Microbial Glycobiology: Structures, Relevance and Applications

Table of Contents

Cover image

Copyright

Dedication

List of Contributors

Preface

Chapter 1. Overview of the glycosylated components of the bacterial cell envelope

1. Introduction – The Bacterial Cell Envelope Encountering Environmental Challenges

2. The Gram-Negative Cell Envelope

3. The Gram-Positive Cell Envelope

4. The Mycobacterial Cell Envelope

5. The Archaeal Cell Envelopes and S-Layers

6. Conclusions

Chapter 2. Bacterial cell envelope peptidoglycan

1. Introduction

2. Structural Variation in Bacterial Peptidoglycan

3. Biophysical Properties of Peptidoglycan

4. The Molecular Architecture of Peptidoglycan

5. Conclusions

Chapter 3. Core region and lipid A components of lipopolysaccharides

1. Introduction

2. General Structural Features of the Lipid A Molecule

3. Lipopolysaccharides of Mammalian Pathogenic Bacteria: The Case of B. Cepacia Complex

4. Plant Pathogenic Agrobacterium and Xanthomonas LPS and the Activation of Innate Immune Response in Plants

5. Structural Elucidation of Lipid A

6. General Structural Features of the Core Region

7. Core Structures of Various Bacteria

8. Conclusions

Chapter 4. O-Specific polysaccharides of Gram-negative bacteria

1. Introduction

2. Composition of O-PSs

3. Repetitive O-PS Structures

4. Non-Repetitive Motifs

5. Conclusions

Chapter 5. Teichoic acids, lipoteichoic acids and related cell wall glycopolymers of Gram-positive bacteria

1. Introduction

2. Teichoic Acid Structures

2.2. Lipoteichoic acid (LTA) and other M-CWGs

3. Biosynthesis of WTAs and LTA

3.1. WTA biosynthesis in B. subtilis and S. aureus

3.2. LTA biosynthesis in S. aureus

3.3. Incorporation of d-alanine into WTAs and LTA

4. Roles of WTAs and LTA in Bacterial Physiology

5. Teichoic Acids and Host Cell Receptor Interaction

6. Conclusions and Perspectives

Chapter 6. Bacterial capsular polysaccharides and exopolysaccharides

1. Introduction

2. Carbohydrate Components of Capsular and Exo-Polysaccharides

3. Non-Carbohydrate Substituents of Capsular and Exopolysaccharides

4. Structure Overview of Bacterial Polysaccharides

5. Polysaccharide Shapes

6. Biological Functions of Capsular and Exopolysaccharides

7. Exopolysaccharides of the Burkholderia Cepacia Complex: A Case Study

8. Conclusion

Chapter 7. Bacterial surface layer glycoproteins and non-classical secondary cell wall polymers

1. Introduction

2. Bacterial and Archaeal S-Layers

3. General Features of Glycosylated S-Layer Proteins

4. Genetics

5. Biosynthesis

6. The Non-Classical Group of Secondary Cell Wall Polymers

7. Outlook

Chapter 8. Glycosylation of bacterial and archaeal flagellins

1. Introduction

2. Flagellar Glycan Structures

3. Structural Analysis of Flagellar Glycans

4. Flagellar Glycan Biosynthetic Pathways

5. Conclusions and Future Perspectives

Chapter 9. Glycosylated components of the mycobacterial cell wall

1. Characteristic Features of Mycobacterium spp.

2. The Mycobacterial Envelope

3. The Mycobacterial Cell Wall Skeleton: The Mycolylarabinogalactan-PG Complex

4. The Soluble Cross-Species Glycoconjugates of the Mycobacterial Cell Wall

5. The Species and Sub-Species Specific Soluble Glycoconjugates of Mycobacterial Cell Walls

6. Conclusions

Chapter 10. Glycoconjugate structure and function in fungal cell walls

1. Introduction

2. Overall Structure

3. Wall Polysaccharides

4. Cell Wall Glycoproteins of Ascomycetous Fungi

5. Characteristics of Fungal Cell Wall Glycoproteins

6. The Mannoprotein Glycans

7. Functions of Wall Glycoproteins

8. Dynamics of the Fungal Wall Proteome

9. Conclusions

Chapter 11. Cytoplasmic carbohydrate molecules

1. Introduction

2. Occurrence, Distribution and Function of Glycogen

3. Structure of Glycogen

4. Biosynthesis of Glycogen

5. Degradation of Glycogen

6. Occurrence and Distribution of Trehalose

7. Biosynthesis of Trehalose

8. Functions of Trehalose

9. Conclusions

Chapter 12. Glycosylated compounds of parasitic protozoa

1. Introduction

2. The Surface Coats of Parasitic Protozoa – An Overview

3. Protein-Linked and Free GPI Glycolipids

4. N-linked glycans

5. O-Linked Glycans

6. Phosphoglycosylation

7. Parasite Cyst Wall Polysaccharides

8. Intracellular Reserve Glycans

9. Conclusions

Chapter 13. Analytical approaches towards the structural characterization of microbial wall glycopolymers

1. Introduction

2. Isolation and Purification of Bacterial Glycan Structures

3. NMR Techniques Employed for Structural Characterization of Glycans

4. Mass Spectrometry of Glycans

5. Conclusions

Chapter 14. Single-molecule characterization of microbial polysaccharides

1. Introduction

2. Atomic Force Microscopy and its Application to Microbial PSs

3. Measurements of Mechanical Properties of Single PSs

4. Atomic Force Microscopy as a Tool to Investigate Function of Microbial PSs

5. Single-Molecule Studies of Microbial PSs Using Optical Techniques

6. Conclusions

Chapter 15. Viral surface glycoproteins in carbohydrate recognition

1. Introduction

2. Influenza Virus

3. Parainfluenza

4. Dengue Virus

5. Rotavirus

6. Conclusions

Chapter 16. Biosynthesis of bacterial peptidoglycan

1. Introduction

2. Assembly of the Monomer Unit

3. Translocation of the monomer unit

4. Polymerization of the Monomer Unit

5. Variations in Peptidoglycan Biosynthesis

6. In Vivo Functioning of the Monomer Unit Assembly

7. In Vivo Functioning of the Polymerization Process

8. Inhibition of Peptidoglycan Biosynthesis

9. Concluding Remarks

Chapter 17. Biosynthesis and membrane assembly of lipid A

1. Introduction

2. The Constitutive Lipid a Biosynthetic Pathway

3. Transport

4. Modification of the Kdo-Lipid a Domain of LPS

5. Conclusions

Chapter 18. Biosynthesis of O-antigen chains and assembly

1. Introduction

2. The E. coli K-12 (O16) O-Antigen

3. S. Enterica LT2 and a Family of O-Antigens

4. Initial Transferases that Initiate O-Antigen Synthesis

5. Overview of the Wzx/Wzy Pathway

6. The ABC Transporter Pathway

7. The Synthase Pathway

8. Conclusions

Chapter 19. Biosynthesis of cell wall teichoic acid polymers

1. Introduction

2. Model of Teichoic Acid Biosynthesis

3. Biosynthesis of WTA Precursors

4. Studying Membrane Activities

5. Linkage Unit Glycosyltransferases

6. Teichoic Acid Polymer Biosynthesis

7. Outstanding Issues

8. Conclusions

Chapter 20. Biosynthesis and assembly of capsular polysaccharides

1. Introduction

2. Biosynthesis and Transport of CPSs Across the Inner Membrane

3. Capsular Polysaccharide Export Across the Outer Membrane

4. Bridging the Gap Between the Inner and Outer Membranes

4.3. The Wza-Wzc interactions in Wzy-dependent CPS systems

5. Cell-Surface Attachment of the CPS

6. Conclusions

Chapter 21. Biosynthesis of the mycobacterial cell envelope components

1. Mycobacterial Glycosyltransferases

2. Biochemistry and Genetics of Peptidoglycan (PG) Synthesis

3. Biochemistry and Genetics of AG Synthesis

4. Biosynthesis and Genetics of the Phosphatidylinositol- (PI-) Containing Phosphatidylinositol-Mannosides (PIMs), LMs and LAMs

