160 results on '"Brennan RG"'
Search Results
2. Thiamine deficiency in sheep exported live by sea
- Author
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THOMAS, KW, primary, KELLY, AP, additional, BEERS, PT, additional, and BRENNAN, RG, additional
- Published
- 1990
- Full Text
- View/download PDF
3. Structures of trehalose-6-phosphate synthase, Tps1, from the fungal pathogen Cryptococcus neoformans : A target for antifungals.
- Author
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Washington EJ, Zhou Y, Hsu AL, Petrovich M, Tenor JL, Toffaletti DL, Guan Z, Perfect JR, Borgnia MJ, Bartesaghi A, and Brennan RG
- Subjects
- Fungal Proteins metabolism, Fungal Proteins genetics, Fungal Proteins chemistry, Models, Molecular, Humans, Catalytic Domain, Crystallography, X-Ray, Cryptococcus neoformans enzymology, Cryptococcus neoformans metabolism, Cryptococcus neoformans genetics, Glucosyltransferases metabolism, Glucosyltransferases genetics, Antifungal Agents pharmacology, Antifungal Agents chemistry, Antifungal Agents metabolism, Trehalose metabolism, Trehalose analogs & derivatives, Trehalose biosynthesis
- Abstract
Invasive fungal diseases are a major threat to human health, resulting in more than 1.5 million annual deaths worldwide. The arsenal of antifungal therapeutics remains limited and is in dire need of drugs that target additional biosynthetic pathways that are absent from humans. One such pathway involves the biosynthesis of trehalose. Trehalose is a disaccharide that is required for pathogenic fungi to survive in their human hosts. In the first step of trehalose biosynthesis, trehalose-6-phosphate synthase (Tps1) converts UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate. Here, we report the structures of full-length Cryptococcus neoformans Tps1 (CnTps1) in unliganded form and in complex with uridine diphosphate and glucose-6-phosphate. Comparison of these two structures reveals significant movement toward the catalytic pocket by the N terminus upon ligand binding and identifies residues required for substrate binding, as well as residues that stabilize the tetramer. Intriguingly, an intrinsically disordered domain (IDD), which is conserved among Cryptococcal species and closely related basidiomycetes, extends from each subunit of the tetramer into the "solvent" but is not visible in density maps. We determined that the IDD is not required for C. neoformans Tps1-dependent thermotolerance and osmotic stress survival. Studies with UDP-galactose highlight the exquisite substrate specificity of CnTps1. In toto, these studies expand our knowledge of trehalose biosynthesis in Cryptococcus and highlight the potential of developing antifungal therapeutics that disrupt the synthesis of this disaccharide or the formation of a functional tetramer and the use of cryo-EM in the structural characterization of CnTps1-ligand/drug complexes., Competing Interests: Competing interests statement:The authors declare no competing interest.
- Published
- 2024
- Full Text
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4. How c-di-GMP controls progression through the Streptomyces life cycle.
- Author
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Gallagher KA, Tschowri N, Brennan RG, Schumacher MA, and Buttner MJ
- Subjects
- Signal Transduction, Streptomyces metabolism, Streptomyces growth & development, Streptomyces genetics, Cyclic GMP metabolism, Cyclic GMP analogs & derivatives, Spores, Bacterial growth & development, Spores, Bacterial metabolism, Spores, Bacterial genetics, Gene Expression Regulation, Bacterial, Bacterial Proteins metabolism, Bacterial Proteins genetics
- Abstract
Members of the antibiotic-producing bacterial genus Streptomyces undergo a complex developmental life cycle that culminates in the production of spores. Central to control of this cell differentiation process is signaling through the second messenger 3', 5'-cyclic diguanylic acid (c-di-GMP). So far, three proteins that are directly controlled by c-di-GMP in Streptomyces have been functionally and structurally characterized: the key developmental regulators BldD and σ
WhiG , and the glycogen-degrading enzyme GlgX. c-di-GMP signals through BldD and σWhiG , respectively, to control the two most dramatic transitions of the Streptomyces life cycle, the formation of the reproductive aerial hyphae and their differentiation into spore chains. Later in development, c-di-GMP activates GlgX-mediated degradation of glycogen, releasing stored carbon for spore maturation., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)- Published
- 2024
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5. Structures of trehalose-6-phosphate synthase, Tps1, from the fungal pathogen Cryptococcus neoformans : a target for novel antifungals.
- Author
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Washington EJ, Zhou Y, Hsu AL, Petrovich M, Tenor JL, Toffaletti DL, Guan Z, Perfect JR, Borgnia MJ, Bartesaghi A, and Brennan RG
- Abstract
Invasive fungal diseases are a major threat to human health, resulting in more than 1.5 million annual deaths worldwide. The arsenal of antifungal therapeutics remains limited and is in dire need of novel drugs that target additional biosynthetic pathways that are absent from humans. One such pathway involves the biosynthesis of trehalose. Trehalose is a disaccharide that is required for pathogenic fungi to survive in their human hosts. In the first step of trehalose biosynthesis, trehalose-6-phosphate synthase (Tps1) converts UDP-glucose and glucose-6-phosphate to trehalose-6-phosphate. Here, we report the structures of full-length Cryptococcus neoformans Tps1 (CnTps1) in unliganded form and in complex with uridine diphosphate and glucose-6-phosphate. Comparison of these two structures reveals significant movement towards the catalytic pocket by the N-terminus upon ligand binding and identifies residues required for substrate-binding, as well as residues that stabilize the tetramer. Intriguingly, an intrinsically disordered domain (IDD), which is conserved amongst Cryptococcal species and closely related Basidiomycetes, extends from each subunit of the tetramer into the "solvent" but is not visible in density maps. We determined that the IDD is not required for C. neoformans Tps1-dependent thermotolerance and osmotic stress survival. Studies with UDP-galactose highlight the exquisite substrate specificity of CnTps1. In toto , these studies expand our knowledge of trehalose biosynthesis in Cryptococcus and highlight the potential of developing antifungal therapeutics that disrupt the synthesis of this disaccharide or the formation of a functional tetramer and the use of cryo-EM in the structural characterization of CnTps1-ligand/drug complexes., Significance Statement: Fungal infections are responsible for over a million deaths worldwide each year. Biosynthesis of a disaccharide, trehalose, is required for multiple pathogenic fungi to transition from the environment to the human host. Enzymes in the trehalose biosynthesis pathway are absent in humans and, therefore, are potentially significant targets for novel antifungal therapeutics. One enzyme in the trehalose biosynthesis is trehalose-6-phosphate synthase (Tps1). Here, we describe the cryo-electron microscopy structures of the CnTps1 homo-tetramer in the unliganded form and in complex with a substrate and a product. These structures and subsequent biochemical analysis reveal key details of substrate-binding residues and substrate specificity. These structures should facilitate structure-guided design of inhibitors against CnTps1.
- Published
- 2024
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6. A single amino acid in the Salmonella effector SarA/SteE triggers supraphysiological activation of STAT3 for anti-inflammatory target gene expression.
- Author
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Gaggioli MR, Jones AG, Panagi I, Washington EJ, Loney RE, Muench JH, Brennan RG, Thurston TLM, and Ko DC
- Abstract
Non-typhoidal Salmonella enterica cause an estimated 1 million cases of gastroenteritis annually in the United States. These serovars use secreted protein effectors to mimic and reprogram host cellular functions. We previously discovered that the secreted effector SarA ( Salmonella anti-inflammatory response activator; also known as SteE) was required for increased intracellular replication of S . Typhimurium and production of the anti-inflammatory cytokine interleukin-10 (IL-10). SarA facilitates phosphorylation of STAT3 through a region of homology with the host cytokine receptor gp130. Here, we demonstrate that a single amino acid difference between SarA and gp130 is critical for the anti-inflammatory bias of SarA-STAT3 signaling. An isoleucine at the pY+1 position of the YxxQ motif in SarA (which binds the SH2 domain in STAT3) causes increased STAT3 phosphorylation and expression of anti-inflammatory target genes. This isoleucine, completely conserved in ~4000 Salmonella isolates, renders SarA a better substrate for tyrosine phosphorylation by GSK-3. GSK-3 is canonically a serine/threonine kinase that nonetheless undergoes tyrosine autophosphorylation at a motif that has an invariant isoleucine at the pY+1 position. Our results provide a molecular basis for how a Salmonella secreted effector achieves supraphysiological levels of STAT3 activation to control host genes during infection., Competing Interests: Declaration of interests: The authors declare no competing interests.
- Published
- 2024
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7. Hormonal steroids induce multidrug resistance and stress response genes in Neisseria gonorrhoeae by binding to MtrR.
- Author
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Hooks GM, Ayala JC, Holley CL, Dhulipala V, Beggs GA, Perfect JR, Schumacher MA, Shafer WM, and Brennan RG
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- Bacterial Proteins genetics, Bacterial Proteins metabolism, Drug Resistance, Multiple, Steroids metabolism, Anti-Bacterial Agents pharmacology, Anti-Bacterial Agents metabolism, Neisseria gonorrhoeae genetics, Neisseria gonorrhoeae metabolism, Repressor Proteins genetics, Repressor Proteins metabolism
- Abstract
Transcriptional regulator MtrR inhibits the expression of the multidrug efflux pump operon mtrCDE in the pathogenic bacterium Neisseria gonorrhoeae. Here, we show that MtrR binds the hormonal steroids progesterone, β-estradiol, and testosterone, which are present at urogenital infection sites, as well as ethinyl estrogen, a component of some hormonal contraceptives. Steroid binding leads to the decreased affinity of MtrR for cognate DNA, increased mtrCDE expression, and enhanced antimicrobial resistance. Furthermore, we solve crystal structures of MtrR bound to each steroid, thus revealing their binding mechanisms and the conformational changes that induce MtrR., (© 2024. The Author(s).)
- Published
- 2024
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8. Hormonal steroids bind the Neisseria gonorrhoeae multidrug resistance regulator, MtrR, to induce a multidrug binding efflux pump and stress-response sigma factor.
- Author
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Hooks GM, Ayala JC, Beggs GA, Perfect JR, Schumacher MA, Shafer WM, and Brennan RG
- Abstract
Overexpression of the multidrug efflux pump MtrCDE, a critical factor of multidrug-resistance in Neisseria gonorrhoeae , the causative agent of gonorrheae, is repressed by the transcriptional regulator, MtrR (multiple transferable resistance repressor). Here, we report the results from a series of in vitro experiments to identify innate, human inducers of MtrR and to understand the biochemical and structural mechanisms of the gene regulatory function of MtrR. Isothermal titration calorimetry experiments reveal that MtrR binds the hormonal steroids progesterone, β-estradiol, and testosterone, all of which are present at significant concentrations at urogenital infection sites as well as ethinyl estrogen, a component of some birth control pills. Binding of these steroids results in decreased affinity of MtrR for cognate DNA, as demonstrated by fluorescence polarization-based assays. The crystal structures of MtrR bound to each steroid provided insight into the flexibility of the binding pocket, elucidated specific residue-ligand interactions, and revealed the conformational consequences of the induction mechanism of MtrR. Three residues, D171, W136 and R176 are key to the specific binding of these gonadal steroids. These studies provide a molecular understanding of the transcriptional regulation by MtrR that promotes N. gonorrhoeae survival in its human host.
- Published
- 2023
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9. Single-cell genome-wide association reveals that a nonsynonymous variant in ERAP1 confers increased susceptibility to influenza virus.
- Author
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Schott BH, Wang L, Zhu X, Harding AT, Ko ER, Bourgeois JS, Washington EJ, Burke TW, Anderson J, Bergstrom E, Gardener Z, Paterson S, Brennan RG, Chiu C, McClain MT, Woods CW, Gregory SG, Heaton NS, and Ko DC
- Abstract
During pandemics, individuals exhibit differences in risk and clinical outcomes. Here, we developed single-cell high-throughput human in vitro susceptibility testing (scHi-HOST), a method for rapidly identifying genetic variants that confer resistance and susceptibility. We applied this method to influenza A virus (IAV), the cause of four pandemics since the start of the 20
th century. scHi-HOST leverages single-cell RNA sequencing (scRNA-seq) to simultaneously assign genetic identity to cells in mixed infections of cell lines of European, African, and Asian origin, reveal associated genetic variants for viral burden, and identify expression quantitative trait loci. Integration of scHi-HOST with human challenge and experimental validation demonstrated that a missense variant in endoplasmic reticulum aminopeptidase 1 ( ERAP1 ; rs27895) increased IAV burden in cells and human volunteers. rs27895 exhibits population differentiation, likely contributing to greater permissivity of cells from African populations to IAV. scHi-HOST is a broadly applicable method and resource for decoding infectious-disease genetics., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing interests.- Published
- 2022
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10. Molecular dissection of the glutamine synthetase-GlnR nitrogen regulatory circuitry in Gram-positive bacteria.
