcin b17 as gyrase poisons
PLOS ONE, 2013Co-Authors: Frederic Collin, Robert E Thompson, Katrina A Jolliffe, Richard J Payne, Anthony MaxwellAbstract:Fluoroquinolones are very important drugs in the clinical antiBacterial arsenal; their success is principally due to their mode of action: the stabilisation of a gyrase-DNA intermediate (the cleavage complex), which triggers a chain of events leading to cell death. Microcin B17 (MccB17) is a modified peptide Bacterial Toxin that acts by a similar mode of action, but is unfortunately unsuitable as a therapeutic drug. However, its structure and mechanism could inspire the design of new antiBacterial compounds that are needed to circumvent the rise in Bacterial resistance to current antibiotics. Here we describe the investigation of the structural features responsible for MccB17 activity and the identification of fragments of the Toxin that retain the ability to stabilise the cleavage complex.
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the action of the Bacterial Toxin microcin b17 on dna gyrase
Biochimie, 2007Co-Authors: William M Parks, Andrew R Bottrill, Olivier A Pierrat, Marcus C Durrant, Anthony MaxwellAbstract:Microcin B17 (MccB17) is a peptide-based Bacterial Toxin that targets DNA gyrase, the Bacterial enzyme that introduces supercoils into DNA. The site and mode of action of MccB17 on gyrase are unclear. We review what is currently known about MccB17-gyrase interactions and summarise approaches to understanding its mode of action that involve modification of the Toxin. We describe experiments in which treatment of the Toxin at high pH leads to the deamidation of two asparagine residues to aspartates. The modified Toxin was found to be inactive in vivo and in vitro, suggesting that the Asn residues are essential for activity. Following on from these studies we have used molecular modelling to suggest a 3D structure for microcin B17. We discuss the implications of this model for MccB17 action and investigate the possibility that it binds metal ions.
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the action of the Bacterial Toxin microcin b17 insight into the cleavage religation reaction of dna gyrase
Journal of Biological Chemistry, 2003Co-Authors: Olivier A Pierrat, Anthony MaxwellAbstract:We have examined the effects of the Bacterial Toxin microcin B17 (MccB17) on the reactions of Escherichia coli DNA gyrase. MccB17 slows down but does not completely inhibit the DNA supercoiling and relaxation reactions of gyrase. A kinetic analysis of the cleavage-religation equilibrium of gyrase was performed to determine the effect of the Toxin on the forward (cleavage) and reverse (religation) reactions. A simple mechanism of two consecutive reversible reactions with a nicked DNA intermediate was used to simulate the kinetics of cleavage and religation. The action of MccB17 on the kinetics of cleavage and religation was compared with that of the quinolones ciprofloxacin and oxolinic acid. With relaxed DNA as substrate, only a small amount of gyrase cleavage complex is observed with MccB17 in the absence of ATP, whereas the presence of the nucleotide significantly enhances the effect of the Toxin on both the cleavage and religation reactions. In contrast, ciprofloxacin, oxolinic acid, and Ca2+ show lesser dependence on ATP to stabilize the cleavage complex. MccB17 enhances the overall rate of DNA cleavage by increasing the forward rate constant (k 2) of the second equilibrium. In contrast, ciprofloxacin increases the amount of cleaved DNA by a combined effect on the forward and reverse rate constants of both equilibria. Based on these results and on the observations that MccB17 only slowly inhibits the supercoiling and relaxation reactions, we suggest a model of the interaction of MccB17 with gyrase.
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The antibiotic microcin B17 is a DNA gyrase poison: characterisation of the mode of inhibition.
Journal of molecular biology, 2001Co-Authors: Jonathan G. Heddle, Stephen J. Blance, Deborah B. Zamble, Florian Hollfelder, Deborah Ann Miller, Lois M. Wentzell, Christopher T. Walsh, Anthony MaxwellAbstract:Abstract Microcin B17 is a 3.1-kDa bactericidal peptide; the putative target of this antibiotic is DNA gyrase. Microcin B17 has no detectable effect on gyrase-catalysed DNA supercoiling or relaxation activities in vitro and is unable to stabilise DNA cleavage in the absence of nucleotides. However, in the presence of ATP, or the non-hydrolysable analogue 5′-adenylyl β,γ-imidodiphosphate, microcin B17 stabilises a gyrase-dependent DNA cleavage complex in a manner reminiscent of quinolones, Ca 2+ , or the Bacterial Toxin CcdB. The pattern of DNA cleavage produced by gyrase in the presence of microcin B17 is different from that produced by quinolones and more closely resembles Ca 2+ -mediated cleavage. Several gyrase mutants, including well-known quinolone-resistant mutants, are cross resistant to microcin-induced DNA cleavage. We suggest that microcin exerts its effects through a mechanism that has similarities to those of both the Bacterial Toxin CcdB and the quinolone antiBacterial agents.
