The antimicrobial mechanism of action of 3,4-methylenedioxy-βββ-nitropropene A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy Kylie Suzanne White B. App. Sci. School of Applied Science Science Engineering and Technology Portfolio RMIT UNIVERSITY March 2008 Declaration I certify that except where due acknowledgement has been made, the work is that of the author alone; the work has not been submitted previously, in whole or in part, to qualify for any other academic award; the content of the thesis is the result of work which has been carried out since the official commencement date of the approved research program; and, any editorial work, paid or unpaid, carried out by a third party is acknowledged. __________________ Kylie Suzanne White __________________ ii Acknowledements I would firstly like to thank my supervisors Gina Nicoletti and Robert Borland for their confidence, support and invaluable guidance throughout. In regards to the chlamydia work I would like to thank Robert Alexander for his generosity with his time, for use of the lab and for providing and maintaining the C. trachomatis clinical strain and McCoy cell monolayers. Thankyou to everyone in the lab RCH for making me feel welcome during my visits. I would also like to thank those in the department who have helped me along the way, particularly Viraj Perera for use of the Campylobacter mutant and Tien Huynh for help with quantitative PCR. Thanks also to Priya, Nitin, Eddie and Stephane for their suggestions for the Microarray work. A big thankyou to Thu for her friendship, making sure I had enough media and being there to tend to cultures when I could not. I would like to thank everyone, past and present that have offered their friendship in the lab and in the department. You have provided a supportive and enjoyable environment in which to undertake this, at times daunting, project. It is much appreciated. Please accept my apologies, for there are too many of you to name, but I hope you know who you are. Many thanks to my family and friends who have supported me, even when they have no idea what I’m talking about and struggle to understand why, especially Stuart who has survived me for many stressful years. No doubt you have seen many sides to me during these tumultuous years, hopefully I have grown for the better. Lastly, thanks to BioDiem Ltd. for supplying the compound and all of their financial support, without which this project would not have been possible. iii Table of Contents Page TABLE OF CONTENTS IV LIST OF ABBREVIATIONS XI LIST OF FIGURES XIV LIST OF TABLES XVI ABSTRACT 1 CHAPTER 1 GENERAL INTRODUCTION 2 1.1 AIM OF THIS RESEARCH 2 1.1.1 Significance of the project 2 1.1.2 Plan of investigation for mechanism studies 3 1.1.3 Summary of chapter contents 5 1.2 BDM-I 6 1.2.1 Benzyl nitroalkenes 9 1.3 THE NEED FOR NEW ANTIMICROBIAL AGENTS 10 1.3.1 Therapeutic anti-infective agents 11 Antiseptics 12 1.3.2 Development of anti-infective drugs 13 New approved drugs 14 1.3.3 Novel cellular targets for drugs 16 Protein phosphatases 16 Bacterial phosphatases 17 CHAPTER 2 ANTIBACTERIAL ACTIVITY OF BDM-I 19 2.1 INTRODUCTION 19 iv 2.2 ACTIVITY SPECTRUM FOR BDM-I: MINIMUM INHIBITORY AND MINIMUM MICROBICIDAL CONCENTRATIONS 21 2.2.1 Background 21 2.2.2 Materials and Methods 22 2.2.3 Results 24 2.2.4 Discussion 24 2.3 ACTIVITY AGAINST AN LOS-DEFICIENT MUTANT 26 2.3.1 Background 26 2.3.2 Materials and Methods 27 2.3.3 Results 27 2.3.4 Discussion 28 2.4 EFFECT OF THIOL -CONTAINING COMPOUNDS ON INHIBITORY ACTIVITY 29 2.4.1 Background 29 2.4.2 Materials and Methods 30 2.4.3 Results 30 2.4.4 Discussion 31 2.5 IN VITRO ACTIVITY AGAINST CHLAMYDIA TRACHOMATIS 33 2.5.1 Background 33 2.5.2 Materials and Methods 35 2.5.3 Results 37 2.5.4 Discussion 39 2.6 KILL RATES 41 2.6.1 Background 41 Rate of Kill 41 Effect of BDM-I on S. aureus at different population growth rates 41 2.6.2 Materials and Methods 42 Rate of Kill 42 Effect of BDM-I on S. aureus at different population growth rates 43 2.6.3 Results 44 v Rate of Kill 44 Effect of BDM-I on S. aureus at different population growth rates 45 2.6.4 Discussion 48 Rate of Kill 48 Effect of BDM-I on S. aureus at different poopulation growth rates 49 2.7 INTERACTIONS WITH ANTIBIOTICS 50 2.7.1 Background 50 2.7.2 Materials and Methods 51 Synergism or Antagonism of BDM-I with