- Author
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Travis BA, Peck JV, Salinas R, Dopkins B, Lent N, Nguyen VD, Borgnia MJ, Brennan RG, and Schumacher MA
- Subjects
- Bacillus subtilis metabolism, Bacterial Proteins metabolism, Glutamine metabolism, Glutamate-Ammonia Ligase genetics, Glutamate-Ammonia Ligase metabolism, Nitrogen metabolism
- Abstract
How bacteria sense and respond to nitrogen levels are central questions in microbial physiology. In Gram-positive bacteria, nitrogen homeostasis is controlled by an operon encoding glutamine synthetase (GS), a dodecameric machine that assimilates ammonium into glutamine, and the GlnR repressor. GlnR detects nitrogen excess indirectly by binding glutamine-feedback-inhibited-GS (FBI-GS), which activates its transcription-repression function. The molecular mechanisms behind this regulatory circuitry, however, are unknown. Here we describe biochemical and structural analyses of GS and FBI-GS-GlnR complexes from pathogenic and non-pathogenic Gram-positive bacteria. The structures show FBI-GS binds the GlnR C-terminal domain within its active-site cavity, juxtaposing two GlnR monomers to form a DNA-binding-competent GlnR dimer. The FBI-GS-GlnR interaction stabilizes the inactive GS conformation. Strikingly, this interaction also favors a remarkable dodecamer to tetradecamer transition in some GS, breaking the paradigm that all bacterial GS are dodecamers. These data thus unveil unique structural mechanisms of transcription and enzymatic regulation., (© 2022. The Author(s).)
- Published
- 2022
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11. The nucleotide messenger (p)ppGpp is an anti-inducer of the purine synthesis transcription regulator PurR in Bacillus.
- Author
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Anderson BW, Schumacher MA, Yang J, Turdiev A, Turdiev H, Schroeder JW, He Q, Lee VT, Brennan RG, and Wang JD
- Subjects
- Binding Sites, Gene Expression Regulation, Bacterial, Bacillus subtilis metabolism, Bacterial Proteins metabolism, DNA, Bacterial metabolism, DNA-Binding Proteins metabolism, Guanosine Pentaphosphate metabolism, Guanosine Tetraphosphate metabolism, Repressor Proteins metabolism
- Abstract
The nucleotide messenger (p)ppGpp allows bacteria to adapt to fluctuating environments by reprogramming the transcriptome. Despite its well-recognized role in gene regulation, (p)ppGpp is only known to directly affect transcription in Proteobacteria by binding to the RNA polymerase. Here, we reveal a different mechanism of gene regulation by (p)ppGpp in Firmicutes: (p)ppGpp directly binds to the transcription factor PurR to downregulate purine biosynthesis gene expression upon amino acid starvation. We first identified PurR as a receptor of (p)ppGpp in Bacillus anthracis. A co-structure with Bacillus subtilis PurR reveals that (p)ppGpp binds to a PurR pocket reminiscent of the active site of phosphoribosyltransferase enzymes that has been repurposed to serve a purely regulatory role, where the effectors (p)ppGpp and PRPP compete to allosterically control transcription. PRPP inhibits PurR DNA binding to induce transcription of purine synthesis genes, whereas (p)ppGpp antagonizes PRPP to enhance PurR DNA binding and repress transcription. A (p)ppGpp-refractory purR mutant in B. subtilis fails to downregulate purine synthesis genes upon amino acid starvation. Our work establishes the precedent of (p)ppGpp as an effector of a classical transcription repressor and reveals the key function of (p)ppGpp in regulating nucleotide synthesis through gene regulation, from soil bacteria to pathogens., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2022
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12. Evolution of a σ-(c-di-GMP)-anti-σ switch.
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Schumacher MA, Gallagher KA, Holmes NA, Chandra G, Henderson M, Kysela DT, Brennan RG, and Buttner MJ
- Subjects
- Actinobacteria genetics, Crystallography, X-Ray, Cyclic GMP analogs & derivatives, Fimbriae, Bacterial, Gene Expression Regulation, Bacterial, Models, Molecular, Protein Binding, Protein Conformation, Protein Domains, Sigma Factor genetics, Streptomyces genetics, Actinobacteria metabolism, Sigma Factor metabolism, Streptomyces metabolism
- Abstract
Filamentous actinobacteria of the genus Streptomyces have a complex lifecycle involving the differentiation of reproductive aerial hyphae into spores. We recently showed c-di-GMP controls this transition by arming a unique anti-σ, RsiG, to bind the sporulation-specific σ, WhiG. The Streptomyces venezuelae RsiG-(c-di-GMP)
2 -WhiG structure revealed that a monomeric RsiG binds c-di-GMP via two E(X)3 S(X)2 R(X)3 Q(X)3 D repeat motifs, one on each helix of an antiparallel coiled-coil. Here we show that RsiG homologs are found scattered throughout the Actinobacteria. Strikingly, RsiGs from unicellular bacteria descending from the most basal branch of the Actinobacteria are small proteins containing only one c-di-GMP binding motif, yet still bind their WhiG partners. Our structure of a Rubrobacter radiotolerans (RsiG)2 -(c-di-GMP)2 -WhiG complex revealed that these single-motif RsiGs are able to form an antiparallel coiled-coil through homodimerization, thereby allowing them to bind c-di-GMP similar to the monomeric twin-motif RsiGs. Further data show that in the unicellular actinobacterium R. radiotolerans , the (RsiG)2 -(c-di-GMP)2 -WhiG regulatory switch controls type IV pilus expression. Phylogenetic analysis indicates the single-motif RsiGs likely represent the ancestral state and an internal gene-duplication event gave rise to the twin-motif RsiGs inherited elsewhere in the Actinobacteria. Thus, these studies show how the anti-σ RsiG has evolved through an intragenic duplication event from a small protein carrying a single c-di-GMP binding motif, which functions as a homodimer, to a larger protein carrying two c-di-GMP binding motifs, which functions as a monomer. Consistent with this, our structures reveal potential selective advantages of the monomeric twin-motif anti-σ factors., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)- Published
- 2021
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13. Structures of Neisseria gonorrhoeae MtrR-operator complexes reveal molecular mechanisms of DNA recognition and antibiotic resistance-conferring clinical mutations.
- Author
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Beggs GA, Ayala JC, Kavanaugh LG, Read TD, Hooks GM, Schumacher MA, Shafer WM, and Brennan RG
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- Binding Sites, Gene Expression Regulation, Bacterial, Mutation, Protein Binding, Bacterial Proteins genetics, Bacterial Proteins metabolism, DNA, Bacterial metabolism, Drug Resistance, Multiple, Bacterial genetics, Neisseria gonorrhoeae genetics, Neisseria gonorrhoeae metabolism, Repressor Proteins genetics, Repressor Proteins metabolism
- Abstract
Mutations within the mtrR gene are commonly found amongst multidrug resistant clinical isolates of Neisseria gonorrhoeae, which has been labelled a superbug by the Centers for Disease Control and Prevention. These mutations appear to contribute to antibiotic resistance by interfering with the ability of MtrR to bind to and repress expression of its target genes, which include the mtrCDE multidrug efflux transporter genes and the rpoH oxidative stress response sigma factor gene. However, the DNA-recognition mechanism of MtrR and the consensus sequence within these operators to which MtrR binds has remained unknown. In this work, we report the crystal structures of MtrR bound to the mtrCDE and rpoH operators, which reveal a conserved, but degenerate, DNA consensus binding site 5'-MCRTRCRN4YGYAYGK-3'. We complement our structural data with a comprehensive mutational analysis of key MtrR-DNA contacts to reveal their importance for MtrR-DNA binding both in vitro and in vivo. Furthermore, we model and generate common clinical mutations of MtrR to provide plausible biochemical explanations for the contribution of these mutations to multidrug resistance in N. gonorrhoeae. Collectively, our findings unveil key biological mechanisms underlying the global stress responses of N. gonorrhoeae., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2021
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14. Structural Basis for Virulence Activation of Francisella tularensis.
- Author
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Travis BA, Ramsey KM, Prezioso SM, Tallo T, Wandzilak JM, Hsu A, Borgnia M, Bartesaghi A, Dove SL, Brennan RG, and Schumacher MA
- Subjects
- Escherichia coli genetics, Escherichia coli metabolism, DNA-Directed RNA Polymerases genetics, DNA-Directed RNA Polymerases metabolism, Francisella tularensis genetics, Francisella tularensis metabolism, Francisella tularensis pathogenicity, Promoter Regions, Genetic, Sigma Factor genetics, Sigma Factor metabolism, Virulence Factors genetics, Virulence Factors metabolism
- Abstract
The bacterium Francisella tularensis (Ft) is one of the most infectious agents known. Ft virulence is controlled by a unique combination of transcription regulators: the MglA-SspA heterodimer, PigR, and the stress signal, ppGpp. MglA-SspA assembles with the σ
70 -associated RNAP holoenzyme (RNAPσ70 ), forming a virulence-specialized polymerase. These factors activate Francisella pathogenicity island (FPI) gene expression, which is required for virulence, but the mechanism is unknown. Here we report FtRNAPσ70 -promoter-DNA, FtRNAPσ70 -(MglA-SspA)-promoter DNA, and FtRNAPσ70 -(MglA-SspA)-ppGpp-PigR-promoter DNA cryo-EM structures. Structural and genetic analyses show MglA-SspA facilitates σ70 binding to DNA to regulate virulence and virulence-enhancing genes. Our Escherichia coli RNAPσ70- homodimeric EcSspA structure suggests this is a general SspA-transcription regulation mechanism. Strikingly, our FtRNAPσ70 -(MglA-SspA)-ppGpp-PigR-DNA structure reveals ppGpp binding to MglA-SspA tethers PigR to promoters. PigR in turn recruits FtRNAP αCTDs to DNA UP elements. Thus, these studies unveil a unique mechanism for Ft pathogenesis involving a virulence-specialized RNAP that employs two (MglA-SspA)-based strategies to activate virulence genes., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)- Published
- 2021
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15. Optimization of a Noncanonical Anti-infective: Interrogation of the Target Binding Pocket for a Small-Molecule Inhibitor of Escherichia coli Polysaccharide Capsule Expression.
- Author
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Arshad M, Beggs GA, Brennan RG, and Seed PC
- Subjects
- Anti-Bacterial Agents pharmacology, Binding Sites, Drug Resistance, Microbial, Ligands, Polysaccharides, Protein Binding, Escherichia coli genetics, Escherichia coli metabolism, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism
- Abstract
We previously identified a small-molecule inhibitor of capsule biogenesis (designated DU011) and identified its target as MprA, a MarR family transcriptional repressor of multidrug efflux pumps. Unlike other proposed MprA ligands, such as salicylate and 2,4-dinitrophenol (DNP), DU011 does not alter Escherichia coli antibiotic resistance and has significantly enhanced inhibition of capsule expression. We hypothesized that the potency and the unique action of DU011 are due to novel interactions with the MprA binding pocket and the conformation assumed by MprA upon binding DU011 relative to other ligands. To understand the dynamics of MprA-DU011 interaction, we performed hydrogen-deuterium exchange mass spectrometry (HDX-MS); this suggested that four peptide regions undergo conformational changes upon binding DU011. We conducted isothermal calorimetric titration (ITC) to quantitatively characterize MprA binding to DU011 and canonical ligands and observed a distinct two-site binding isotherm associated with the binding reaction of MprA to DU011; however, salicylate and DNP showed a one-site binding isotherm with lower affinity. To elucidate the binding pocket(s) of MprA, we selected single point mutants of MprA that included mutated residues predicted to be within the putative binding pocket (Q51A, F58A, and E65D) as well as on or near the DNA-binding domain (L81A, S83T, and T86A). Our ITC studies suggest that two of the tested MprA mutants had lower affinity for DU011: Q51A and F58A. In addition to elucidating the MprA binding pocket for DU011, we studied the binding of these mutants to salicylate and DNP to reveal the binding pockets of these canonical ligands., (Copyright © 2020 American Society for Microbiology.)
