Discovery of novel antibiotics via Streptomyces sporulation by Scott McAuley A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Biochemistry University of Toronto c Copyright 2019 by Scott McAuley Abstract Discovery of novel antibiotics via Streptomyces sporulation Scott McAuley Doctor of Philosophy Graduate Department of Biochemistry University of Toronto 2019 Streptomyces are filamentous bacteria known for producing a wide range of bioactive molecules. In addition to being nature's chemists they have a unique multicellular life- cycle that ends with the sporulation of aerial hyphae. I used this unique Streptomyces development cycle, specifically sporulation, as a screening platform to discover novel bi- ologically active molecules and characterized their mechanisms of action. Min-1 inhibits the growth of a range of Gram-positive bacteria, is active against the cell envelope, and induces a short cell phenotype in B. subtilis. Another molecule, EN-7, inhibits bacterial gyrase and is active against extensively resistant Staphylococcus aureus in addition to other Gram-positive pathogens. In addition to investigating these specific molecules, I developed a high-throughput screen to identify molecules that disrupt the Gram-positive bacterial membrane and found that Streptomyces venezuelae sporulation is highly sensi- tive to multiple forms of DNA damage. ii Contents 1 Introduction 1 1.1 The challenges of antibiotic discovery and development . 1 1.2 Identifying novel antimicrobials and their mechanism of action . 3 1.2.1 Identifying bioactive molecules through growth inhibition . 4 1.2.2 Identifying mechanism of action through reporter screens and assays 7 1.2.3 Target-based screening . 13 1.2.4 Novel screening approaches . 14 1.2.5 Conclusions . 16 1.3 Small molecule impact on bacterial morphology . 17 1.3.1 Activators of the SOS response disrupt the cell cycle . 18 1.3.2 Cell envelope determines cell shape . 21 1.3.3 Direct disruption of FtsZ disrupts the cell cycle . 23 1.3.4 Nutrient availability impacts both growth and the cell cycle . 25 1.3.5 Cytological profiling identifies antibiotic mechanisms of action . 27 1.3.6 Conclusion . 27 1.4 Thesis objectives and outline . 28 2 Membrane activity profiling of small molecule B. subtilis growth in- hibitors utilizing novel duel-dye fluorescence assay 29 2.1 Abstract . 29 2.2 Introduction . 30 2.3 Results and Discussion . 32 2.3.1 High-throughput assay for determining impact on membrane po- tential and permeability . 32 2.3.2 Screen for biologically active small molecules against B. subtilis . 34 2.3.3 Membrane disruption by biologically active small molecules . 36 2.4 Conclusion . 40 iii 3 A chemical inhibitor of cell growth reduces cell size in Bacillus subtilis 46 3.1 Abstract . 46 3.2 Introduction . 47 3.3 Results and Discussion . 48 3.3.1 Min-1 inhibits Streptomyces sporulation at sub-inhibitory concen- trations . 48 3.3.2 Min-1 inhibits growth of Gram-positive bacteria and reduces cell length of B. subtilis .......................... 49 3.3.3 Structural analogs of Min-1 have altered effects on growth rate and cell length . 51 3.3.4 Min-1 disrupts coordination between growth and FtsZ ring assembly 55 3.3.5 Min-1 targets the cell envelope . 56 3.3.6 Min-1 is a novel inhibitor of bacterial growth . 60 4 Discovery of a novel DNA gyrase-targeting antibiotic through the chem- ical perturbation of Streptomyces venezuelae sporulation 62 4.1 Abstract . 62 4.2 Introduction . 63 4.3 Results . 64 4.3.1 The Streptomyces venezuelae sporulation program is sensitive to DNA damage . 64 4.3.2 Novel small molecule sporulation inhibitors . 66 4.3.3 EN-7 targets DNA gyrase . 68 4.4 Discussion . 75 5 Concluding Remarks 79 5.1 Thesis Summary . 79 5.2 Future Directions . 80 5.2.1 Investigating mechanism of Streptomyces sporulation inhibition via DNA damage . 80 5.2.2 Additional approaches for elucidating Min-1's target and mecha- nism of action . 80 5.2.3 Identify mechanism of gyrase inhibition for EN-7 . 81 5.2.4 Creating high-thoughput method of screening Streptomyces sporu- lation inhibition . 81 5.2.5 Additional small molecule screens against bacterial development . 81 5.3 Conclusion . 82 iv 6 Materials and Methods 83 6.1 General Experimental Procedures . 83 6.1.1 Strains and plasmids . 83 6.2 Growth inhibition assays . 83 6.2.1 Broth dilution assay . 83 6.2.2 Streptomyces sporulation inhibition . 83 6.2.3 Superficial S. aureus skin infection model . 85 6.3 PAINs assays . 85 6.3.1 Duel-dye membrane disruption screen . 85 6.3.2 Dynamic Light Scattering . 