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Microbial Adhesins, Agglutinins & Toxins Victor Nizet, MD UCSD School of Medicine May 11, 2004 Essentials of Glycobiology Lecture 26.

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Presentation on theme: "Microbial Adhesins, Agglutinins & Toxins Victor Nizet, MD UCSD School of Medicine May 11, 2004 Essentials of Glycobiology Lecture 26."— Presentation transcript:

1 Microbial Adhesins, Agglutinins & Toxins Victor Nizet, MD UCSD School of Medicine May 11, 2004 Essentials of Glycobiology Lecture 26

2 Microbial Adherence to Host Epithelium 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 Most microorganisms express more than one type of adhesive factor

3 “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 host cell membrane adhesin receptor

4 Pili (“hair”) and Fimbriae (“Threads”) Lateral mobility of adhesin structure in bacterial membrane provides a Velcro TM -like effect

5 Pili/Fimbriae Host glycolipid or glycoprotein Host cell surface protein/carbohydrate Host cell membrane Actin polymerization Intimin Pedestal Tip adhesin Major subunit (pili) Host  -integrin Afimbrial adhesins Secreted Hp 90 P

6 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 Bacterium

7 Microbial Binding to Glycoproteins Glycoprotein glycans are displaced away from the membrane compared to glycolipids, which may make them less effective as microbial receptors O Ser/Thr N Asn N-LINKED CHAIN O-LINKED CHAIN GLYCOSPHINGOLIPID OUTSIDE INSIDE S = Sialic acid CELL MEMBRANE

8 Measuring Adhesin-Receptor Interactions Hemagglutination Use mutant cells or nutritionally manipulate composition Competition experiments with soluble carbohydrates Remove receptor with exoglycosidases Regenerate different receptor with glycosyltransferase. Bacteria Binding + _ Cell Binding Assays

9 Binding Measurements  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 Thin-layer chromatography Polyacrylamide gel electrophoresis Host glycoproteins Host glycolipids Bacterial overlay

10 Adhesin Protein Bacterial Species Target Tissue Carbohydrate Ligand on Host Cell PapG (P-pilus)Escherichia coliUrinary Gal  4Gal  - in glycolipids SfaS (S-pilus)Escherichia coliG.I. Tract Sia  3Gal  4Glc  Cer FimH (Type 1 pilus) Escherichia coliG.I. TractMannose-oligosaccharides HifE Haemophilus influenzae RespiratorySialylyganglioside-GM1 FHABordetella pertussisRespiratorySulfated glycolipids, heparin BabAHelicobacter pyloriStomach [Fuc  2]Gal  3[Fuc  4]GlcNAc (Le b )- Hs AntigenStreptococcus gordoniiRespiratory  2-3-linked Sia-containing receptors Opc adhesinNeisseria meningitidesRespiratoryHeparin sulfate proteoglycans PsaAStrep. pneumoniaeRespiratoryN-acetyl hexosamine galactose EfaAEnterococcus faecalisG.I. TractD-galactose or L-fucose + glycans Examples of Bacterial Adhesins Binding Host Glycans

11 Electron microscopic image of E. coli expressing surface pili adhesive tip host cell tip receptor pilus new adhesive tip tip receptor alternate host cell pilus surface localization fiber formation assembly of pilus organelle adhesin units at end of pilus

12 Structure of Two E. coli Pili Subunits PapG FimH Glycan binding site PapG+ E. coli binding to bladder epithelium ureter bladder cell membrane P pilus Glycoprotein receptor

13 Bordetella pertussis : Agent of “Whooping Cough” WT FHA - Epithelial cell adherence Filamentous hemagglutinin (FHA) bacteria cilia nonciliated cells

14 H. Pylori surface BabA protein (blood group antigen-binding adhesin) Binds to carbohydrate blood-group antigen Lewis B (LeB) on MUC5AC glycoprotein expressed in mucus- producing gastric epithelium Helicobacter pylori

15 How host glycans may affect the destiny of H. pylori colonization: Hooper & Gordon (2001) Glycobiology 11:1R

16 Nov-Apr Year-round Apr-Nov 1917 PANDEMIC Influenza Acute repiratory tract infection spread from person-to-person by respiratory droplets. ~ 20,000 deaths and110,000 hospitalizations in U.S. annually. Enveloped, single-stranded RNA virus of family orthomyxoviridae. Typical symptoms are fever, dry cough, sore throat, runny or stuffy nose, headache, muscle aches,and extreme fatigue.