- Published
- 2020
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16. When is a transcription factor a NAP?
- Author
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Dorman CJ, Schumacher MA, Bush MJ, Brennan RG, and Buttner MJ
- Subjects
- Binding Sites, Biological Evolution, Gene Expression Regulation, Bacterial, Genome, Bacterial, Protein Conformation, Bacterial Proteins physiology, DNA-Binding Proteins physiology, Streptomyces physiology, Transcription Factors physiology
- Abstract
Proteins that regulate transcription often also play an architectural role in the genome. Thus, it has been difficult to define with precision the distinctions between transcription factors and nucleoid-associated proteins (NAPs). Anachronistic descriptions of NAPs as 'histone-like' implied an organizational function in a bacterial chromatin-like complex. Definitions based on protein abundance, regulatory mechanisms, target gene number, or the features of their DNA-binding sites are insufficient as marks of distinction, and trying to distinguish transcription factors and NAPs based on their ranking within regulatory hierarchies or positions in gene-control networks is also unsatisfactory. The terms 'transcription factor' and 'NAP' are ad hoc operational definitions with each protein lying along a spectrum of structural and functional features extending from highly specific actors with few gene targets to those with a pervasive influence on the transcriptome. The Streptomyces BldC protein is used to illustrate these issues., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2020
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17. Crystal structure of an Escherichia coli Hfq Core (residues 2-69)-DNA complex reveals multifunctional nucleic acid binding sites.
- Author
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Orans J, Kovach AR, Hoff KE, Horstmann NM, and Brennan RG
- Subjects
- Binding Sites, Crystallography, X-Ray, DNA metabolism, Escherichia coli Proteins metabolism, Host Factor 1 Protein metabolism, Models, Molecular, Protein Binding, RNA, Double-Stranded chemistry, RNA, Double-Stranded metabolism, RNA, Messenger chemistry, DNA chemistry, Escherichia coli Proteins chemistry, Host Factor 1 Protein chemistry
- Abstract
Hfq regulates bacterial gene expression post-transcriptionally by binding small RNAs and their target mRNAs, facilitating sRNA-mRNA annealing, typically resulting in translation inhibition and RNA turnover. Hfq is also found in the nucleoid and binds double-stranded (ds) DNA with a slight preference for A-tracts. Here, we present the crystal structure of the Escherichia coli Hfq Core bound to a 30 bp DNA, containing three 6 bp A-tracts. Although previously postulated to bind to the 'distal' face, three statistically disordered double stranded DNA molecules bind across the proximal face of the Hfq hexamer as parallel, straight rods with B-DNA like conformational properties. One DNA duplex spans the diameter of the hexamer and passes over the uridine-binding proximal-face pore, whereas the remaining DNA duplexes interact with the rims and serve as bridges between adjacent hexamers. Binding is sequence-independent with residues N13, R16, R17 and Q41 interacting exclusively with the DNA backbone. Atomic force microscopy data support the sequence-independent nature of the Hfq-DNA interaction and a role for Hfq in DNA compaction and nucleoid architecture. Our structure and nucleic acid-binding studies also provide insight into the mechanism of sequence-independent binding of Hfq to dsRNA stems, a function that is critical for proper riboregulation., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2020
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18. MarR family proteins are important regulators of clinically relevant antibiotic resistance.
- Author
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Beggs GA, Brennan RG, and Arshad M
- Subjects
- Anti-Bacterial Agents chemistry, Escherichia coli Proteins metabolism, Microbial Sensitivity Tests, Models, Molecular, Repressor Proteins metabolism, Anti-Bacterial Agents pharmacology, Drug Resistance, Multiple, Bacterial drug effects, Escherichia coli drug effects, Escherichia coli Proteins antagonists & inhibitors, Repressor Proteins antagonists & inhibitors
- Abstract
There has been a rapid spread of multidrug-resistant (MDR) bacteria across the world. MDR efflux transporters are an important mechanism of antibiotic resistance in many pathogens among both Gram positive and Gram negative bacteria. These pumps can recognize a variety of chemically and structurally different compounds, including innate and clinically administered antibiotics. Intriguingly, these efflux pumps are often regulated by transcription factors that themselves bind a diverse set of substrates thereby allowing them to regulate the expression of their cognate MDR efflux pumps. One significant family of such transcription factors is the Multiple antibiotic resistance Repressor (MarR) family. Members of this family are well conserved across different bacterial species and in some cases are known to regulate vital bacterial functions. This review focusses on the role of MarR family transcriptional factors in antibiotic resistance within a select group of clinically relevant pathogens., (© 2019 The Protein Society.)
- Published
- 2020
- Full Text
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19. c-di-GMP Arms an Anti-σ to Control Progression of Multicellular Differentiation in Streptomyces.
- Author
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Gallagher KA, Schumacher MA, Bush MJ, Bibb MJ, Chandra G, Holmes NA, Zeng W, Henderson M, Zhang H, Findlay KC, Brennan RG, and Buttner MJ
- Subjects
- Amino Acid Sequence, Bacterial Proteins genetics, Cyclic GMP metabolism, Cyclic GMP physiology, DNA-Binding Proteins metabolism, Gene Expression Regulation, Bacterial genetics, Protein Domains, RNA, Bacterial metabolism, Spores, Bacterial metabolism, Streptomyces genetics, Cyclic GMP analogs & derivatives, Sigma Factor metabolism, Streptomyces metabolism
- Abstract
Streptomyces are our primary source of antibiotics, produced concomitantly with the transition from vegetative growth to sporulation in a complex developmental life cycle. We previously showed that the signaling molecule c-di-GMP binds BldD, a master repressor, to control initiation of development. Here we demonstrate that c-di-GMP also intervenes later in development to control differentiation of the reproductive hyphae into spores by arming a novel anti-σ (RsiG) to bind and sequester a sporulation-specific σ factor (σ
WhiG ). We present the structure of the RsiG-(c-di-GMP)2 -σWhiG complex, revealing an unusual, partially intercalated c-di-GMP dimer bound at the RsiG-σWhiG interface. RsiG binds c-di-GMP in the absence of σWhiG , employing a novel E(X)3 S(X)2 R(X)3 Q(X)3 D motif repeated on each helix of a coiled coil. Further studies demonstrate that c-di-GMP is essential for RsiG to inhibit σWhiG . These findings reveal a newly described control mechanism for σ-anti-σ complex formation and establish c-di-GMP as the central integrator of Streptomyces development., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2020
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20. The Salmonella Secreted Effector SarA/SteE Mimics Cytokine Receptor Signaling to Activate STAT3.
- Author
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Gibbs KD, Washington EJ, Jaslow SL, Bourgeois JS, Foster MW, Guo R, Brennan RG, and Ko DC
- Subjects
- Cell Line, Cytokine Receptor gp130 metabolism, Cytokines metabolism, Humans, Immunity, Innate, Receptors, Cytokine metabolism, STAT3 Transcription Factor immunology, Salmonella, Signal Transduction, Bacterial Proteins metabolism, Glycogen Synthase Kinase 3 metabolism, Molecular Mimicry immunology, STAT3 Transcription Factor metabolism, Trans-Activators metabolism
- Abstract
Bacteria masterfully co-opt and subvert host signal transduction. As a paradigmatic example, Salmonella uses two type-3 secretion systems to inject effector proteins that facilitate Salmonella entry, establishment of an intracellular niche, and modulation of immune responses. We previously demonstrated that the Salmonella anti-inflammatory response activator SarA (Stm2585, GogC, PagJ, SteE) activates the host transcription factor STAT3 to drive expression of immunomodulatory STAT3-targets. Here, we demonstrate-by sequence, function, and biochemical measurement-that SarA mimics the cytoplasmic domain of glycoprotein 130 (gp130, IL6ST). SarA is phosphorylated at a YxxQ motif, facilitating binding to STAT3 with greater affinity than gp130. Departing from canonical gp130 signaling, SarA function is JAK-independent but requires GSK-3, a key regulator of metabolism and development. Our results reveal that SarA undergoes host phosphorylation to recruit a STAT3-activating complex, circumventing cytokine receptor activation. Effector mimicry of gp130 suggests GSK-3 can regulate normal cytokine signaling, potentially enabling metabolic and immune crosstalk., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2020
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21. Structural, Biochemical, and In Vivo Characterization of MtrR-Mediated Resistance to Innate Antimicrobials by the Human Pathogen Neisseria gonorrhoeae .
- Author
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Beggs GA, Zalucki YM, Brown NG, Rastegari S, Phillips RK, Palzkill T, Shafer WM, Kumaraswami M, and Brennan RG
- Subjects
- Binding Sites, Chenodeoxycholic Acid metabolism, Crystallography, X-Ray, Humans, Models, Molecular, Neisseria gonorrhoeae chemistry, Neisseria gonorrhoeae metabolism, Protein Binding, Taurodeoxycholic Acid metabolism, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Drug Resistance, Multiple, Bacterial, Neisseria gonorrhoeae pathogenicity, Repressor Proteins chemistry, Repressor Proteins metabolism
- Abstract
Neisseria gonorrhoeae responds to host-derived antimicrobials by inducing the expression of the mtrCDE -encoded multidrug efflux pump, which expels microbicides, such as bile salts, fatty acids, and multiple extrinsically administered drugs, from the cell. In the absence of these cytotoxins, the TetR family member MtrR represses the mtrCDE genes. Although antimicrobial-dependent derepression of mtrCDE is clear, the physiological inducers of MtrR are unknown. Here, we report the crystal structure of an induced form of MtrR. In the binding pocket of MtrR, we observed electron density that we hypothesized was N -cyclohexyl-3-aminopropanesulfonic acid (CAPS), a component of the crystallization reagent. Using the MtrR-CAPS structure as an inducer-bound template, we hypothesized that bile salts, which bear significant chemical resemblance to CAPS, are physiologically relevant inducers. Indeed, characterization of MtrR-chenodeoxycholate and MtrR-taurodeoxycholate interactions, both in vitro and in vivo , revealed that these bile salts, but not glyocholate or taurocholate, bind MtrR tightly and can act as bona fide inducers. Furthermore, two residues, W136 and R176, were shown to be important in binding chenodeoxycholate but not taurodeoxycholate, suggesting different binding modes of the bile salts. These data provide insight into a crucial mechanism utilized by the pathogen to overcome innate human defenses. IMPORTANCE Neisseria gonorrhoeae causes a significant disease burden worldwide, and a meteoric rise in its multidrug resistance has reduced the efficacy of antibiotics previously or currently approved for therapy of gonorrheal infections. The multidrug efflux pump MtrCDE transports multiple drugs and host-derived antimicrobials from the bacterial cell and confers survival advantage on the pathogen within the host. Transcription of the pump is repressed by MtrR but relieved by the cytosolic influx of antimicrobials. Here, we describe the structure of induced MtrR and use this structure to identify bile salts as physiological inducers of MtrR. These findings provide a mechanistic basis for antimicrobial sensing and gonococcal protection by MtrR through the derepression of mtrCDE expression after exposure to intrinsic and clinically applied antimicrobials., (Copyright © 2019 Beggs et al.)
- Published
- 2019
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22. Control of meristem determinacy by trehalose 6-phosphate phosphatases is uncoupled from enzymatic activity.
