Antimicrobial Peptides from Membrane-Interacting Stretches of Bacterial Proteins

Antimicrobial Peptides from Membrane-Interacting Stretches of Bacterial Proteins A thesis submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY BY KARABI SAIKIA Department of Biosciences and Bioengineering Indian Institute of Technology Guwahati Guwahati - 781039, India June 2019 Dedicated to my parents TH-2072_126106010 INDIAN INSTITUTE OF TECHNOLOGY GUWAHATI DEPARTMENT OF BIOSCIENCES AND BIOENGINEERING DECLARATION The research work presented in this thesis is an original work carried out by me in the Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, India, for the degree of the Doctor of Philosophy. Research work was executed under the guidance of my supervisor Dr. Nitin Chaudhary, and no work has been submitted, in part or whole, for any other degree to any other University/Institute. As per the standards of reporting research findings, due acknowledgements have been made wherever the work described is based on the findings of other researchers. Karabi Saikia Date: TH-2072_126106010 INDIAN INSTITUTE OF TECHNOLOGY GUWAHATI DEPARTMENT OF BIOSCIENCES AND BIOENGINEERING CERTIFICATE This is to certify that the work incorporated in the thesis titled “Antimicrobial Peptides from Membrane-Interacting Stretches of Bacterial Proteins” submitted by Ms Karabi Saikia for the award of the degree of Doctor of Philosophy is an authentic record of the research work carried out under my guidance in the Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, India. The work is original and has not been submitted in part or full for any other degree of any other University/Institute. Dr. Nitin Chaudhary (Supervisor) Date: TH-2072_126106010 Table of Contents Table of Contents i Abbreviations iv Acknowledgement v Synopsis vii List of publications x CHAPTER 1 Introduction and Literature Review 1 1.1 Introduction 3 1.2 Distinctive features of antimicrobial peptides 4 1.3 A historical perspective 6 1.4 Sequences and structures of antimicrobial peptides 9 1.4.1 Alpha-helical peptides 9 1.4.2 β-sheet peptides 11 1.4.3 Circular peptides 13 1.4.4 α-β structured peptides 15 1.4.5 Non- α-β structured peptides 17 1.4.6 Peptides rich in specific amino acids 18 1.4.7 Anionic peptides 19 1.5 Antimicrobial peptide databases 19 1.6 Mechanism of action 21 1.6.1 Membrane-disruptive peptides 22 1.6.1.1 The barrel stave model 22 1.6.1.2 The toroidal pore model 23 1.6.1.3 The carpet model 24 1.6.2 Non-membrane-disruptive peptides 25 1.7 Selectivity of antimicrobial peptides towards Gram-positive or Gram-negative bacteria 26 1.8 Immunomodulatory functions of antimicrobial peptides 26 1.9 Antimicrobial resistance against antimicrobial peptides 27 1.10 Challenges in developing AMPs as antibiotics 30 1.11 Antimicrobial peptides as therapeutics 32 1.12 Hypothesis: membrane-binding stretches in microbial proteins can turn out to be potential AMPs 35 CHAPTER 2 Materials and Methods 37 2.1 Materials 39 2.2 Peptides 39 2.2.1 Peptide synthesis 39 i TH-2072_126106010 2.2.2 Peptide purification and characterization 40 2.2.3 Peptide solutions and concentration estimation 40 2.3 Surface activity of peptides 41 2.4 Preparation of small unilamellar vesicles 41 2.5 Steady-state tryptophan fluorescence 41 2.6 Tryptophan fluorescence quenching 42 2.7 Outer membrane permeabilization assay 42 2.8 Inner membrane permeabilization assay 43 2.9 Circular dichroism spectroscopy 43 2.10 Antibacterial assay 44 2.10.1 Colony count method 44 2.10.2 Broth microdilution method 44 2.10.3 Antimycobacterial assay 45 2.11 Antifungal assay 45 2.11.1 Colony count method 45 2.11.2 Broth microdilution method 45 2.12 Salt sensitivity assay 46 2.12.1 Colony count method 46 2.12.2 Broth microdilution method 46 2.13 Hemolytic assay 46 2.14 FESEM analysis 47 2.15 Killing kinetics assay 47 CHAPTER 3 Antimicrobial Peptides from N-terminal Membrane-Binding Region of E. coli MreB Protein 49 3.1 Summary 51 3.2 Results 52 3.2.1 Peptides 52 3.2.2 Surface activity and membrane binding of the peptides 54 3.2.3 Tryptophan fluorescence 56 3.2.4 Tryptophan fluorescence quenching 57 3.2.5 Antimicrobial assay 58 3.2.6 Salt sensitivity 59 3.2.7 Outer membrane permeabilization assay 61 3.2.8 Inner membrane permeabilization assay 61 3.2.9 Circular dichroism spectroscopy 62 3.2.10 Hemolytic assay 63 3.2.11 FESEM analysis 64 3.2.12 Killing kinetics assay 65 ii TH-2072_126106010 3.3 Conclusion 67 Chapter 4 Antimicrobial Peptides from C-terminal Amphipathic Region of E. coli FtsA Protein 69 4.1 Summary 71 4.2 Results 71 4.2.1 Peptides 71 4.2.2 Tryptophan fluorescence 74 4.2.3 Tryptophan quenching 76 4.2.4 Antimicrobial assay 77 4.2.5 Salt sensitivity assay 78 4.2.6 Outer membrane permeabilization assay 79 4.2.7 Inner membrane permeabilization assay 80 4.2.8 Circular dichroism 81 