Bacterial toxins

Disease

function of susceptibilityof host

relates to mechanism ofbacterial pathogenesis

immune competent/compromised

immunizationsage

trauma genetics

antimicrobial therapy

secretion of factors (toxins) direct host cell manipulation

Bacterial toxin studies

I. Disease mechanism II. Insight into protein designIII. Tool to manipulate / study eukaryotic cell

functionIV. Vaccine productionV. Disease therapyVI. Biological warfare

Types of bacterial toxins

modulate cellular activity cytolytic

cell receptor interactiontype III secretion ‘effectors’

bacterial toxins mimic eukaryotic cell processes~

function as precise tools for manipulating eukaryotic cell processes

I. Disease mechanism

Diseases caused by bacterial toxins:

• Diphtheria • Tetanus• Botulism• Anthrax • Cholera• Pertussis “whooping cough”• Gas gangrene• Toxic shock syndrome• Enterohemorrhagic E. coli - O157:H7• Necrotizing fasciitis “flesh-eating bacteria”

1888 - Disease mechanism related to toxin production

Emile Roux (1853-1933) Alexandre Yersin (1863-1943)

Diphtheria - prototype toxigenic disease

natural infections ~ only in humans

disease begins in upper respiratory tract with colonization of epithelial cells of pharynx

pseudomembrane = hallmark of disease

associated with degenerative changes in nerves, heart muscle, kidneys, other organs - mortality 50% if untreated

toxin - reaches all parts of body via bloodstream

Diphtheria disease

(1799 - suspected that George Washington died of diphtheria at age 67)

1821 - Pierre Bretonneau - diphtheritis (pseudomembrane)

1884 - Loeffler cultured organism - linked disease to soluble poison

Diphtheria

1890 - Diphtheria anti-toxin produced

Nobel Prize in Medicine 1901

Emil Adolf von Behring1854 - 1917

Shibasaburo Kitasato1852 - 1931

II. Insight into protein design

Studying toxin function

• purify protein - develop antibodies• clone and sequence gene - identify consensus sequence

patterns• identify molecular mechanism of action - enzymatic reaction• map function / functional domains (mutational studies)

• determine crystallographic structure

• determine protein function within cellular context

CH2

CH2

COOH

E

CH2

COOH

DCH2

OH

OH

CH2CH2

Y F S

CH2

A

Types of bacterial toxins

modulate cellular activity cytolytic

cell receptor interactiontype III secretion ‘effectors’

Cell modulating toxins ~

ADP-ribosyltransferase

S SA-subunit B-subunit

L enzyme activity / receptor binding / internalization intracellular trafficking

diphtheria toxin - prototype A-B toxin

ADP-ribosyltransferase reaction

O

O

N

CH2

CH2

PP

Adenine

CONH2

Toxin

O

OCH2

CH2

PP

Adenine

Cellular Target - EF2

+

NH

CONH2

ADP-ribosylated protein

Nicotinamide

NAD-glycohydrolase ADP-ribosyltransferase

(Choe et al., 1992)

Diphtheria toxin structure

1951 - Freeman identified toxin gene within a lysogenic -phage - transfer of phage between C. diphtheria produces toxigenic strain

Diphtheria toxin - production & regulation

(gsbs.utmb.edu/microbook/images/fig32_3.JPG)

Toxin regulated by iron

Diagnosis - growth on selective (tellurite) medium - forms black colonies Use immunological tests for toxin production

Binding of DT B-subunit to receptor - precursor to heparin-binding epidermal growth factor

Furin cleaves A-B-subunits

Endocytic vesicle fuses with lysosome

Low pH of phagolysozome - A-subunit translocated into cell cytoplasm

A-subunit ADP-ribosylates EF2 - inhibition of protein synthesis

Potent toxin - 1 molecule kills a cell (www.biken.osaka-u.ac.jp/.../ project/pro09.html)

Receptor mediated endocytosis (RME)

Internalization of diphtheria toxin

Cholera

1854 - Filippo Pacini identified comma- shaped bacillus organism

1884 - Robert Koch identified cholera bacillus, Vibrio cholerae (maintained credit for discovery until 1965) 

early epidemiology - England

1826-1837 - cholera epidemic William Farr theory - cholera spread by

“miasma” in air

William Farr (1807-1883) chief statistician Office of the Registrar-General -

campaigned for better sanitary conditions

Graph illustrating Farr's elevation theory in Langmuir AD.