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Claeys H, Vi SL, Xu X, Satoh-Nagasawa N, Eveland AL, Goldshmidt A, Feil R, Beggs GA, Sakai H, Brennan RG, Lunn JE, and Jackson D
- Subjects
- Flowers growth & development, Gene Expression Regulation, Developmental, Gene Expression Regulation, Plant, Meristem enzymology, Meristem growth & development, Phosphoric Monoester Hydrolases metabolism, Plant Proteins metabolism, Zea mays metabolism, Meristem metabolism, Phosphoric Monoester Hydrolases physiology, Plant Proteins physiology, Zea mays growth & development
- Abstract
Meristem fate is regulated by trehalose 6-phosphate phosphatases (TPPs), but their mechanism of action remains mysterious. Loss of the maize TPPs RAMOSA3 and TPP4 leads to reduced meristem determinacy and more inflorescence branching. However, analysis of an allelic series revealed no correlation between enzymatic activity and branching, and a catalytically inactive version of RA3 complements the ra3 mutant. Together with their nuclear localization, these findings suggest a moonlighting function for TPPs.
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- 2019
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23. The MerR-like protein BldC binds DNA direct repeats as cooperative multimers to regulate Streptomyces development.
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Schumacher MA, den Hengst CD, Bush MJ, Le TBK, Tran NT, Chandra G, Zeng W, Travis B, Brennan RG, and Buttner MJ
- Subjects
- Bacterial Proteins chemistry, Bacterial Proteins genetics, Base Sequence, Binding Sites genetics, DNA, Bacterial chemistry, DNA, Bacterial genetics, DNA, Bacterial metabolism, DNA-Binding Proteins chemistry, DNA-Binding Proteins genetics, Gene Expression Regulation, Bacterial, Gene Expression Regulation, Developmental, Genes, Bacterial, Models, Molecular, Nucleic Acid Conformation, Promoter Regions, Genetic, Protein Structure, Quaternary, Regulon, Repetitive Sequences, Nucleic Acid, Repressor Proteins genetics, Static Electricity, Streptomyces coelicolor genetics, Streptomyces coelicolor growth & development, Bacterial Proteins metabolism, DNA-Binding Proteins metabolism, Streptomyces coelicolor metabolism
- Abstract
Streptomycetes are notable for their complex life cycle and production of most clinically important antibiotics. A key factor that controls entry into development and the onset of antibiotic production is the 68-residue protein, BldC. BldC is a putative DNA-binding protein related to MerR regulators, but lacks coiled-coil dimerization and effector-binding domains characteristic of classical MerR proteins. Hence, the molecular function of the protein has been unclear. Here we show that BldC is indeed a DNA-binding protein and controls a regulon that includes other key developmental regulators. Intriguingly, BldC DNA-binding sites vary significantly in length. Our BldC-DNA structures explain this DNA-binding capability by revealing that BldC utilizes a DNA-binding mode distinct from MerR and other known regulators, involving asymmetric head-to-tail oligomerization on DNA direct repeats that results in dramatic DNA distortion. Notably, BldC-like proteins radiate throughout eubacteria, establishing BldC as the founding member of a new structural family of regulators.
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- 2018
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24. Dissection of the molecular circuitry controlling virulence in Francisella tularensis .
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Cuthbert BJ, Ross W, Rohlfing AE, Dove SL, Gourse RL, Brennan RG, and Schumacher MA
- Subjects
- Adhesins, Bacterial chemistry, Adhesins, Bacterial genetics, Bioterrorism prevention & control, Cells, Cultured, Crystallography, DNA-Binding Proteins chemistry, DNA-Binding Proteins genetics, DNA-Directed RNA Polymerases metabolism, Gene Expression Regulation, Bacterial, Guanosine Tetraphosphate genetics, Humans, Macrophages metabolism, Protein Conformation, Transcription, Genetic, Virulence genetics, Adhesins, Bacterial metabolism, DNA-Binding Proteins metabolism, Francisella tularensis pathogenicity, Genomic Islands genetics, Guanosine Tetraphosphate metabolism, Tularemia microbiology
- Abstract
Francisella tularensis, the etiological agent of tularemia, is one of the most infectious bacteria known. Because of its extreme pathogenicity, F. tularensis is classified as a category A bioweapon by the US government. F. tularensis virulence stems from genes encoded on the Francisella pathogenicity island (FPI). An unusual set of Francisella regulators-the heteromeric macrophage growth locus protein A (MglA)-stringent starvation protein A (SspA) complex and the DNA-binding protein pathogenicity island gene regulator (PigR)-activates FPI transcription and thus is essential for virulence. Intriguingly, the second messenger, guanosine-tetraphosphate (ppGpp), which is produced during infection, is also involved in coordinating Francisella virulence; however, its role has been unclear. Here we identify MglA-SspA as a novel ppGpp-binding complex and describe structures of apo- and ppGpp-bound MglA-SspA. We demonstrate that MglA-SspA, which binds RNA polymerase (RNAP), also interacts with the C-terminal domain of PigR, thus anchoring the (MglA-SspA)-RNAP complex to the FPI promoter. Furthermore, we show that MglA-SspA must be bound to ppGpp to mediate high-affinity interactions with PigR. Thus, these studies unveil a novel pathway different from those described previously for regulation of transcription by ppGpp. The data also indicate that F. tularensis pathogenesis is controlled by a highly interconnected molecular circuitry in which the virulence machinery directly senses infection via a small molecule stress signal., (© 2017 Cuthbert et al.; Published by Cold Spring Harbor Laboratory Press.)
- Published
- 2017
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25. Structural and In Vivo Studies on Trehalose-6-Phosphate Synthase from Pathogenic Fungi Provide Insights into Its Catalytic Mechanism, Biological Necessity, and Potential for Novel Antifungal Drug Design.
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Miao Y, Tenor JL, Toffaletti DL, Maskarinec SA, Liu J, Lee RE, Perfect JR, and Brennan RG
- Subjects
- Antifungal Agents isolation & purification, Aspergillus fumigatus enzymology, Aspergillus fumigatus genetics, Aspergillus fumigatus pathogenicity, Biocatalysis, Biofilms growth & development, Candida albicans enzymology, Candida albicans genetics, Candida albicans pathogenicity, Catalytic Domain, Glucosyltransferases genetics, Humans, Hyphae physiology, Mutation, Protein Binding, Trehalose analogs & derivatives, Virulence, Antifungal Agents chemistry, Antifungal Agents metabolism, Drug Design, Glucosyltransferases chemistry, Glucosyltransferases metabolism
- Abstract
The disaccharide trehalose is critical to the survival of pathogenic fungi in their human host. Trehalose-6-phosphate synthase (Tps1) catalyzes the first step of trehalose biosynthesis in fungi. Here, we report the first structures of eukaryotic Tps1s in complex with substrates or substrate analogues. The overall structures of Tps1 from Candida albicans and Aspergillus fumigatus are essentially identical and reveal N- and C-terminal Rossmann fold domains that form the glucose-6-phosphate and UDP-glucose substrate binding sites, respectively. These Tps1 structures with substrates or substrate analogues reveal key residues involved in recognition and catalysis. Disruption of these key residues severely impaired Tps1 enzymatic activity. Subsequent cellular analyses also highlight the enzymatic function of Tps1 in thermotolerance, yeast-hypha transition, and biofilm development. These results suggest that Tps1 enzymatic functionality is essential for the fungal stress response and virulence. Furthermore, structures of Tps1 in complex with the nonhydrolyzable inhibitor, validoxylamine A, visualize the transition state and support an internal return-like catalytic mechanism that is generalizable to other GT-B-fold retaining glycosyltransferases. Collectively, our results depict key Tps1-substrate interactions, unveil the enzymatic mechanism of these fungal proteins, and pave the way for high-throughput inhibitor screening buttressed and guided by the current structures and those of high-affinity ligand-Tps1 complexes. IMPORTANCE Invasive fungal diseases have emerged as major threats, resulting in more than 1.5 million deaths annually worldwide. This epidemic has been further complicated by increasing resistance to all major classes of antifungal drugs in the clinic. Trehalose biosynthesis is essential for the fungal stress response and virulence. Critically, this biosynthetic pathway is absent in mammals, and thus, the two enzymes that carry out trehalose biosynthesis, namely, trehalose-6-phosphate synthase (Tps1) and trehalose-6-phosphate phosphatase (Tps2), are prominent targets for antifungal intervention. Here, we report the first eukaryotic Tps1 structures from the pathogenic fungi Candida albicans and Aspergillus fumigatus in complex with substrates, substrate analogues, and inhibitors. These structures reveal key protein-substrate interactions, providing atomic-level scaffolds for structure-guided drug design of novel antifungals that target Tps1., (Copyright © 2017 Miao et al.)
- Published
- 2017
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26. The Streptomyces master regulator BldD binds c-di-GMP sequentially to create a functional BldD2-(c-di-GMP)4 complex.
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Schumacher MA, Zeng W, Findlay KC, Buttner MJ, Brennan RG, and Tschowri N
- Subjects
- Binding Sites, Crystallography, X-Ray, Cyclic GMP chemistry, Hydrogen Bonding, Models, Molecular, Protein Binding, Protein Domains, Protein Stability, Protein Structure, Quaternary, Bacterial Proteins chemistry, Cyclic GMP analogs & derivatives, Repressor Proteins chemistry, Streptomyces
- Abstract
Streptomyces are ubiquitous soil bacteria that undergo a complex developmental transition coinciding with their production of antibiotics. This transition is controlled by binding of a novel tetrameric form of the second messenger, 3΄-5΄ cyclic diguanylic acid (c-di-GMP) to the master repressor, BldD. In all domains of life, nucleotide-based second messengers allow a rapid integration of external and internal signals into regulatory pathways that control cellular responses to changing conditions. c-di-GMP can assume alternative oligomeric states to effect different functions, binding to effector proteins as monomers, intercalated dimers or, uniquely in the case of BldD, as a tetramer. However, at physiological concentrations c-di-GMP is a monomer and little is known about how higher oligomeric complexes assemble on effector proteins and if intermediates in assembly pathways have regulatory significance. Here, we show that c-di-GMP binds BldD using an ordered, sequential mechanism and that BldD function necessitates the assembly of the BldD2-(c-di-GMP)4 complex., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2017
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27. Central Role of the Trehalose Biosynthesis Pathway in the Pathogenesis of Human Fungal Infections: Opportunities and Challenges for Therapeutic Development.
- Author
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Thammahong A, Puttikamonkul S, Perfect JR, Brennan RG, and Cramer RA
- Subjects
- Antifungal Agents adverse effects, Antifungal Agents therapeutic use, Aspergillus fumigatus genetics, Aspergillus fumigatus metabolism, Aspergillus fumigatus pathogenicity, Candida albicans drug effects, Candida albicans genetics, Candida albicans metabolism, Candida albicans pathogenicity, Cryptococcus neoformans genetics, Cryptococcus neoformans metabolism, Cryptococcus neoformans pathogenicity, Drug Discovery, Fungi genetics, Glucosyltransferases genetics, Host-Pathogen Interactions, Humans, Phosphoric Monoester Hydrolases genetics, Virulence, Virulence Factors genetics, Virulence Factors metabolism, Antifungal Agents pharmacology, Biosynthetic Pathways genetics, Fungi drug effects, Fungi metabolism, Invasive Fungal Infections microbiology, Invasive Fungal Infections therapy, Trehalose biosynthesis
- Abstract
Invasive fungal infections cause significant morbidity and mortality in part due to a limited antifungal drug arsenal. One therapeutic challenge faced by clinicians is the significant host toxicity associated with antifungal drugs. Another challenge is the fungistatic mechanism of action of some drugs. Consequently, the identification of fungus-specific drug targets essential for fitness in vivo remains a significant goal of medical mycology research. The trehalose biosynthetic pathway is found in a wide variety of organisms, including human-pathogenic fungi, but not in humans. Genes encoding proteins involved in trehalose biosynthesis are mechanistically linked to the metabolism, cell wall homeostasis, stress responses, and virulence of Candida albicans , Cryptococcus neoformans , and Aspergillus fumigatus . While there are a number of pathways for trehalose production across the tree of life, the TPS/TPP (trehalose-6-phosphate synthase/trehalose-6-phosphate phosphatase) pathway is the canonical pathway found in human-pathogenic fungi. Importantly, data suggest that proteins involved in trehalose biosynthesis play other critical roles in fungal metabolism and in vivo fitness that remain to be fully elucidated. By further defining the biology and functions of trehalose and its biosynthetic pathway components in pathogenic fungi, an opportunity exists to leverage this pathway as a potent antifungal drug target. The goal of this review is to cover the known roles of this important molecule and its associated biosynthesis-encoding genes in the human-pathogenic fungi studied to date and to employ these data to critically assess the opportunities and challenges facing development of this pathway as a therapeutic target., (Copyright © 2017 American Society for Microbiology.)
