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Author: Brendan Rayhan Amer
Publisher:
ISBN:
Category :
Languages : en
Pages : 164
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Book Description
Many species of pathogenic Gram-positive bacteria have become resistant to commonly used antibiotics, creating a pressing need for the development of novel antimicrobial therapies. These pathogenic bacteria are surrounded by a complex peptidoglycan cell wall that is decorated with a range of macromolecules such as surface proteins, protein-based oligomeric pili, and surface glycopolymers. These structures enable the microbe to effectively interact with their environment, and in pathogenetic bacteria are frequently virulence factors that are involved in: immune system modulation, bacterial adhesion, nutrient acquisition, and spore formation. Two distinct enzyme superfamilies are primarily responsible for covalently attaching macromolecules to the Gram-positive cell wall: sortase enzymes that attach proteins and assemble pili, and LytR-Psr-CpsA (LCP) enzymes that attach glycopolymers. This dissertation presents my efforts to discover a sortase inhibitor, structural and mechanistic investigations of both sortase and LCP enzymes, and the development of a new sortase mediated ligation technology. I describe our efforts to find inhibitors against the S. aureus sortase transpeptidase. We utilized both experimental and computational methods to discover potent inhibitor scaffolds. This led to structure-activity studies that not only improved inhibitor potency, but also yielded molecules that can inhibit bacterial protein display in vivo, and appear to be non-toxic to human cells. Sortase enzymes have also proven to be valuable biotechnological conjugation tools. I describe the development of a novel sortase-mediated ligation tool. We demonstrate a 15-fold rate enhancement for existing sortase mediated ligation strategies. Additionally, I discuss studies on sortase pilus assembly and display. To gain insight into the molecular mechanism of sortase pilus assembly, we reconstituted an archetypal Gram-positive pilus assembly process in vitro. This has allowed us to generate molecular models of various intermediates of pilus assembly and define the molecular determinants of this assembly process. We have identified novel features on both the sortase and pilin precursor proteins that are essential for pilus covalent isopeptide assembly. In the concluding chapter of this dissertation, structural work characterizing the unique LCP enzyme that mediates surface protein glycosylation is presented. Work on this project reveals that LCP enzymes use a conserved phosphotransferase mechanism to attach glycopolymers to surface displayed proteins, and this may be a mechanism for actinobacteria wall teichoic acid display, a key macromolecule in the bacterial cell envelope. Combined, this work serves to further define the mechanism of Gram-positive bacterial macromolecular surface display, improved sortase-mediated bioconjugation strategies, and steps towards novel anti-infective sortase inhibitors.
Author: Brendan Rayhan Amer
Publisher:
ISBN:
Category :
Languages : en
Pages : 164
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Book Description
Many species of pathogenic Gram-positive bacteria have become resistant to commonly used antibiotics, creating a pressing need for the development of novel antimicrobial therapies. These pathogenic bacteria are surrounded by a complex peptidoglycan cell wall that is decorated with a range of macromolecules such as surface proteins, protein-based oligomeric pili, and surface glycopolymers. These structures enable the microbe to effectively interact with their environment, and in pathogenetic bacteria are frequently virulence factors that are involved in: immune system modulation, bacterial adhesion, nutrient acquisition, and spore formation. Two distinct enzyme superfamilies are primarily responsible for covalently attaching macromolecules to the Gram-positive cell wall: sortase enzymes that attach proteins and assemble pili, and LytR-Psr-CpsA (LCP) enzymes that attach glycopolymers. This dissertation presents my efforts to discover a sortase inhibitor, structural and mechanistic investigations of both sortase and LCP enzymes, and the development of a new sortase mediated ligation technology. I describe our efforts to find inhibitors against the S. aureus sortase transpeptidase. We utilized both experimental and computational methods to discover potent inhibitor scaffolds. This led to structure-activity studies that not only improved inhibitor potency, but also yielded molecules that can inhibit bacterial protein display in vivo, and appear to be non-toxic to human cells. Sortase enzymes have also proven to be valuable biotechnological conjugation tools. I describe the development of a novel sortase-mediated ligation tool. We demonstrate a 15-fold rate enhancement for existing sortase mediated ligation strategies. Additionally, I discuss studies on sortase pilus assembly and display. To gain insight into the molecular mechanism of sortase pilus assembly, we reconstituted an archetypal Gram-positive pilus assembly process in vitro. This has allowed us to generate molecular models of various intermediates of pilus assembly and define the molecular determinants of this assembly process. We have identified novel features on both the sortase and pilin precursor proteins that are essential for pilus covalent isopeptide assembly. In the concluding chapter of this dissertation, structural work characterizing the unique LCP enzyme that mediates surface protein glycosylation is presented. Work on this project reveals that LCP enzymes use a conserved phosphotransferase mechanism to attach glycopolymers to surface displayed proteins, and this may be a mechanism for actinobacteria wall teichoic acid display, a key macromolecule in the bacterial cell envelope. Combined, this work serves to further define the mechanism of Gram-positive bacterial macromolecular surface display, improved sortase-mediated bioconjugation strategies, and steps towards novel anti-infective sortase inhibitors.