5. Biosynthesis and Genetics of the Glycopeptidolipids

6. Biosynthesis and Genetics of the Phthiocerol-Containing Lipids

7. Biosynthesis of the Trehalose-Containing Glycolipids

8. Conclusions

Chapter 22. Biosynthesis of fungal and yeast glycans

1. Introduction

2. Precursors for Glycan Synthesis

3. Fungal Protein Glycosylation

4. Fungal Glycolipids

5. Fungal Cell Wall Polymers

6. Intracellular Glycans

7. Exopolysaccharides

8. Conclusions

Chapter 23. Chemical synthesis of bacterial lipid A

1. Introduction

2. Early Chemical Syntheses of Bacterial Lipid A

3. Improved Synthesis of Lipid A Analogues

4. Synthesis of Lipid A Containing an Unsaturated Fatty Acyl Group

5. Concluding Remarks

Chapter 24. Chemical synthesis of the core oligosaccharide of bacterial lipopolysaccharide

1. Introduction

2. Synthesis of 3-Deoxy-d-Manno-Oct-2-Ulosonic Acid (Kdo)- and d-Glycero-D-Talo-Oct-2-Ulosonic Acid (Ko)-Containing Core Structures

3. Synthesis of Heptose-Containing Core Structures

4. Synthesis of Phosphorylated Core Units

5. Synthesis of Outer Core Units

6. Concluding Remarks

Chapter 25. Chemical synthesis of lipoteichoic acid and derivatives

1. Introduction

2. Van Boom’s Synthesis of S. Aureus LTA Type I

3. Kusumoto’s Synthesis of LTA Fragments from Enterococcus hirae and Streptococcus Pyogenes

4. Schmidt’s Synthesis of LTA Type I from S. Aureus

5. Conclusion

Chapter 26. Chemical synthesis of parasitic glycoconjugates and phosphoglycans

1. Introduction

2. Chemical Synthesis of Parasitic Glycoconjugates (GPI Anchors)

3. Chemical Synthesis of Parasitic Phosphoglycans of Leishmania

4. Conclusions and Future Perspectives

Chapter 27. Bacterial lectin-like interactions in cell recognition and adhesion

1. Introduction

2. Mannose-Specific Bacterial Lectins

3. Fucose-Specific Bacterial Lectins

4. Galactose and GalNAc-Specific Bacterial Lectins

5. N-Acetylglucosamine-Specific Bacterial Lectins

6. Tissue Tropism of UPEC

7. Utilizing Glycan Array Technology to Identify and Characterize Novel Bacteria–Carbohydrate Interactions

8. Inhibitors of Lectin-Mediated Adhesion of Bacteria to Host Cells

9. Conclusions

Chapter 28. Lectin-like interactions in virus–cell recognition

1. Introduction

2. Making it Stick: Env Mediates HIV Attachment and Entry into Host Cells

3. Promotion of HIV Capture, Trans-Infection and Dissemination by Dc-Sign – The Paradigm Revisited

4. Binding of HIV to DC-SIGN on B-Cells and Platelets – Modulation of Immune Responses and Trans-Infection of T-Cells

5. Impact of DC-SIGN Polymorphisms on the Susceptibility to HIV Infection

6. Langerin on Langerhans Cells – Barrier Against HIV Transmission?

7. DC-SIGNR and LSECtin – Consequences of HIV Capture by Vascular Endothelial Cells

8. Conclusions

Chapter 29. Sialic acid-specific microbial lectins

1. Introduction

2. Role of Sialic Acid-Specific Microbial Lectins in Host Cell Attachment

3. Conserved Binding Site of Sialic Acid-Specific Microbial Lectins

4. Sialic Acid-Binding Domain Associated with Bacterial Sialic Acid Transport Systems

5. Conclusions

Chapter 30. Bacterial toxins and their carbohydrate receptors at the host–pathogen interface

1. Introduction

2. Toxin Receptor GSL-Binding

3. Intracellular Trafficking of GSLs

4. Intracellular Toxin Traffic

5. Glycosphingolipid Receptors and Toxin-Induced Pathology

6. Toxin-GSL-Mediated Signalling

7. Gb3 and Drug Resistance

8. Verotoxin1 as an Anti-Neoplastic

9. Verotoxin Opens a Window for Human Immunodeficiency Virus (HIV) Therapy

10. Conclusions

Chapter 31. Toll-like receptor recognition of lipoglycans, glycolipids and lipopeptides

1. Introduction

2. Toll-Like Receptors in Vertebrates

3. TLR-2

4. TLR-4

5. Conclusions

Chapter 32. NOD receptor recognition of peptidoglycan

1. Biological Activities of Peptidoglycan: An Historical Perspective

2. The Nod-Like Proteins: Receptors of the Innate Immune System

3. Structural Requirements of PG for Nod Proteins Detection

4. The Role of Nod Proteins in Sensing of Bacterial Infections

5. Peptidoglycan Metabolism and Modulation of Nod-Dependent Responses

6. Conclusions and Perspectives

Chapter 33. Microbial interaction with mucus and mucins

1. Introduction

2. Overall Effect of Epithelial Colonization on Mucus and Mucins

3. The Role of Microbes in the Normal Turnover of Mucus Gels

4. Microbial Interactions with Membrane-Associated Mucins

5. Microbial Binding and Homing to Secreted Mucins

6. Microbial Penetration of Mucus Gels

7. Microbially Mediated Re-Programming of Host Glycosylation

8. Pathologies of Mucus Turnover, Abnormal Colonization, and Biofilm Formation

9. Conclusions

Chapter 34. Mannose–fucose recognition by DC-SIGN

1. Introduction

2. DC-SIGN Structure and Expression

3. Selective Recognition of Man- and Fuc-Containing Glycans by DC-SIGN

4. In Vivo Function and Role in Dendritic Cell Biology of DC-SIGN

5. Pathogens Target DC-SIGN to Subvert Host Immune Responses

6. Conclusions

Chapter 35. Host surfactant proteins in microbial recognition

1. Introduction – General Overview on Surfactant Proteins

2. Genomic Organization

3. Molecular Structure

4. Biosynthesis and Tissue Distribution of SP-A and SP-D

5. Regulation of Gene Expression

6. Putative Receptors for SP-A and SP-D

7. Diverse Functions of SP-A and SP-D

8. Clinical Applications and Significance

9. Conclusions

Chapter 36. T-Cell recognition of microbial lipoglycans and glycolipids

1. Introduction

2. Structure and Cell Biology of CD1 Antigen-Presenting Molecules

3. Structure of Glycolipid Antigens

4. Presentation of Lipid Antigens

5. Priming, Expansion and Generation of Memory Glycolipid-Specific T-Cells

6. Effector Functions of Glycolipid-Specific T-Cells

7. Conclusions

Chapter 37. Extracellular polymeric substances in microbial biofilms

1. Introduction – Biofilm Systems

2. Extracellular Polymeric Substances (EPS) in Biofilms

3. Nature and Appearance of Microbial EPS Structures

4. The Biofilm Matrix and its Literature Re-Examined

5. Issues Concerning EPS Function in Biofilm Systems

6. EPS Functionality – A Novel Perspective

7. Conclusions

Chapter 38. Physicochemical properties of microbial glycopolymers

1. Introduction

2. Lipopolysaccharides

3. Rhamnolipids

4. Mycobacterial Glycopolymers

5. Future Outlook

Chapter 39. Microbial biofilm-related polysaccharides in biofouling and corrosion