- Published
- 2017
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28. Trehalose pathway as an antifungal target.
- Author
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Perfect JR, Tenor JL, Miao Y, and Brennan RG
- Subjects
- Animals, Antifungal Agents chemistry, Antifungal Agents isolation & purification, Antifungal Agents therapeutic use, Enzyme Inhibitors chemistry, Fluconazole pharmacology, Fluconazole therapeutic use, Fungi enzymology, Glucosyltransferases antagonists & inhibitors, Glucosyltransferases genetics, Glucosyltransferases metabolism, Humans, Mycoses drug therapy, Antifungal Agents pharmacology, Drug Discovery, Enzyme Inhibitors pharmacology, Fungi drug effects, Metabolic Networks and Pathways drug effects, Trehalose metabolism
- Abstract
With an increasing immunocompromised population which is linked to invasive fungal infections, it is clear that our present 3 classes of antifungal agents may not be sufficient to provide optimal management to these fragile patients. Furthermore, with widespread use of antifungal agents, drug-resistant fungal infections are on the rise. Therefore, there is some urgency to develop the antifungal pipeline with the goal of new antifungal agent discovery. In this review, a simple metabolic pathway, which forms the disaccharide, trehalose, will be characterized and its potential as a focus for antifungal target(s) explained. It possesses several important features for development of antifungal agents. First, it appears to have fungicidal characteristics and second, it is broad spectrum with importance across both ascomycete and basidiomycete species. Finally, this pathway is not found in mammals so theoretically specific inhibitors of the trehalose pathway and its enzymes in fungi should be relatively non-toxic for mammals. The trehalose pathway and its critical enzymes are now in a position to have directed antifungal discovery initiated in order to find a new class of antifungal drugs.
- Published
- 2017
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29. Structures of trehalose-6-phosphate phosphatase from pathogenic fungi reveal the mechanisms of substrate recognition and catalysis.
- Author
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Miao Y, Tenor JL, Toffaletti DL, Washington EJ, Liu J, Shadrick WR, Schumacher MA, Lee RE, Perfect JR, and Brennan RG
- Subjects
- Biocatalysis, Candida albicans chemistry, Candida albicans genetics, Candida albicans metabolism, Cryptococcus neoformans chemistry, Cryptococcus neoformans genetics, Fungal Proteins genetics, Fungal Proteins metabolism, Gene Expression Regulation, Fungal, Phosphoric Monoester Hydrolases genetics, Phosphoric Monoester Hydrolases metabolism, Substrate Specificity, Sugar Phosphates chemistry, Sugar Phosphates metabolism, Trehalose analogs & derivatives, Trehalose chemistry, Trehalose metabolism, Candida albicans enzymology, Cryptococcus neoformans enzymology, Fungal Proteins chemistry, Phosphoric Monoester Hydrolases chemistry
- Abstract
Trehalose is a disaccharide essential for the survival and virulence of pathogenic fungi. The biosynthesis of trehalose requires trehalose-6-phosphate synthase, Tps1, and trehalose-6-phosphate phosphatase, Tps2. Here, we report the structures of the N-terminal domain of Tps2 (Tps2NTD) from Candida albicans, a transition-state complex of the Tps2 C-terminal trehalose-6-phosphate phosphatase domain (Tps2PD) bound to BeF3 and trehalose, and catalytically dead Tps2PD(D24N) from Cryptococcus neoformans bound to trehalose-6-phosphate (T6P). The Tps2NTD closely resembles the structure of Tps1 but lacks any catalytic activity. The Tps2PD-BeF3-trehalose and Tps2PD(D24N)-T6P complex structures reveal a "closed" conformation that is effected by extensive interactions between each trehalose hydroxyl group and residues of the cap and core domains of the protein, thereby providing exquisite substrate specificity. Disruption of any of the direct substrate-protein residue interactions leads to significant or complete loss of phosphatase activity. Notably, the Tps2PD-BeF3-trehalose complex structure captures an aspartyl-BeF3 covalent adduct, which closely mimics the proposed aspartyl-phosphate intermediate of the phosphatase catalytic cycle. Structures of substrate-free Tps2PD reveal an "open" conformation whereby the cap and core domains separate and visualize the striking conformational changes effected by substrate binding and product release and the role of two hinge regions centered at approximately residues 102-103 and 184-188. Significantly, tps2Δ, tps2NTDΔ, and tps2D705N strains are unable to grow at elevated temperatures. Combined, these studies provide a deeper understanding of the substrate recognition and catalytic mechanism of Tps2 and provide a structural basis for the future design of novel antifungal compounds against a target found in three major fungal pathogens.
- Published
- 2016
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30. HipBA-promoter structures reveal the basis of heritable multidrug tolerance.
- Author
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Schumacher MA, Balani P, Min J, Chinnam NB, Hansen S, Vulić M, Lewis K, and Brennan RG
- Subjects
- Catalytic Domain, Crystallography, X-Ray, DNA-Binding Proteins genetics, Down-Regulation genetics, Drug Resistance, Multiple, Bacterial drug effects, Drug Tolerance genetics, Escherichia coli genetics, Escherichia coli pathogenicity, Escherichia coli Proteins chemistry, Escherichia coli Proteins genetics, Gene Expression Regulation, Bacterial genetics, Humans, Models, Molecular, Mutation genetics, Operon genetics, Phenotype, Protein Multimerization, Protein Structure, Tertiary genetics, Transcription, Genetic genetics, Urinary Bladder microbiology, Urinary Bladder pathology, Urinary Tract Infections drug therapy, Urinary Tract Infections microbiology, Anti-Bacterial Agents pharmacology, DNA-Binding Proteins metabolism, Drug Resistance, Multiple, Bacterial genetics, Escherichia coli drug effects, Escherichia coli metabolism, Escherichia coli Proteins metabolism, Promoter Regions, Genetic genetics
- Abstract
Multidrug tolerance is largely responsible for chronic infections and caused by a small population of dormant cells called persisters. Selection for survival in the presence of antibiotics produced the first genetic link to multidrug tolerance: a mutant in the Escherichia coli hipA locus. HipA encodes a serine-protein kinase, the multidrug tolerance activity of which is neutralized by binding to the transcriptional regulator HipB and hipBA promoter. The physiological role of HipA in multidrug tolerance, however, has been unclear. Here we show that wild-type HipA contributes to persister formation and that high-persister hipA mutants cause multidrug tolerance in urinary tract infections. Perplexingly, high-persister mutations map to the N-subdomain-1 of HipA far from its active site. Structures of higher-order HipA-HipB-promoter complexes reveal HipA forms dimers in these assemblies via N-subdomain-1 interactions that occlude their active sites. High-persistence mutations, therefore, diminish HipA-HipA dimerization, thereby unleashing HipA to effect multidrug tolerance. Thus, our studies reveal the mechanistic basis of heritable, clinically relevant antibiotic tolerance.
- Published
- 2015
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31. Structural and Biochemical Characterization of the Francisella tularensis Pathogenicity Regulator, Macrophage Locus Protein A (MglA).
- Author
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Cuthbert BJ, Brennan RG, and Schumacher MA
- Subjects
- Amino Acid Sequence, Binding Sites, Chromatography, Gel, Cross-Linking Reagents metabolism, DNA-Directed RNA Polymerases metabolism, Ligands, Malates metabolism, Models, Molecular, Molecular Sequence Data, Protein Binding, Protein Multimerization, Structural Homology, Protein, Tularemia microbiology, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Francisella tularensis pathogenicity, Macrophages microbiology
- Abstract
Francisella tularensis is one of the most infectious bacteria known and is the etiologic agent of tularemia. Francisella virulence arises from a 33 kilobase (Kb) pathogenicity island (FPI) that is regulated by the macrophage locus protein A (MglA) and the stringent starvation protein A (SspA). These proteins interact with both RNA polymerase (RNAP) and the pathogenicity island gene regulator (PigR) to activate FPI transcription. However, the molecular mechanisms involved are not well understood. Indeed, while most bacterial SspA proteins function as homodimers to activate transcription, F. tularensis SspA forms a heterodimer with the MglA protein, which is unique to F. tularensis. To gain insight into MglA function, we performed structural and biochemical studies. The MglA structure revealed that it contains a fold similar to the SspA protein family. Unexpectedly, MglA also formed a homodimer in the crystal. Chemical crosslinking and size exclusion chromatography (SEC) studies showed that MglA is able to self-associate in solution to form a dimer but that it preferentially heterodimerizes with SspA. Finally, the MglA structure revealed malate, which was used in crystallization, bound in an open pocket formed by the dimer, suggesting the possibility that this cleft could function in small molecule ligand binding. The location of this binding region relative to recently mapped PigR and RNAP interacting sites suggest possible roles for small molecule binding in MglA and SspA•MglA function.
- Published
- 2015
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32. Mutations within the mepA operator affect binding of the MepR regulatory protein and its induction by MepA substrates in Staphylococcus aureus.
- Author
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Schindler BD, Seo SM, Birukou I, Brennan RG, and Kaatz GW
- Subjects
- Computational Biology, DNA, Bacterial, DNA, Intergenic, Endopeptidases genetics, Inverted Repeat Sequences, Models, Molecular, Mutation, Nucleic Acid Conformation, Protein Binding, Protein Conformation, Staphylococcus aureus genetics, Endopeptidases metabolism, Gene Expression Regulation, Bacterial physiology, Gene Expression Regulation, Enzymologic physiology, Staphylococcus aureus metabolism
- Abstract
The expression of mepA, encoding the Staphylococcus aureus MepA multidrug efflux protein, is repressed by the MarR homologue MepR. Repression occurs through binding of two MepR dimers to an operator with two homologous and closely approximated pseudopalindromic binding sites (site 1 [S1] and site 2 [S2]). MepR binding is impeded in the presence of pentamidine, a MepA substrate. The effects of various mepA operator mutations on MepR binding were determined using electrophoretic mobility shift assays and isothermal titration calorimetry, and an in vivo confirmation of the effects observed was established for a fully palindromic operator mutant. Altering the S1-S2 spacing by 1 to 4 bp severely impaired S2 binding, likely due to a physical collision between adjacent MepR dimers. Extension of the spacing to 9 bp eliminated the S1 binding-mediated DNA allostery required for efficient S2 binding, consistent with positive cooperative binding of MepR dimers. Binding of a single dimer to S1 was maintained when S2 was disrupted, whereas disruption of S1 eliminated any significant binding to S2, also consistent with positive cooperativity. Palindromization of binding sites, especially S2, enhanced MepR affinity for the mepA operator and reduced MepA substrate-mediated MepR induction. As a result, the on-off equilibrium between MepR and its binding sites was shifted toward the on state, resulting in less free MepR being available for interaction with inducing ligand. The selective pressure(s) under which mepA expression is advantageous likely contributed to the accumulation of mutations in the mepA operator, resulting in the current sequence from which MepR is readily induced by MepA substrates., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)
- Published
- 2015
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33. Glutathione activates virulence gene expression of an intracellular pathogen.
