1. Bacterial toxins 1

2. Disease function of susceptibility of host relates to mechanism of
bacterial pathogenesis
immune competent/compromised
immunizations
age
trauma
genetics
antimicrobial therapy
secretion of factors (toxins)
direct host cell manipulation

3. Bacterial toxin studies
I. Disease mechanism
II. Insight into protein design
III. Tool to manipulate / study eukaryotic cell function
IV. Vaccine production
V. Disease therapy
VI. Biological warfare
Roux/Yersin demonstrated that heat labile toxin in fluid phase of dihtheria cultures caused all symptoms of diphtheria when injected into animal models 2

4. Types of bacterial toxins
modulate cellular activity
cytolytic
cell receptor interaction
type III secretion ‘effectors’
bacterial toxins mimic eukaryotic cell processes ~
function as precise tools
for manipulating eukaryotic cell processes 4

5. I. Disease mechanism

6. 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” 3

7. Diphtheria - prototype toxigenic disease
Disease mechanism related to toxin production
Emile Roux ( )
Alexandre Yersin ( )

8. Diphtheria 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
( suspected that George Washington died of diphtheria at age 67)

9. Diphtheria 1821 - Pierre Bretonneau - diphtheritis (pseudomembrane)
Loeffler cultured organism -
linked disease to soluble poison
Gave rise to the term virulence factor
Diphthera = membrane (pseudomembrane = hallmark of disease)

10. 1890 - Diphtheria anti-toxin produced
Emil Adolf von Behring
Shibasaburo Kitasato
Nobel Prize in Medicine 1901

11. II. Insight into protein design

12. 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)
CH2
COOH E D OH Y F S A
determine crystallographic structure
determine protein function within cellular context

13. Types of bacterial toxins
modulate cellular activity
cytolytic
cell receptor interaction
type III secretion ‘effectors’ 4

14. Cell modulating toxins ~
S S
A-subunit
B-subunit
L enzyme activity / receptor binding / internalization
intracellular trafficking
diphtheria toxin - prototype A-B toxin
ADP-ribosyltransferase

15. ADP-ribosyltransferase reaction
CH2 P
Adenine
CONH2
Toxin
Cellular Target - EF2 + H
ADP-ribosylated protein
Nicotinamide
NAD-glycohydrolase ADP-ribosyltransferase

16. Diphtheria toxin structure
(Choe et al., 1992)

17. Diphtheria toxin - production & regulation
Freeman identified toxin gene within a lysogenic b-phage -
transfer of phage between C. diphtheria produces toxigenic strain
Diagnosis - growth on selective (tellurite) medium - forms black colonies
Use immunological tests for toxin production
(gsbs.utmb.edu/microbook/images/fig32_3.JPG)
Toxin regulated by iron

18. Internalization of diphtheria toxin
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
( project/pro09.html)
Receptor mediated endocytosis (RME)

19. Cholera History early epidemiology - England
cholera epidemic
William Farr theory - cholera spread by
“miasma” in air
William Farr ( ) chief statistician Office of the Registrar-General - campaigned for better sanitary conditions
John Snow, Anesthesiologist
Photograph, 1857, in Gordis L.
Epidemiology, WB Saunders,
Philadelphia, 1996
second cholera epidemic
(10,675 deaths in 1853)
1854 (Aug 31) deaths in 3 days ~ Broad St.
Dr. John Snow - traced spread of ‘poison’ to
sewage-tainted water pump on Broad Street
History
Graph illustrating Farr's elevation theory in Langmuir AD. Bacteriological Review 25, 174, 1961
Filippo Pacini identified comma- shaped bacillus organism
Robert Koch identified cholera bacillus, Vibrio cholerae (maintained credit for discovery until 1965) 

20. Cholera Bacteriology Transmission Disease Vibrio cholerae
motile, gram-negative curved rod
facultative anaerobe
non-lactose fermentor
oxidase positive
grows in salt & fresh water
Transmission
contaminated water
raw seafood
Disease
severe watery diarrhea - ‘rice water stool’
(UCLA Department of Epidemiology website)

21. 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 dehydration
hypotension
collapse of the circulatory system
mortality rates high in children
bacteria eventually washes out - self-limiting
( img/news/cholera_victim..