1. Introduction – Biofilm Systems

2. Biofilm-Related PSs in Biofouling and Corrosion

3. Biofouling Causing Environmental and Industrial Problems

4. Microbially Influenced Corrosion (MIC)

5. Conclusions

Chapter 40. Microbial glycosylated components in plant disease

1. Introduction

2. Induced Defence Responses in Plants

3. The Diverse Roles of Bacterial LPSs in Plant Disease

4. Bacterial PG as a MAMP

5. The Multiple Roles of Exopolysaccharides (EPSs)

6. Bacterial Cyclic Glucan and Suppression of Host Defences

7. Fungal Chitin and Oomycete Glucan as MAMPs

8. Concluding Remarks

Chapter 41. Antigenic variation of microbial surface glycosylated molecules

1. Introduction

2. Protein-Linked Glycosylation

3. Lipid-Linked Glycosylation

4. Capsules

5. Future Perspectives

Chapter 42. Phase variation of bacterial surface glycosylated molecules in immune evasion

1. Introduction

2. Molecular Mimicry of Human Antigens by the LOSs of Pathogenic Neisseria and Haemophilus

3. The LOS of N. gonorrhoeae Mimics Human Paragloboside and Serves as a Ligand to the Asialoglycoprotein Receptor

4. The LOS of NTHi Mimics the Human Platelet-Activating Factor (PAF) and can Act as a Ligand for the Platelet-Activating Factor Receptor (PAF-R) on Airway Epithelial Cells

5. Conclusions

Chapter 43. Molecular mimicry of host glycosylated structures by bacteria

1. Introduction

2. Expression of Ganglioside Mimicry by C. Jejuni

3. C. Jejuni Ganglioside Mimicry and Pathogenic Anti-Ganglioside Antibodies

4. Relevance of Molecular Micmicry in the Pathogenesis of GBS

5. Expression of Le Antigens in H. Pylori LPS

6. Roles of LPS-Expressed Le Antigens in H. Pylori Pathogenesis

7. Conclusions and Future Outlook

Chapter 44. Role of microbial glycosylation in host cell invasion

1. Introduction

2. LPSs and LOSs in Cell Invasion

3. Role of Bacterial Capsules in Invasiveness

4. Role of Protein Glycosylation in Invasion

5. Conclusions

Chapter 45. Exopolysaccharides produced by lactic acid bacteria in food and probiotic applications

1. Introduction

2. The EPSs from LAB

3. Technological Applications of EPS from LAB in Dairy Products

4. Health Benefits of EPS from LAB and Bifidobacteria for Probiotic Applications

5. Concluding Remarks and Future Trends

Chapter 46. Industrial exploitation by genetic engineering of bacterial glycosylation systems

1. Introduction

2. N-Glycosylation Pathway in C. Jejuni

3. Pilin O-Glycosylation in P. Aeruginosa

4. Pilin O-Glycosylation in Neisseria Spp.

5. Recombinant Protein N- and O-Glycosylation in E. Coli

6. Towards a New Era in Glyco-Engineering

7. Conclusions

Chapter 47. Glycomimetics as inhibitors in anti-infection therapy

1. Introduction

2. Replacement of the Ring Oxygen

3. Replacement of the Glycosidic Oxygen

4. Mimicking Specific Functional Groups within Sugars – Sialylmimetics

5. Glycomimetics in Current Clinical Use as Anti-Infectives

6. Conclusions and Future Directions

Chapter 48. Bacterial polysaccharide vaccines

1. Introduction

2. Capsular Polysaccharide–Protein Conjugates

3. Synthetic Glycoconjugate Vaccines

4. Peptide-Mimetics of Polysaccharide Epitopes

5. Conclusions

Chapter 49. Immunomodulation by zwitterionic polysaccharides

1. Introduction

2. Immunomodulation by Polysaccharides

3. Zwitterionic Polysaccharides (ZPSs)

4. Activation of T-cells by ZPSs Through Innate and Adaptive Immunomodulation

5. Biological Impact of ZPS-Induced T-Cell Activation

6. Potential for ZPSs as Therapeutics

7. Conclusions

Chapter 50. Future potential of glycomics in microbiology and infectious diseases

1. Introduction

2. Advances in Carbohydrate Analytical Techniques

3. Future Outlook

Index

Copyright

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Dedication

To all those who contributed, knowingly or unknowingly, to this endeavour.

List of Contributors

Nawaf Al-Maharik

College of Life Sciences, Division of Biological Chemistry and Drug Discovery, University of Dundee, Dundee, UK

Heidi Annuk

School of Natural Sciences, National University of Ireland, Galway, Ireland

Michael A. Apicella

Department of Microbiology, University of Iowa, Iowa City, Iowa, USA

Ben J. Appelmelk

Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, The Netherlands

Ivo G. Boneca

Group Biology and Genetics of the Bacterial Cell Wall, Institut Pasteur, Paris, France

Petra Born

Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle upon Tyne, UK

Klaus Brandenburg

Division of Biophysics, Department of Immunochemistry and Biochemical Microbiology, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Borstel, Germany

Patrick J. Brennan

Department of Microbiology, Immunology and Pathology, College of Veterinary and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA

Volker Briken

Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA

Eric D. Brown

Department of Biochemistry and Biomedical Sciences and the Michael G. De Groote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada

Stephen D. Carrington

Veterinary Science Centre, University College Dublin, Belfield, Dublin, Ireland

Paola Cescutti

Dipartimento di Scienze della Vita, Università di Trieste, Trieste, Italy

Delphi Chatterjee

Department of Microbiology, Immunology and Pathology, College of Veterinary and Biomedical Sciences, Colorado State University, Fort Collins, Colorado, USA

Marguerite Clyne

Health Science Centre, School of Medicine and Medical Science, University College Dublin, Belfield, Dublin, Ireland

Richard M. Cooper

Department of Biology and Biochemistry, University of Bath, Bath, UK

Anthony P. Corfield

Mucin Research Group, University of Bristol, Bristol, UK

Monica M. Cunneen

School of Molecular and Microbial Biosciences, University of Sydney, New South Wales, Australia

Piet W.J. de Groot

Swammerdam Institute of Life Sciences, University of Amsterdam, Amsterdam, The Netherlands

Gennaro De Libero

Experimental Immunology, Department of Research, University Hospital, University of Basel, Basel, Switzerland

Clara G. de los Reyes-Gavilán

Departamento de Microbiología y Bioquímica de Productos Lácteos, Instituto de Productos Lácteos de Asturias, Consejo Superior de Investigaciones Científicas, Villaviciosa, Asturias, Spain

Tamara L. Doering

Washington University School of Medicine, St. Louis, Missouri, USA

Max Dow

BIOMERIT Research Centre, Department of Microbiology, University College Cork, Cork, Ireland

Nicole N. Driessen

Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, The Netherlands

Jeffrey C. Dyason

Institute for Glycomics, Gold Coast Campus, Griffith University, Queensland, Australia

Eva Maria Egelseer

Zentrum für NanoBiotechnologie, Universität für Bodenkultur Wien, Vienna, Austria

Alan D. Elbein

Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA

Mario F. Feldman

Alberta Ingenuity Centre for Carbohydrate Science, Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada

Eamonn Fitz Patrick

Veterinary Science Centre, University College Dublin, Belfield, Dublin, Ireland

Yukari Fujimoto

Department of Chemistry, Graduate School of Science, Osaka University, Osaka, Japan

Koichi Fukase

Department of Chemistry, Graduate School of Science, Osaka University, Osaka, Japan

Patrick Garidel

Institut für Physikalische Chemie, Martin-Luther-Universität Halle-Wittenberg, Halle/Saale, Germany

Jeroen Geurtsen

Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, The Netherlands

Marlyn Gonzalez

Department of Biology, Brooklyn College of City University of New York, Brooklyn, New York, USA

