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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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Microbial Adherence to Host Epithelium
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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
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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
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adhesin
receptor
host cell membrane
Pili (“hair”) and Fimbriae (“Threads”)
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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
_
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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
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surface localization
fiber formation
Electron microscopic image of E. coli
expressing surface pili
pilus
pilus
adhesive
tip
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host cell
new
adhesive
tip
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alternate
host cell
Structure of Two E. coli Pili
Subunits
Glycan
binding site
bladder
ureter
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cell membrane
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PapG
PapG+ E. coli binding
to bladder epithelium
FimH
Glycoprotein
receptor
Bordetella pertussis : Agent of “Whooping Cough”
Filamentous
hemagglutinin
(FHA)
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WT
bacteria
cilia
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nonciliated cells
FHA Epithelial cell adherence
Helicobacter pylori
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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 mucusproducing gastric epithelium
How host glycans
may affect the destiny
of H. pylori
colonization:
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Hooper & Gordon (2001)
Glycobiology 11:1R
Influenza
• Acute repiratory tract infection
spread from person-to-person by
respiratory droplets.
1917 PANDEMIC
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• ~ 20,000 deaths and110,000
hospitalizations in U.S. annually.
• Enveloped, single-stranded RNA
virus of family orthomyxoviridae.
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• 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
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Hemagglutinin
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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
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–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
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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
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NH2
Oseltamivir carboxylate
(a sialic acid analogue)
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Malaria (Plasmodium) Infections
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P. vivax merozoite
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Duffy binding
protein
Duffy blood
group antigen
glycoprotein
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Sialic acid residues
on glycophorin A
EBA-175
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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.
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• Left untreated, death may occur rapidly,
especially in young children.
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Cholera Toxin: Structural Features
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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
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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
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?
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BOTULINUM TOXIN BINDING
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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
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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
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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
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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
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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
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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
Immunostaining with
peroxidase-conjugated
Ulex europaeus
agglutinin Type 1 for
Fuca1-2Gal epitopes
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Hooper & Gordon (2001) Glycobiology 11:1R