22. Virulence factors Vibrio cholerae motility / chemotaxis - flagella
adherence - Tcp (toxin coregulated pili)
encoded on pathogenicity island
origin - filamentous phage (VPIF)
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
( 07/stories/ f.htm)

23. Cholera toxin
(Sixma et al., 1991)
AB5 toxin
B-subunit
A-subunit

24. Cellular mechanism of action of cholera toxin
(From A. Salyers, D. Whitt, 2002)
GM1
receptor

25. Other ADP-ribosylating toxins
(Gi)
(M. Wilson, R. McNab, B. Henderson, Bacterial Disease Mechanisms, 2002)

26. Comparison of ADP-ribosylating proteins
Bacterial ADPRT toxins - A subunit sequence
Diphtheria Toxin Group
2  Loop Active site loop     7
DT 18SSYHGTKPGYVDSIQKG IQKPKSGTQGNYDDDWKG .FYST DNKYDAAGYSVDNE 146SVEYINN
ETA437VGYHGTFLEAAQSIVFG G GVRARS..Q.DLDAIWRG .FYIAG DAL..AYGYAQDQE 551RLETILG
Cholera Toxin Group
CT 4KLYRADSRPPDEIKQSG GLMPRGQSEYFDRGTQMNINLYDHARGTQTGFVRHDDG YVSTS ISLRSAHLVGQTILS 110EQEVSAL
LTI 4KLYRADSRPPDEIKRSG GLMPRGHNEYFDRGTQMNINLYDHARGTQTGFVRYDDG YVSTS LSLRSAHLAGQSILS 110EQEVSAL
PT 6TVYRYDSRPPEDVFQNG F TAWGNNDNVLDHLTGRSCQVGSSNSA FVSTS SSRRYTE.VYLEHRM 127QSEYLAH
EXS316KTFRGTRGG DAFNAVEEGKVGHDDG YLSTS LNPGVARSF.GQGTI 379EKEILYN
EXT319KTFRGTQGR DAFEAVKEGQVGHDAG YLSTS RDPSVARSFAGQGTI 383EQEILYD
MTX 94RLLRWDRRPPNDIFLNG F IPRVTNQNLSPVEDTHLLNYLRTNSPSI FVSTT RARYNNLGLEITPWT 195EDEITFP
CI 333IVYR..RSGPQEFGL TLTSPEYDFNKIENIDAFKEKWEGKVITYPN FISTS IGSVNMSAFAKRKII 419EYEVLLN
C3D 85ILFRGDDPAYLG PEFQDKILNKDGTINRDVFEQVKAKFLKKDRTEYG YISTS LMS.AQFGGRPIVTK 171QLEVLLP
C31 85ILFRGDDPAYLG TEFQNTLLNSNGTINKTAFEKAKAKFLNKDRLEYG YISTS LMNVSQFAGRPIITK 172QLEMLLP
EDN 85YVYRLLNLDYLTSIVG. FTNEDLYKLQQTNNGQYDENLVRKLNNVMNSRIYREDG YSSTQ LVSGAAVGGRPIELR 181QQEVLLP
CHE131NVFRGVRGT RFTA.QQGTVVRFGQ FTSTS LQKKVAEFFGLDTFF 192EDEVLIP
CHB131YVYRGVRG RFMT.QRGKSVRFGQ.FTSSS LRKEATVNFGQDTLF 192EDEVLIP
M61123SVYRGTNV RFRYTGKG.SVRFGH FASSS LNRSVATSSPFFNGQ 187EEEVLIP
HMT154QVFRGVHGL RFRPAGPRATVRLGG FASAS LKHVAAQQFGEDTFF 216EEEVLIP
Eukaryotic ADPRT proteins

27. Conserved NAD-binding cleft structure
Diphtheria toxin A-subunit
E. coli HLT / Exotoxin A
(Sixma et al. 1991)

28. Types of cellular activity modulating toxins
A-B toxins
S S
A-subunit
B-subunit
L enzyme activity / receptor binding / internalization
intracellular trafficking
Enzyme activity Toxin
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 DNT
adenylate cyclase - Bordetella and Pseudomonas adenylate cyclase
Zn-endopeptidase - botulinum and tetanus neurotoxins

29. Types of bacterial toxins
modulate cellular activity
cytolytic
cell receptor interaction
type III secretion ‘effectors’ 4

30. Cytolytic - membrane damaging toxins
Streptolysin-like structure
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) 5

31. Streptolysin O pore
(Sekiya et al, J. Bact. 1993)

32. Types of bacterial toxins
modulate cellular activity
cytolytic
cell receptor interaction
type III secretion ‘effectors’ 4

33. Cell receptor interaction toxins
Hormone-like
E. coli STa - heat-stable toxin (guanylin hormone-like)
stimulates guanylate cyclase
18-19 aa (processed peptide) - structure stabilized by disulfide bond
ST1a
ST1b
[H2O]
[CL- ]
[Na+]
(From A. Salyers, D. Whitt, 2002)
Superantigens (26-28 kDa)
Staph enterotoxins
A, B, C1, C2, C3, D, E, TSST-1
Strep enterotoxins
SpeA, SpeB