Jorge E Galan - One of the best experts on this subject based on the ideXlab platform.
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Alternate subunit assembly diversifies the function of a Bacterial Toxin
Nature Communications, 2019Co-Authors: Casey C. Fowler, Gabrielle Stack, Xuyao Jiao, Maria Lara-tejero, Jorge E GalanAbstract:Bacterial Toxins with an AB_5 architecture consist of an active (A) subunit inserted into a ring-like platform comprised of five delivery (B) subunits. Salmonella Typhi, the cause of typhoid fever, produces an unusual A_2B_5 Toxin known as typhoid Toxin. Here, we report that upon infection of human cells, S . Typhi produces two forms of typhoid Toxin that have distinct delivery components but share common active subunits. The two typhoid Toxins exhibit different trafficking properties, elicit different effects when administered to laboratory animals, and are expressed using different regulatory mechanisms and in response to distinct metabolic cues. Collectively, these results indicate that the evolution of two typhoid Toxin variants has conferred functional versatility to this virulence factor. More broadly, this study reveals a new paradigm in Toxin biology and suggests that the evolutionary expansion of AB_5 Toxins was likely fueled by the plasticity inherent to their structural design coupled to the functional versatility afforded by the combination of homologous Toxin components. Salmonella Typhi produces the typhoid Toxin. Here, Fowler et al. show that S . Typhi produces two forms of typhoid Toxin that are differentially regulated and display different trafficking properties and different effects when administered to laboratory animals.
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Alternate subunit assembly diversifies the function of a Bacterial Toxin
bioRxiv, 2019Co-Authors: Casey C. Fowler, Gabrielle Stack, Xuyao Jiao, Maria Lara-tejero, Jorge E GalanAbstract:Bacterial Toxins with an AB% architecture are central to Bacterial pathogenesis. Functionally diverse and evolutionarily distant, AB5 Toxins adopt synonymous structures in which a discrete domain of the Toxin9s active (A) subunit is inserted into a ring-like platform comprised of five delivery (B) subunits. Salmonella Typhi, the cause of typhoid fever, produces an unusual A2B5 Toxin known as typhoid Toxin, a major virulence factor. Here, we report that upon infection of human cells, S. Typhi produces two forms of typhoid Toxin that have distinct delivery components but share common active subunits. We demonstrate that the two typhoid Toxins exhibit substantially different trafficking properties, elicit markedly different effects when administered to laboratory animals, and are expressed in response to different regulatory mechanisms and distinct metabolic cues. Collectively, these results indicate that the evolution of two typhoid Toxin variants has conferred functional versatility to this virulence factor. More broadly, this study reveals a new paradigm in Toxin biology and suggests that the evolutionary expansion of AB5 Toxins was likely fueled by the remarkable plasticity inherent to their structural design coupled to the functional versatility afforded by the combination of homologous Toxin components.
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host adaptation of a Bacterial Toxin from the human pathogen salmonella typhi
Cell, 2014Co-Authors: Lingquan Deng, Jeongmin Song, Jorge E Galan, Xiang Gao, Jiawei Wang, Xi Chen, Nissi Varki, Yuko Naitomatsui, Ajit VarkiAbstract:Salmonella Typhi is an exclusive human pathogen that causes typhoid fever. Typhoid Toxin is a S. Typhi virulence factor that can reproduce most of the typhoid fever symptoms in experimental animals. Toxicity depends on Toxin binding to terminally sialylated glycans on surface glycoproteins. Human glycans are unusual because of the lack of CMAH, which in other mammals converts N-acetylneuraminic acid (Neu5Ac) to N-glycolylneuraminic acid (Neu5Gc). Here, we report that typhoid Toxin binds to and is toxic toward cells expressing glycans terminated in Neu5Ac (expressed by humans) over glycans terminated in Neu5Gc (expressed by other mammals). Mice constitutively expressing CMAH thus displaying Neu5Gc in all tissues are resistant to typhoid Toxin. The atomic structure of typhoid Toxin bound to Neu5Ac reveals the structural bases for its binding specificity. These findings provide insight into the molecular bases for Salmonella Typhi’s host specificity and may help the development of therapies for typhoid fever.