I. Darren Grice

Institute for Glycomics, Gold Coast Campus, Griffith University, Queensland, Australia

Patricia Guerry

Enteric Diseases Department, Naval Medical Research Center, Silver Spring, Maryland, USA

Thomas Gutsmann

Division of Biophysics, Department of Immunochemistry and Biochemical Microbiology, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Borstel, Germany

Holger Heine

Division of Innate Immunity, Department of Immunology and Cell Biology, Borstel Research Center, Leibniz-Center for Medicine and Biosciences, Borstel, Germany

Joanne Heng

Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia

Otto Holst

Division of Structural Biochemistry, Department of Immunochemistry and Biochemical Microbiology, Borstel Research Center, Leibniz-Center for Medicine and Biosciences, Borstel, Germany

Harold. J Jennings

Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada

Michael P. Jennings

School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Queensland, Australia

Margaret I. Kanipes

Department of Chemistry, North Carolina Agricultural and Technical State University, Greensboro, North Carolina, USA

Dennis L. Kasper

Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA

Wafa Khamri

Department of Nephrology and Transplantation, King’s College London School of Medicine, Guy’s Hospital Campus, London, UK

Milton J. Kiefel

Institute for Glycomics, Gold Coast Campus, Griffith University, Queensland, Australia

Dionne C.G. Klein

Biophysics and Medical Technology, Department of Physics, The Norwegian University of Science and Technology, Trondheim

Department of Cancer Research and Molecular Medicine, The Norwegian University of Science and Technology, Trondheim, Norway

Frans M. Klis

Swammerdam Institute of Life Sciences, University of Amsterdam, Amsterdam, The Netherlands

Yuriy A. Knirel

N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia

Thomas Kohler

Cellular and Molecular Microbiology Section, Medical Microbiology Department, University of Tübingen, Tübingen, Germany

Paul Kosma

Department of Chemistry, University of Natural Resources and Applied Life Sciences Vienna, Vienna, Austria

Emir Kulauzovic

Cellular and Molecular Microbiology Section, Medical Microbiology Department, University of Tübingen, Tübingen, Germany

Shoichi Kusumoto

Suntory Institute for Bioorganic Research, Osaka, Japan

John R. Lawrence

Aquatic Ecosystem Protection Research Division, Science and Technology Branch, Environment Canada, Saskatoon, Saskatchewan, Canada

Clifford A. Lingwood

The Hospital for Sick Children, Molecular Structure & Function Programme, Toronto, Ontario, Canada

Peter N. Lipke

Department of Biology, Brooklyn College of City University of New York, Brooklyn, New York, USA

Susan M. Logan

Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada

Radia Mahfoud

The Hospital for Sick Children, Molecular Structure & Function Programme, Toronto, Ontario, Canada

Malcolm J. McConville

Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia

Rachel M. McLoughlin

Channing Laboratory, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, USA

Paul Messner

Zentrum für NanoBiotechnologie, Universität für Bodenkultur Wien, Vienna, Austria

Antonio Molinaro

Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, Napoli, Italy

Anthony P. Moran

School of Natural Sciences, National University of Ireland, Galway, Ireland

Institute for Glycomics, Gold Coast Campus, Griffith University, Queensland, Australia

Thomas Naderer

Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia

Thomas R. Neu

Department of River Ecology, Helmholtz Centre for Environmental Research – UFZ, Magdeburg, Germany

Mari-Anne Newman

Department of Plant Biology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark

Andrei V. Nikolaev

College of Life Sciences, Division of Biological Chemistry and Drug Discovery, University of Dundee, Dundee, UK

Christian M. Pedersen

Fachbereich Chemie, Universität Konstanz, Konstanz, Germany

Mark P. Pereira

Department of Biochemistry and Biomedical Sciences and the Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada

Andreas Peschel

Cellular and Molecular Microbiology Section, Medical Microbiology Department, University of Tübingen, Tübingen, Germany

Stefan Pöhlmann

Institute of Virology, Hannover Medical School, Hannover, Germany

Robert A. Pon

Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada

Stuart A. Ralph

Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria, Australia

Peter R. Reeves

School of Molecular and Microbial Biosciences, University of Sydney, New South Wales, Australia

Anne N. Reid

Microbiology Research Division, Bureau of Microbial Hazards, Food Directorate, Health Products and Food Branch, Health Canada, Ottawa, Ontario, Canada

Colm J. Reid

Veterinary Science Centre, University College Dublin, Belfield, Dublin, Ireland

Morgann C. Reilly

Washington University School of Medicine, St Louis, Missouri, USA

Sabine Riekenberg

Division of Innate Immunity, Department of Immunology and Cell Biology, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, Borstel, Germany

Patricia Ruas-Madiedo

Departamento de Microbiología y Bioquímica de Productos Lácteos, Instituto de Productos Lácteos de Asturias, Consejo Superior de Investigaciones Científicas, Villaviciosa, Asturias, Spain

Nuria Salazar

Departamento de Microbiología y Bioquímica de Productos Lácteos, Instituto de Productos Lácteos de Asturias, Consejo Superior de Investigaciones Científicas, Villaviciosa, Asturias, Spain

Christina Schäffer

Zentrum für NanoBiotechnologie, Universität für Bodenkultur Wien, Vienna, Austria

Richard R. Schmidt

Fachbereich Chemie, Universität Konstanz, Konstanz, Germany

Ian C. Schoenhofen

Institute for Biological Sciences, National Research Council, Ottawa, Ontario, Canada

Marit Sletmoen

Biophysics and Medical Technology, Department of Physics, The Norwegian University of Science and Technology, Trondheim, Norway

Uwe B. Sleytr

Zentrum für NanoBiotechnologie, Universität für Bodenkultur Wien, Vienna, Austria

Evelyn C. Soo

Institute for Marine Biosciences, National Research Council, Halifax, Nova Scotia, Canada

Imke Steffen

Institute of Virology, Hannover Medical School, Hannover, Germany

Daniel C. Stein

Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland, USA

Bjørn T. Stokke

Biophysics and Medical Technology, Department of Physics, The Norwegian University of Science and Technology, Trondheim, Norway

Christine M. Szymanski

Department of Bio-logical Sciences, University of Alberta, Edmonton, Alberta, Canada

Joe Tiralongo

Institute for Glycomics, Gold Coast Campus, Griffith University, Queensland, Australia

Jennifer A. Tee

College of Life Sciences, Division of Biological Chemistry and Drug Discovery, University of Dundee, Dundee, UK

Mark R. Thursz

Gastroenterology and Hepatology Research, Faculty of Medicine, Imperial College, St Mary’s Campus, London, UK

M. Stephen Trent

Section of Molecular Genetics and Microbiology, University of Texas, Austin, Texas, USA

Theodros S. Tsegaye

Institute of Virology, Hannover Medical School, Hannover, Germany

Jean van Heijenoort

Institut de Biochimie et Biophysique Moléculaire et Cellulaire, Université Paris-Sud, Orsay, France

Waldemar Vollmer

Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle upon Tyne, UK

Mark von Itzstein

Institute for Glycomics, Gold Coast Campus, Griffith University, Queensland, Australia

Jennifer C. Wilson

Institute for Glycomics, Gold Coast Campus, Griffith University, Queensland, Australia

Guoqing Xia

Cellular and Molecular Microbiology Section, Medical Microbiology Department, University of Tübingen, Tübingen, Germany

Preface

Anthony P. Moran

Glycobiology can be said to be sweet biology. The full appreciation of the role of sugars, glycomolecules and glycosylated structures and their biological functions has been a more recent one compared to that of nucleic acids and proteins, particularly in the specialization of microbiology and related fields. Understanding has grown that monosaccharides represent an alphabet of biological information similar to amino acids and nucleic acids, but with a greater, and potentially unsurpassed, coding capacity. Though it has been predicted that micro-organisms can synthesize more sugar building blocks than their eukaryotic counterparts, e.g. for bacteria it is considered to be six-fold greater, this extensive coding capacity impacts the biological functioning of microbial molecules and also influences the interaction of microbes with their environment, including host structures.

