Open Research Online The Open University’s repository of research publications and other research outputs A Genomic and Proteomic Investigation Into the Clostridium botulinum Neurotoxin Complex Thesis How to cite: Ashton, Philip Matthew (2014). A Genomic and Proteomic Investigation Into the Clostridium botulinum Neurotoxin Complex. PhD thesis The Open University. For guidance on citations see FAQs. c 2014 The Author Version: Version of Record Copyright and Moral Rights for the articles on this site are retained by the individual authors and/or other copyright owners. For more information on Open Research Online’s data policy on reuse of materials please consult the policies page. oro.open.ac.uk A Genomic and Proteomic Investigation into the Clostridium botuiinum Neurotoxin Complex Philip Matthew Ashton BSc Affiliated Research Centre - Health Protection Agency This work is offered in part fulfilment of a PhD in Biological Sciences in November 2013 ProQuest Number: 13835686 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 13835686 Published by ProQuest LLC(2019). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 Acknolwedgements I would like to thank all the people whose forebearance and understanding was so heavily relied upon during the writing of this thesis. Specificially Nikki Rockell for the first 4 years and Cat Anscombe for the last 6 months, which are obviously the most important. I would also like to thank: • Fgr proteomic assistance, Min Fang, Tom Gaulton, Raju Misra and Russell Jones. • For microbiological assistance, Ijay Nwafor, Charles Ohai and David Wooldridge. • For bioinformatic assistance, Raju Misra, Anthony Underwood, Richard Myers and Tim Dallman. I could not have finished (or started!) this PhD without the support, advice, and guidance of my three excellent supervisors Kathie Grant, Mike Peck and Saheer Gharbia. I would also like to thank the people who have helped me get where I am today over the longer term. Specifically, my father Richard and brother James. 2 Contents 1. Introduction ................ ....... 11 1.1. Clostridium - The Genus .............. .......................................................11 1.2. Clostridium - The Pathogens .......................................................12 1.3. Botulism and Clostridium botulinum....... .......................................... 14 1.4. Botulism Epidemiology ....................... .............. ...........................18 1.4.1. Food borne botulism ........ 18 1.4.2. Wound botulism ........................................................ ...24 1.4.3. Infant botulism .............. 28 1.4.4. Other forms of botulism ; ......................................... 32 1.4.5. Symptoms, diagnosis and treatment of botulism .............................34 1.5. Botulinum Toxin .............. 40 1.5.1. Bacterial toxin complexes...... ................ .........................................42 1.5.2. Botulinum toxin and the associated non-toxin proteins ..................43 1.5.3. The role of the ANTPs in BoNT intoxication ..................................49 1.5.4. Major steps in botulinum neurotoxin action ...................................56 1.5.5. Evolution of BoNT and the associated non-toxic proteins .... 66 1.5.6. Regulation of bont and the Clostridium botulinum transcriptome .... 67 1.6. Research aims ................ .........................................................71 2. Methods ............................ ..:........... 72 2.1. In silico investigation of the botulinum toxin complex and C. botulinum proteome ......... 72 2.1.1. In silico investigation of botulinum neurotoxin and the neurotoxin associated proteins .................... 72 2.1.2. Protein sub cellular location... ................................. 73 2.2. Microbiology ..................................... 76 2.2.1. Strain list .................................... :........................:........:...................76 2.2.2. Growth conditions... ............................................ 77 2.2.3. Growth curves ...................................... 77 2.3. Investigation of the proteome of C. botulinum ..........................................78 2.3.1. Protein precipitation .................................................................... .....78 2.3.2. Determination of protein concentration... ........................ 79 2.3.3. Endopeptidase immunoassay for measuring BoNT activity ............80 2.3.4. 1D Sodium Dodecyl Sulphate Polyacrylamide Gel electrophoresis. 81 2.3.5. In-gel digestion ............................... 81 2.3.6. LC-MS analysis of peptide fragment mixtures ...................... 82 2.3.7. Comparison with MvirDB .....................................................84 2.3.8. Calculation of extracellular protein cost ....................................... 85 2.4. Investigation of toxin complex gene type and genomic background of botulinum toxin producing strains ................ ........................................ 85 2.4.1. DNA extraction .................................................................. ............85 2.4.2. PCR to determine toxin type and toxin complex type ..................... 86 2.4.3. fAFLP analysis of clinical strains ...................................................87 2.5. Analysis of transcription in C. botulinum ......................................88 2.5.1. Extraction of RNA from C. botulinum culture .......................... .......88 2.5.2. DNase treatment .................. ...................................................... 90 2.5.3. RNA quantity and purity analysis .................... .........................91 2.5.4. Analysis of RNA quality by agarose gel electrophoresis ................ 91 2.5.5. Analysis of RNA quality using Agilent Bioanalyser ......................... 91 2.5.6. Reverse Transcription .......................................................... 92 2.5.7. Quantitative PCR (qPCR).... ..........................................................92 2.5.8. Relative gene expression analysis ..... 95 2.5.9. Purification of RNA using phenol:chloroform:isoamylalcohol ...........95 2.5.10. Taking of samples for RNA-seq .... 96 2.5.11. Reduction of rRNA before preparation of RNA-seq libraries.... .....96 2.5.12. SOLiD sequencing of samples .......................................................97 2.5.13. Analysis of RNA-seq data ............................................... 97 2.6. Primers. ............ 100 3. Results ............................... 101 3.1. In silico investigation of the botulinum toxin complex and C. botulinum proteome ........................... 101 3.1.1. In silico investigation of botulinum neurotoxin and the neurotoxin associated proteins ......................................... ....105 3.1.2. Identification of P-47 family gene clusters in non-C. botulinum species and their association with genes encoding putative toxin proteins ........... 131 3.1.3. Prediction of supernatant proteins of C. botulinum................. ......154 3.1.4. Summary of findings of in silico investigation .................................156 3.2. Proteomic investigation of C. botulinum to establish protein profiles associated with toxin producing strains..... ................ 157 3.2.1. C. botulinum and C. sporogenes growth curves. ............................158 3.2.2. Optimisation of protein precipitation from C. botulinum culture supernatant ........................................ 160 3.2.3. Determination of toxin concentration in the culture supernatant using endopeptidase assay ............... 168 3.2.4. Detection and identification of botulinum neurotoxin and other proteins in C. botulinum culture supernatant by LC-MS/MS .............. ..171 3.2.5. Comparison of predicted and experimentally identified supernatant proteins of C. botulinum A1 ATCC 19397 .................................................. 188 3.2.6. Extracellular protein cost as an indicator of involvement in virulence 191 3.2.7. Identification of toxin complex proteins in clinical strains of C. botulinum by LC-MS/MS ............. .................................................. 196 3.2.8. Summary of findings of proteomic investigation .............. 206 3.3. Genomic diversity and toxin complex type of clinical isolates causing botulism in the UK ............................................................................. ............207 3.3.1. Investigating genomic diversity of isolates of causing different forms of botulism in the UK .............................................................................. 208 3.3.2. Determination of the toxin complex type of clinical strains of C. botulinum .................................. : . 212 3.3.3. Summary of findings of genomic diversity, toxin complex type and form of botulism .....................................................................................215 3.4. RNA-Seq transcriptome profiling to investigate C. botulinum toxin complex expression....... ................................................................................ ..............216