Clostridium botulinum

Toxin: The Neuromuscular Wonder Drug

Amy Malhowski Biology 360 March 30, 2005 Figure taken from: http://www.consultingroom.com/Aesthetics/Products/Product_Display.asp?ID=1

Public Perception of Botulinum Toxin

Bioterrorism!

Figures taken from: http://www.safetycentral.com/bottoxfacin.html

http://archives.cnn.com/2002/ALLPOLITICS/06/12/bush.terror/

And of course…Botox® Aka “The Fountain of Youth”

Figures taken from:

Outline of Talk

•

Historical background of C. botulinum

• Transmission of Botulinum toxin • Molecular pathogenesis • Therapeutic uses of Botulinum toxin • Concluding remarks

What is Botulism?

• Results in flaccid paralysis of muscles • Caused by toxin produced from

C. botulinum

• Three types – via route of entry of bacteria – Foodborne, infant, wound • Mainly exists as foodborne outbreaks • Now have bioterrorism threat

The History of Botulinum Toxin

• Coined “botulism” in 1700s from

botulus

(sausage) after an outbreak from consumption of improperly cooked sausage • Published 1 st case studies on botulinum intoxication • Accurately described neurological symptoms • 1 st to propose therapeutic use of toxin Figure adapted from: Erbguth, 2004.

Symptoms of Botulism

Figure taken from Caya,

et al.

, 2004.

Finding the Culprit

• Emile Pierre van Ermengem – – 1 st to connect botulism to bacterium isolated from raw, salted pork & postmortem tissues of botulism victims – Sucessfully isolated bacterium, naming it

Bacillus botulinus

Clostridium botulinum

• Strict anaerobe • Gram-positive • Bacillus (rod) shape • Ubiquitous in terrestrial environment • Virulence factor = Botulinum toxin – Released under specific conditions Figure taken from http://www.jhsph.edu/Publications/Special/cover2.htm

Botulism and Bioterrorism

• Botulinum toxin attempted use as biological weapon during WWII – aborted when toxin did not affect test animals (donkeys) • Great potential in toxicity • BoNT no longer considered good biological weapon

Mass Producing Botulinum Toxin

• Fort Detrick (1946) – bioweapon research – 1 st time mass produce toxin • Production process- grow, crystallize • 1972 – Nixon terminates all research on biological warfare agents • Research continues – 1979 – Schantz produces batch 79-11; used until 1997 • 1991 – several batches made – Botox ® by Allergan Inc.

So what?

Importance of

C. botulinum

Research

• Bioterrorism/outbreaks • Kerner – brought about idea of therapeutics • Most recent work – harnessing BoNT as therapeutic agent for neuromuscular disorders

Outline of Talk

• • Historical background of

C. botulinum

Transmission of Botulinum toxin

• Molecular pathogenesis • Therapeutic uses of Botulinum toxin • Impediments in Treatment • Concluding remarks

Transmission of Botulinum Toxin

• Most commonly via improperly cooked food • Conditions to produce toxin not completely understood • Complex route of transmission – Ingestion/injection – Neurotoxin produced as progenitor complex – Absorbed into tissue  circulates blood – Docks onto receptors of neuron  up acetylcholine  paralysis transcytosis  binds

Classes of Botulinum Toxin

• Seven different subtypes of botulinum toxin – A, B, C1, D, E, F, and G • Same general mechanism for muscular paralysis • Vary in structure, target site, & toxicity • Only two manufactured for commercial use – A and B

Target Proteins of Botulinum Toxins

Serotype A B C1 D Cellular Substrate

SNAP-25 VAMP/Synaptobrevin Cellubrevin Syntaxin 1A, 1B SNAP-25 VAMP/Synaptobrevin (18, 181) Cellubrevin (181)

Target Cleavage Site

Gln197-Arg198 Gln76-Phe77 Gln59-Phe60?

Lys253-Ala254 Lys252-253 Lys59-Leu60 Ala67-Asp68 Lys42-Leu43?

Arg180-Ile181

E F G

SNAP-25(46) VAMP/Synaptobrevin (181, 182) Cellubrevin VAMP/Synaptobrevin Figure adapted from: Aoki. 2004. Curr Med Chem. 11: 3085-3092.

Gln58-Lys59 Gln41-Lys42?