The concept behind this book is to present, in an easy-to-read format, reviews of the important central aspects of microbial glycobiology, i.e. the study of carbohydrates as related to the biology of microorganisms. The importance of substitution of proteins by sugars (glycosylation) and the role played by glycosylated structures (glycoproteins, glycopeptides, glycolipids, lipoglycans, glycoconjugates, etc.) in disease development, immune recognition, and environmental processes have become well-established. Moreover, from the viewpoint of biotechnology industry, glycobiology, microbial glycobiology and microbial glycomics are important components. Microbial, especially bacterial, glycomes represent an excellent toolbox for glycobiologists to understand the fundamentals of glycosylation pathways, to develop new techniques for glycobiology, and to exploit glycosylation pathways for development of novel diagnostics and therapeutics. Despite the relevance of glycobiology in medical and environmental microbiology, and the potential to exploit this knowledge for industrial and medical processes, this field has only begun to be fully appreciated. In particular, glycomics – the applied biology and chemistry of the structures and functions of carbohydrates – and microbial glycomics – glycomics as related to microbial components – have become recognized as areas of emerging technological development. For instance, this area has been highlighted in the Massachusetts Institute of Technology Review as one of ten emerging technologies that will have a significant influence in the near future. Furthermore, it has been commented that the field of microbial glycobiology could fuel a revolution in biology and industry and aid biomedical development and drug discovery.

Indeed, the number of publications in this field has risen dramatically in recent years, making it extremely difficult for even the most diligent reader to stay abreast of progress. Additionally, in many areas of microbial glycobiology well-based and extensive reviews are lacking. Thus, we considered there was a major need to provide a book reviewing the range of topics relevant to microbial glycobiology since no such book has previously been available. It is our hope that this text distills the most important cutting-edge findings in the field to produce a timely and definitive overview, providing a useful introduction to the subject for new researchers, as well as an invaluable reference for experienced ones. Our goal has been to create a state-of-the-art compendium and to delineate the knowns and unknowns in the field.

Since microbial glycobiology represents a multidisciplinary and emerging area with implications for a range of basic and applied research fields, as well as having industrial, medical and biotechnological implications, care has been taken in the choice of topics to be covered. This volume cannot attempt to be completely comprehensive, but we believe, the central concepts and areas of intensive investigation have been covered as have aspects of the glycobiology of bacteria, viruses, fungi and protozoa. The approach has been to cover and link knowledge among microbiologists, synthetic and analytical chemists, biomedical and biopharmaceutical scientists and biotechnologists. The first section of the book introduces readers to the nature, structures and functions of glycomolecules and glycosylated components of microorganisms and infectious agents. This includes not only such components of bacteria but also those from viruses, fungi and protozoa. In the next section, the genetics and biosynthesis of these components, as well as the ability to chemically synthesize a number of these molecules are reviewed. The interaction with and recognition by the host of these molecules is considered subsequently, as are both the environmental and medical relevance of microbial glycosylated structures and glycomolecules. Finally, the biotechnological and medical applications of microbial glycosylation and glycosylated molecules is explored. Collectively, the chapters present basic science understanding of these molecules through to the applied science of exploitation and applications of microbial glycosylation, both industrially and biomedically.

We have been fortunate to have been joined by our colleagues, leaders in the field, who have contributed their ideas, experiences and insights in a free and open manner to yield what we believe are insightful, concise and stimulating chapters in a review format. The editors sincerely thank the numerous contributors of these chapters. Furthermore, special thanks is deserved by Mari Moran and Sharon Ackerman for their expert secretarial assistance during manuscript preparation at different stages of this project. Finally, and by no means least, we thank Lisa Tickner and Christine Minihawe for her unwavering support in making this text a reality, Kristi Anderson for her editorial skills, and Claire Hutchins, Caroline Jones and their team for their efforts during the production phase.

On behalf of the editors

Chapter 1. Overview of the glycosylated components of the bacterial cell envelope

Otto Holst, Anthony P. Moran and Patrick J. Brennan

Summary

Within this chapter, the various types of bacterial cell envelope and their carbohydrate-related molecules, as well as the glycoprotein S-layers that can be found in all bacteria except mycobacteria, are introduced. Whereas each of the Gram-positive, Gram-negative and mycobacterial cell envelopes possess a general and typical architecture, the archaeal cell envelope shows a broader variety of constructions and may be comprised of only a membrane. Apart from S-layers, carbohydrate-containing macromolecules like lipopolysaccharides, peptidoglycan, lipoteichoic acids, teichoic acids, capsule polysaccharides, lipoarabinomannan and others are briefly described. This chapter refers to other, more detailed, subsequent chapters that summarize the chemistry and biological function of the cell envelope macromolecules.

Keywords: Bacterial cell envelope; Lipopolysaccharide; Lipoteichoic acid; Teichoic acid; Lipoarabinomannan; Arabinogalactan; Peptidoglycan; Polysaccharide; Glycolipid; Glycoprotein

1. Introduction – The Bacterial Cell Envelope Encountering Environmental Challenges

Bacteria of various species populate most of the environments encountered on Earth. As a group, these microorganisms cope with very low, even down to −4°C, temperatures (psycrophilic bacteria), medium range temperatures (mesophilic bacteria), warm to hot temperatures (thermophilic bactera) or very hot, up to 115°C, temperatures (hyperthermophilic bacteria). Also, they exhibit an ability to survive and withstand an impressive range of pH, between pH 0.7 and 9 (acido- and alkaliphilic bacteria), to survive high pressures (deep sea, barophilic bacteria), or they may need or have to survive high or higher salt concentrations (halotolerant and halophilic bacteria) in their environments. Moreover, they may need to live and proliferate in eukaryotic hosts (symbiotic or pathogenic bacteria). In addition, bacterial species have developed the means to overcome longer periods of dryness and/or starvation or exposure to strong UV radiation. In order to survive in a particular niche, bacteria have developed a large variety of intracellular physiological adaptations; also, most possess an outer cellular barrier, the cell envelope, by which they communicate with and protect themselves from the environment they inhabit (Seltmann and Holst, 2001).

In nearly all genera of the domain Bacteria and in several of those of the domain archaeal, this layer is present which, by definition, consists of the cytoplasmic membrane (CM), the cell wall and, if present, outer layers such as capsules or sheaths. The outer barrier of other genera of the domain archaeal is formed by a particular membrane structure which contains ether-linked lipids that span the whole membrane and are highly resistant to temperature and pH (Seltmann and Holst, 2001).

When considering the broad variety of environments inhabited and encountered by bacteria, it is quite astonishing that only a few general architectural forms of cell envelope have evolved to cope with these varied conditions. The common architectural principle of the CM and cell wall is present in all bacterial envelopes in the four great variations of the envelope, i.e. Gram-negative bacterial, Gram-positive bacterial, mycobacterial and archaeal, and which may possess further adaptation even at the species level. Importantly, all cell envelopes possess a significant proportion of carbohydrate-containing constituents. This chapter serves to introduce the various types of cell envelope and their carbohydrate-related molecules and the glycoprotein S-layers that can be found in all bacteria except mycobacteria. As subsequent chapters will present more detailed reviews of the cell envelope-related macromolecules, their synthesis and applications, extensive literature will not be cited here, merely the relevant chapters will be indicated.