Bacteriological Review 25, 174, 1961

John Snow, AnesthesiologistPhotograph, 1857, in Gordis L. Epidemiology, WB Saunders, Philadelphia, 1996

1846-1853 - second cholera epidemic (10,675 deaths in 1853)

1854 (Aug 31) - 127 deaths in 3 days ~ Broad St. Dr. John Snow - traced spread of ‘poison’ to sewage-tainted water pump on Broad Street

History

BacteriologyVibrio cholerae

motile, gram-negative curved rod facultative anaerobe

non-lactose fermentoroxidase positive

grows in salt & fresh water

Transmissioncontaminated water

raw seafood

Diseasesevere watery diarrhea - ‘rice water stool’

(UCLA Department of Epidemiology website)

Cholera

Cholera-disease

bacterium attaches to intestinal epithelial cells produces - cholera toxin

rapid onset - can cause severe diarrhea (20 L water loss / day)

massive fluid loss - severe dehydrationhypotension

collapse of the circulatory systemmortality rates high in children

bacteria eventually washes out - self-limiting (www.cameroon-info.net/ img/news/cholera_victim..

Virulence factors

(www.hinduonnet.com/.../ 07/stories/0807048f.htm)

motility / chemotaxis - flagella

adherence - Tcp (toxin coregulated pili) encoded on pathogenicity island origin - filamentous phage (VPI) Tcp = receptor for CTX phage

enterotoxin - cholera toxin, A-B toxin encoded on CTX phage

neuraminidase - removes sialic acid from oligosaccharides on epithelial cells - resemble cholera toxin receptor - GM1 ganglioside

Vibrio cholerae

Cholera toxin

(Sixma et al., 1991)

AB5 toxin

B-subunit

A-subunit

(From A. Salyers, D. Whitt, 2002)

GM1

receptor

Cellular mechanism of action of cholera toxin

Other ADP-ribosylating toxins

(M. Wilson, R. McNab, B. Henderson, Bacterial Disease Mechanisms, 2002)

(Gi)

Comparison of ADP-ribosylating proteins

Diphtheria Toxin Group 2 2 Loop Active site loop 3 3 7

DT 18SSYHGTKPGYVDSIQKG ....................IQKPKSGTQGNYDDDWKG .FYST DNKYDAAGYSVDNE 146SVEYINNETA437VGYHGTFLEAAQSIVFG G...................GVRARS..Q.DLDAIWRG .FYIAG DAL..AYGYAQDQE 551RLETILG

Cholera Toxin GroupCT 4KLYRADSRPPDEIKQSG GLMPRGQSEYFDRGTQMNINLYDHARGTQTGFVRHDDG YVSTS ISLRSAHLVGQTILS 110EQEVSALLTI 4KLYRADSRPPDEIKRSG GLMPRGHNEYFDRGTQMNINLYDHARGTQTGFVRYDDG YVSTS LSLRSAHLAGQSILS 110EQEVSALPT 6TVYRYDSRPPEDVFQNG F...........TAWGNNDNVLDHLTGRSCQVGSSNSA FVSTS SSRRYTE.VYLEHRM 127QSEYLAHEXS316KTFRGTRGG........ ......................DAFNAVEEGKVGHDDG YLSTS LNPGVARSF.GQGTI 379EKEILYNEXT319KTFRGTQGR.......... ....................DAFEAVKEGQVGHDAG YLSTS RDPSVARSFAGQGTI 383EQEILYDMTX 94RLLRWDRRPPNDIFLNG F.........IPRVTNQNLSPVEDTHLLNYLRTNSPSI FVSTT RARYNNLGLEITPWT 195EDEITFPCI 333IVYR..RSGPQEFGL.. .......TLTSPEYDFNKIENIDAFKEKWEGKVITYPN FISTS IGSVNMSAFAKRKII 419EYEVLLNC3D 85ILFRGDDPAYLG..... ...PEFQDKILNKDGTINRDVFEQVKAKFLKKDRTEYG YISTS LMS.AQFGGRPIVTK 171QLEVLLPC31 85ILFRGDDPAYLG..... ...TEFQNTLLNSNGTINKTAFEKAKAKFLNKDRLEYG YISTS LMNVSQFAGRPIITK 172QLEMLLPEDN 85YVYRLLNLDYLTSIVG. FTNEDLYKLQQTNNGQYDENLVRKLNNVMNSRIYREDG YSSTQ LVSGAAVGGRPIELR 181QQEVLLP