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structural basis for the reversible activation of a rho protein by the Bacterial Toxin sope
The EMBO Journal, 2002Co-Authors: Gretel Buchwald, Jorge E Galan, Andrea Friebel, Wolfdietrich Hardt, Alfred Wittinghofer, Klaus ScheffzekAbstract:The Bacterial enteropathogen Salmonella typhimurium employs a type III secretion system to inject Bacterial Toxins into the host cell cytosol. These Toxins transiently activate Rho family GTP-binding protein-dependent signaling cascades to induce cytoskeletal rearrangements. One of these translocated Salmonella Toxins, SopE, can activate Cdc42 in a Dbl-like fashion despite its lack of sequence similarity to Dbl-like proteins, the Rho-specific eukaryotic guanine nucleotide exchange factors. To elucidate the mechanism of SopE-mediated guanine nucleotide exchange, we have analyzed the structure of the complex between a catalytic fragment of SopE and Cdc42. SopE binds to and locks the switch I and switch II regions of Cdc42 in a conformation that promotes guanine nucleotide release. This conformation is strikingly similar to that of Rac1 in complex with the eukaryotic Dbl-like exchange factor Tiam1. However, the catalytic domain of SopE has an entirely different architecture from that of Tiam1 and interacts with the switch regions via different amino acids. Therefore, SopE represents the first example of a non-Dbl-like protein capable of inducing guanine nucleotide exchange in Rho family proteins.
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a Bacterial Toxin that controls cell cycle progression as a deoxyribonuclease i like protein
Science, 2000Co-Authors: Maria Laratejero, Jorge E GalanAbstract:Many Bacterial pathogens encode a multisubunit Toxin, termed cytolethal distending Toxin (CDT), that induces cell cycle arrest, cytoplasm distention, and, eventually, chromatin fragmentation and cell death. In one such pathogen, Campylobacter jejuni, one of the subunits of this Toxin, CdtB, was shown to exhibit features of type I deoxyribonucleases. Transient expression of this subunit in cultured cells caused marked chromatin disruption. Microinjection of low amounts of CdtB induced cytoplasmic distention and cell cycle arrest. CdtB mutants with substitutions in residues equivalent to those required for catalysis or magnesium binding in type I deoxyribonucleases did not cause chromatin disruption. CDT holoToxin containing these mutant forms of CdtB did not induce morphological changes or cell cycle arrest.
Scott A. Strobel - One of the best experts on this subject based on the ideXlab platform.
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the positively charged active site of the Bacterial Toxin rele causes a large shift in the general base pka
Biochemistry, 2020Co-Authors: David A Hiller, Brian F Dunican, Sunitha Nallur, Joseph A Piccirilli, Scott A. StrobelAbstract:The Bacterial Toxin RelE cleaves mRNA in the ribosomal A site. Although it shares a global fold with other microbial RNases, the active site contains several positively charged residues instead of histidines and glutamates that are typical of ribonucleases. The pH dependences of wild-type and mutant RelE indicate it uses general acid-base catalysis, but either the general acid (proposed to be R81) or the general base must have a substantially downshifted pKa. However, which group is shifted cannot be determined using available structural and biochemical data. Here, we use a phosphorothiolate at the scissile phosphate to remove the need for a general acid. We show this modification rescues nearly all of the defect of the R81A mutation, supporting R81 as the general acid. We also find that the observed pKa of the general base is dependent on the charge of the side chain at position 81. This indicates that positive charge in the active site contributes to a general base pKa downshifted by more than 5 units. Although this modestly reduces the effectiveness of general acid-base catalysis, it is strongly supplemented by the role of the positive charge in stabilizing the transition state for cleavage. Furthermore, we show that the ribosome is required for cleavage but not binding of mRNA by RelE. Ribosome functional groups do not directly contact the scissile phosphate, indicating that positioning and charge interactions dominate RelE catalysis. The unusual RelE active site catalyzes phosphoryl transfer at a rate comparable to those of similar enzymes, but in a ribosome-dependent fashion.
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Bacterial Toxin rele a highly efficient ribonuclease with exquisite substrate specificity using atypical catalytic residues
Biochemistry, 2013Co-Authors: Meghan A Griffin, Jared H. Davis, Scott A. StrobelAbstract:The Toxin RelE is a ribosome-dependent endoribonuclease implicated in diverse cellular processes, including persistence. During amino acid starvation, RelE inhibits translation by cleaving ribosomal A-site mRNA. Although RelE is structurally similar to other microbial endoribonucleases, the active-site amino acid composition differs substantially and lacks obvious candidates for general acid–base functionality. Highly conserved RelE residues (Lys52, Lys54, Arg61, Arg81, and Tyr87) surround the mRNA scissile phosphate, and specific 16S rRNA contacts further contribute to substrate positioning. We used a single-turnover kinetic assay to evaluate the catalytic importance of individual residues in the RelE active site. Within the context of the ribosome, RelE rapidly cleaves A-site mRNA at a rate similar to those of traditional ribonucleases. Single-turnover rate constants decreased between 102- and 106-fold for the RelE active-site mutants of Lys52, Lys54, Arg61, and Arg81. RelE may principally promote catal...