2. The Gram-Negative Cell Envelope

The Gram-negative bacterial world contains a broad variety of genera that may comprise only harmless species (e.g. phototrophic bacteria of genera like Rhodobacter or Rhodomicrobium) or both harmless and human- or animal-pathogenic species (e.g. Escherichia coli or Acinetobacter spp.) or plant-pathogenic species (e.g. Erwinia and Xanthomonas spp.). Independent of this, all Gram-negative bacteria possess a cell envelope of the same general architecture which is schematically depicted in Figure 1.1. The periplasmic space is present on top of the CM and contains peptidoglycan (PG), also known as murein, as a major constituent. This macromolecular sacculus is composed of sugar chains built from alternating 2-acetamido-2-deoxy-

d

-glucopyranose (GlcpNAc) and 2-acetamido-3-O-[(R)-1-carboxyethyl]-2-deoxy-

d

-glucopyranose, also termed N-acetylmuramic acid (MurNAc), residues which carry, and can be cross-linked by, smaller peptides – the amino acid composition of which varies in different species. The PG sacculus represents a rigid layer that determines the form of the bacterial cell and is important for osmotic stability and acts as a protective barrier. However, the PG sacculus is not a completely closed wall that inhibits transport of small molecules (e.g. nutrients) or their excretion. Although very stable, the PG matrix represents a mesh with holes large enough to guarantee the flow of molecules, including those that have to be transported to the outer membrane (OM). The three-dimensional architecture of PG has been an issue of discussion for a long time. The first model, which many scientists consider represents the correct one, is built from sugar chains that run in a parallel direction to the CM and which are interlinked by short peptide stems that are oriented perpendicular to this membrane. About ten years ago, an alternative, called the scaffold model, was proposed with perpendicular sugar chains connected by peptides that run parallel to the CM. It should be noted that there is no clear proof for either model to date. Importantly, the Gram-negative PG is a rather thin construction making its examination difficult. A detailed review of PG is given in Chapter 2 and of its biosynthesis in Chapter 16.

Apart from PG, the periplasmic space contains various smaller molecules like mono- and oligosaccharides, amino acids and peptides, as well as the biosynthetic precursors and degradation products of PG. The concentration of all these substances is rather high, thus the periplasmic space represents a highly viscous solution that can be considered a gel-like matrix.

Outside the periplasmic space is located a second membrane, the OM, which had been considered unique to Gram-negative bacteria for a long time. Nevertheless, the presence of an analogous structure in mycobacteria has been postulated for quite some time and whose presence has finally been verified recently (see below) (Hoffmann et al., 2008). Both leaflets of the OM represent lipid bilayers that are organized asymmetrically, i.e. the inner leaflet is composed of different molecules compared to the outer one. In the case of the OM of Gram-negative bacteria, the inner leaflet is comprised of phospholipids, whereas the outer leaflet is mainly constructed from lipopolysaccharides (LPSs) (Holst and Müller-Loennies, 2007). In some cases, polysaccharide capsules are present that may be anchored by a lipid into the OM and this is also true for the enterobacterial common antigen (ECA) of enterobacteria. In addition, the OM contains various proteins (outer membrane proteins, OMPs), constituting up to 50% of the membrane and which often interact with LPS molecules, thereby yielding particular lipid–protein structural units. Several of the OMPs are channel-formers and are involved in diffusion of ions (e.g. diffusion of phosphate through PhoE channels in E. coli) and transport of mono- and small oligosaccharides (e.g. specific-channel forming proteins, like LamB that is involved in transport of maltose and maltodextrins) and, thus, they play important roles in the uptake of nutrients. Other OMPs represent structural proteins like OmpA and related proteins in various enterobacterial species and Braun’s lipoprotein, also known as PG-associated lipoprotein, in E. coli and related species. The latter is covalently linked to PG and has its lipid moiety embedded in the inner leaflet of the OM, thus interconnecting PG and OM and providing structural stability.

Molecules of LPS are of high relevance not only for Gram-negative bacteria but also for an infected, eukaryotic host. In bacteria, these molecules are part of the protective barrier shielding the microbes from dangerous environmental compounds, like bile salts in the gut or antibiotics in sensu latu. In contrast, domains within LPS, specifically the core region and the O-specific polysaccharide, can also act as receptors for bacteriophages, thereby contributing indirectly to the destruction of the bacterial cell in such cases. In pathogenic bacteria that infect either humans or animals or plants, LPSs represent very important virulence factors. Moreover, this family of molecules is also called the endotoxins of Gram-negative bacteria; however, their toxicity is highly dependent on their structural properties, and it should be noted that not all LPSs are toxic molecules, not even those from pathogens. Importantly, in many chronically infecting bacterial species (e.g. Helicobacter pylori, Porphyromonas gingivalis, etc.), LPSs are of low toxic and immunological activity and this attribute is considered to aid the development of chronicity (Moran, 2007).

Chemically, LPSs, as exemplified by those of the Enterobacteriaceae, are lipoglycans. Three regions within LPS can be distinguished, according to their chemical structure, biosynthesis, genetics and function, i.e. the lipid A component (that anchors the whole molecule in the OM and which represents the endotoxically active moiety of toxic LPS), the core region which is covalently linked to lipid A and which may modulate lipid A toxicity and, finally, the O-specific polysaccharide which is covalently linked to the core region and represents a highly antigenic structure which is also called the O-antigen. Whereas the core region is comprised of an oligosaccharide of up to ≈15 sugars, the O-antigen is a polysaccharide mostly composed of repeating units containing 2–8 monosaccharide residues. In general, between one and about 40 repeating units are found, but this glycan may also be much longer. When all three regions are present in LPS, the resulting high-molecular-mass molecule is referred to as smooth- (S-) form LPS and is normally encountered in wild-type enterobacterial strains. A low-molecular-mass, rough- (R-) form LPS that lacks the O-antigen, thus consisting only of the lipid A and core regions, occurs in many laboratory-derived enterobacterial strains. Resembling this phenotype, but still a member of the LPS family of molecules, are the so-called lipo-oligosaccharide (LOS) molecules encountered in the wild-type strains of certain mucosal pathogens in particular (e.g. Bordetella pertussis, Haemophilus influenzae and Campylobacter jejuni). These molecules, composed of core and lipid A regions, exhibit greater structural variability in the core oligosaccharide than encountered in enterobacterial R-form LPS. Chemical and structural details of LPSs are discussed in Chapter 3 and Chapter 4, their biosyntheses in Chapter 17 and Chapter 18, chemical syntheses in Chapter 24 and Chapter 25 and functional aspects in Chapter 31.

As mentioned earlier, capsules composed of capsular polysaccharides (CPSs) may be anchored to the OM. Generally, it is considered that this is achieved by a glycerolipid structure, however, such a structure has only been identified in a few cases (e.g. E. coli, C. jejuni) (Schmidt and Jann, 1982; Corcoran et al., 2006). In other cases, CPSs have been found linked to the core region of R-form LPS, yielding an S-form-like LPS structure which is referred to as KLPS. Thus, probably all CPSs represent or are analogous to lipoglycans that, in many cases, contain repeating units like O-antigens. Consequently, both structures can only be distinguished by their genetics.

Rather than considering capsules of CPS as amorphous structures, since they are composed of 90% water they provide a gel-like hydrated mesh that protects the bacteria from dehydration. Polysaccharide capsules are present in many pathogenic bacteria (e.g. E. coli, Klebsiella pneumoniae, C. jejuni, etc.), in which CPSs function as important virluence factors, including aiding bacterial evasion of the host’s defence system. The CPSs often represent antigenic structures called K-antigens and they are also involved in biofilm production and/or influence bacterial colonization. In plant–bacterial interactions, CPSs play also an important role, independent of whether the process is pathogenic or symbiotic.

