Spore Germination Apparatus in Clostridium botulinum Group I and II Hannah Pye MSc by Research University of East Anglia Quadram Institute Bioscience September 2017 This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. i ABSTRACT Spore germination is a significant step in the transformation of dormant spores into exponentially dividing vegetative cells, and in the case of Clostridium botulinum in the formation of the deadly botulinum neurotoxin. The ability of nutrient and non-nutrient germinants to induce germination of strains Af84 (C. botulinum Group I) and Eklund 17B (C. botulinum Group II) was established by measuring the change in optical density and by microscopic observation. Germination of strains Af84 and Eklund 17B was initiated by the nutrient germinants L-alanine and L-cysteine and the non-nutrient germinants dodecylamine (DDA) and lysozyme, but not by calcium dipicolinic acid (CaDPA). Heating spores of strain Af84 for 4 hours at 95°C delivered a 3-log reduction in viability. Heat damaged spores of strain Af84 could not be recovered using lysozyme, L-alanine or DDA. Thermal death of spores of strain Eklund 17B occurred within 2 minutes of heating at 85°C, and resulted in a 5-log reduction in viability. The presence of lysozyme (10μg/ml) increased the recovery of heat damaged spores of strain Eklund 17B, however, the presence of L- alanine and DDA had no effect. The timing of DPA release in relation to cortex hydrolysis is still unknown in C. botulinum, however this study found that DPA is released within 18 hours of germinant addition in strain Af84 and within 50 minutes for strain Eklund 17B. In addition, the average DPA concentration per spore of strains Af84 and Eklund 17B was shown to be 1.9pg and 2.1pg, respectively. Results obtained indicate that part of the germination apparatus in Group II strain Eklund 17B is damaged by heating, rather than the spore DNA. Understanding the mechanisms involved in spore germination can improve the control of botulinum neurotoxin forming clostridia, prevent foodborne botulism, and thereby contribute to the microbiological safety of foods. ii CONTENTS ABSTRACT ................................................................................................................................... ii LIST OF FIGURES AND TABLES......................................................................................................... vi ABBREVIATIONS ........................................................................................................................ viii ACKNOWLEDGEMENTS ................................................................................................................. ix 1. Introduction ......................................................................................................................... 1 1.1 Botulinum Neurotoxins ................................................................................................. 2 1.2 Botulism ......................................................................................................................... 5 1.2.1 History of Botulism ................................................................................................. 5 1.2.2 Foodborne Botulism ............................................................................................... 6 1.2.3 Wound Botulism ..................................................................................................... 8 1.2.4 Infant Botulism ....................................................................................................... 9 1.3 Aetiology ...................................................................................................................... 10 1.3.1 Regular neurotransmission at a synapse ............................................................. 10 1.3.2 Mode of action of botulinum toxin ...................................................................... 11 1.4 Uses of Botulinum Toxin ............................................................................................. 11 1.4.1 Therapeutic ........................................................................................................... 11 1.4.2 Biological Weapon ................................................................................................ 13 1.5 Endospores .................................................................................................................. 14 1.5.1 Sporulation ........................................................................................................... 14 1.5.2 Spore Structure and Function .............................................................................. 15 1.5.3 Germination .......................................................................................................... 18 1.6 Experimental Rationale ............................................................................................... 22 1.7 Aims ............................................................................................................................. 24 2. Materials & Methods ......................................................................................................... 25 2.1 Preparation of spores .................................................................................................. 25 iii 2.2 Assessing the inhibitory effect of lysozyme on growth from spores of C. botulinum Group I and II ..................................................................................................................... 26 2.3 Preparation of germinant solutions ............................................................................ 26 2.4 Germination assay measuring change in optical density ........................................... 27 2.5 Germination assay monitoring percentage of phase-dark spores ............................ 28 2.6 Removal of spore coats using dithiothreitol .............................................................. 28 2.7 Removal of spore coats using sodium thioglycolate .................................................. 28 2.8 Thermal death of spores of C. botulinum Group I strain Af84 and Group II strain Eklund 17B ......................................................................................................................... 29 2.9 Heat Inactivation and decoating of spores ................................................................. 29 2.10 Recovery of heat inactivated spores with lysozyme ................................................ 30 2.11 Recovery of heat inactivated spores with L-alanine ................................................ 30 2.12 Recovery of heat inactivated spores with dodecylamine ........................................ 31 2.13 Monitoring CaDPA release using Terbium Fluorescence ......................................... 31 2.14 Quantifying the DPA content per spore of C. botulinum Group I strain Af84 and Group II strain Eklund 17B................................................................................................. 32 3. Results ................................................................................................................................ 33 3.1 Thermal death of spores of C. botulinum Group I strain Af84 and Group II strain Eklund 17B ......................................................................................................................... 33 3.2 L-alanine and L-cysteine germinate spores of C. botulinum Group I and II .............. 35 3.3 Exogenous CaDPA does not induce germination in C. botulinum Group I strain Af84 or Group II strain Eklund 17B at 30°C ............................................................................... 36 3.4 Exogenous CaDPA did not induce germination of coated or decoated spores of C. botulinum Group I strain Af84 or Group II strain Eklund 17B at 45°C ............................. 37 3.5 Germination assay using microscopic analyses of spores of C. botulinum Group I strain Af84 and Group II strain Eklund 17B gave similar results to those using the Bioscreen ........................................................................................................................... 38 3.6 DDA induced germination in spores of C. botulinum Group I strain Af84 and Group II strain Eklund 17B ............................................................................................................... 39 iv 3.7 Lysozyme does not have an inhibitory effect on growth from spores of C. botulinum Group I strain Af84 or Group II strain Eklund 17B ............................................................ 40 3.8 Lysozyme induces germination of decoated spores of C. botulinum Group I strain Af84 and Group II strain Eklund 17B ................................................................................. 41 3.9 Lysozyme recovers heat-damaged spores of C. botulinum Group II strain Eklund 17B but not spores of Group I strain Af84 ............................................................................... 43 3.10 Spores of C. botulinum Group I strain Af84 and Group II strain Eklund 17B cannot be recovered with DDA after heat inactivation ............................................................... 45 3.11 Spores of C. botulinum Group I strain Af84