Presentation is loading. Please wait.

Presentation is loading. Please wait.

Microbial Adhesins, Agglutinins & Toxins

Similar presentations

Presentation on theme: "Microbial Adhesins, Agglutinins & Toxins"— Presentation transcript:

1 Microbial Adhesins, Agglutinins & Toxins
Victor Nizet, MD UCSD School of Medicine May 11, 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 VelcroTM-like effect

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

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 Microbial Binding to Glycoproteins
= Sialic acid N-LINKED CHAIN O-LINKED CHAIN GLYCOSPHINGOLIPID S O N Ser/Thr Asn OUTSIDE CELL MEMBRANE Shiga toxin binds to both glycolipids and glycoproteins - Only binding to glycolipids causes cell death INSIDE Glycoprotein glycans are displaced away from the membrane compared to glycolipids, which may make them less effective as microbial receptors

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

9 Polyacrylamide gel electrophoresis
Thin-layer chromatography Polyacrylamide gel electrophoresis Host glycoproteins glycolipids Bacterial overlay 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
Protein Bacterial Species Target Tissue Carbohydrate Ligand on Host Cell PapG (P-pilus) Escherichia coli Urinary Gala4Galb- in glycolipids SfaS (S-pilus) G.I. Tract Siaa3Galb4GlcbCer FimH (Type 1 pilus) Mannose-oligosaccharides HifE Haemophilus influenzae Respiratory Sialylyganglioside-GM1 FHA Bordetella pertussis Sulfated glycolipids, heparin BabA Helicobacter pylori Stomach [Fuca2]Galb3[Fuca4]GlcNAc (Leb)- Hs Antigen Streptococcus gordonii a2-3-linked Sia-containing receptors Opc adhesin Neisseria meningitides Heparin sulfate proteoglycans PsaA Strep. pneumoniae N-acetyl hexosamine galactose EfaA Enterococcus faecalis D-galactose or L-fucose + glycans

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

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

13 Bordetella pertussis : Agent of “Whooping Cough”
Filamentous hemagglutinin (FHA) WT FHA - Epithelial cell adherence 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. 1917 PANDEMIC Nov-Apr Year-round Apr-Nov

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

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 a2,6 sialic acids Flu B binds to a2,3 sialic acids Flu C prefers 9-O-acetylated sialic acids

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

21 Influenza: Interactions with Sialic Acid

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 Oseltamivir carboxylate (a sialic acid analogue)

24 Malaria (Plasmodium) Infections

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

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 Proposed Receptor Sequence
Examples of Glycosphingolipid Receptors for Bacterial Toxins Toxin Microorganism Tissue Proposed Receptor Sequence Cholera toxin Vibrio cholerae Small intestine Galb3GalNAcb4(NeuAca3)Galb4GlcbCer (GM1 ganglioside) Heat-labile toxin Escherichia coli Intestine Tetanus toxin Clostridium tetani Nerve membrane G1b gangliosides (GT1b most efficient) Botulinum toxin Clostridium botulinum (+NeuAca8)NeuAca3Galb3GalNacb4 (NeuAca8NeuAca3)Galb4GlcbCer Toxin A Clostridium difficile Large intestine GalNAcb3Galb4GlcNacb3Galb4GlcbCer Shiga toxin Shigella dysenteriae Gala4GalbCer or Gala4Galb4GlcbCer

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

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

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 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 C-terminus binds to receptor, e.g., Toxin A binds to Galb1,4GlcNAc Hydrophobic segment in central region of protein may mediate membrane translocation, uptake occurs through endosome in a pH dependent manner N-terminus contains glucosyltransferase domain Binding Catalytic Translocation

37 Large Clostridial Cytotoxins
Modification of target proteins by glucosylation Targets include Rho (cytoskeletal organization), Ras (growth control), Rac, cdc42 and other GTPases Glucosylation of specific Thr residue involved in nucleotide binding and coordination of Mg2+ Busch & Aktories (2000) COSB 10:528

38 Microbes that Bind Proteoglycans on Host Tissue
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 Plasmodium falciparum (circumsporozootes) Heaptocytes, placenta Leishmania amazonensi (amastigotes) Macrophages, fibroblasts, epithelium Herpes simplex virus (HSV) Mucosal surfaces of mouth, eyes, genital tract Dengue flavivirus Macrophages? HIV-1 T lymphocytes

39 Herpes Simplex Virus Infection

40 Herpes Simplex Entry gC Binding 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 Cell membrane Cell surface proteoglycans (heparan-sulfate) 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
Foot and Mouth disease virus

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. Dengue virus causes hemorrhagic fever Filamentous protein on surface binds GAG Open cleft - clamp

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 Foot and Mouth disease virus

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

Download ppt "Microbial Adhesins, Agglutinins & Toxins"

Similar presentations

Ads by Google