A special case of CPS is that of ECA where the structure comprises the repeating unit →3)-α-

d

-FucpNAc-(1→4)-β-

d

-ManpNAcA-(1→4)-α-

d

-GlcpNAc-(1→ (where

d

-FucpNAc, 2-acetamido-2-deoxy-

d

-fucopyranose;

d

-ManpNAcA, pyranosidic 2-acetamido-2-deoxy-

d

-mannuronic acid;

d

-GlcpNAc, 2-acetamido-2-deoxy-

d

-glucoyranose). Other polysaccharide structures belong to the so-called exocellular polymeric structures, also termed exopolysaccharides. Such molecules are often found in the surrounding milieu of bacteria (e.g. in the culture supernatant) and have importance in biofilm formation or in a variety of bacteria–host interactions. Mostly, exopolysaccharides are composed of repeating units of oligosaccharides. Chapter 6 provides an overview of CPS and these related molecules.

Importantly, all bacterial (lipo)glycans represent structures that are located in or on the outer bacterial surface and are synthesized in the cytosol and at the inner leaflet of the CM. Thus, bacteria have to deal with the great challenge of transport of hydro- or amphiphilic macromolecules through their lipid-like cell walls.

In the biosynthesis of LPS, which is best understood in E. coli, the syntheses of lipid A and the core oligosaccharide are genetically and biochemically connected. Beginning with uridine diphosphate- (UDP-) GlcpNAc, the tetra-acylated and bisphosphorylated lipid A precursor lipid IVa is synthesized in a series of six steps. Prior to the addition of the fifth and sixth fatty acid, two residues of 3-deoxy-

d

-manno-oct-2-ulopyranosonic acid (Kdo) are transferred by one Kdo-transferase and yield the disaccharide α-Kdo-(2→4)-α-Kdo which is linked to position C-6′ of β-

d

-GlcpNAcyl-3-O-Acyl-4-P-(1→6)-α-

d

-GlcpNAcyl-3-O-Acyl-1-P. Subsequently, the core region is completed by a series of glycosyltransferases that transfer activated monosaccharide precursors. This all occurs at the inner leaflet of the CM. Such completed R-form LPS is then transported (or flipped) from the inner to the outer leaflet of the CM, a process which is achieved by the adenosine triphosphate- (ATP-) binding cassette (ABC)-transporter, MsbA. There, on the periplasmic side, the completed O-antigen is transferred to the core oligosaccharide and the completed LPS is then transferred to the OM. The molecular basis for this translocation process still remains an enigma (for more details see Chapter 17).

The biosynthesis of heteropolymeric O-antigen and of many such CPSs (e.g. E. coli capsule groups 1 and 4) follow the same general principle (see Chapter 18). The repeating units, built by the sequential action of glycosyltransferases, are linked to an undecaprenyl-phosphate carrier lipid. Lipid-linked repeats are then flipped across the CM and polymerized on the periplasmic side. The O-antigen (and some CPS) are attached to lipid A-core molecules by a ligase.

With regard to the further transport of CPS to the surface, a number of participating components involved in the translocation of group 1 capsules and their interaction have been elucidated recently, confirming the earlier concept of zones of adhesion, or Bayer’s fusion sites, published in the early 1980s (Bayer, 1981). These CPS molecules are transported to the surface via a complex of the two proteins Wzc and Wza, the latter of which is a channel-forming protein. Biosynthesis and export of E. coli group 2 and 3 capsules proceeds differently. Briefly, the polysaccharides are completely synthesized at the cytosolic surface of the CM, transferred to a diacylglycerophosphate anchor and the whole lipoglycan is translocated through the CM, by the ABC-transporter called KpsM, and through the periplasm and OM, by the protein complex of KpsE and KpsD. A review of the biosynthesis and assembly of CPSs is presented in Chapter 20.

3. The Gram-Positive Cell Envelope

As in Gram-negative bacteria, the Gram-positive group of bacteria comprise many harmless species and several important pathogenic ones, e.g. Streptococcus pneumoniae, Staphylococcus aureus, Enterococcus faecalis and Clostridium tetani. The Gram-positive cell envelope (Figure 1.2) is organized more simply than that of Gram-negative bacteria, but is as effective. The cell wall, layered on top of the CM, is mainly composed of a thick layer of PG, generally 20–80nm thick, compared to a≈7nm PG in Gram-negative bacteria. The general chemical structure of this PG is similar to that of Gram-negative bacteria. In addition, the overall conformation, i.e. 3-dimensional architecture of this PG, is discussed using the same bases of the two models mentioned above for Gram-negative PG.

The Gram-positive cell envelope does not contain a second membrane, i.e. the outer membrane as seen in Gram-negative bacteria. However, CPSs are often present in Gram-positive bacteria and represent, in many cases of pathogenic species, important virulence factors with similar functions as those mentioned for Gram-negative species. Moreover, the structures of CPSs of Gram-positive and -negative bacteria are generally similar. Again, in the former bacterial group, polysaccharide capsules are layered over the PG, but it remains unclear whether they are connected to the cell wall and, if so, how this is achieved. As far as has been investigated, Gram-positive CPSs are synthesized in a similar manner as E. coli group 1 capsules. Moreover, Gram-positive bacteria also produce exopolysaccharides which often are components of biofilms.

Three major carbohydrate structures are present in the Gram-positive cell envelope, namely the lipoteichoic acids (LTAs), wall teichoic acids (WTAs) and wall polysaccharides (WPSs). Several LTAs and WTAs are structurally very similar in their polymeric chain, which does not represent a classical polysaccharide but is, instead, produced from phosphodiester-bridged alditol units (of glycerol or ribitol) that are often substituted by sugars. Both macromolecules may contain

d

-alanine (

d

-Ala) which, in LTA, may substitute both the sugar(s) (as in E. faecalis) and the alditol residues. In LTA, this polymeric chain is linked to a short oligosaccharide which serves as a linker and binds the chain to a diacylglycerophosphate lipid anchor that is embedded in the CM. Thus, LTA molecules protrude through the PG matrix and reach the outer surface. The LTAs are considered immunomodulatory constituents of Gram-positive bacteria that react with cells of the innate immune system via Toll-like receptor (TLR-) 2 and induce cytokine expression. However, this view is heavily debated (Zähringer et al., 2008).

In WTA, the polymeric chain is also bound to a linker, which is a disaccharide composed of 2-acetamido-2-deoxy-mannopyranose (ManpNAc) and GlcpNAc that is covalently linked by a phosphodiester bridge to C-6 of MurNAc of PG. Both WTAs and LTAs play important roles in interactions with host receptors, maintaining cation homeostasis and providing physicochemical surface properties.

The biosyntheses of WTAs and LTAs occur in cyclic processes. In the case of WTA of Bacillus subtilis, the binding unit is synthesized first, linked to a lipid carrier. Subsequently, the glycerophosphate units are attached by a polymerase and part of this chain is subsequently substituted with glucopyranose (

d

-Glcp). The polymer is transported from the inner to the outer leaflet of the CM by means of a two-stage ABC-transporter. Then, other free hydroxyl-groups of the glycerol are substituted by

d

-Ala. Finally, the completed molecule is linked to MurNAc of PG. On the other hand, the biosynthesis of LTA begins with that of the lipid anchor, i.e. 1,2-diacylglycerol (e.g. in S. aureus), which is substituted with two residues of

d

-Glcp. To this linker, glycerophosphate units are attached up to the required chain length. Any further substituents can be incorporated during or after chain elongation. Chapter 5, Chapter 19 and Chapter 25 describe the structures and syntheses, both biological and chemical, of WTA and LTA in detail.

Another class of polysaccharides, WPS, namely the teichuronic acids, are usually synthesized in greater amounts under phosphate starvation. They are polysaccharides composed of at least one residue of uronic acid, the negative charge of which helps to overcome the lack of phosphate charges, e.g. in metal cation binding. Teichuronic acids are also linked by a phosphodiester bridge to MurNAc of PG via a linker structure. Their biosynthesis occurs in a cyclic process which involves a lipid carrier to which the linker is synthesized first, followed by transfer of the monosaccharide units and then polymerization.