Author: Orlando Edward Martinez
Publisher:
ISBN:
Category :
Languages : en
Pages : 223
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Book Description
Pathogenic Gram-positive bacteria have acquired resistance to many commonly used antibiotics. One potential strategy to combat this problem is to develop small molecule therapeutics that target bacterial virulence mechanisms which enable pathogens to cause disease. The bacterial cell surface contains many virulence factors that have key roles in host-pathogen interactions, but the mechanisms by which they are synthesized and displayed remains incompletely understood. The work described in this dissertation is concentrated on understanding how bacteria synthesize wall teichoic acid (WTA) glycopolymers. WTAs are peptidoglycan-anchored alditol-phosphate polymers that have important functions in host immune system evasion, biofilm formation, and antibiotic resistance, among other roles. This research is specifically focused on the TagA glycosyltransferase, which catalyzes the first committed enzymatic step in WTA biosynthesis. Small molecule TagA inhibitors might function as novel antivirulence therapeutics because cells that lack the enzyme in methicillin-resistant Staphylococcus aureus (MRSA) maintain viability but become re-sensitized to [beta]-lactam antibiotics. The research described in this dissertation advances our understanding of the enzymatic mechanism of TagA and its role in controlling the architecture of the bacterial cell wall. Chapter 1 surveys the biosynthetic pathways that are used to produce peptidoglycan and WTA polymers in Bacillus subtilis and S. aureus. Chapter 2 details the determination of the first crystal structure of the soluble portion of TagA from Thermoanaerobacter italicus, which is shown to adopt a novel glycosyltransferase structural fold. This study identified two conserved residues in TagA that are important for catalysis and demonstrated that C-terminal tail residues in the enzyme are essential for enzymatic activity in vitro and membrane association in cells. Chapter 3 describes the construction of a solubility-enhanced TagA variant and crystal structures of this enzyme bound to its native substrate, UDP-ManNAc, and an epimer of the substrate, UDP-GlcNAc. These structures provide insight into stereospecific protein-ligand contacts that confer substrate specificity. Molecular dynamics simulations of full-length TagA models with and without its bound substrates are also presented which demonstrate that UDP-ManNAc stabilizes construction of the enzyme's active site through interactions with key catalytic residues in the C-terminal tail. Collectively, these data suggest a model of enzyme function in which membrane association via the C-terminal tail triggers a conformational change in TagA that is further stabilized by stereospecific contacts with its UDP-ManNAc substrate. Lastly, Chapter 4 details ongoing progress in capturing the active, monomeric form of the protein for structural investigations and studying the influence of TagA activity on cell morphology in B. subtilis using transmission electron microscopy. This work contributes to our knowledge of WTA biogenesis in bacteria and lays a foundation for the structure-guided development of TagA-specific inhibitors that could function as antivirulence agents.
Author: Jan Löwe
Publisher: Springer
ISBN: 331953047X
Category : Science
Languages : en
Pages : 457
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Book Description
This book describes the structures and functions of active protein filaments, found in bacteria and archaea, and now known to perform crucial roles in cell division and intra-cellular motility, as well as being essential for controlling cell shape and growth. These roles are possible because the cytoskeletal and cytomotive filaments provide long range order from small subunits. Studies of these filaments are therefore of central importance to understanding prokaryotic cell biology. The wide variation in subunit and polymer structure and its relationship with the range of functions also provide important insights into cell evolution, including the emergence of eukaryotic cells. Individual chapters, written by leading researchers, review the great advances made in the past 20-25 years, and still ongoing, to discover the architectures, dynamics and roles of filaments found in relevant model organisms. Others describe one of the families of dynamic filaments found in many species. The most common types of filament are deeply related to eukaryotic cytoskeletal proteins, notably actin and tubulin that polymerise and depolymerise under the control of nucleotide hydrolysis. Related systems are found to perform a variety of roles, depending on the organisms. Surprisingly, prokaryotes all lack the molecular motors associated with eukaryotic F-actin and microtubules. Archaea, but not bacteria, also have active filaments related to the eukaryotic ESCRT system. Non-dynamic fibres, including intermediate filament-like structures, are known to occur in some bacteria.. Details of known filament structures are discussed and related to what has been established about their molecular mechanisms, including current controversies. The final chapter covers the use of some of these dynamic filaments in Systems Biology research. The level of information in all chapters is suitable both for active researchers and for advanced students in courses involving bacterial or archaeal physiology, molecular microbiology, structural cell biology, molecular motility or evolution. Chapter 3 of this book is open access under a CC BY 4.0 license.
Author: J.-M. Ghuysen
Publisher: Elsevier
ISBN: 0080860877
Category : Science
Languages : en
Pages : 607
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Book Description
Studies of the bacterial cell wall emerged as a new field of research in the early 1950s, and has flourished in a multitude of directions. This excellent book provides an integrated collection of contributions forming a fundamental reference for researchers and of general use to teachers, advanced students in the life sciences, and all scientists in bacterial cell wall research. Chapters include topics such as: Peptidoglycan, an essential constituent of bacterial endospores; Teichoic and teichuronic acids, lipoteichoic acids, lipoglycans, neural complex polysaccharides and several specialized proteins are frequently unique wall-associated components of Gram-positive bacteria; Bacterial cells evolving signal transduction pathways; Underlying mechanisms of bacterial resistance to antibiotics.