Bacterial ADPRT toxins - A subunit sequence

CHE131NVFRGVRGT........ .......................RFTA.QQGTVVRFGQ FTSTS LQKKVAEFFGLDTFF 192EDEVLIPCHB131YVYRGVRG......... .......................RFMT.QRGKSVRFGQ.FTSSS LRKEATVNFGQDTLF 192EDEVLIPM61123SVYRGTNV......... .......................RFRYTGKG.SVRFGH FASSS LNRSVATSSPFFNGQ 187EEEVLIPHMT154QVFRGVHGL........ .......................RFRPAGPRATVRLGG FASAS LKHVAAQQFGEDTFF 216EEEVLIP

Eukaryotic ADPRT proteins

Conserved NAD-binding cleft structure

(Sixma et al. 1991)

E. coli HLT / Exotoxin A

Diphtheria toxin A-subunit

Types of cellular activity modulating toxins

A-B toxins

ADP-ribosyltransferase - DT, ETA, CT, PT, C2 NAD-glycohydrolase - shiga toxin, Stx (ricin)glucosyltransferase - C. difficile (Toxin A, B), C. sordellii (LT)deamidase - CNF1, Bordetella DNTadenylate cyclase - Bordetella and Pseudomonas

adenylate cyclase Zn-endopeptidase - botulinum and tetanus neurotoxins

Enzyme activity Toxin

S SA-subunit B-subunit

L enzyme activity / receptor binding / internalization intracellular trafficking

Types of bacterial toxins

modulate cellular activity cytolytic

cell receptor interactiontype III secretion ‘effectors’

Cytolytic - membrane damaging toxins

• Phospholipase C C. perfringes alpha (80 kDa) “gas gangrene”

• Surfactant S. aureus delta (5 kDa)

• cholesterol dependent cytolysins (CDCs) pore forming toxins

Streptolysin (60 kDa)

Streptolysin-like structure

(Sekiya et al, J. Bact. 1993)

Streptolysin O pore

Types of bacterial toxins

modulate cellular activity cytolytic

cell receptor interactiontype III secretion ‘effectors’

Cell receptor interaction toxins

[H2O]

[CL- ][Na+]

(From A. Salyers, D. Whitt, 2002)

Superantigens (26-28 kDa)Staph enterotoxins A, B, C1, C2, C3, D, E, TSST-1Strep enterotoxins SpeA, SpeB

Hormone-likeE. coli STa - heat-stable toxin

(guanylin hormone-like) stimulates guanylate cyclase

18-19 aa (processed peptide) - structure stabilized by disulfide bond

ST1a

ST1b

Types of bacterial toxins

modulate cellular activity cytolytic

cell receptor interaction type III secretion ‘effectors’

Type III secretion effectors

PseudomonasExoS

GAP ADPRT

GAP tyrosine phosphataseSalmonellaSptP

GAPYersiniaYopE

GTP

GDP

PI

GTP active

GDP-inactive

cell targets

(GAP) (GEF)Rho Rac

Cdc42

III. Vaccine production

1891 - first anti-toxin given to diphtheritic child passive immune protection

1923 - Ramon introduced diphtheria toxoid vaccine

Diphtheria vaccine

Current immunization protocol for diphtheria:

5 doses of DTaP (diphtheria, tetanus, acellular pertussis)2, 4, 6, 12-15 months

4-6 years

Td (tetanus, diphtheria) (3-4 times less diphtheriatoxoid than in DTaP formulation) (new TdaP vaccine)

11-16 years - then every 10 years

Decreasing prevalence of

diphtheria in US with DPT vaccine

>157,000 cases5,000 deaths

Massive immunization

Toxin vaccine development

Toxoid vaccine - treatment of purified toxin with formaldehyde (e.g. diphtheria and tetanus vaccines)

Recombinant toxin vaccines - mutant, enzymatically inactive forms of toxins (e.g. inactivated ctxA gene with B-subunit)

S SA-subunit B-subunit

L enzyme activity / receptor binding / internalization intracellular trafficking

Combinatorial vaccines - more than one antigen (e.g. acellular pertussis vaccine - non-toxic form of toxin + fimbrial antigen)

IV. Tool to manipulate and studyeukaryotic cell function

Cellular targets of bacterial toxins

GTP

GDP

PI

GTP active

GDP-inactive

Effectors

(GAP) (GEF)

