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
BINDING & ENTRY
BUDDING & RELEASE

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)
NH2 HN OH

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?

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. 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