4.2.9 Hemolytic assay 83 4.2.10 FESEM analysis 84 4.3 Conclusion 84 Chapter 5 Aromatic Residue-Rich Antimicrobial Peptide Derived from C-terminal Region of B. subtilis LCI Peptide 87 5.1 Summary 89 5.2 Results 90 5.2.1 Peptide 90 5.2.2 Antibacterial activity 90 5.2.3 Salt sensitivity assay 91 5.2.4 Steady state tryptophan fluorescence 92 5.2.5 Tryptophan fluorescence quenching 92 5.2.6 Membrane permeabilization assay 93 5.2.7 Circular dichroism 94 5.2.8 Hemolytic assay 95 5.2.9 FESEM analysis 95 5.3 Conclusion 96 Chapter 6 Conclusion, Discussion, and Possibilities 99 REFERENCES 109 iii TH-2072_126106010 Abbreviations AMP Antimicrobial peptide CHL Cholesterol DIPEA N,N-diisopropylethylamine DiSC3(5) 3,3′-dipropylthiadicarbocyanine iodide DMF Dimethylformamide EDTA Ethylenediaminetetraacetic acid Fmoc Fluorenylmethyloxycarbonyl chloride FESEM Field emission scanning electron microscope HBTU N,N,Nʹ,Nʹ-tetramethyl-O-(1H-benzotriazol-1-yl)uranium hexafluorophosphate HCCA α-Cyano-4-hydroxycinnamic acid HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) HOBt 1-hydroxybenzotriazole hydrate HPLC High performance liquid chromatography M Molar MALDI-TOF Matrix-assisted laser desorption ionization-time of flight MDR Multidrug resistant MIC Minimum inhibitory concentration MRSA Methicillin-resistant S. aureus NPN N-phenyl-1-naphthylamine OD Optical density POPC 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine POPE 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine POPG 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) SUVs Small unilamellar vesicles TB Tuberculosis TFA Trifluoroacetic acid TFE Trifluoroethanol XDR Extensively drug resistant iv TH-2072_126106010 Acknowledgement Completion of this doctoral thesis was possible with the support of some wonderful people in my life. I would like to express my sincere gratitude to all of them. Firstly, I would like to convey my heartfelt gratitude to my supervisor Dr. Nitin Chaudhary for his incredible support and inspiration throughout my research period. It was my privilege to work and learn under his guidance. I would always be grateful, for his kindness, patience and trust that he bestowed on me. I would like to thank my committee chairperson Dr. Vibin Ramakrishnan, for his insightful comments and valuable suggestions related to my work. I am also grateful to my committee members Dr. Sachin Kumar and Dr. Ganesh Natarajan for their guidance and encouragement during my research period. I thank Professor Vishal Trivedi for his generous help and suggestions regarding my research work. Faculty members of the Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati have been very kind enough to extend their help at various phases of my research, and I do hereby acknowledge all of them. My heartfelt appreciation to all my lab members; Sravani, Debika, Vinay, Anshuman, Feba, Nethi, Anirban, Pranav and Shubhra from the core of my heart, for their love, support and unfailing help during my research period. I will always cherish the wonderful time spent with them. I would like to take this opportunity to thank my friends and juniors from Molecular Informatics and Design Lab; Sajitha, Prakash, Gaurav J, Gaurav P, Ruchika, Jahnu, Vivek, Anjali, and Franklin for their help and encouragement throughout my research period. I acknowledge Central Instrument Facility (CIF), Indian Institute of Technology Guwahati for providing me with electron microscopy and MALDI spectrometry facilities as an invaluable tool for my research work. v TH-2072_126106010 I acknowledge Department of Chemistry, Indian Institute of Technology Guwahati, for permitting me to carry out CD spectroscopic experiments in the department. I would like to thank Dr. R. Nagaraj, Centre for Cellular and Molecular Biology, Hyderabad for their generous help during my course of research work. My sincere thanks to Dr. Gopaljee Jha, National Institute of Plant Genome Research, New Delhi, India, for providing me the Xanthomonas oryzae pv. oryzae bacterium. I am thankful to Dr. Basil Mathew, Centre for Cellular and Molecular Biology, Hyderabad for his valuable suggestions and help with my microbiology work. I would like to express my sincere gratitude to the non-teaching staffs of the Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati for their kind cooperation and support during this period. Above all, I thank my family for their love, affection, and trust on me. I don’t have enough words to express my gratitude towards my parents for giving me the freedom to chase my dreams, my husband Devabrat, who stood by me in all my ups and downs, and my sisters Aditi and Manjeera whose love and support was immeasurable in my journey. My research would not have been possible without their encouragement and blessings.

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