microbial adhesins, agglutinins & toxins victor nizet, md ucsd school of medicine may 11, 2004...

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



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 thebacterial cell wall

host cell membrane

adhesinreceptor

Pili (“hair”) and Fimbriae (“Threads”)





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

+

_





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



Electron microscopic image of E. coli

expressing surface pili





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



PapG FimH

Glycanbinding site

PapG+ E. coli bindingto bladder epithelium



ureter

bladder

cell membrane

P pilus

Glycoproteinreceptor

Bordetella pertussis : Agent of “Whooping Cough”







WT

FHA -

Epithelial cell adherence

Filamentoushemagglutinin

(FHA)

bacteria

cilia

nonciliated cells



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



How host glycans may affect the destiny of H. pylori colonization:



Hooper & Gordon (2001)Glycobiology 11:1R



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.





Hemagglutinin

Ion Channel

Lipid Envelope

Neuraminidase(sialidase)

Capsid

RNP

Structure of Influenza Virus



Variation of Influenza Viruses

Point Mutations of Hemagglutinin

and/or Neuraminidase Gene(Antigenic Drift)

GeneticReassortment

(Antigenic Shift)

Human H2N2

Avian H3N8

Human H3N2



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



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)



Oseltamivir carboxylate(a sialic acid analogue)

O

O

NH2

O

HN

OH



Malaria (Plasmodium) Infections















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





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



Cholera Toxin: Structural Features



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)







?



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









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

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









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





Gut Microflora Regulate Intestinal Glycans

Hooper & Gordon (2001) Glycobiology 11:1R





Immunostaining withperoxidase-conjugatedUlex europaeusagglutinin Type 1 forFuc1-2Gal epitopes

microbial adhesins, agglutinins & toxins victor nizet, md ucsd school of medicine may 11, 2004...

Documents

microbial adherence

microbial adhesins

host epithelium adherence

surface glycolipids

host cellsmany adhesins

strength of binding

adhesins of microbes

different receptor