85 6.4 Miscroscopy Methods . 86 6.4.1 Scanning electron miscroscopy . 86 6.4.2 B. subtilis cell length measurement and FtsZ immunofluorescence labeling . 86 6.5 Reporter assays . 87 6.6 Isolation and characterization of resistant mutants . 87 6.6.1 Resistant mutant generation . 87 6.6.2 Sequencing resistant mutants . 87 6.6.3 S. aureus allelic exchange . 88 6.7 Gyrase inhibition . 88 6.7.1 E. coli gyrase supercoiling . 88 6.7.2 S. aureus gyrase supercoiling . 89 6.7.3 E. coli topoisomerase IV decatenation . 89 6.7.4 S.aureus topoisomerase IV decatenation . 89 6.7.5 S.aureus gyrase cleavage . 89 7 Appendix 1: A chemical inhibitor of cell growth reduces cell size in Bacillus subtilis 90 7.1 Results and Discussion . 90 7.1.1 Min-1 does not inhibit B. subtilis sporulation . 90 7.1.2 Min-1 activates B. subtilis PywaC-lux reporter . 91 7.1.3 Unable to generate Min-1 resistant mutants . 91 7.1.4 Min-1 potentiates some known antibiotics . 92 7.1.5 Identifying sensitive strains using a B. subtilis knockdown library 93 7.1.6 Impact of Min-1 on HEK293 viability . 96 v 8 Appendix 2: Discovery of a novel DNA gyrase-targeting antibiotic through the chemical perturbation of Streptomyces venezuelae sporu- lation 98 8.1 Results and Discussion . 98 8.1.1 Growth inhibition and reporter activation of S. venezuelae sporu- lation screen hits . 98 8.1.2 Aggregation activity of S. venezuelae sporulation screen hits . 101 8.1.3 Gyrase S. aureus antisense strains show resistance to EN-7 . 101 8.1.4 EN-7 does not reduce S. aureus bacterial load in a skin mouse infection model . 104 Bibliography 106 vi List of Tables 1.1 Reporter strains used to identify antibiotic MOA . 10 1.2 Cell morphologies induced by cell wall inhibiting antibiotics . 24 3.1 Minimum inhibitory concentration of Min-1 . 50 4.1 Table of gyrase mutations found in EN-7 resistant mutants . 68 6.1 Table of strains used in studies . 84 7.1 Impact of Min-1 on activity of known antibiotics . 94 7.2 B. subtilis CRISPRi hits on xylose induction with Min-1 treatment . 96 8.1 Growth inhibition of S. venezuelae sporulation screen hits . 100 8.2 Growth inhibition of S. venezuelae sporulation screen hits . 102 vii List of Figures 1.1 Simplified screening processes for discovering new antibiotics and uncov- ering their mechanism of action . 4 1.2 Examples of bioactive molecules containing PAIN substructures . 5 1.3 Whole-cell reporter assays for determining MOA . 9 1.4 Chemical-genetic assays for determining MOA . 12 1.5 Streptomyces lifecycle . 16 1.6 Small molecules activate the SOS response and disrupts the cell cycle . 19 2.1 Chemical and optical properties of TO-PRO-3 iodide and DiOC2(3) . 33 2.2 Effect of known nisin, CCCP, and vancomycin on TO-PRO-3 iodide and DiOC2(3) fluorescence . 35 2.3 Screen of bioactive synthetic molecules for membrane activity . 37 2.4 Membrane activity screen summary . 39 2.5 Concentration dependent impact of bioactive molecules on TO-PRO-3 io- dide and DiOC2(3) fluorescence . 40 2.6 Membrane permeability hits . 42 2.7 Membrane potential hits . 43 2.8 Membrane potential hits, cont. 44 2.9 Calculated logP values for bioactive compound library . 45 3.1 Effect of Min-1 on S. venezuelae development . 49 3.2 Cell length and growth rate effects of Min-1 on B. subtilis . 52 3.3 Structure and activity of Min-1 analogs against B. subtilis and S. aureus 53 3.4 Effect of Min-1 analogs on B. subtilis growth and cell length . 54 3.5 Min-1 disrupts coordination between growth and FtsZ ring assembly in- dependent of known cell size mechanisms . 57 3.6 Min-1 targets the cell envelope . 59 3.7 Impact of supplemental magnesium on Min-1 activity . 60 viii 4.1 Streptomyces venezuelae sporulation is sensitive to DNA damage . 65 4.2 Chemical structures and induced S. venezuelae phenotypes . 66 4.3 Identification of EN-7 as an inhibitor of S. venezuale and Gram-positive pathogens . 67 4.4 EN-7 inhibits growth of extensively resistant S. aureus . 69 4.5 Allelic Exchange of EN-7 resistance mutations into S. aureus . 71 4.6 in-vitro inhibition of S. aureus gyrase by EN-7 . 72 4.7 Cross-resistance of EN-7 resistant strains with other gyrase inhibitors . 73 4.8 Chemical structures of EN-7 and other investigational gyrase inhibitors . 74 4.9 Analysis of EN-7 resistance gyrase mutations . 76 7.1 Min-1 does not disrupt B. subtilis sporulation . 91 7.2 Min-1 activates B. subtilis PywaC-lux reporter strain . 92 7.3 Min-1 potentiates known antibiotics . 93 7.4 Impact of Min-1 on growth of a B. subtilis CRISPRi essential knock-down library . ..