Ala81-Ala82

Outline of Talk

• Historical background of

C. botulinum

• Transmission of Botulinum toxin •

Molecular pathogenesis

• Therapeutic uses of Botulinum toxin • Impediments in Treatment • Concluding remarks

Molecular Pathogenesis of BoNT

BoNT synthesized as single-chain polypeptide (inactive form) Polypeptide cleaved by protease to create dichain structure (active form) BoNT binds to epithelium, transcytosed, reaches general circulation Receptor-mediated endocytosis at peripheral cholinergic nerve endings In cytosol, toxin cleaves target, blocking neurotransmitter release = flaccid paralysis

Major Steps in BoNT Action

Figure taken from: Simpson. 2004. Annu. Rev. Pharmacol. Toxicol. 44: 161-193.

Botulinum Toxin Type A

Aoki. 2004. Curr Med Chem. 11: 3085-3092.

Simpson. 2004. Annu. Rev. Pharmacol. Toxicol. 44: 161-193.

Figure taken from: Arnon,

et al.

2001.

Uses of Botulinum Toxin

• Bioterrorism agent – Category A • Local paralytic agent – Botox® • Therapeutic agent – Neuromuscular disorders – Pain management

BoNT as Local Paralytic Agent

• Use Botulinum toxin type A (Botox®) • Many cosmetic uses • Few clinical side effects • Fast acting – 6 hrs post injection • Effects last 3-6 months • Serial injections required to maintain results

BoNT/A Induces Local Paralysis

• Local effects = dose dependent • Injection site affects physical outcome Before Botox® After Botox® Figures adapted from: Mendez-Eastman. 2003. Plast. Surg. Nurs. 23: 64-70.

Outline of Talk

• Historical background of

C. botulinum

• Transmission of Botulinum toxin • • Molecular pathogenesis

Therapeutic uses of Botulinum toxin

• Impediments in Treatment • Concluding remarks

BoNT as a Therapeutic Agent

• Botox® used for aesthetics  therapeutic use in neuromuscular disorders • BoNT/A = Botox ® from Allergan, Inc.

• BoNT/B = MYOBLOC™ from Elan Pharmaceuticals

BoNT as Therapeutic Agent in Neuromuscular Disorders

• Purified BoNT/A = Botox ® • Treat medical conditions characterized by muscle hyperactivity/spasm – blepharospasm, strabismus, cervical dystonia, glabellar lines, spastic dystonia, limb spasticity, tremors, chronic anal fissure, hyperhidrosis, etc.

• Currently only FDA approved for 4 disorders – Blepharospasm (aka focal dystonia) – Strabismus – Cervical dystonia – Hyperhidrosis

BoNT/A & Muscle Hyperactivity Cervical Dystonia (CD)

• CD – involuntary contractions of neck and shoulder muscles • FDA approved injections with BoNT/A (2000) • BoNT/A is injected into affected muscles to reduce muscle contraction • BoNT/A effectively reduces muscle spasticity and pain associated with CD

Cervical Dystonia Study with Botox® by Allergan, Inc.

• Phase 3 randomized, multi-center, double blind, placebo-controlled study on treatment of CD with Botox ® (1998) • 170 subjects (88 in Botox® group, 82 in placebo group), analyzed until 10 wks post injection • Study suggested that majority of patients had beneficial response by 6 th week

Cervical Dystonia Study with BoNT/A as Dysport®

• A multicenter, double-blind, randomized, controlled trial with Dysport® to treat CD in the USA (2005) • Patients (80) randomly assigned to receive Dysport® (500U) or placebo • Dysport was significantly more effective than placebo at weeks 4, 8, and 12 • Dysport group had 38% with positive treatment response, with median duration of response of 18.5 weeks

BoNT/A & Pain Management

• Testing BoNT use in controlling pain associated disorders • Data suggests BoNT acts in complex manner – not just controlling overactive muscle • Appears that BoNT inhibits the release of neurotransmitters (glutamate and substance P) involved in pain transmission

Peripheral and Central Nervous System Sensitization

Figure taken from: Aoki, 2003.

Botulinum Toxin A Affects Sensitization of PNS & CNS

Figure taken from: Aoki, 2003.

Antinociceptive Activity of BoNT/A

• Acute pain (phase 1) is not relieved by BoNT/A • Inflammatory pain (phase II) is relieved by BoNT/A • Increasing doses decrease phase II pain appreciably • Antinociceptive activity is maintained longer with higher dose of BoNT/A Figure taken from: Aoki, 2003.

BoNT/A Injection Reduces Formalin-induced Pain

• Upon formalin challenge 5 days post-injection, dose dependent decrease in Glut release is observed • Injection of BoNT/A prevents increase of formalin induced Glut release Figure taken from: Aoki, 2003.

BoNT/A Reduces Pain

A) Antinociceptive Activity of BoNT/A in formalin-challenged rats.