Author: Bruce Alberts
Publisher:
ISBN: 9780815332183
Category : Cytology
Languages : en
Pages : 0
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Book Description
Author: Kenneth R. Boheler
Publisher: Humana Press
ISBN: 9781493975518
Category : Science
Languages : en
Pages : 348
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Book Description
This volume provides readers with the latest techniques and tools to assess modifications and functions of the surfaceome. The chapters in this book are divided into 4 sections: discovery-based approaches to surfaceome content; targeted approaches for surfaceome content; cell-based function analyses related to surfaceome content; and computational approaches in surfaceome studies. Section 1 focuses on discovery-based approaches for cataloging surfaceome content that analyses the surfaceome of bacteria, avian embryos, and mammalian systems. Section 2 discusses methods that over-express specific targets in Sf9 cells and generate bi-specific antibodies for targeting cancer and somatic cells. Section 3 explores voltage dependent sodium channels and high-content electrophysiological analyses. The final section looks at the new web-based platform known as targets-search. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Comprehensive and thorough, Surfaceome: Methods and Protocols assists in the study of cell surface protein biology and function. It is a valuable resource for all researchers interested in this field.
Author: Tony Romeo
Publisher: Springer Science & Business Media
ISBN: 3540754180
Category : Medical
Languages : en
Pages : 302
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Book Description
Throughout the biological world, bacteria thrive predominantly in surface-attached, matrix-enclosed, multicellular communities or biofilms, as opposed to isolated planktonic cells. This choice of lifestyle is not trivial, as it involves major shifts in the use of genetic information and cellular energy, and has profound consequences for bacterial physiology and survival. Growth within a biofilm can thwart immune function and antibiotic therapy and thereby complicate the treatment of infectious diseases, especially chronic and foreign device-associated infections. Modern studies of many important biofilms have advanced well beyond the descriptive stage, and have begun to provide molecular details of the structural, biochemical, and genetic processes that drive biofilm formation and its dispersion. There is much diversity in the details of biofilm development among various species, but there are also commonalities. In most species, environmental and nutritional conditions greatly influence biofilm development. Similar kinds of adhesive molecules often promote biofilm formation in diverse species. Signaling and regulatory processes that drive biofilm development are often conserved, especially among related bacteria. Knowledge of such processes holds great promise for efforts to control biofilm growth and combat biofilm-associated infections. This volume focuses on the biology of biofilms that affect human disease, although it is by no means comprehensive. It opens with chapters that provide the reader with current perspectives on biofilm development, physiology, environmental, and regulatory effects, the role of quorum sensing, and resistance/phenotypic persistence to antimicrobial agents during biofilm growth.
Author: Michael L. Vasil
Publisher: American Society for Microbiology Press
ISBN: 1555816762
Category : Science
Languages : en
Pages : 1189
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Book Description
A comprehensive compendium of scholarly contributions relating to bacterial virulence gene regulation. • Provides insights into global control and the switch between distinct infectious states (e.g., acute vs. chronic). • Considers key issues about the mechanisms of gene regulation relating to: surface factors, exported toxins and export mechanisms. • Reflects on how the regulation of intracellular lifestyles and the response to stress can ultimately have an impact on the outcome of an infection. • Highlights and examines some emerging regulatory mechanisms of special significance. • Serves as an ideal compendium of valuable topics for students, researchers and faculty with interests in how the mechanisms of gene regulation ultimately affect the outcome of an array of bacterial infectious diseases.
Author: Vincent A. Fischetti
Publisher:
ISBN:
Category : Bacterial vaccines
Languages : en
Pages : 760
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Book Description
This book is the only single volume to deal with all aspects of gram–positive pathogens. It addresses the mechanisms of gram–positive bacterial pathogenicity, including the current knowledge on gram–positive structure and mechanisms of antibiotic resistance. Emphasizing streptococci, staphylococci, listeria, and spore–forming pathogens, Gram–Positive Pathogens includes chapters written by many of the leading researchers in these areas. The chapters systematically dissect these organisms biologically, genetically, and immunologically in an attempt to understand the strategies used by these bacteria to cause human disease.
Author:
Publisher: Academic Press
ISBN: 0080860567
Category : Science
Languages : en
Pages : 643
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Book Description
Established almost 30 years ago, Methods in Microbiology is the most prestigious series devoted to techniques and methodology in the field. Now totally revamped, revitalized, with a new format and expanded scope, Methods in Microbiology will continue to provide you with tried and tested, cutting-edge protocols to directly benefit your research. Focuses on the methods most useful for the microbiologist interested in the way in which bacteria cause disease Includes section devoted to 'Approaches to characterising pathogenic mechanisms' by Stanley Falkow Covers safety aspects, detection, identification and speciation Includes techniques for the study of host interactions and reactions in animals and plants Describes biochemical and molecular genetic approaches Essential methods for gene expression and analysis Covers strategies and problems for disease control