G-proteins

(DT, ETA, CT, PTC. difficile (Toxin A, B),

C. sordellii (LT) CNF1, Bordetella DNT)

Actin - cytoskeletal structure

(Clostridium C2, Iota toxin)

Use of toxins to study G-protein function

(From A. Salyers, D. Whitt, 2002)

GM1

receptor

cholera toxin

V. Disease therapy

Botulinum toxin

(K. Turton, J. Chaddock, K.R. Acharya, TRENDS in Biochemical Sciences, 2002)

absorbed - from intestine - spreads by bloodstreambinds - receptor on motor neuron of peripheral nervous system

internalized - by receptor mediated endocytosis (RME)vesicle acidification - releases LC into motor neuronLC - cleaves SNARE proteins - not SNARE complex

result - inhibition of acetylcholine release - prevents muscle contractionflaccid paralysis

Botulinum toxin

(Synaptobrevin)

Comparison - tetanus & botulinum toxin activity

(science.cancerresearchuk.org/images/flat/sch)i

Mammalian motor neuron & trafficking pathways of BoNTs (in blue) and TeNT (in red). Microtubule are in brown and actin filaments in green. Red crosses - sites of inhibition of neurotransmitter release.

(Adapted from Lalli et al. Trends Microbiol 2003; 11: 31)

BoNT - acts on PNS

inhibits release of stimulatoryneurotransmitter(acetylcholine)

at peripheral nerve endings

- flaccid paralysis -

TeNT - acts on CNS

inhibits release of inhibitory neurotransmitters

(glycine / aminobutyric acid)at interneuronal junctions

- spastic paralysis -

RAT VAMP1 50 VNVDKVLERDQKLSELDDRADALQAGASVFESSAAKLKRKYWW

RAT VAMP2 48 VNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWW

BoNT/F BoNT/D BoNT/G

BoNT/BTeNT

botulinum & tetanus toxin sites of proteolysis

1989 - USDA licensed Botox for treatment of muscle disorders(treat by injecting toxin into hyperactive muscle)

Botulinum toxin - use as a therapeutic agent

Botox

Use - injection of low dose of BoNT - localized paralysis at site of injection

relates to extended duration of BoNT effectsBoNT/A (months) > BoNT/E (weeks)

treatment extended from peripheral to autonomic nervous system

hyperhidrosis (sweating), myofascial pain, migraine headache

future uses - designer therapeuticstargeting of LC to non-neuronal cells

use of HC in transport of large polypeptides / DNA / enzymes / drugs

Botox injections to remove wrinkles

Prior to botulinum toxin injections

Subject relaxed Subject frowning

After botulinum toxin injections

Subject relaxed Subject attempting to frown

Complications of Botox injections

‘droopy eyelid’ (ptosis) - botulinum toxin reaching eyelid muscle

develop immunity to toxin (rare) - use of more purified toxin

- use of different antigenic type (BoNT/B)

Design of toxins for therapeutic uses

S SA-subunit B-subunit

L enzyme activity / receptor binding / internalization intracellular trafficking

S SA-subunit B-subunit

L enzyme activity / altered receptor binding altered acellular trafficking

receptor for granulocyte-macrohage colony-stimulating factortargets myeloid leukemia cells - linked to toxin

VH-VL - linked to toxin

Cell-specific cytotoxicity

VI. Biological warfare

Category A Diseases/AgentsThe U.S. public health system and primary healthcare providers must be prepared to address various biological agents, including pathogens that are rarely seen in the United States. High-priority agents include organisms that pose a risk to national security because they

* can be easily disseminated or transmitted from person to person;* result in high mortality rates and have the potential for major public health impact;* might cause public panic and social disruption; and* require special action for public health preparedness.

Bioterrorism agents - Biodefense

Category A agents» Anthrax (Bacillus anthracis)» Botulism (Clostridium botulinum toxin)» Plague (Yersinia pestis)» Smallpox (variola major)» Tularemia (Francisella tularensis)» Viral hemorrhagic fevers (filoviruses [e.g., Ebola, Marburg]

and arenaviruses [e.g., Lassa, Machupo])

Treatment of toxin mediated diseases

anti-toxin antibodies (passive)anti-toxin vaccines (active)humoral immune response

Ineffective therapyantibiotics

innate immune response

Concepts - bacterial toxins

• types of bacterial toxins• toxin design / mechanism• use as a tool to manipulate / study eukaryotic cells• functional similarity to eukaryotic cell proteins• vaccine design• use in disease therapy• use in biological warfare