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Microbial Adhesins, Agglutinins & Toxins Victor Nizet, MD UCSD School of Medicine May 11, 2004 Essentials of Glycobiology Lecture 26 QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Microbial Adherence to Host Epithelium QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Adherence to skin or mucosal surfaces is an fundamental characteristic of the normal human microflora Mucosal adherence is also an essential first step in the pathogenesis of many important infectious diseases QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Most microorganisms express more than one type of adhesive factor “Adhesins”: Microbial Proteins that Mediate Adhesion to Host Cells • • • • Many adhesins are lectins Some bind to terminal sugars, others bind to internal carbohydrate sequences Direct adherence interactions: (surface glycolipids,glycoproteins, or glycosaminoglycans) Indirect adherence interactions: (matrix glycoproteins, mucin) adhesins in the bacterial cell wall QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. adhesin receptor host cell membrane Pili (“hair”) and Fimbriae (“Threads”) QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Lateral mobility of adhesin structure in bacterial membrane provides a VelcroTM-like effect Pili/Fimbriae Intimin Pedestal Host b-integrin Major subunit (pili) Tip adhesin Host glycolipid or glycoprotein Actin polymerization Host cell surface protein/carbohydrate Host cell membrane P Secreted Hp 90 Afimbrial adhesins Host Cell Receptors • Animal cells express “receptors” (carbohydrate ligands) for adhesins of microbes • Receptors can be glycolipids, glycoproteins, or proteoglycans • Tissue tropism is determined by the array of adhesin-receptor pairs Microbial Binding to Glycoproteins = Sialic acid N-LINKED CHAIN O-LINKED CHAIN GLYCOSPHINGOLIPID S O Ser/Thr N Asn OUTSIDE CELL MEMBRANE INSIDE Glycoprotein glycans are displaced away from the membrane compared to glycolipids, which may make them less effective as microbial receptors Measuring Adhesin-Receptor Interactions Hemagglutination Cell Binding Assays + Binding _ QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Bacteria • Use mutant cells or nutritionally manipulate composition • Competition experiments with soluble carbohydrates • Remove receptor with exoglycosidases • Regenerate different receptor with glycosyltransferase Bacterial overlay Binding Measurements Host glycoproteins Host glycolipids Overlay methods: Challenge microorganisms to bind immobilized carbohydrate receptors Can use tissue sections, TLC plates, PAGE blots Using a centrifuge, you can measure the strength of binding in g-force Polyacrylamide gel electrophoresis Thin-layer chromatography Examples of Bacterial Adhesins Binding Host Glycans Adhesin Protein Bacterial Species Target Tissue Carbohydrate Ligand on Host Cell PapG (P-pilus) Escherichia coli Urinary Gala4Galb- in glycolipids SfaS (S-pilus) Escherichia coli G.I. Tract Siaa3Galb4GlcbCer FimH (Type 1 Escherichia coli G.I. Tract Mannose-oligosaccharides Haemophilus Respiratory Sialylyganglioside-GM1 pilus) HifE influenzae FHA Bordetella pertussis Respiratory Sulfated glycolipids, heparin BabA Helicobacter pylori Stomach [Fuca2]Galb3[Fuca4]GlcNAc (Leb)- Hs Antigen Streptococcus gordonii Respiratory a2-3-linked Sia-containing receptors Opc adhesin Neisseria meningitides Respiratory Heparin sulfate proteoglycans assembly of pilus organelle adhesin units at end of pilus QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. surface localization fiber formation Electron microscopic image of E. coli expressing surface pili pilus pilus adhesive tip QuickTime™ and a tip receptor TIFF (Uncompressed) decompressor are needed to see this picture. host cell new adhesive tip QuickTime™ and a tip receptor TIFF (Uncompressed) decompressor are needed to see this picture. alternate host cell Structure of Two E. coli Pili Subunits Glycan binding site bladder ureter QuickTime™ and a P pilus TIFF (Uncompressed) decompressor are needed to see this picture. cell membrane QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. PapG PapG+ E. coli binding to bladder epithelium FimH Glycoprotein receptor Bordetella pertussis : Agent of “Whooping Cough” Filamentous hemagglutinin (FHA) QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompress ed) dec ompres sor are needed to s ee this pic ture. WT bacteria cilia QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. nonciliated cells FHA Epithelial cell adherence Helicobacter pylori QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. H. Pylori surface BabA protein (blood group antigen-binding adhesin) Binds to carbohydrate blood-group antigen Lewis B (LeB) on MUC5AC glycoprotein expressed in mucusproducing gastric epithelium How host glycans may affect the destiny of H. pylori colonization: QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Hooper & Gordon (2001) Glycobiology 11:1R Influenza • Acute repiratory tract infection spread from person-to-person by respiratory droplets. 