B) Subcutaneous BoNT/A injection reduces formalin-induced glutamate release in rat paw in a formalin-challenged inflammatory pain animal model.

Figures taken from: Aoki, 2003.

Conclusions on Therapeutics

• BoNT mechanism = specific • Uses are diverse – Local flaccid paralysis – Reducing muscle spasticity – Reducing pain • Currently, BoNT therapy on muscle disorders and associated pain

Outline of Talk

• Historical background of

C. botulinum

• Transmission of Botulinum toxin • Molecular pathogenesis • Therapeutic uses of Botulinum toxin •

Impediments in Treatment

• Concluding remarks

Impediments in Treating with BoNT

• FDA approval pending for many disorders • Fleeting effects – need repeated injections • Socioeconomics – less expensive than surgery but not permanent • Social constraints – – not up to snuff on research – stigma in using deadly toxin for good use

Concluding Remarks

• Toxin = great therapeutic agent!

• Research to understand mechanism of release of BoNT from

C. botulinum

• Impediments in therapeutics • Future with Botox® is bright!

And remember… Sometimes wrinkles aren’t all that bad!

Thank you!

• Chris White-Ziegler • My readers: Caitlin Reed & Natalia Grob • Bio 360 students Figure taken from: http://www.jwolfe.clara.net/Humour/MedMiscel.htm

• • • • • • • • •

References

Aoki, K.R. 2001. A Comparison of the Safety Margins of Botulinum Neurotoxin Serotypes A, B, and F in Mice. Toxicon. 39: 1815-1820.

Aoki, K.R. 2003. Evidence for Antinociceptive Activity of Botulinum Toxin Type A in Pain Management. Headache. 43: S9-S15.

Aoki, K.R. 2004. Botulinum Toxin: A Successful Therapeutic Protein.

Curr.

Med. Chem. 11: 3085-3092.

Bossi, P., A. Tegnell, A. Baka, F. Van Loock, J. Hendriks, A. Werner, H. Maidhof, and G. Gouvras. 2004. Bichat Guidelines for the Clinical Management of Botulism and Bioterrorism-related Botulism. Eurosurveillance. 9: 1-4.

Breldenbach, M.A. and A.T. Brunger. 2004. Substrate Recognition Strategy for Botulinum Neurotoxin Serotype A. Nature. 432: 925-929.

Casadevall, A., E. Dadachova, and L. Pirofski. 2004. Passive Antibody Therapy for Infectious Diseases. Nat Rev Microbiol. 2: 695-703.

Casadevall, A. and L. Pirofski. 2004. The Weapon Potential of a Microbe. Trends Microbiol. 12: 259-263.

Caya, J.G., R. Agoi, and J.E. Miller. 2004. Ramifications of Botulinum Toxin.

Clostridium botulinum

and the Clinical Laboratorian: A Detailed Review of Botulism, Including Biological Warfare Arch Pathol Lab Med. 128: 653-662.

CDC Botulism Emergency Preparedness & Response. http://www.bt.cdc.gov/agent/botulism/

• • • • •

References

Chaddock, J.A., J.R. Purkiss, L.M. Friis, J.D. Broadbridge, M.J. Duggan, S.J. Fooks, C.C. Shone, C.P. Quinn, and K.A. Foster. Inhibition of Vesicular Secretion In Both Neuronal and Nonneuronal Cells by a Retargeted Endopeptidase Derivative of

Clostridium botulinum

Type A. Infect. Immun. 68: 2587-2593.

Coffield, J.A., N.M. Bakry, A.B. Maksymowych, and L.L. Simpson. 1999. Characterization of a Vertebrate Neuromuscular Junction That Demonstrates Selective Resistance to Botulinum Toxin.

J. Pharmacol.

Exp. Ther. 289: 1509-1516.

Coffield, J.A., N.M. Bakry, R.-d. Zhang, J. Carlson, L.G. Gomella, and L.L. Simpson. 1997.

In Vitro

Characterization of Botulinum Toxin Types A, C and D Action on Human Tissues: Combined Electrophysiologic, Pharmacologic and Molecular Biologic Approaches.

J. Pharmacol.

Exp. Ther. 280: 1489-1498.

Cui, M., S. Khanijou, J. Rubino, and K.R. Aoki. 2004. Subcutaneous Administration of Botulinum Toxin A Reduces Formalin-Induced Pain. Pain. 107: 125-133.