17 Hemagglutinin Ion Channel Lipid Envelope Neuraminidase (sialidase) Capsid RNP Structure of Influenza Virus

18 Variation of Influenza Viruses Point Mutations of Hemagglutinin and/or Neuraminidase Gene (Antigenic Drift) Genetic Reassortment (Antigenic Shift) Human H2N2 Avian H3N8 Human H3N2

19 Influenza Hemagglutinin Binds Sialic Acid –Flu A binds to  2,6 sialic acids –Flu B binds to  2,3 sialic acids –Flu C prefers 9-O-acetylated sialic acids

20 Influenza HA-Mediated Membrane Fusion Target membrane Viral membrane Target membrane Low pH Crystal structures Predicted anchors Neutral form Low pH form HA2 HA1 HA2 Fusion peptide Fusion peptide

21 BUDDING & RELEASE BINDING & ENTRY Influenza: Interactions with Sialic Acid

22 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. Influenza: Why the Neuraminidase? (explanation for handout)

23 Oseltamivir carboxylate (a sialic acid analogue) O O NH 2 O HN OH

24 Malaria (Plasmodium) Infections

25 P. vivax merozoite Duffy blood group antigen glycoprotein Duffy binding protein P. falciparum merozoite Sialic acid residues on glycophorin A EBA-175

26 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 populations. Malaria Invasion of Host Erythrocytes (explanation for handout)

27 ToxinMicroorganismTissueProposed Receptor Sequence Cholera toxinVibrio choleraeSmall intestine Gal  3GalNAc  4(NeuAc  3)Gal  4Gl c  Cer (GM1 ganglioside) Heat-labile toxin Escherichia coliIntestine Gal  3GalNAc  4(NeuAc  3)Gal  4Gl c  Cer (GM1 ganglioside) Tetanus toxinClostridium tetani Nerve membrane G 1b gangliosides (G T1b most efficient) Botulinum toxin Clostridium botulinum Nerve membrane (+NeuAc  8)NeuAc  3Gal  3GalNac  4 (NeuAc  8NeuAc  3)Gal  4Glc  Cer Toxin AClostridium difficileLarge intestine GalNAc  3Gal  4GlcNac  3Gal  4Glc  Cer Shiga toxinShigella dysenteriaeLarge intestineGal  4Gal  Cer or Gal  4Gal  4Glc  Cer Examples of Glycosphingolipid Receptors for Bacterial Toxins

28 Cholera Acute bacterial infection caused by ingestion of water contaminated with Vibrio cholerae 01 or Sudden watery diarrhea and vomiting can result in severe dehydration. Left untreated, death may occur rapidly, especially in young children.

29 AB5 Hexameric Assembly Cholera Toxin: Structural Features

30 Cholera Toxin Receptor: GM1 Ganglioside GM1

31 Cholera Toxin A-subunit B-subunits (5) GM1 GTP-binding protein Adenylate cyclase GM1 NAD + ADP-Ribose cAM P ATP

32 Cholera Toxin Biologic Effect CT receptor (GM1 ) Adenylate cyclase Cholera toxin A subunit Neutral NaCl Absorption AT P Cholera toxin Anion Secretion phosphorylation (+) (-) protein

33 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. Cholera Toxin Mechanism of Action (explanation for handout)

34 ? Clostridium Botulinum Toxin: A Paralytic

35 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 Second receptor: Postulated to be integral membrane protein BOTULINUM TOXIN BINDING

36 Toxins A and B from Clostridium difficile (antibiotic- associated diarrhea, pseudomembranous colitis) Hemorrhagic and lethal toxins of C. sordellii and  - toxin of C. novyi (enterotoxemia and gas gangrene) These toxins turn out to be glucosyltransferases Large Clostridial Cytotoxins BindingCatalyticTranslocation

37 Modification of target proteins by glucosylation Targets include Rho (cytoskeletal organization), Ras (growth control), Rac, cdc42 and other GTPases Large Clostridial Cytotoxins Busch & Aktories (2000) COSB 10:528

38 MicrobeTarget Tissue Bordetella pertussisCiliated epithelium in respiratory tract Chlamydia trachomatisEyes, genital tract, respiratory epithelium Haemophilus influenzaeRespiratory epithelium Borrelia burgdorferiEndothelium, epithelium, extracellular matrix Neisseria gonorrheaGenital tract Staphylococcus aureusConnective tissues, epithelial cells Mycobacterium tuberculosisRespiratory epithelium Plasmodium falciparum (circumsporozootes) Heaptocytes, placenta Leishmania amazonensi (amastigotes)Macrophages, fibroblasts, epithelium Herpes simplex virus (HSV)Mucosal surfaces of mouth, eyes, genital tract Dengue flavivirusMacrophages? HIV-1T lymphocytes Microbes that Bind Proteoglycans on Host Tissue

39 Herpes Simplex Virus Infection

40 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) gC gD HVEM/TNF/NGF receptor family Membrane fusion gB and others (gH - gL) Penetration Uncoat genome Nuclear pore Virus-mediated Intracellular transport  TIF Nucleus Viral DNA

41 Flaviviruses: Dengue and West Nile

42 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.

43 Foot & Mouth Disease Virus 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

44 Gut Microflora Regulate Intestinal Glycans Hooper & Gordon (2001) Glycobiology 11:1R Immunostaining with peroxidase-conjugated Ulex europaeus agglutinin Type 1 for Fuc  1-2Gal epitopes


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