Microbial Adhesins,Agglutinins &
Toxins
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Victor Nizet, MDUCSD School of Medicine
May 11, 2004
Essentials of GlycobiologyLecture 26
Microbial Adherence to Host Epithelium
Adherence to skin or mucosalsurfaces 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
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Most microorganisms express more than one type of adhesive factor
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“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)
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adhesins in thebacterial cell wall
host cell membrane
adhesinreceptor
Pili (“hair”) and Fimbriae (“Threads”)
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Lateral mobility of adhesin structure in bacterial membrane provides a VelcroTM-like
effect
Pili/Fimbriae
Host glycolipidor glycoprotein
Host cell surfaceprotein/carbohydrate
Host cell membrane
Actinpolymerization
Intimin
Pedestal
Tip adhesinMajor subunit(pili)
Host -integrin
Afimbrialadhesins
SecretedHp 90
P
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
Microbial Binding to Glycoproteins
Glycoprotein glycans are displaced away from the membrane compared to glycolipids, which may make them less effective as microbial receptors
OSer/Thr
NAsn
N-LINKED CHAINN-LINKED CHAIN
O-LINKED CHAINO-LINKED CHAIN
GLYCOSPHINGOLIPIDGLYCOSPHINGOLIPID
OUTSIDE
INSIDE
S
= Sialic acid
CELLMEMBRANE
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
.
BacteriaB
ind
ing
+
_
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Cell Binding Assays
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
chromatographyPolyacrylamide
gel electrophoresis
Hostglycoproteins
Hostglycolipids
Bacterial overlay
AdhesinProtein
BacterialSpecies
TargetTissue
Carbohydrate Ligandon Host Cell
PapG (P-pilus) Escherichia coli Urinary Gal4Gal- in glycolipids
SfaS (S-pilus) Escherichia coli G.I. Tract Sia3Gal4GlcCer
FimH (Type 1
pilus)
Escherichia coli G.I. Tract Mannose-oligosaccharides
HifE Haemophilus
influenzae
Respirator
y
Sialylyganglioside-GM1
FHA Bordetella pertussis Respirator
y
Sulfated glycolipids, heparin
BabA Helicobacter pylori Stomach [Fuc2]Gal3[Fuc4]GlcNAc
(Leb)-
Hs Antigen Streptococcus
gordonii
Respirator
y
2-3-linked Sia-containing
receptors
Opc adhesin Neisseria
meningitides
Respirator
y
Heparin sulfate
proteoglycans
PsaA Strep. pneumoniae Respirator
y
N-acetyl hexosamine
galactose
EfaA Enterococcus
faecalis
G.I. Tract D-galactose or L-fucose +
glycans
Examples of Bacterial Adhesins Binding Host Glycans
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Electron microscopic image of E. coli
expressing surface pili
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adhesive tip
host cell
tipreceptor
pilus newadhesive tip
tipreceptor
alternatehost cell
pilus
surface localization
fiber formation
assembly of pilus organelle
adhesin unitsat end of pilus
Structure of Two E. coli Pili Subunits
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PapG FimH
Glycanbinding site
PapG+ E. coli bindingto bladder epithelium
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ureter
bladder
cell membrane
P pilus
Glycoproteinreceptor
Bordetella pertussis : Agent of “Whooping Cough”
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WT
FHA -
Epithelial cell adherence
Filamentoushemagglutinin
(FHA)
bacteria
cilia
nonciliated cells
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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
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How host glycans may affect the destiny of H. pylori colonization:
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Hooper & Gordon (2001)Glycobiology 11:1R
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Nov-Apr
Year-round
Apr-Nov
1917 PANDEMIC
Influenza• Acute repiratory tract infectionspread from person-to-person by respiratory droplets.
• ~ 20,000 deaths and110,000hospitalizations in U.S. annually.
• Enveloped, single-stranded RNA virus of family orthomyxoviridae.
