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Bacterial toxins

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.

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Bacterial toxins

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  1. Bacterial toxins

  2. 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

  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

  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

  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”

  7. 1888 - Disease mechanism related to toxin production Alexandre Yersin (1863-1943) Emile Roux (1853-1933) Diphtheria - prototype toxigenic disease

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

  9. 1821 - Pierre Bretonneau - diphtheritis (pseudomembrane) 1884 - Loeffler cultured organism - linked disease to soluble poison Diphtheria

  10. Emil Adolf von Behring 1854 - 1917 Shibasaburo Kitasato 1852 - 1931 1890 - Diphtheria anti-toxin produced Nobel Prize in Medicine 1901

  11. II. Insight into protein design

  12. OH COOH COOH CH2 CH2 OH CH2 D E A CH2 CH2 CH2 CH2 Y F S 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

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

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

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

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

  17. Toxin regulated by iron (gsbs.utmb.edu/microbook/images/fig32_3.JPG) Diphtheria toxin - production & regulation 1951 - 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

  18. 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) Internalization of diphtheria toxin Receptor mediated endocytosis (RME)

  19. early epidemiology - England 1826-1837 - cholera epidemic William Farr theory -cholera spread by “miasma” in air 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 William Farr (1807-1883) 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 Graph illustrating Farr's elevation theory in Langmuir AD. Bacteriological Review 25, 174, 1961 Cholera History 1854 - Filippo Pacini identified comma- shaped bacillus organism 1884 -Robert Kochidentified cholera bacillus, Vibrio cholerae (maintained credit for discovery until 1965)

  20. Bacteriology 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) Cholera

  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 (www.cameroon-info.net/ img/news/cholera_victim..

  22. Vibrio cholerae motility / chemotaxis-flagella adherence -Tcp (toxincoregulatedpili) 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 (www.hinduonnet.com/.../ 07/stories/0807048f.htm) Virulence factors

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

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

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

  26. Eukaryotic ADPRT proteins CHE131NVFRGVRGT........ .......................RFTA.QQGTVVRFGQ FTSTSLQKKVAEFFGLDTFF 192EDEVLIP CHB131YVYRGVRG......... .......................RFMT.QRGKSVRFGQ.FTSSSLRKEATVNFGQDTLF 192EDEVLIP M61123SVYRGTNV......... .......................RFRYTGKG.SVRFGH FASSSLNRSVATSSPFFNGQ 187EEEVLIP HMT154QVFRGVHGL........ .......................RFRPAGPRATVRLGGFASASLKHVAAQQFGEDTFF 216EEEVLIP Comparison of ADP-ribosylating proteins Bacterial ADPRT toxins - A subunit sequence Diphtheria Toxin Group 22LoopActive siteloop33 7 DT 18SSYHGTKPGYVDSIQKG ....................IQKPKSGTQGNYDDDWKG .FYSTDNKYDAAGYSVDNE146SVEYINN ETA437VGYHGTFLEAAQSIVFG G...................GVRARS..Q.DLDAIWRG .FYIAG DAL..AYGYAQDQE551RLETILG Cholera Toxin Group CT 4KLYRADSRPPDEIKQSG GLMPRGQSEYFDRGTQMNINLYDHARGTQTGFVRHDDGYVSTSISLRSAHLVGQTILS 110EQEVSAL LTI 4KLYRADSRPPDEIKRSG GLMPRGHNEYFDRGTQMNINLYDHARGTQTGFVRYDDGYVSTSLSLRSAHLAGQSILS 110EQEVSAL PT 6TVYRYDSRPPEDVFQNG F...........TAWGNNDNVLDHLTGRSCQVGSSNSA FVSTS SSRRYTE.VYLEHRM 127QSEYLAH EXS316KTFRGTRGG........ ......................DAFNAVEEGKVGHDDGYLSTSLNPGVARSF.GQGTI379EKEILYN EXT319KTFRGTQGR.......... ....................DAFEAVKEGQVGHDAGYLSTS RDPSVARSFAGQGTI383EQEILYD MTX 94RLLRWDRRPPNDIFLNG F.........IPRVTNQNLSPVEDTHLLNYLRTNSPSI FVSTT RARYNNLGLEITPWT 195EDEITFP CI 333IVYR..RSGPQEFGL.. .......TLTSPEYDFNKIENIDAFKEKWEGKVITYPN FISTSIGSVNMSAFAKRKII 419EYEVLLN C3D 85ILFRGDDPAYLG..... ...PEFQDKILNKDGTINRDVFEQVKAKFLKKDRTEYGYISTSLMS.AQFGGRPIVTK171QLEVLLP C31 85ILFRGDDPAYLG..... ...TEFQNTLLNSNGTINKTAFEKAKAKFLNKDRLEYGYISTSLMNVSQFAGRPIITK172QLEMLLP EDN 85YVYRLLNLDYLTSIVG. FTNEDLYKLQQTNNGQYDENLVRKLNNVMNSRIYREDGYSSTQ LVSGAAVGGRPIELR181QQEVLLP

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

  28. S S A-subunit B-subunit Lenzyme activity / receptor binding / internalization intracellular trafficking Types of cellular activity modulating toxins A-B toxins Enzyme activityToxin 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’

  30. Streptolysin-like structure 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)

  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’

  33. [H2O] [CL- ] [Na+] 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 (From A. Salyers, D. Whitt, 2002) Cell receptor interaction toxins 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’

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

  36. III. Vaccine production

  37. Diphtheria vaccine 1891 - first anti-toxin given to diphtheritic child passive immune protection 1923 - Ramon introduced diphtheria toxoid 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 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. 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 Lenzyme activity / receptor binding / internalization intracellular trafficking Combinatorial vaccines - more than one antigen (e.g. acellular pertussis vaccine - non-toxic form of toxin + fimbrial antigen) Toxin vaccine development

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

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

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

  44. V. Disease therapy

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

  46. (Synaptobrevin) Botulinum toxin 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. TeNT - acts onCNS inhibits release of inhibitory neurotransmitters (glycine / g-aminobutyric acid) at interneuronal junctions - spastic paralysis - 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 onPNS inhibits release of stimulatory neurotransmitter (acetylcholine) at peripheral nerve endings - flaccid paralysis - (science.cancerresearchuk.org/images/flat/sch)i Comparison - tetanus & botulinum toxin activity

  48. BoNT/F BoNT/D BoNT/G RAT VAMP150VNVDKVLERDQKLSELDDRADALQAGASVFESSAAKLKRKYWW RAT VAMP248VNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYWW BoNT/B TeNT botulinum & tetanus toxin sites of proteolysis

  49. Botulinum toxin - use as a therapeutic agent 1989 - 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

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