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Bacterial Toxin RelE: A Highly Efficient Ribonuclease with Exquisite Substrate Specificity Using Atypical Catalytic Residues
2013Co-Authors: Meghan A. Griffin, Jared H. Davis, Scott A. StrobelAbstract:The Toxin RelE is a ribosome-dependent endoribonuclease implicated in diverse cellular processes, including persistence. During amino acid starvation, RelE inhibits translation by cleaving ribosomal A-site mRNA. Although RelE is structurally similar to other microbial endoribonucleases, the active-site amino acid composition differs substantially and lacks obvious candidates for general acid–base functionality. Highly conserved RelE residues (Lys52, Lys54, Arg61, Arg81, and Tyr87) surround the mRNA scissile phosphate, and specific 16S rRNA contacts further contribute to substrate positioning. We used a single-turnover kinetic assay to evaluate the catalytic importance of individual residues in the RelE active site. Within the context of the ribosome, RelE rapidly cleaves A-site mRNA at a rate similar to those of traditional ribonucleases. Single-turnover rate constants decreased between 102- and 106-fold for the RelE active-site mutants of Lys52, Lys54, Arg61, and Arg81. RelE may principally promote catalysis via transition-state charge stabilization and leaving-group protonation, in addition to achieving in-line substrate positioning in cooperation with the ribosome. This kinetic analysis complements structural information to provide a foundation for understanding the molecular mechanism of this atypical endoribonuclease
Brice Felden - One of the best experts on this subject based on the ideXlab platform.
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Cross-Regulations between Bacterial Toxin-AntiToxin Systems: Evidence of an Interconnected Regulatory Network?
Trends in Microbiology, 2020Co-Authors: Camille Riffaud, Marie-laure Pinel-marie, Brice FeldenAbstract:Toxin-antiToxin (TA) systems are ubiquitous among bacteria and include stable Toxins whose toxicity can be counteracted by RNA or protein antiToxins. They are involved in multiple functions that range from stability maintenance for mobile genetic elements to stress adaptation. Bacterial chromosomes frequently have multiple homologues of TA system loci, and it is unclear why there are so many of them. In this review we focus on cross-regulations between TA systems, which occur between both homologous and nonhomologous systems, from similar or distinct types, whether encoded from plasmids or chromosomes. In addition to being able to modulate RNA expression levels, cross-regulations between these systems can also influence their toxicity. This suggests the idea that they are involved in an interconnected regulatory network.
Olivier A Pierrat - One of the best experts on this subject based on the ideXlab platform.
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the action of the Bacterial Toxin microcin b17 on dna gyrase
Biochimie, 2007Co-Authors: William M Parks, Andrew R Bottrill, Olivier A Pierrat, Marcus C Durrant, Anthony MaxwellAbstract:Microcin B17 (MccB17) is a peptide-based Bacterial Toxin that targets DNA gyrase, the Bacterial enzyme that introduces supercoils into DNA. The site and mode of action of MccB17 on gyrase are unclear. We review what is currently known about MccB17-gyrase interactions and summarise approaches to understanding its mode of action that involve modification of the Toxin. We describe experiments in which treatment of the Toxin at high pH leads to the deamidation of two asparagine residues to aspartates. The modified Toxin was found to be inactive in vivo and in vitro, suggesting that the Asn residues are essential for activity. Following on from these studies we have used molecular modelling to suggest a 3D structure for microcin B17. We discuss the implications of this model for MccB17 action and investigate the possibility that it binds metal ions.
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the action of the Bacterial Toxin microcin b17 insight into the cleavage religation reaction of dna gyrase
Journal of Biological Chemistry, 2003Co-Authors: Olivier A Pierrat, Anthony MaxwellAbstract:We have examined the effects of the Bacterial Toxin microcin B17 (MccB17) on the reactions of Escherichia coli DNA gyrase. MccB17 slows down but does not completely inhibit the DNA supercoiling and relaxation reactions of gyrase. A kinetic analysis of the cleavage-religation equilibrium of gyrase was performed to determine the effect of the Toxin on the forward (cleavage) and reverse (religation) reactions. A simple mechanism of two consecutive reversible reactions with a nicked DNA intermediate was used to simulate the kinetics of cleavage and religation. The action of MccB17 on the kinetics of cleavage and religation was compared with that of the quinolones ciprofloxacin and oxolinic acid. With relaxed DNA as substrate, only a small amount of gyrase cleavage complex is observed with MccB17 in the absence of ATP, whereas the presence of the nucleotide significantly enhances the effect of the Toxin on both the cleavage and religation reactions. In contrast, ciprofloxacin, oxolinic acid, and Ca2+ show lesser dependence on ATP to stabilize the cleavage complex. MccB17 enhances the overall rate of DNA cleavage by increasing the forward rate constant (k 2) of the second equilibrium. In contrast, ciprofloxacin increases the amount of cleaved DNA by a combined effect on the forward and reverse rate constants of both equilibria. Based on these results and on the observations that MccB17 only slowly inhibits the supercoiling and relaxation reactions, we suggest a model of the interaction of MccB17 with gyrase.