• Typical symptoms are fever,dry cough, sore throat, runnyor stuffy nose, headache, muscle aches,and extreme fatigue.
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Hemagglutinin
Ion Channel
Lipid Envelope
Neuraminidase(sialidase)
Capsid
RNP
Structure of Influenza Virus
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Variation of Influenza Viruses
Point Mutations of Hemagglutinin
and/or Neuraminidase Gene(Antigenic Drift)
GeneticReassortment
(Antigenic Shift)
Human H2N2
Avian H3N8
Human H3N2
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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
Influenza HA-Mediated Membrane Fusion
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Target membrane
Viral membrane
Viral membrane
Target membrane
Low pH
Crystal structures
Predicted anchors
Neutralform Low pH
form
HA2
HA1
HA1
HA2
Fusionpeptide
Fusionpeptide
BUDDING & RELEASEBINDING & ENTRY
Influenza: Interactions with Sialic Acid
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)
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Oseltamivir carboxylate(a sialic acid analogue)
O
O
NH2
O
HN
OH
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Malaria (Plasmodium) Infections
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P. vivax merozoite
Duffy bloodgroup antigenglycoprotein
Duffy bindingprotein
P. falciparum merozoite
Sialic acid residueson glycophorin A
EBA-175
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)
Toxin Microorganism Tissue Proposed Receptor Sequence
Cholera toxin Vibrio cholerae Small
intestine
Gal3GalNAc4(NeuAc3)Gal4
GlcCer (GM1 ganglioside)
Heat-labile
toxin
Escherichia coli Intestine Gal3GalNAc4(NeuAc3)Gal4
GlcCer (GM1 ganglioside)
Tetanus toxin Clostridium tetani Nerve
membrane
G1b gangliosides (GT1b most
efficient)
Botulinum
toxin
Clostridium
botulinum
Nerve
membrane
(+NeuAc8)NeuAc3Gal3GalN
ac4
(NeuAc8NeuAc3)Gal4GlcCe
r
Toxin A Clostridium
difficile
Large
intestine
GalNAc3Gal4GlcNac3Gal4G
lcCer
Shiga toxin Shigella
dysenteriae
Large
intestine
Gal4GalCer or
Gal4Gal4GlcCer
Examples of Glycosphingolipid Receptors for Bacterial Toxins
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Cholera• Acute bacterial infection causedby ingestion of water contaminatedwith 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.
AB5 Hexameric Assembly
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Cholera Toxin: Structural Features
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Cholera Toxin Receptor: GM1
Ganglioside GM1
Cholera ToxinA-subunit
B-subunits (5)
GM1
GTP-binding protein
Adenylate cyclase
GM1
NAD+
ADP-Ribose
ADP-RibosecAMP
ATP
Cholera Toxin Biologic Effect
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CT receptor(GM1 )
Adenylate
cyclase
Cholera toxin
A subunit
Neutral NaCl
Absorption
ATP
Cholera toxin
Anion Secretion phosphorylati
on
(+)
(-)
protein
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)
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?
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Clostridium Botulinum Toxin: A Paralytic
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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
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
BindingCatalytic Translocation
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
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
(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
Microbes that Bind Proteoglycans on Host Tissue
Herpes Simplex Virus Infection
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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
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Binding
Binding
Cell membrane Cell surfaceproteoglycans(heparan-sulfate)
gC
gD
HVEM/TNF/NGFreceptor family
Membrane fusiongB and others (gH - gL)
Penetration Uncoat genome
Nuclear pore
Virus-mediatedIntracellular transport
TIF
Nucleus
Viral DNA
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Flaviviruses: Dengue and West Nile
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
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
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Gut Microflora Regulate Intestinal Glycans
Hooper & Gordon (2001) Glycobiology 11:1R
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Immunostaining withperoxidase-conjugatedUlex europaeusagglutinin Type 1 forFuc1-2Gal epitopes
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