- Author
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Reniere ML, Whiteley AT, Hamilton KL, John SM, Lauer P, Brennan RG, and Portnoy DA
- Subjects
- Allosteric Regulation drug effects, Bacterial Proteins metabolism, DNA metabolism, Gene Expression Regulation, Bacterial drug effects, Glutathione pharmacology, Intracellular Space drug effects, Listeria monocytogenes drug effects, Macrophages metabolism, Mutation genetics, Peptide Termination Factors metabolism, Protein Binding, Selection, Genetic genetics, Suppression, Genetic genetics, Virulence genetics, Gene Expression Regulation, Bacterial genetics, Glutathione metabolism, Intracellular Space metabolism, Intracellular Space microbiology, Listeria monocytogenes genetics, Listeria monocytogenes pathogenicity
- Abstract
Intracellular pathogens are responsible for much of the world-wide morbidity and mortality due to infectious diseases. To colonize their hosts successfully, pathogens must sense their environment and regulate virulence gene expression appropriately. Accordingly, on entry into mammalian cells, the facultative intracellular bacterial pathogen Listeria monocytogenes remodels its transcriptional program by activating the master virulence regulator PrfA. Here we show that bacterial and host-derived glutathione are required to activate PrfA. In this study a genetic selection led to the identification of a bacterial mutant in glutathione synthase that exhibited reduced virulence gene expression and was attenuated 150-fold in mice. Genome sequencing of suppressor mutants that arose spontaneously in vivo revealed a single nucleotide change in prfA that locks the protein in the active conformation (PrfA*) and completely bypassed the requirement for glutathione during infection. Biochemical and genetic studies support a model in which glutathione-dependent PrfA activation is mediated by allosteric binding of glutathione to PrfA. Whereas glutathione and other low-molecular-weight thiols have important roles in redox homeostasis in all forms of life, here we demonstrate that glutathione represents a critical signalling molecule that activates the virulence of an intracellular pathogen.
- Published
- 2015
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34. Recognition of U-rich RNA by Hfq from the Gram-positive pathogen Listeria monocytogenes.
- Author
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Kovach AR, Hoff KE, Canty JT, Orans J, and Brennan RG
- Subjects
- Amino Acid Motifs, Crystallography, X-Ray, Fluorescence Polarization, Host Factor 1 Protein chemistry, Listeria monocytogenes genetics, Mutation genetics, Protein Binding, Protein Conformation, RNA, Messenger genetics, RNA, Small Nuclear chemistry, RNA, Small Nuclear genetics, Tryptophan chemistry, Tryptophan genetics, Tryptophan metabolism, Gene Expression Regulation, Bacterial, Host Factor 1 Protein metabolism, Listeria monocytogenes metabolism, RNA, Messenger metabolism, RNA, Small Nuclear metabolism
- Abstract
Hfq is a post-transcriptional regulator that binds U- and A-rich regions of sRNAs and their target mRNAs to stimulate their annealing in order to effect translation regulation and, often, to alter their stability. The functional importance of Hfq and its RNA-binding properties are relatively well understood in Gram-negative bacteria, whereas less is known about the RNA-binding properties of this riboregulator in Gram-positive species. Here, we describe the structure of Hfq from the Gram-positive pathogen Listeria monocytogenes in its RNA-free form and in complex with a U6 oligoribonucleotide. As expected, the protein takes the canonical hexameric toroidal shape of all other known Hfq structures. The U6 RNA binds on the "proximal face" in a pocket formed by conserved residues Q9, N42, F43, and K58. Additionally residues G5 and Q6 are involved in protein-nucleic and inter-subunit contacts that promote uracil specificity. Unlike Staphylococcus aureus (Sa) Hfq, Lm Hfq requires magnesium to bind U6 with high affinity. In contrast, the longer oligo-uridine, U16, binds Lm Hfq tightly in the presence or absence of magnesium, thereby suggesting the importance of additional residues on the proximal face and possibly the lateral rim in RNA interaction. Intrinsic tryptophan fluorescence quenching (TFQ) studies reveal, surprisingly, that Lm Hfq can bind (GU)3G and U6 on its proximal and distal faces, indicating a less stringent adenine-nucleotide specificity site on the distal face as compared to the Gram-positive Hfq proteins from Sa and Bacillus subtilis and suggesting as yet uncharacterized RNA-binding modes on both faces., (© 2014 Kovach et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
- Published
- 2014
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35. Tetrameric c-di-GMP mediates effective transcription factor dimerization to control Streptomyces development.
- Author
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Tschowri N, Schumacher MA, Schlimpert S, Chinnam NB, Findlay KC, Brennan RG, and Buttner MJ
- Subjects
- Amino Acid Sequence, Bacterial Proteins chemistry, Crystallography, X-Ray, Cyclic GMP metabolism, Dimerization, Models, Molecular, Molecular Sequence Data, Sequence Alignment, Spores, Bacterial metabolism, Streptomyces cytology, Transcription Factors chemistry, Bacterial Proteins metabolism, Cyclic GMP analogs & derivatives, Streptomyces growth & development, Streptomyces metabolism, Transcription Factors metabolism
- Abstract
The cyclic dinucleotide c-di-GMP is a signaling molecule with diverse functions in cellular physiology. Here, we report that c-di-GMP can assemble into a tetramer that mediates the effective dimerization of a transcription factor, BldD, which controls the progression of multicellular differentiation in sporulating actinomycete bacteria. BldD represses expression of sporulation genes during vegetative growth in a manner that depends on c-di-GMP-mediated dimerization. Structural and biochemical analyses show that tetrameric c-di-GMP links two subunits of BldD through their C-terminal domains, which are otherwise separated by ~10 Å and thus cannot effect dimerization directly. Binding of the c-di-GMP tetramer by BldD is selective and requires a bipartite RXD-X8-RXXD signature. The findings indicate a unique mechanism of protein dimerization and the ability of nucleotide signaling molecules to assume alternative oligomeric states to effect different functions., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
36. Structural mechanism of transcription regulation of the Staphylococcus aureus multidrug efflux operon mepRA by the MarR family repressor MepR.
- Author
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Birukou I, Seo SM, Schindler BD, Kaatz GW, and Brennan RG
- Subjects
- Bacterial Proteins metabolism, DNA, Bacterial metabolism, Gene Expression Regulation, Bacterial, Models, Molecular, Nucleic Acid Conformation, Protein Binding, Protein Structure, Secondary, Repressor Proteins metabolism, Transcription, Genetic, Bacterial Proteins chemistry, DNA, Bacterial chemistry, Operator Regions, Genetic, Repressor Proteins chemistry, Staphylococcus aureus genetics
- Abstract
The multidrug efflux pump MepA is a major contributor to multidrug resistance in Staphylococcus aureus. MepR, a member of the multiple antibiotic resistance regulator (MarR) family, represses mepA and its own gene. Here, we report the structure of a MepR-mepR operator complex. Structural comparison of DNA-bound MepR with 'induced' apoMepR reveals the large conformational changes needed to allow the DNA-binding winged helix-turn-helix motifs to interact with the consecutive major and minor grooves of the GTTAG signature sequence. Intriguingly, MepR makes no hydrogen bonds to major groove nucleobases. Rather, recognition-helix residues Thr60, Gly61, Pro62 and Thr63 make sequence-specifying van der Waals contacts with the TTAG bases. Removing these contacts dramatically affects MepR-DNA binding activity. The wings insert into the flanking minor grooves, whereby residue Arg87, buttressed by Asp85, interacts with the O2 of T4 and O4' ribosyl oxygens of A23 and T4. Mutating Asp85 and Arg87, both conserved throughout the MarR family, markedly affects MepR repressor activity. The His14':Arg59 and Arg10':His35:Phe108 interaction networks stabilize the DNA-binding conformation of MepR thereby contributing significantly to its high affinity binding. A structure-guided model of the MepR-mepA operator complex suggests that MepR dimers do not interact directly and cooperative binding is likely achieved by DNA-mediated allosteric effects.
- Published
- 2014
- Full Text
- View/download PDF
37. Mapping Hfq-RNA interaction surfaces using tryptophan fluorescence quenching.
- Author
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Robinson KE, Orans J, Kovach AR, Link TM, and Brennan RG
- Subjects
- 5' Untranslated Regions, Amino Acid Motifs, Binding Sites, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Fluorescence, Host Factor 1 Protein genetics, Host Factor 1 Protein metabolism, Listeria monocytogenes, Models, Molecular, Mutation, Protein Binding, RNA chemistry, Staphylococcus aureus, Escherichia coli Proteins chemistry, Host Factor 1 Protein chemistry, RNA metabolism, Tryptophan chemistry
- Abstract
Hfq is a posttranscriptional riboregulator and RNA chaperone that binds small RNAs and target mRNAs to effect their annealing and message-specific regulation in response to environmental stressors. Structures of Hfq-RNA complexes indicate that U-rich sequences prefer the proximal face and A-rich sequences the distal face; however, the Hfq-binding sites of most RNAs are unknown. Here, we present an Hfq-RNA mapping approach that uses single tryptophan-substituted Hfq proteins, all of which retain the wild-type Hfq structure, and tryptophan fluorescence quenching (TFQ) by proximal RNA binding. TFQ properly identified the respective distal and proximal binding of A15 and U6 RNA to Gram-negative Escherichia coli (Ec) Hfq and the distal face binding of (AA)3A, (AU)3A and (AC)3A to Gram-positive Staphylococcus aureus (Sa) Hfq. The inability of (GU)3G to bind the distal face of Sa Hfq reveals the (R-L)n binding motif is a more restrictive (A-L)n binding motif. Remarkably Hfq from Gram-positive Listeria monocytogenes (Lm) binds (GU)3G on its proximal face. TFQ experiments also revealed the Ec Hfq (A-R-N)n distal face-binding motif should be redefined as an (A-A-N)n binding motif. TFQ data also demonstrated that the 5'-untranslated region of hfq mRNA binds both the proximal and distal faces of Ec Hfq and the unstructured C-terminus.
- Published
- 2014
- Full Text
- View/download PDF
38. The molecular mechanisms of allosteric mutations impairing MepR repressor function in multidrug-resistant strains of Staphylococcus aureus.
- Author
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Birukou I, Tonthat NK, Seo SM, Schindler BD, Kaatz GW, and Brennan RG
- Subjects
- Anti-Bacterial Agents pharmacology, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Binding Sites, Drug Resistance, Multiple, Bacterial, Gene Expression Regulation, Bacterial, Humans, Models, Molecular, Protein Binding, Repressor Proteins chemistry, Repressor Proteins metabolism, Staphylococcus aureus chemistry, Staphylococcus aureus drug effects, Staphylococcus aureus genetics, Bacterial Proteins genetics, Mutation, Missense, Repressor Proteins genetics, Staphylococcal Infections microbiology, Staphylococcus aureus metabolism
- Abstract
Unlabelled: Overexpression of the Staphylococcus aureus multidrug efflux pump MepA confers resistance to a wide variety of antimicrobials. mepA expression is controlled by MarR family member MepR, which represses mepA and autorepresses its own production. Mutations in mepR are a primary cause of mepA overexpression in clinical isolates of multidrug-resistant S. aureus. Here, we report crystal structures of three multidrug-resistant MepR variants, which contain the single-amino-acid substitution A103V, F27L, or Q18P, and wild-type MepR in its DNA-bound conformation. Although each mutation impairs MepR function by decreasing its DNA binding affinity, none is located in the DNA binding domain. Rather, all are found in the linker region connecting the dimerization and DNA binding domains. Specifically, the A103V substitution impinges on F27, which resolves potential steric clashes via displacement of the DNA binding winged-helix-turn-helix motifs that lead to a 27-fold reduction in DNA binding affinity. The F27L substitution forces F104 into an alternative rotamer, which kinks helix 5, thereby interfering with the positioning of the DNA binding domains and decreasing mepR operator affinity by 35-fold. The Q18P mutation affects the MepR structure and function most significantly by either creating kinks in the middle of helix 1 or completely unfolding its C terminus. In addition, helix 5 of Q18P is either bent or completely dissected into two smaller helices. Consequently, DNA binding is diminished by 2,000-fold. Our structural studies reveal heretofore-unobserved allosteric mechanisms that affect repressor function of a MarR family member and result in multidrug-resistant Staphylococcus aureus., Importance: Staphylococcus aureus is a major health threat to immunocompromised patients. S. aureus multidrug-resistant variants that overexpress the multidrug efflux pump mepA emerge frequently due to point mutations in MarR family member MepR, the mepA transcription repressor. Significantly, the majority of MepR mutations identified in these S. aureus clinical isolates are found not in the DNA binding domain but rather in a linker region, connecting the dimerization and DNA binding domains. The location of these mutants underscores the critical importance of a properly functioning allosteric mechanism that regulates MepR function. Understanding the dysregulation of such allosteric MepR mutants underlies this study. The high-resolution structures of three such allosteric MepR mutants reveal unpredictable conformational consequences, all of which preclude cognate DNA binding, while biochemical studies emphasize their debilitating effects on DNA binding affinity. Hence, mutations in the linker region of MepR and their structural consequences are key generators of multidrug-resistant Staphylococcus aureus.
