bacterial toxins
DESCRIPTION
Bacterial toxins. function of susceptibility of host. relates to mechanism of bacterial pathogenesis. Disease. immune competent/compromised immunizations age trauma genetics antimicrobial therapy. secretion of factors (toxins) direct host cell manipulation. Bacterial toxin studies. - PowerPoint PPT PresentationTRANSCRIPT
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
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
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
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
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
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
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
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)
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
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
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