1917 PANDEMIC QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. • ~ 20,000 deaths and110,000 hospitalizations in U.S. annually. • Enveloped, single-stranded RNA virus of family orthomyxoviridae. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. • Typical symptoms are fever, dry cough, sore throat, runny or stuffy nose, headache, muscle aches,and extreme fatigue. Nov-Apr Year-round Apr-Nov Structure of Influenza Virus Ion Channel QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Hemagglutinin QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. RNP Capsid Lipid Envelope Neuraminidase (sialidase) Variation of Influenza Viruses Point Mutations of Hemagglutinin and/or Neuraminidase Gene (Antigenic Drift) Human H2N2 Human H3N2 Genetic Reassortment (Antigenic Shift) Avian H3N8 Influenza Hemagglutinin Binds Sialic Acid QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. –Flu A binds to a2,6 sialic acids –Flu B binds to a2,3 sialic acids –Flu C prefers 9-O-acetylated sialic acids Influenza HA-Mediated Membrane Fusion Target membrane HA1 HA1 Low pH HA2 Fusion peptide Target membrane Fusion peptide HA2 Viral membrane Neutral form QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Viral membrane Low pH form Crystal structures Predicted anchors Influenza: Interactions with Sialic Acid BINDING & ENTRY BUDDING & RELEAS Influenza: Why the Neuraminidase? (explanation for handout) Neuraminidase (NA) is found in the envelope of the influenza virus. It degrades sialic acid. However, sialic acid serves as the eukaryotic cell receptor for the hemagglutinin (HA) of influenza virus. Is this not a paradox? A balance between HA and NA activities is necessary because of the complex life cycle of influenza. Remember that sialic acid is found in mucus, and is also present in the envelope of the influenza virus as it buds from the infected host cell membrane. The mucus could act as a nonproductive receptor for the virus, while the sialic acid in the envelope would cause auto-agglutination mediated by the hemagglutinin. Also without neuraminidase, budding viruses would stick to the host cell and not be released to infect other host cells. Neuraminidase acts to circumvent these competing reactions while not being so active as to destroy the cell surface receptor. O O OH HN O QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. NH2 Oseltamivir carboxylate (a sialic acid analogue) QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Malaria (Plasmodium) Infections QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. P. vivax merozoite QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Duffy binding protein Duffy blood group antigen glycoprotein QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Sialic acid residues on glycophorin A EBA-175 QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. P. falciparum merozoite Malaria Invasion of Host Erythrocytes (explanation for handout) The human malaria parasite, Plasmodium vivax, and the simian malaria parasite, P. knowlesi, are completely dependent on interaction with the Duffy blood group antigen for invasion of human erythrocytes. The Duffy blood group antigen is a 38-kD glycoprotein with seven putative transmembrane segments and 66 extracellular amino acids at the N-terminus. The binding site for P. vivax and P. knowlesi has been mapped to a 35-amino-acid segment of the extracellular region at the N-terminus of the Duffy antigen. Unlike P. vivax, P. falciparum does not use the Duffy antigen as a receptor for invasion. Initial studies identified sialic acid residues of glycophorin A as invasion receptors for P. falciparum. A 175-kD P. falciparum sialic acid binding protein, also known as EBA-175, binds sialic acid residues on glycophorin A during invasion. Some P. falciparum laboratory strains use sialic acid residues on alternative sialo-glycoproteins-such as glycophorin B-as invasion receptors. The use of multiple invasion pathways may provide P. falciparum with a survival advantage when faced with host immune responses or receptor heterogeneity in host Examples of Glycosphingolipid Receptors for Bacterial Toxins Toxin Cholera toxin Microorganism Vibrio cholerae Tissue Small intestine Proposed Receptor Sequence Galb3GalNAcb4(NeuAca3)Galb4Gl cbCer (GM1 ganglioside) Heat-labile Escherichia coli Intestine toxin Tetanus toxin cbCer (GM1 ganglioside) Clostridium tetani Nerve G1b gangliosides (GT1b most membrane efficient) Nerve (+NeuAca8)NeuAca3Galb3GalNacb botulinum membrane 4 (NeuAca8NeuAca3)Galb4GlcbCer Clostridium difficile Large intestine GalNAcb3Galb4GlcNacb3Galb4Glc Botulinum toxin Clostridium Toxin A Galb3GalNAcb4(NeuAca3)Galb4Gl bCer Cholera • Acute bacterial infection caused by ingestion of water contaminated with Vibrio cholerae 01 or 0139. • Sudden watery diarrhea and vomiting can result in severe dehydration. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. • Left untreated, death may occur rapidly, especially in young children. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Cholera Toxin: Structural Features QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. AB5 Hexameric Assembly Cholera Toxin Receptor: GM1 Ganglioside GM1 Cholera Toxin A-subunit B-subunits (5) GM1 GM1 GTP-binding protein Adenylate cyclase NAD+ ADP-Ribose ATP ADP-Ribose cAM Cholera Toxin Biologic Effect Cholera toxin Anion Secretion (+) protein AT P phosphorylation Neutral NaCl Absorption CT receptor (GM1 ) (-) Adenylate cyclase Cholera toxin A subunit QuickTime™ and a Cinepak decompressor are needed to see this picture. Cholera Toxin Mechanism of Action (explanation for handout) Cholera toxin is a protein molecule comprised of a beta subunit (consisting of 5 noncovalently linked molecules) and an alpha subunit (containing 2 peptides, alpha 1 and 2) and having a molecular weight of ~84,000. The 5 beta subunit proteins are arranged in a circular fashion, and appear to be important for the binding of cholera toxin to a specific membrane receptor called GM1-ganglioside, found in the luminal membrane of enterocytes. The alpha 1 subunit then enters the cell by a mechanism which has not been fully defined. The alpha 1 subunit irreversibly activates adenylate cyclase located in the basolateral membrane, initiating the formation of cyclic AMP from ATP. The large increases in cellular cyclic AMP activate a cascade of biochemical events which ultimately cause phosphorylation of several proteins which may be important in the regulation of intestinal salt and water transport or are themselves transport proteins. The final effect is an inhibition of neutral Na/CI absorption and a stimulation of anion secretion, causing luminal accumulation of fluid and diarrhea. Clostridium Botulinum Toxin: A Paralytic QuickTime™ and a TIF F (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. ? Quic kTime™ and a TIFF (Unc ompres sed) dec ompres sor are needed to see this pic ture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. BOTULINUM TOXIN BINDING QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Double receptor model: First receptors are gangliosides with more than one neuraminic acid, e.g. GT1b Type of binding: Lock & Key; Little or no change in conformation of bound botulinum neurotoxin Role: Bring toxin into proximity with second receptor QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. Second receptor: Postulated to be integral membrane protein Large Clostridial Cytotoxins Toxins A and B from Clostridium difficile (antibioticassociated diarrhea, pseudomembranous colitis) Hemorrhagic and lethal toxins of C. sordellii and atoxin of C. novyi (enterotoxemia and gas gangrene) These toxins turn out to be glucosyltransferases Catalytic Translocation Binding Large Clostridial Cytotoxins Modification of target proteins by glucosylation Targets include Rho (cytoskeletal organization), Ras (growth control), Rac, cdc42 and other GTPases Busch & Aktories (2000) COSB 10:528 Microbes that Bind Proteoglycans on Host Tissue Microbe Target Tissue Bordetella pertussis Ciliated epithelium in respiratory tract Chlamydia trachomatis Eyes, genital tract, respiratory epithelium Haemophilus influenzae Respiratory epithelium Borrelia burgdorferi Endothelium, epithelium, extracellular matrix Neisseria gonorrhea Genital tract Staphylococcus aureus Connective tissues, epithelial cells Mycobacterium tuberculosis Respiratory epithelium Plasmodium falciparum Heaptocytes, placenta (circumsporozootes) Leishmania amazonensi (amastigotes) Macrophages, fibroblasts, epithelium Herpes simplex virus (HSV) Mucosal surfaces of mouth, eyes, genital tract Dengue flavivirus Macrophages? Herpes Simplex Virus Infection QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. gC Herpes Simplex Entry • Herpes simplex virus uses heparan sulfate as a coreceptor, infection requires both proteoglycan and a protein receptor of the HVE class • Fusion of the viral envelope with the host membrane also requires heparan sulfate and other viral proteins Binding Cell membrane Cell surface proteoglycans (heparan-sulfate) Binding gD QuickTime™ and a TIFF (Uncompressed) decompressor HVEM/TNF/NGF receptor family to see this picture. are needed gB and others (gH - gL) Membrane fusion Penetration Uncoat genome Nuclear pore Virus-mediated Intracellular transport Nucleus aTIF Viral DNA Flaviviruses: Dengue and West Nile QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Flavivirus Adhesin Model E-glycoprotein is the viral hemagglutinin and mediates host cell binding. Example of a relatively non-specific binding site (hydrophilic FG region), which interacts with many heparan sulfate sequences with variable affinity Exogenous heparin can block flavirus infectivity. Foot & Mouth Disease Virus QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Depression that defines binding site for heparin is made up of segments from all three major capsid proteins Fry et al. (1999) EMBO J 18:543 QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Gut Microflora Regulate Intestinal Glycans Immunostaining with peroxidase-conjugated Ulex europaeus agglutinin Type 1 for Fuca1-2Gal epitopes QuickTime™ and a TIFF (LZW) decompressor are needed to see this picture. QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture. Hooper & Gordon (2001) Glycobiology 11:1R