- Published
- 2013
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- View/download PDF
39. Functional consequences of substitution mutations in MepR, a repressor of the Staphylococcus aureus MepA multidrug efflux pump gene.
- Author
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Schindler BD, Seo SM, Jacinto PL, Kumaraswami M, Birukou I, Brennan RG, and Kaatz GW
- Subjects
- Amino Acid Sequence, Anti-Bacterial Agents pharmacology, Bacterial Proteins genetics, Binding Sites, Drug Resistance, Multiple, Bacterial genetics, Genes, MDR genetics, Models, Molecular, Mutation, Protein Conformation, Reverse Transcriptase Polymerase Chain Reaction, Staphylococcus aureus genetics, Amino Acid Substitution, Bacterial Proteins metabolism, Gene Expression Regulation, Bacterial physiology, Genes, MDR physiology, Staphylococcus aureus metabolism
- Abstract
The expression of mepA, encoding the Staphylococcus aureus MepA multidrug efflux protein, is repressed by the MarR homologue MepR. MepR dimers bind differently to operators upstream of mepR and mepA, with affinity being greatest at the mepA operator. MepR substitution mutations may result in mepA overexpression, with A103V most common in clinical strains. Evaluation of the functional consequences of this and other MepR substitutions using a lacZ reporter gene assay revealed markedly reduced repressor activity in the presence of Q18P, F27L, G97E, and A103V substitutions. Reporter data were generally supported by susceptibility and efflux assays, and electrophoretic mobility shift assays (EMSAs) confirmed compromised affinities of MepR F27L and A103V for the mepR and mepA operators. One mutant protein contained two substitutions (T94P and T132M); T132M compensated for the functional defect incurred by T94P and also rescued that of A103V but not F27L, establishing it as a limited-range suppressor. The function of another derivative with 10 substitutions was minimally affected, and this may be an extreme example of suppression involving interactions among several residues. Structural correlations for the observed functional effects were ascertained by modeling mutations onto apo-MepR. It is likely that F27L and A103V affect the protein-DNA interaction by repositioning of DNA recognition helices. Negative functional consequences of MepR substitution mutations may result from interference with structural plasticity, alteration of helical arrangements, reduced protein-cognate DNA affinity, or possibly association of MepR protomers. Structural determinations will provide further insight into the consequences of these and other mutations that affect MepR function, especially the T132M suppressor.
- Published
- 2013
- Full Text
- View/download PDF
40. Studies of IscR reveal a unique mechanism for metal-dependent regulation of DNA binding specificity.
- Author
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Rajagopalan S, Teter SJ, Zwart PH, Brennan RG, Phillips KJ, and Kiley PJ
- Subjects
- Crystallography, X-Ray, Escherichia coli chemistry, Escherichia coli metabolism, Models, Biological, Models, Molecular, Nucleic Acid Conformation, Protein Binding, Substrate Specificity, DNA, Bacterial metabolism, Escherichia coli enzymology, Escherichia coli Proteins chemistry, Escherichia coli Proteins metabolism, Metals metabolism, Transcription Factors chemistry, Transcription Factors metabolism
- Abstract
IscR from Escherichia coli is an unusual metalloregulator in that both apo and iron sulfur (Fe-S)-IscR regulate transcription and exhibit different DNA binding specificities. Here, we report structural and biochemical studies of IscR suggesting that remodeling of the protein-DNA interface upon Fe-S ligation broadens the DNA binding specificity of IscR from binding the type 2 motif only to both type 1 and type 2 motifs. Analysis of an apo-IscR variant with relaxed target-site discrimination identified a key residue in wild-type apo-IscR that, we propose, makes unfavorable interactions with a type 1 motif. Upon Fe-S binding, these interactions are apparently removed, thereby allowing holo-IscR to bind both type 1 and type 2 motifs. These data suggest a unique mechanism of ligand-mediated DNA site recognition, whereby metallocluster ligation relocates a protein-specificity determinant to expand DNA target-site selection, allowing a broader transcriptomic response by holo-IscR.
- Published
- 2013
- Full Text
- View/download PDF
41. Serine substitution of proline at codon 151 of TP53 confers gain of function activity leading to anoikis resistance and tumor progression of head and neck cancer cells.
- Author
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Xie TX, Zhou G, Zhao M, Sano D, Jasser SA, Brennan RG, and Myers JN
- Subjects
- Animals, Blotting, Western, Carcinoma, Squamous Cell metabolism, Carcinoma, Squamous Cell pathology, Cell Line, Tumor, Codon, Disease Progression, Head and Neck Neoplasms metabolism, Head and Neck Neoplasms pathology, Mice, Mice, Nude, Neoplasms, Experimental, Reverse Transcriptase Polymerase Chain Reaction, Squamous Cell Carcinoma of Head and Neck, Anoikis genetics, Carcinoma, Squamous Cell genetics, DNA, Neoplasm genetics, Head and Neck Neoplasms genetics, Mutation, Proline genetics, Serine genetics, Tumor Suppressor Protein p53 genetics
- Abstract
Objectives/hypothesis: Mutation of the TP53 gene occurs in more than half of cases of head and neck squamous cell carcinoma (HNSCC). However, little is known about how specific TP53 mutations affect tumor progression. The objective of this study is to determine the gain of function of mutant p53 with a proline-to-serine substitution at codon 151., Study Design: Laboratory-based study., Methods: A panel of HNSCC cell lines was determined with anoikis assays, and orthotopic mouse experiments were performed. TP53 was sequenced. The shRNA knockdown and overexpression approaches were used for testing mutant p53 functions. The crystal structure of the p53 protein was analyzed using an in silico approach., Results: An anoikis-resistant cell line, Tu138, was found to have a proline-to-serine substitution at codon 151 of TP53, which results in loss of wild-type p53 transcriptional activity. Moreover, the mutant p53 was shown to promote anoikis resistance and soft agar growth. Using an in silico approach based on the crystal structure of wild-type p53 protein, substitution of proline by serine at position 151 would create a cavity in a hydrophobic pocket, the loss of van der Waals contacts, and the thermodynamically unfavorable placement of a polar group, the hydroxyl oxygen atom of the serine, within a hydrophobic region, all of which likely cause a locally altered structure., Conclusions: Our data suggest that mutation at position 151 leads to a structural alteration, which results in significant functional changes in the p53 protein that impact tumor progression., (Copyright © 2012 The American Laryngological, Rhinological and Otological Society, Inc.)
- Published
- 2013
- Full Text
- View/download PDF
42. Structural mechanism of Staphylococcus aureus Hfq binding to an RNA A-tract.
- Author
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Horstmann N, Orans J, Valentin-Hansen P, Shelburne SA 3rd, and Brennan RG
- Subjects
- Amino Acid Motifs, Gram-Positive Bacteria, Host Factor 1 Protein metabolism, Lysine chemistry, Models, Molecular, Protein Binding, Purines chemistry, Purines metabolism, Host Factor 1 Protein chemistry, Poly A chemistry, RNA chemistry, Staphylococcus aureus
- Abstract
Hfq is a post-transcriptional regulator that plays a key role in bacterial gene expression by binding AU-rich sequences and A-tracts to facilitate the annealing of sRNAs to target mRNAs and to affect RNA stability. To understand how Hfq from the Gram-positive bacterium Staphylococcus aureus (Sa) binds A-tract RNA, we determined the crystal structure of an Sa Hfq-adenine oligoribonucleotide complex. The structure reveals a bipartite RNA-binding motif on the distal face that is composed of a purine nucleotide-specificity site (R-site) and a non-discriminating linker site (L-site). The (R-L)-binding motif, which is also utilized by Bacillus subtilis Hfq to bind (AG)(3)A, differs from the (A-R-N) tripartite poly(A) RNA-binding motif of Escherichia coli Hfq whereby the Sa Hfq R-site strongly prefers adenosine, is more aromatic and permits deeper insertion of the adenine ring. R-site adenine-stacking residue Phe30, which is conserved among Gram-positive bacterial Hfqs, and an altered conformation about β3 and β4 eliminate the adenosine-specificity site (A-site) and create the L-site. Binding studies show that Sa Hfq binds (AU)(3)A ≈ (AG)(3)A ≥ (AC)(3)A > (AA)(3)A and L-site residue Lys33 plays a significant role. The (R-L) motif is likely utilized by Hfqs from most Gram-positive bacteria to bind alternating (A-N)(n) RNA.
- Published
- 2012
- Full Text
- View/download PDF
43. Role of unusual P loop ejection and autophosphorylation in HipA-mediated persistence and multidrug tolerance.
- Author
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Schumacher MA, Min J, Link TM, Guan Z, Xu W, Ahn YH, Soderblom EJ, Kurie JM, Evdokimov A, Moseley MA, Lewis K, and Brennan RG
- Subjects
- Adenosine Triphosphate chemistry, Adenosine Triphosphate metabolism, Amino Acid Motifs, Escherichia coli chemistry, Escherichia coli Proteins metabolism, Phosphorylation physiology, Protein Serine-Threonine Kinases metabolism, Serine chemistry, Serine metabolism, Drug Resistance, Multiple, Bacterial physiology, Escherichia coli enzymology, Escherichia coli Proteins chemistry, Protein Serine-Threonine Kinases chemistry
- Abstract
HipA is a bacterial serine/threonine protein kinase that phosphorylates targets, bringing about persistence and multidrug tolerance. Autophosphorylation of residue Ser150 is a critical regulatory mechanism of HipA function. Intriguingly, Ser150 is not located on the activation loop, as are other kinases; instead, it is in the protein core, where it forms part of the ATP-binding "P loop motif." How this buried residue is phosphorylated and regulates kinase activity is unclear. Here, we report multiple structures that reveal the P loop motif's exhibition of a remarkable "in-out" conformational equilibrium, which allows access to Ser150 and its intermolecular autophosphorylation. Phosphorylated Ser150 stabilizes the "out state," which inactivates the kinase by disrupting the ATP-binding pocket. Thus, our data reveal a mechanism of protein kinase regulation that is vital for multidrug tolerance and persistence, as kinase inactivation provides the critical first step in allowing dormant cells to revert to the growth phenotype and to reinfect the host., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
44. GQ-16, a novel peroxisome proliferator-activated receptor γ (PPARγ) ligand, promotes insulin sensitization without weight gain.
- Author
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Amato AA, Rajagopalan S, Lin JZ, Carvalho BM, Figueira AC, Lu J, Ayers SD, Mottin M, Silveira RL, Souza PC, Mourão RH, Saad MJ, Togashi M, Simeoni LA, Abdalla DS, Skaf MS, Polikparpov I, Lima MC, Galdino SL, Brennan RG, Baxter JD, Pitta IR, Webb P, Phillips KJ, and Neves FA
- Subjects
- 3T3-L1 Cells, Animals, Cyclin-Dependent Kinase 5 genetics, Cyclin-Dependent Kinase 5 metabolism, Drug Evaluation, Preclinical, Humans, Hypoglycemic Agents chemistry, Hypoglycemic Agents pharmacokinetics, Ligands, Mice, NIH 3T3 Cells, PPAR gamma genetics, PPAR gamma metabolism, Phosphorylation drug effects, Phosphorylation genetics, Protein Structure, Secondary, Thiazolidinediones chemistry, Thiazolidinediones pharmacokinetics, U937 Cells, Hypoglycemic Agents pharmacology, PPAR gamma agonists, Thiazolidinediones pharmacology, Weight Gain
- Abstract
The recent discovery that peroxisome proliferator-activated receptor γ (PPARγ) targeted anti-diabetic drugs function by inhibiting Cdk5-mediated phosphorylation of the receptor has provided a new viewpoint to evaluate and perhaps develop improved insulin-sensitizing agents. Herein we report the development of a novel thiazolidinedione that retains similar anti-diabetic efficacy as rosiglitazone in mice yet does not elicit weight gain or edema, common side effects associated with full PPARγ activation. Further characterization of this compound shows GQ-16 to be an effective inhibitor of Cdk5-mediated phosphorylation of PPARγ. The structure of GQ-16 bound to PPARγ demonstrates that the compound utilizes a binding mode distinct from other reported PPARγ ligands, although it does share some structural features with other partial agonists, such as MRL-24 and PA-082, that have similarly been reported to dissociate insulin sensitization from weight gain. Hydrogen/deuterium exchange studies reveal that GQ-16 strongly stabilizes the β-sheet region of the receptor, presumably explaining the compound's efficacy in inhibiting Cdk5-mediated phosphorylation of Ser-273. Molecular dynamics simulations suggest that the partial agonist activity of GQ-16 results from the compound's weak ability to stabilize helix 12 in its active conformation. Our results suggest that the emerging model, whereby "ideal" PPARγ-based therapeutics stabilize the β-sheet/Ser-273 region and inhibit Cdk5-mediated phosphorylation while minimally invoking adipogenesis and classical agonism, is indeed a valid framework to develop improved PPARγ modulators that retain antidiabetic actions while minimizing untoward effects.