De Paiva, A., F.A. Meunier, J. Molgo, K.R. Aoki, and J.O. Dolly. 1999. Functional Repair of Motor Endplates After Botulinum Neurotoxin Type A Poisoning: Biphasic Switch of Synaptic Activity Between Nerve Sprouts and their Parent Terminals. Proc. Natl. Acad. Sci. USA. 96: 3200-3205.

• • • • •

References

Erbguth, F.J. 2004. Historical Notes on Botulism,

Clostridium botulinum

, Botulinum Toxin, and the Idea of the Therapeutic Use of the Toxin. Mov Dis. 19: S2-S6.

Fernandez-Salas, E., H. Ho, P. Garay, L.E. Steward, and K.R. Aoki. 2004. Is the Light Chain Subcellular Localization an Important Factor in Botulinum Toxin Duration of Action? Mov. Dis. 19: S23-S34.

Fernandez-Salas, E., L.E. Steward, H. Ho, P.E. Garay, S.W. Sun, M.A. Gilmore, J.V. Ordas, J. Wang, J. Francis, and K.R. Aoki. 2004. Plasma Membrane Localization Signals in the Light Chain of Botulinum Neurotoxin. Proc. Natl. Acad. Sci. USA. 101: 3208-3213.

Foran, P.G., N. Mohammed, G.O. Lisk, S. Nagwaney, G.W. Lawrence, E. Johnson, L. Smith, K.R. Aoki, and J.O. Dolly. 2003. Evaluation of the Therapeutic Usefulness of Botulinum Neurotoxin B, C1, E, and F Compared with the Long Lasting Type A: Basis for Distinct Durations of Inhibition of Exocytosis in Central Neurons. J. Biol. Chem. 278: 1363-1371.

Franciosa, G., M. Pourshaban, A. De Luca, A. Buccino, B. Dallapiccola, and P. Aureli. 2004. Identification of Type A, B, E, and F Botulinum Neurotoxin Genes and of Botulinum Neurotoxigenic Clostridia by Denaturing High-Performance Liquid Chromatography.

App Env

Microbiol. 70: 4170-4176.

• • • • • • •

References

Hall, Y.H.J., J.A. Chaddock, H.J. Moulsdale, E.R. Kirby, F.C.G. Alexander, J.D. Marks, and K.A. Foster. 2004. Novel Application of an Antibodies. J. Immun. Met. 288: 55-60.

in vitro

Technique to the Detection and Quantification of Botulinum Neurotoxin Hong, B., L. Jiang, Y. Hu, D. Fang, and H. Guo. 2004.

J

.

Met. 58: 403-411.

Microbiol.

Jathoul, A.P., J.L. Holley, and H.S. Garmory. 2004. Efficacy of DNA Vaccines Expressing the Type F Botulinum Toxin Hc Fragment Using Different Promoters. Vaccine. 22: 3942-3946.

Johnston, M.D., S. Lawson, and J.A. Otter. 2005. Evaluation of Hydrogen Peroxide Vapour as a Method for the Decontamination of Surfaces Contaminated with

Clostridium botulinum

Spores.

J. Microbiol. Met.

60:

403-411.

Lacy, D.B., W. Tepp, A.C. Cohen, B.R. DasGupta, and R.C. Stevens. 1998. Crystal Structure of Botulinum Neurotoxin Type A and Implications for Toxicity. Nat. Struct. Biol. 5: 898-902.

Lovenklev, J. E. Holst, E. Borch, and P. Radstrom. 2004. Relative Neurotoxin Gene Expression in

Clostridium botulinum

Type B, Determined Using Quantitative Reverse Transcription-PCR.

App Env

Microbiol. 70: 2919-2927.

Maksymowych, A.B. and L.L. Simpson. 1998. Binding and Transcytosis of Botulinum Neurotoxin by Polarized Human Colon Carcinoma Cells.

J.

Biol. Chem. 273: 21950-21957.

References

• • • • • • • Maksymowych, A.B., M. Reinhard, C.J. Malizio, M.C. Goodnough, E.A. Johnson, and L.L. Simpson. 1999. Pure Botulinum Neurotoxin Is Absorbed from the Stomach and Small Intestine and Produces Peripheral Neuromuscular Blockade. Infect. Immun. 67: 4708-4712.

Marvaud, J.C., S. Raffestin, M. Gibert, and M.R. Popoff. 2000. Regulation of the Toxigenesis in

Clostridium botulinum

and

Clostridium tetani

.

Biol. Cell.

92:

455-457.

McLaughlin, J.B., J. Sobel, T. Lynn, E. Funk, and J.P. Middaugh. 2004. Botulism Type E Outbreak Associated with Eating a Beached Whale, Alaska. Emerg. Infect. Dis. 10: 1685-1687.