4. The Mycobacterial Cell Envelope

Many mycobacteria live in soil and water and are harmless members of the genus, but three species are well known as human and animal pathogens, i.e. Mycobacterium tuberculosis, Mycobacterium leprae and the Mycobacterium avium–Mycobacterium intracellulare complex. In the host, these species live intracellularly, in particular in the phagosome of the most-feared defensive cell of the innate immune system, the macrophage. Such intracellular mycobacteria inhibit phagosome–lysosome fusion of macrophages and may survive for an extended period intracellularly. For this lifestyle, mycobacteria need a well-constructed protection barrier that renders them resistant to the various defence activities of eukaryotic hosts. Although mycobacteria yield slightly positive reactions in the Gram stain, the mycobacterial cell envelope (Figure 1.3) possesses more Gram-negative than -positive features, i.e. a less extensive PG matrix and an outer lipid bilayer which has been identified as like an outer membrane, the mycobacterial outer membrane (MOM) (Hoffmann et al., 2008). Apart from such general similarities, the mycobacterial cell envelope represents a unique barrier that should be clearly distinguishable from other bacterial membrane barriers. The PG matrix is located on top of the CM and is organized similarly to those of Gram-positive and -negative bacteria.

As in other cell envelopes, the mycobacterial barrier contains several important glycan structures, namely; lipoarabinomannan (LAM) and its biosynthetic precursors [the lipomannan (LM) and the phosphatidylinositol-mannosides (PIM)], the mycoloyl-arabinogalactan (MAG) complex and a capsular structure which comprises the polysaccharide arabinomannan (AM) (Daffé and Draper, 1998; Wittkowski et al., 2007) and glucan (with proteins). Detailed overviews of the structures and functions of these components and their biosynthesis are presented in Chapter 9 and Chapter 21.

The LAM is a lipoglycan which is anchored in the CM by a phosphoinositol moiety that carries as characteristic fatty acids 9-hexadecenoic acid (16:1, palmitoleic acid) and 10-methyl-octadecanoic acid (tuberculostearic acid). In particular, bound to the inositol residue is an α-(1→6)-linked

d

-mannan main chain with α-(1→2)-linked mannopyranose (Manp) branches that are substituted by a longer branched arabinan composed of arabinofuranose (Araf) residues at the second last Manp residue. The whole molecule is anchored in the CM by its lipid moiety and protrudes through the PG to the outside of the cell wall, similar to the LTA of Gram-positive bacteria. Here, at its non-reducing end, it may carry Man residues that substitute the terminal Ara sugars in monosaccharidic and disaccharidic substitutions. There is evidence also that part of the LAM is anchored in the MOM, probably in the outer leaflet, but proof to date is lacking. Mycobacterial LAM has been reported to possess important functions in the pathogenesis of mycobacteria, e.g. induction of phagocytosis of these microbes, phagosome–lysosome fusion or activation of innate immune responses. Nevertheless, some of the reports in these areas are conflicting.

The biosynthesis of LAM has not yet been completely elucidated. As a general pathway, it is believed that biosynthesis starts with PIM and that chain elongation to obtain the mannan chain proceeds from the Manp residue at O-6 of the inositol that builds up to yield LM. The molecule known as PIM4, PIM containing Manp residues, is the likely precursor here. In the last step, the arabinan, a main chain of α-(1→5)-linked

d

-Araf residues that may be branched at the non-reducing end, is produced and linked to LM.

The MAG is, as are the TA of Gram-positive bacteria, covalently linked via a disaccharide linker composed of

l

-rhamnopyranose (

l

-Rhap) and

d

-GlcpNAc and a phosphodiester bridge to C-6 of MurNAc of PG. The linker is substituted by a galactan chain, which is furnished exclusively from

d

-galactofuranose (

d

-Galf) residues in alternating β-(1→5) and β-(1→6)-linkages, a polysaccharide feature that is rarely found in Nature. Quite early in this chain, branches of arabinan chains, i.e. α-(1→5)-linked

d

-Araf residues, are present that protrude to the outer surface of the cell wall. At their reducing ends, a so-called hexa-arabinosyl motif is present as a branched structure, the terminal arabinose residues of which are substituted at C-5 by very long chain (up to 90 C-atoms) α-alkyl-α-hydroxy-fatty acids, the so-called mycolic acids that occur in a broad range of structural varieties (Barry et al., 1998). These mycolic acids form the inner leaflet of the MOM, whereas the outer leaflet is believed to be composed of various lipids, including glycolipids, different types of which are present in different mycobacterial species (e.g. mycobacterial LOSs and glycopeptidolipids). The MAG complex, together with the MOM, represents cell envelope structures of mycobacteria that are highly important for cell wall architecture and rigidity, as well as protection. Thus, MAG (and LAM) biosynthesis represent potentially important drug targets.

The biosynthesis of the arabinogalactan proceeds stepwise, beginning with the linker and followed by the galactan chain which are linked to a decaprenylphosphoryl (C50-P) lipid. The arabinan chains are attached by the sequential addition of Araf residues that are transported as C50-P-Araf units.

The mycobacterial capsule has a different composition to those of Gram-negative and -positive bacteria; namely, it is not a purely polysaccharide capsule, but contains proteins, including glycoproteins, and mainly two polysaccharides, an arabinomannan and a glucan. The arabinomannan possesses a similar structure to the polysaccharide of LAM. So far, it remains unclear how this glycan is synthesized and transported to the outer surface. Although it has been reported several times to the contrary, a recent investigation shows that this molecule has no immunomodulatory properties. Instead, it has been shown to activate natural killer cells that then destroy tumour cells. Thus, the AM possesses cytotoxic properties. The glucan is structurally similar to glycogen, but no particular biological functions have been described so far.

5. The Archaeal Cell Envelopes and S-Layers

As mentioned above, the archaeal cell envelope structures cannot be classified as a common architectural type as is the case with the other envelope types described thus far. Instead, the cell envelope shows a number of structural variants which are adapted to the particular or extreme living conditions.

Archaea can be subdivided into the kingdoms Euryarchaeota and Crenarchaeota, any further classification of which occurs according to the physiological properties of the organism in question. All members possess a membrane which, in several cases, consists of a tetra-ether lipid that spans the whole width of the membrane, i.e. no bilayer is present. Here, this membrane represents the whole cell envelope. A so-called pseudomurein represents the cell-stabilizing component in Gram-positive Archaea. In other genera, a relatively thick layer of acidic polysaccharides constitute the cell wall. Finally, nearly all Archaea, and also many Gram-negative and -positive Eubacteria, contain cell surface layers (S-layers) which thus represent common structures of the prokaryotic cell envelope. S-layers appear in lattice-like arrangements that are formed by a self-assembly process and are attached to the bacterial cell wall by certain cell wall polymers. Also, the outer region of quite many S-layer proteins is often substituted by covalently linked glycan chains and, thus, glycoproteins are formed. The understanding of the mode of interaction between S-layer (glyco)proteins and secondary cell wall polymers is believed will pave the way towards the production of novel nanopatterned biomaterials for various applications in the fields of biomedicine and nanobiotechnology. Chapter 7 details the structure, function and applications of S-layer structures.

6. Conclusions

The cell envelope represents the outermost layer of the bacterial cell which has as general functions the protection of the cell, communication with the environment, maintenance of cellular shape, stability and rigidity of the cell, as well as allowing appropriate metabolism, growth and division of the cell. Important components of the cell envelope are carbohydrate-based and carbohydrate-containing macromolecules that contribute significantly to all of these functions, independent of which variation of the cell envelope occurs, i.e. Gram-positive, -negative, mycobacterial or archaeal. The structures, syntheses and functions of all these macromolecules are described