- Published
- 2012
- Full Text
- View/download PDF
45. Regulation of the Escherichia coli HipBA toxin-antitoxin system by proteolysis.
- Author
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Hansen S, Vulić M, Min J, Yen TJ, Schumacher MA, Brennan RG, and Lewis K
- Subjects
- Base Sequence, DNA Primers, Proteolysis, Antitoxins metabolism, Bacterial Toxins metabolism, Escherichia coli metabolism
- Abstract
Bacterial populations produce antibiotic-tolerant persister cells. A number of recent studies point to the involvement of toxin/antitoxin (TA) modules in persister formation. hipBA is a type II TA module that codes for the HipB antitoxin and the HipA toxin. HipA is an EF-Tu kinase, which causes protein synthesis inhibition and dormancy upon phosphorylation of its substrate. Antitoxins are labile proteins that are degraded by one of the cytosolic ATP-dependent proteases. We followed the rate of HipB degradation in different protease deficient strains and found that HipB was stabilized in a lon(-) background. These findings were confirmed in an in vitro degradation assay, showing that Lon is the main protease responsible for HipB proteolysis. Moreover, we demonstrated that degradation of HipB is dependent on the presence of an unstructured carboxy-terminal stretch of HipB that encompasses the last 16 amino acid residues. Further, substitution of the conserved carboxy-terminal tryptophan of HipB to alanine or even the complete removal of this 16 residue fragment did not alter the affinity of HipB for hipBA operator DNA or for HipA indicating that the major role of this region of HipB is to control HipB degradation and hence HipA-mediated persistence.
- Published
- 2012
- Full Text
- View/download PDF
46. The crystal structure of the TetR family transcriptional repressor SimR bound to DNA and the role of a flexible N-terminal extension in minor groove binding.
- Author
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Le TB, Schumacher MA, Lawson DM, Brennan RG, and Buttner MJ
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Bacterial Proteins genetics, Bacterial Proteins metabolism, Coumarins chemistry, Crystallography, X-Ray, DNA Footprinting, DNA, Bacterial metabolism, Glycosides chemistry, Models, Molecular, Molecular Sequence Data, Nucleic Acid Conformation, Operator Regions, Genetic, Protein Binding, Repressor Proteins genetics, Repressor Proteins metabolism, Sequence Alignment, Sequence Deletion, Streptomyces antibioticus genetics, Bacterial Proteins chemistry, DNA, Bacterial chemistry, Repressor Proteins chemistry
- Abstract
SimR, a TetR-family transcriptional regulator (TFR), controls the export of simocyclinone, a potent DNA gyrase inhibitor made by Streptomyces antibioticus. Simocyclinone is exported by a specific efflux pump, SimX and the transcription of simX is repressed by SimR, which binds to two operators in the simR-simX intergenic region. The DNA-binding domain of SimR has a classical helix-turn-helix motif, but it also carries an arginine-rich N-terminal extension. Previous structural studies showed that the N-terminal extension is disordered in the absence of DNA. Here, we show that the N-terminal extension is sensitive to protease cleavage, but becomes protease resistant upon binding DNA. We demonstrate by deletion analysis that the extension contributes to DNA binding, and describe the crystal structure of SimR bound to its operator sequence, revealing that the N-terminal extension binds in the minor groove. In addition, SimR makes a number of sequence-specific contacts to the major groove via its helix-turn-helix motif. Bioinformatic analysis shows that an N-terminal extension rich in positively charged residues is a feature of the majority of TFRs. Comparison of the SimR-DNA and SimR-simocyclinone complexes reveals that the conformational changes associated with ligand-mediated derepression result primarily from rigid-body rotation of the subunits about the dimer interface.
- Published
- 2011
- Full Text
- View/download PDF
47. Distinct single amino acid replacements in the control of virulence regulator protein differentially impact streptococcal pathogenesis.
- Author
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Horstmann N, Sahasrabhojane P, Suber B, Kumaraswami M, Olsen RJ, Flores A, Musser JM, Brennan RG, and Shelburne SA 3rd
- Subjects
- Animals, Bacterial Proteins metabolism, Base Sequence, DNA, Bacterial analysis, Disease Models, Animal, Female, Longevity, Mice, Molecular Sequence Data, Mutagenesis, Site-Directed, Polymorphism, Single Nucleotide, Protein Structure, Secondary, RNA, Bacterial analysis, Repressor Proteins metabolism, Streptococcal Infections microbiology, Streptococcus pyogenes pathogenicity, Virulence genetics, Amino Acid Substitution, Amino Acids chemistry, Bacterial Proteins chemistry, Bacterial Proteins genetics, Repressor Proteins chemistry, Repressor Proteins genetics, Streptococcus pyogenes genetics
- Abstract
Sequencing of invasive strains of group A streptococci (GAS) has revealed a diverse array of single nucleotide polymorphisms in the gene encoding the control of virulence regulator (CovR) protein. However, there is limited information regarding the molecular mechanisms by which CovR single amino acid replacements impact GAS pathogenesis. The crystal structure of the CovR C-terminal DNA-binding domain was determined to 1.50 Å resolution and revealed a three-stranded β-sheet followed by a winged helix-turn-helix DNA binding motif. Modeling of the CovR protein-DNA complex indicated that CovR single amino acid replacements observed in clinical GAS isolates could directly alter protein-DNA interaction and impact protein structure. Isoallelic GAS strains that varied by a single amino acid replacement in the CovR DNA binding domain had significantly different transcriptomes compared to wild-type and to each other. Similarly, distinct recombinant CovR variants had differential binding affinity for DNA from the promoter regions of several virulence factor-encoding genes. Finally, mice that were challenged with GAS CovR isoallelic strains had significantly different survival times, which correlated with the transcriptome and protein-DNA binding studies. Taken together, these data provide structural and functional insights into the critical and distinct effects of variation in the CovR protein on GAS pathogenesis.
- Published
- 2011
- Full Text
- View/download PDF
48. Niche-specific contribution to streptococcal virulence of a MalR-regulated carbohydrate binding protein.
- Author
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Shelburne SA 3rd, Sahasrobhajane P, Suber B, Keith DB, Davenport MT, Horstmann N, Kumaraswami M, Olsen RJ, Brennan RG, and Musser JM
- Subjects
- Animals, Bacterial Adhesion, Bacterial Proteins genetics, Binding Sites, Cell Line, Conserved Sequence, DNA, Bacterial genetics, DNA, Bacterial metabolism, Epithelial Cells microbiology, Gene Expression Profiling, Gene Knockout Techniques, Humans, Mice, Oropharynx microbiology, Repressor Proteins genetics, Virulence, Bacterial Proteins metabolism, Gene Expression Regulation, Bacterial, Receptors, Cell Surface metabolism, Repressor Proteins metabolism, Streptococcus pyogenes metabolism, Streptococcus pyogenes pathogenicity, Virulence Factors metabolism
- Abstract
Low G+C Gram-positive bacteria typically contain multiple LacI/GalR regulator family members, which often have highly similar amino-terminal DNA binding domains, suggesting significant overlap in target DNA sequences. The LacI/GalR family regulator catabolite control protein A (CcpA) is a global regulator of the Group A Streptococcus (GAS) transcriptome and contributes to GAS virulence in diverse infection sites. Herein, we studied the role of the maltose repressor (MalR), another LacI/GalR family member, in GAS global gene expression and virulence. MalR inactivation reduced GAS colonization of the mouse oropharynx but did not detrimentally affect invasive infection. The MalR transcriptome was limited to only 25 genes, and a highly conserved MalR DNA-binding sequence was identified. Variation of the MalR binding sequence significantly reduced MalR binding in vitro. In contrast, CcpA bound to the same DNA sequences as MalR but tolerated variation in the promoter sequences with minimal change in binding affinity. Inactivation of pulA, a MalR regulated gene which encodes a cell surface carbohydrate binding protein, significantly reduced GAS human epithelial cell adhesion and mouse oropharyngeal colonization but did not affect GAS invasive disease. These data delineate a molecular mechanism by which hierarchical regulation of carbon source utilization influences bacterial pathogenesis in a site-specific fashion., (© 2011 Blackwell Publishing Ltd.)
- Published
- 2011
- Full Text
- View/download PDF
49. Achieving 0.2% relative expanded uncertainty in ion chromatography analysis using a high-performance methodology.
- Author
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Brennan RG, Butler TA, and Winchester MR
- Abstract
A high-performance (HP) technique that was originally developed for inductively coupled plasma optical emission spectrometry (ICP-OES) has been successfully translated to ion chromatography (IC) to enable analyses with extremely low uncertainty. As an example application of the HP-IC methodology, analyses of several National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs) in the SRM 3180 series of anion standard solutions are reported. The relative expanded uncertainty values expressed at 95% confidence for these analyses range from 0.087% to 0.27% and average 0.18%. Strong correlation between analyte and internal standard anion peak heights or peak areas, as well as the use of a unique drift-correction approach, is shown to be important for attaining such low uncertainty.
- Published
- 2011
- Full Text
- View/download PDF
50. Structures of carbon catabolite protein A-(HPr-Ser46-P) bound to diverse catabolite response element sites reveal the basis for high-affinity binding to degenerate DNA operators.
- Author
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Schumacher MA, Sprehe M, Bartholomae M, Hillen W, and Brennan RG
- Subjects
- Bacillus subtilis genetics, Base Sequence, Binding Sites, Consensus Sequence, Crystallography, X-Ray, DNA, Bacterial chemistry, Models, Molecular, Protein Binding, Response Elements, Bacterial Proteins chemistry, DNA-Binding Proteins chemistry, Operator Regions, Genetic, Phosphoproteins chemistry, Transcription Factors chemistry
- Abstract
In Gram-positive bacteria, carbon catabolite protein A (CcpA) is the master regulator of carbon catabolite control, which ensures optimal energy usage under diverse conditions. Unlike other LacI-GalR proteins, CcpA is activated for DNA binding by first forming a complex with the phosphoprotein HPr-Ser46-P. Bacillus subtilis CcpA functions as both a transcription repressor and activator and binds to more than 50 operators called catabolite response elements (cres). These sites are highly degenerate with the consensus, WTGNNARCGNWWWCAW. How CcpA-(HPr-Ser46-P) binds such diverse sequences is unclear. To gain insight into this question, we solved the structures of the CcpA-(HPr-Ser46-P) complex bound to three different operators, the synthetic (syn) cre, ackA2 cre and gntR-down cre. Strikingly, the structures show that the CcpA-bound operators display different bend angles, ranging from 31° to 56°. These differences are accommodated by a flexible linkage between the CcpA helix-turn-helix-loop-helix motif and hinge helices, which allows independent docking of these DNA-binding modules. This flexibility coupled with an abundance of non-polar residues capable of non-specific nucleobase interactions permits CcpA-(HPr-Ser46-P) to bind diverse operators. Indeed, biochemical data show that CcpA-(HPr-Ser46-P) binds the three cre sites with similar affinities. Thus, the data reveal properties that license this protein to function as a global transcription regulator.
- Published
- 2011
- Full Text
- View/download PDF
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