Microbial Adhesins, Agglutinins & Toxins

1. Microbial Adhesins,Agglutinins & Toxins Victor Nizet, MDUCSD School of MedicineMay 11, 2004Essentials of GlycobiologyLecture 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 in the bacterial cell wall adhesin receptor host cell membrane “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)

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

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

6. Host Cell Receptors Bacterium • 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

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

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

9. Bacterial overlay Host glycoproteins Host glycolipids Thin-layer chromatography Polyacrylamide gel electrophoresis 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

10. Examples of Bacterial Adhesins Binding Host Glycans

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

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

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

14. Helicobacter pylori 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

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

16. Influenza 1917 PANDEMIC • 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. Nov-Apr Year-round Apr-Nov

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

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

19. Influenza Hemagglutinin Binds Sialic Acid • Flu A binds to a2,6 sialic acids • Flu B binds to a2,3 sialic acids • Flu C prefers 9-O-acetylated sialic acids

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

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

22. 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.

23. O O OH HN NH2 O Oseltamivir carboxylate (a sialic acid analogue)

24. Malaria (Plasmodium) Infections

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

26. 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 populations.

27. Examples of Glycosphingolipid Receptors for Bacterial Toxins

28. 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. • Left untreated, death may occur rapidly, especially in young children.

29. Cholera Toxin: Structural Features AB5 Hexameric Assembly

30. Cholera Toxin Receptor: GM1 Ganglioside GM1

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

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

33. 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.

34. ? Clostridium Botulinum Toxin: A Paralytic

35. BOTULINUM TOXIN BINDING 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

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

37. 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

38. Microbes that Bind Proteoglycans on Host Tissue

39. Herpes Simplex Virus Infection

40. gC Binding Herpes Simplex Entry Cell membrane Cell surface proteoglycans (heparan-sulfate) • 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 gD Binding HVEM/TNF/NGF receptor family gB and others (gH - gL) Membrane fusion Penetration Uncoat genome Nuclear pore Virus-mediated Intracellular transport Nucleus aTIF 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 Immunostaining with peroxidase-conjugated Ulex europaeus agglutinin Type 1 for Fuca1-2Gal epitopes Hooper & Gordon (2001) Glycobiology 11:1R