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Bacillus Thuringiensis A University of Sussex DPhil thesis Available online via Sussex Research Online: http://sro.sussex.ac.uk/ This thesis is protected by copyright which belongs to the author. This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Please visit Sussex Research Online for more information and further details Investigation of parasporins, the cytotoxic proteins from the bacterium Bacillus thuringiensis VIDISHA KRISHNAN SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOCHEMISTRY SCHOOL OF LIFE SCIENCES UNIVERSITY OF SUSSEX 2013 Page 1 of 260 Declaration WORK NOT SUBMITTED ELSEWHERE FOR EXAMINATION I hereby declare that this thesis has not been submitted in whole or in part to this or any other University for the award of a degree. Vidisha Krishnan Page 2 of 260 ABSTRACT The cytotoxic activities of proteins from the normally insecticidal Bacillus thuringiensis against human cancer cell lines were investigated. Cry41Aa toxin derived from the Bacillus thuringiensis strain A1462, which shows activity against human cancer cell lines is structurally related to the toxins synthesized by commercially produced transgenic insect-resistant plants, with the exception of an additional C-terminal beta-trefoil ricin domain. To test whether this putative carbohydrate binding domain is responsible for the cytotocidal activity of Cry41Aa against cancer cell lines, we developed an efficient expression system for the toxin and created a deletion mutant lacking the ricin domain. Both Cry41Aa and its deletion mutant were stably expressed and found to have almost identical activities against the HepG2 cancer cell line in vitro. Our results suggest that the acquisition of the ricin domain was not responsible for Cry41Aa having toxicity to human cancer cells and more subtle changes have resulted in the evolution of mammalian cancer cell toxicity. We further attempted to identify those differences that are responsible for the divergence in activity between the cancer-killing toxins and their insecticidal counterparts. We also studied the cell-killing activity of other uncharacterized Bacillus thuringiensis crystal toxins including Cry51 and Cry65 whose targets are not known so far. Page 3 of 260 Acknowledgements I sincerely express my immense gratitude to my supervisor, Dr. Neil Crickmore for his kind and patient guidance and active involvement in all stages of this project from its inception. Neil is not only an outstanding scientist but a great teacher and a wonderful person. I am indebted to him for all that I have learnt and continue to learn from him and I express my profound respect to him. With gratitude I acknowledge Dr. Lynne Mayne for introducing me to Cell Biology skills. My special thanks to Dr. Michelle West for the excellent facilities, expertise and advice. To all her wonderful, approachable and friendly lab members for extending every aid cheerfully and promptly. To Ali who is always in our thoughts. To Mark, Paul, Zenas, Ruchir and the undergrads including Natalie, Laura, Laurence, Heather and everyone with whom I have worked for all the wonderful times that I had with them in the lab. Finally, I would like to thank my wonderful, amazing kids Ash and Babhravi for all their love and support. They always inspire me to do my best. To Krishnan for annoying me for close to two decades—besides for the innumerable print-outs! To my wonderful grand-dad and grand –mum for their unconditional love and providing me the best possible facilities in India and all that I have learnt from them. To all my family and friends who have always been there for me. I wouldn’t have been able to do this without you all! Vidisha. Page 4 of 260 Table of Contents 1. Introduction ............................................................................................................. 11 1.1 General Introduction .......................................................................................... 11 1.2 Bacillus thuringiensis ......................................................................................... 12 1.3 B. thuringiensis taxonomy and genetics ............................................................ 13 1.4 Ecology and prevalence .................................................................................... 15 1.5 Virulence factors ............................................................................................... 16 1.6 Vip toxins .......................................................................................................... 17 1.7 Bt δ-endotoxins ................................................................................................. 18 1.7.1 Cyt δ-endotoxins ......................................................................................... 20 1.8 Cry δ-endotoxins ............................................................................................... 21 1.9 The Mtx Group .................................................................................................. 26 1.10 The Bin group .................................................................................................. 27 1.10.1 The Binary toxins ...................................................................................... 28 1.11 The three-domain Cry toxins ........................................................................... 29 1.12 Split toxins ....................................................................................................... 33 1.13 Mode of action ................................................................................................. 36 1.13.1 Solubilisation and proteolytic activation ..................................................... 37 1.13.2 Receptor binding ....................................................................................... 38 1.14 Bravo-Soberon model...................................................................................... 39 1.15 The “ping-pong” sequential binding model ....................................................... 40 1.16 Zhang-Bulla model .......................................................................................... 40 1.17 Parasporins ..................................................................................................... 44 1.17.1 Parasporin-1 (Cry31A) .............................................................................. 46 1.17.2 Parasporin-2 (Cry46Aa) ............................................................................ 49 1.17.3 Parasporin-3 (Cry41Aa and Cry41Ab) ....................................................... 52 1.17.4 Parasporin-4 (Cry45Aa) ............................................................................ 54 1.17.5 Parasporin-5 (Cry64Aa) ............................................................................ 55 1.17.6 Parasporin-6 (Cry63Aa) ............................................................................ 56 1.18 Present work ................................................................................................... 58 2. Materials and Experimental protocols ..................................................................... 59 2.1 General materials .............................................................................................. 59 2.1.1 Bacterial strains .......................................................................................... 59 2.1.2 Plasmids ..................................................................................................... 61 2.1.3 Insect populations ....................................................................................... 62 2.1.4 Human cancer cell lines .............................................................................. 63 Page 5 of 260 2.1.5 Culture media ............................................................................................. 63 2.1.5.1 Sub-culture Routine ............................................................................. 63 2.1.6 Miscellaneous reagents and kits used for cell assays ................................. 64 2.1.7 Bacterial culture media ............................................................................... 64 2.2 Experimental protocols ...................................................................................... 65 2.2.1 Design of PCR primers for amplification of DNA fragments ......................... 65 2.2.2 PCR amplification of DNA ........................................................................... 65 2.2.3 Purification of PCR amplicons ..................................................................... 66 2.2.3.1 Column purification of the PCR amplicons ............................................ 66 2.2.3.2 Gel isolation of the DNA fragments ....................................................... 66 2.2.4 Ligation of the purified PCR amplicons ....................................................... 67 2.2.5 Transformation of bacterial strains with plasmids ........................................ 67 2.2.5.1 Transformation of E. coli strains by electroporation .............................. 67 2.2.5.2 Transformation of Bacillus thuringiensis strains .................................... 68 2.2.6 Rapid size screen of
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