antibiotics 51 Time-kill interaction between an antibiotic and BDM-I 52 2.7.3 Results 53 Synergism or Antagonism of BDM-I with antibiotics 53 Time-kill interaction between an antibiotic and BDM-I 53 2.7.4 Discussion 54 CHAPTER 3 THE EFFECT OF BDM-I ON CELLULAR MEMBRANES 56 3.1 INTRODUCTION 56 Bacterial membranes 56 Agents disrupting the cell wall or membrane 57 3.2 CYTOPLASMIC MEMBRANE INTEGRITY 58 3.2.1 Background 58 Whole cell lysis 58 Lysis of spheroplasts 59 3.2.2 Materials and Methods 60 Whole cell lysis assay 60 Lysis of Gram-negative spheroplasts 60 3.2.3 Results 62 Whole cell lysis assay 62 Lysis of Gram-negative spheroplasts 62 3.2.4 Discussion 65 Whole cell and spheroplast lysis 65 vi 3.3 PERMEATION OF THE GRAM -NEGATIVE CELL ENVELOPE BY NPN 65 3.3.1 Background 65 3.3.2 Materials and Methods 66 3.3.3 Results 68 3.3.4 Discussion 69 3.4 TRANSMISSION ELECTRON MICROSCOPY OF BDM-I TREATED CELLS 70 3.4.1 Background 70 3.4.2 Materials and Methods 70 3.4.3 Results 71 3.4.4 Discussion 72 CHAPTER 4 EFFECT OF BDM-I ON COLONIAL MORPHOLOGY AND CELL PHYSIOLOGY 76 4.1 INTRODUCTION 76 4.2 INDUCTION OF SMALL COLONY VARIANTS OF S. AUREUS 76 4.2.1 Background 76 4.2.2 Materials and Methods 77 4.2.3 Results 78 4.2.4 Discussion 79 4.3 PRODIGIOSIN PRODUCTION IN SERRATIA MARCESCENS 80 4.3.1 Background 80 4.3.2 Materials and Methods 83 4.3.3 Results 84 4.3.4 Discussion 84 4.4 SWARMING MOTILITY IN PROTEUS SPP . 87 4.4.1 Background 87 4.4.2 Materials and Methods 89 Exposure to BDM-I during surface growth 89 vii Exposure to BDM-I prior to surface growth 90 4.4.3 Results 90 Exposure to BDM-I during surface growth 90 Exposure to BDM-I prior to surface growth 91 4.4.4 Discussion 94 4.5 CAPSULAR POLYSACCHARIDE PRODUCTION IN KLEBSIELLA PNEUMONIAE 96 4.5.1 Background 96 4.5.2 Materials and Methods 98 4.5.3 Results 98 4.5.4 Discussion 99 CHAPTER 5 THE EFFECT OF BDM-I ON CELL FUNCTIONS102 5.1 INTRODUCTION 102 5.2 ATP PRODUCTION BY ATP BIOLUMINESCENCE ASSAY 103 5.2.1 Background 103 5.2.2 Materials and Methods 104 5.2.3 Results 106 5.2.4 Discussion 107 5.3 TRANSCRIPTION /T RANSLATION ASSAY USING CELL -FREE E. COLI SYSTEM 109 5.3.1 Background 109 5.3.2 Materials and Methods 110 5.3.3 Results 111 5.3.4 Discussion 112 5.4 PROTEIN TYROSINE PHOSPHATASE ENZYME ASSAY 113 5.4.1 Background 113 Assayed phosphatases 115 5.4.2 Materials and Methods 117 Assay principle 117 Enzyme titration 118 vi ii Screening assays 119 5.4.3 Results 120 5.4.4 Discussion 124 CHAPTER 6 THE EFFECT OF BDM-I ON GENE EXPRESSION126 6.1 INTRODUCTION 126 6.1.1 DNA Microarray analysis 127 Affymetrix GeneChip ® 128 JCVI CMR Primary annotation database 129 6.2 MATERIALS AND METHODS 130 6.2.1 Selection of treatment parameters 130 6.2.2 Total RNA isolation 131 6.2.3 Labelling and hybridisation 132 6.2.4 Analysis of data 133 Image analysis (GCOS) 133 Absolute analysis (GCOS): Evaluation of data quality 134 Comparison analysis (GCOS): Experiment versus baseline arrays for each replicate 134 Statistical analysis of microarray data 135 Functional analysis of differentially regulated genes 136 6.2.5 Validation of microarray results by quantitative PCR 136 6.3 RESULTS 140 6.3.1 Absolute analysis: Evaluation of data quality 140 6.3.2 Comparison analysis: Experiment versus baseline arrays for each replicate. 143 6.3.3 Statistical analysis of microarray data 144 6.3.4 Functional analysis of differentially regulated genes 145 Comparison of functional categories of genes 146 Comparison of differentially expressed genes 147 Genes differentially regulated at both treatment concentrations 149 Genes differentially regulated at one treatment concentration only 154 6.3.5 Validation of microarray data by quantitative PCR 159 ix 6.4 DISCUSSION 160 CHAPTER 7 CONCLUSONS AND FUTURE DIRECTIONS 165 REFERENCES : 171 APPENDICES 185 Appendix A. Media and buffers 185 Appendix B. Data from samples used for RNA extractions 191 Appendix C. GCOS settings. 198 Appendix D. GCOS Expression Reports 199 Appendix E. GCOS Comparison Reports 209 Appendix F. Pie charts of differentially regulated genes common to both treatment concentrations 221 Appendix G. Pie charts of differentially regulated genes in only one treatment concentration 222 Appendix H: Supplementary Tables - Selected differentially regulated genes and their functional categories as described in JCVI CMR. 224 Appendix I.