34. Types of bacterial toxins
modulate cellular activity
cytolytic
cell receptor interaction
type III secretion ‘effectors’ 4

35. Type III secretion effectors
Pseudomonas
ExoS
GAP ADPRT
GAP tyrosine phosphatase
Salmonella
SptP
GAP
Yersinia
YopE
GTP
GDP PI
GTP active
GDP-inactive
cell targets
(GAP) (GEF)
Rho
Rac
Cdc42

36. III. Vaccine production

37. Diphtheria vaccine
first anti-toxin given to diphtheritic child passive immune protection
Ramon introduced diphtheria toxoid vaccine
Current immunization protocol for diphtheria:
5 doses of DTaP (diphtheria, tetanus, acellular pertussis)
2, 4, 6, months
4-6 years
Td (tetanus, diphtheria) (3-4 times less diphtheria
toxoid than in DTaP formulation) (new TdaP vaccine)
11-16 years - then every 10 years

38. Decreasing prevalence of diphtheria in US with DPT vaccine

39. Massive immunization
>157,000 cases
5,000 deaths

40. 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 S
A-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)

41. IV. Tool to manipulate and study eukaryotic cell function

42. Cellular targets of bacterial toxins
GTP
GDP PI
GTP active
GDP-inactive
Effectors
(GAP) (GEF)
G-proteins
(DT, ETA, CT, PT
C. difficile (Toxin A, B),
C. sordellii (LT) CNF1, Bordetella DNT)
Actin - cytoskeletal structure
(Clostridium C2, Iota toxin)

43. Use of toxins to study G-protein function
cholera toxin
(From A. Salyers, D. Whitt, 2002)
GM1
receptor

44. V. Disease therapy

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

46. Botulinum toxin absorbed - from intestine - spreads by bloodstream
(Synaptobrevin)
absorbed - from intestine - spreads by bloodstream
binds - receptor on motor neuron of peripheral nervous system
internalized - by receptor mediated endocytosis (RME)
vesicle acidification - releases LC into motor neuron
LC - cleaves SNARE proteins - not SNARE complex
result - inhibition of acetylcholine release - prevents muscle contraction
flaccid paralysis

47. 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)
TeNT - acts on CNS
inhibits release of inhibitory neurotransmitters
(glycine / g-aminobutyric acid)
at interneuronal junctions
- spastic paralysis -
BoNT - acts on PNS
inhibits release of stimulatory
neurotransmitter
(acetylcholine)
at peripheral nerve endings
- flaccid paralysis -

48. botulinum & tetanus toxin sites of proteolysis
RAT VAMP1 50 VNVDKVLERDQKLSELDDRADALQAGASVFESSAAKLKRKYWW
RAT VAMP2 48 VNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWW
BoNT/F BoNT/D BoNT/G
BoNT/B
TeNT

49. Botulinum toxin - use as a therapeutic agent
USDA licensed Botox for treatment of muscle disorders
(treat by injecting toxin into hyperactive muscle)

50. Botox Use - injection of low dose of BoNT -
localized paralysis at site of injection
relates to extended duration of BoNT effects
BoNT/A (months) > BoNT/E (weeks)
treatment extended from peripheral to
autonomic nervous system
hyperhidrosis (sweating), myofascial pain, migraine headache
future uses - designer therapeutics
targeting of LC to non-neuronal cells
use of HC in transport of large polypeptides / DNA / enzymes / drugs

51. Prior to botulinum toxin injections
Botox injections to remove wrinkles
Prior to botulinum toxin injections
Subject relaxed Subject frowning
After botulinum toxin injections
Subject relaxed Subject attempting to frown

52. 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)

53. Design of toxins for therapeutic uses
S S
A-subunit
B-subunit
L enzyme activity / receptor binding / internalization
intracellular trafficking
S S
A-subunit
B-subunit
L enzyme activity / altered receptor binding
altered acellular trafficking
Cell-specific cytotoxicity
receptor for granulocyte-macrohage colony-stimulating factor
targets myeloid leukemia cells - linked to toxin
VH-VL - linked to toxin

54. VI. Biological warfare

55. 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])
Category A Diseases/Agents
The 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.

56. Treatment of toxin mediated diseases
anti-toxin antibodies (passive)
anti-toxin vaccines (active)
humoral immune response
Ineffective therapy
antibiotics
innate immune response

57. 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