Mendez-Eastman, S.K. 2003. BOTOX: A Review. Plast Surg Nurs. 23: 64-70.

Merrison, A.F.A., K.E. Chidley, J. Dunnett, and K.A. Sieradzan. 2005. Lesson of the Week: Wound Botulism Associated with Subcutaneous Drug Use. BJM. 325: 1020-1021.

Park, J. and L.L. Simpson. 2003. Inhalational Poisoning by Botulinum Toxin and Inhalation Vaccination with Its Heavy-Chain Component.

Infect &

Immun. 71: 1147-1154.

Park, J. and L.L. Simpson. 2004. Progress Toward Development of an Inhalation Vaccine Against Botulinum Toxin. Expert Rev. Vacc. 3: 477 487.

References

• • • • • • • Purkiss, J., M. Welch, S. Doward, and K. Foster. 2000. Capsaicin-Stimulated Release of Substance P from Cultured Dorsal Root Ganglion Neurons: Involvement of Two Distinct Mechanisms. Biochem. Pharm. 59: 1403 1406.

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Clostridium botulinum

Type A and

Clostridium tetani. Molec.

Microbiol. 55: 235-249.

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References

• • • • • • Simpson, L.L., H. Zepeda, and I. Ohishi. 1988. Partial Characterization of the Enzymatic Activity Associated with the Binary Toxin (Type C 2 ) Produced by Clostridium botulinum. Infect. Immun. 56: 24-27.

Simpson, L.L., J.A. Coffield, and N. Bakry. 1993. Chelation of Zinc Antagonizes the Neuromuscular Blocking Properties of the Seven Serotypes of Botulinum Neurotoxin as well as Tetanus Toxin.

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Mov. Dis.

References

• Yoneda, S., M. Shimazawa, M. Kato, A. Nonoyama, Y. Torii, H. Nishino, N. Sugimoto, S. Kozaki, and H. Hara. 2004. Comparison of the Therapeutic Indexes of Different Molecular Forms of Botulinum Toxin Type A. Eur. J Pharm. 508: 223 229.

• Yowler, B.C. and C. Schengrund. 2004. Glycosphingolipids—Sweets for Botulinum Neurotoxin. Glycoconjugate. 21: 287-293.

• Zhang, L., W. Lin, S. Li, and K.R. Aoki. 2003. Complete DNA Sequences of the Botulinum Neurotoxin Complex of

Clostridium botulinum

Type A-Hall (Allergan) Strain. Gene. 315: 21-32.

Miscellaneous Info.

BoNT in Treating Lower UTIs

• • •

Describe disease What toxin does as treatment After effects of toxin – how change disorder

•

Show mechanism and data to support effects

Genetic Organization of Botulinum Locus in

Clostridium botulinum

5’

ha

operon

ha70 ha17 ha34

RNAP Core BotR/A

botR/A

v

ntnh

ntnh-bont/A operon

bont/A

3’ Figure adapted from: Raffestin, S.,

et al.,

2005. Molec. Microbiol. 55: 235-249.

Cervical Dystonia Study with Botox®

Placebo N=82

Baseline CDSS 9.3

Change in CDSS at week 6 % Patients with Improvement on PGAS Pain Intensity Baseline -0.3

31% 1.8

Change in Pain Intensity at week 6 -0.1

Pain Frequency Baseline 1.9

Change in Pain Frequency at week 6 -0.0

Table adapted from BOTOX ® insert, Allergan, Inc. 1998.

Botox® N=88

9.2

-1.3

51% 1.8

-0.4

1.8

-0.3

95% CI on Difference

(-2.3, 0.3) [a,b] (5%, 34%) [a] (-0.7, -0.2) [c] (-0.5, -0.0) [c]

Transcytosis of BoNT

• BoNT targets gut epithelial – Absorptive enterocytes – M cells of Peyer’s Patches Figures taken from: Simpson. 2004. Annu. Rev. Pharmacol. Toxicol. 44: 161-193.

Major Steps in BoNT Action

Productive Internalization Intracellular Poisoning Binding Low/High Affinity Site Endocytosis Transcytosis Binding (Cleavage Site) Enzymatic Activity (SNAP-25, VAMP Syntaxin) Expose Occult Domains Insert Into Membrane Cross Membrane Reduce Disulfide Bond Chain Separation Renature Light Chain

Dissociation of Zinc Reassociation of Zinc

Figure adapted from: Simpson. 2004. Annu. Rev. Pharmacol. Toxicol. 44: 161-193.