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In vitro Response of Animal and microbial cells to antimicrobial peptides

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Text of In vitro Response of Animal and microbial cells to antimicrobial peptides

  • In vitro response of animal and microbial cells to

    antimicrobial peptides

    A THESIS

    submitted toRashtrasant Tukdoji Maharaj Nagpur University, Nagpur

    For the award of the degree of

    Doctor of Philosophyin

    BIOCHEMISTRY(FACULTY OF SCIENCE)

    Submitted by

    DEOVRAT BEGDE

    DEPARTMENT OF BIOCHEMISTRY

    HISLOP COLLEGE, TEMPLE ROAD, CIVIL LINES,

    NAGPUR.

    MARCH 2012

    Under supervision ofDr. AVINASH UPADHYAY

    Associate ProfessorDepartment of Biochemistry,

    Hislop College, Nagpur

  • DECLARATION

    I hereby declare that the work reported in the Ph.D. thesis entitled In vitro response

    of animal and microbial cells to antimicrobial peptides submitted at

    Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur is an authentic

    record of my work carried out under the supervision of Dr. Avinash Upadhyay. I

    have not submitted this work elsewhere for any other degree or diploma.

    (DEOVRAT BEGDE)Department of Biochemistry

    Hislop College, Temple Road, Civil Lines,Nagpur, Maharashtra, India 440001.

    Date:

  • CERTIFICATE

    This is to certify that the work reported in the Ph.D. thesis entitled In vitro response

    of animal and microbial cells to antimicrobial peptides, submitted by Mr.

    Deovrat Begde at Rashtrasant Tukadoji Maharaj Nagpur University,

    Nagpur is a bonafide record of his original work carried out under my supervision. This

    work has not been submitted elsewhere for any other degree or diploma.

    (Signature of Supervisor)(Dr. Avinash Upadhyay)Department of Biochemistry,

    Hislop College, Temple Road, Civil Lines,Nagpur, Maharashtra, India 440001.

    Date:

  • CERTIFICATE

    (Signature Head of the Institution/ Principal)

    (Dr. Dipti Christian)Principal,

    Hislop College, Temple Road, Civil Lines,Nagpur, Maharashtra, India 440001.

    Date:

    This is to certify that the work reported in the Ph.D. thesis entitled In vitro

    response of animal and microbial cells to antimicrobial peptides, submitted by

    Mr. Deovrat Begde at Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur is a

    bonafide record of his original work carried out at my institute under the supervision

    of Dr. Avinash Upadhyay. All his dues are settled and the College has no objection in

    the submission of the thesis to the University. This work has not been submitted

    elsewhere for any other degree or diploma.

  • This thesis is dedicated to the

    two most beautiful women in my life

    my mother and my wife

  • i

    ACKNOWLEDGEMENTS

    This thesis would not have been possible without the guidance and the help of several individuals who in one way or another contributed and extended their valuable assistance in the preparation and completion of this study.

    First and the foremost, my sincere gratitude to Late Dr. Ashish Bhelwa, Former Principal, Hislop College, Nagpur for making available all the infrastructural facilities and for his everlasting encouragement. I am extremely grateful to Dr. Dipti Christian, Principal, Hislop College, Nagpur and Mr. Sudipta Singh, Secretary, Hislop Education Society, for recognizing my efforts and appreciating my achievements.

    I would be falling short of my words to express my deep sense of gratitude to all my teachers who have taught me Biochemistry and made me realize the importance of this subject in Life Sciences. My most sincere acknowledgements are due to my supervisor, Dr. Avinash Upadhyay, who has been instrumental in developing my interest in Biochemistry. He taught me the fundamental principles of the subject which led the foundation of whatever I today understand as Biochemistry. He has been a true mentor in every sense with immense knowledge not only in his own subject, but almost everything under the sun for that matter. I should also thank him for giving me every possible opportunity to improve my knowledge and to establish in the field of life science. With his able guidance and confidence in me everything became so very simple during the entire course of my dissertation. My words would be really meager to express my utmost gratitude towards him.

    I am also indebted to the senior faculty members of Department of Biochemistry, LIT premises, RTM Nagpur University, Nagpur. Dr. N. V. Shastri, Dr. H. F. Daginawala, Dr. N. Nath and Dr. Aqueel Khan, former heads of the department of Biochemistry, Dr. G. B. Shinde, present head, Dr. Mandakini Patil and special thanks to Dr. Swati Kotwal, Professor, Department of Biochemistry, for her valuable and unbiased suggestions during every uncertainty in my life.

    For the inspiring training sessions and teaching me the protein purification techniques, I am extremely grateful to Dr. Manju Ray, Retired Professor, Indian Association for Cultivation of Science, Kolkata and her enthusiastic team of scholars.

    Acknowledgements to UGC for financially supporting a part of the study.

    My sincere thanks are due to all my colleagues at Hislop School of Biotechnology. I consider myself really fortunate to be a part of such a friendly, learned and intelligent faculty. I would like to thank Mrs. Mashitha Pise and Dr. Jaishree Rudra for their constant encouragement. I am really very

  • ii

    grateful to Dr. Nandita Nashikkar and Mrs. Sunita Bundale for helping me all throughout, for their valuable inputs while designing the experiments and for all the academic and non-academic discussions, which really helped me, grow as an individual. With their blithesome nature they always maintained the working atmosphere cheerful and memorable for me. I would also like to thank all my students at Hislop School of Biotechnology for all the help they are ready to provide me whenever it is required the most. My special thanks are due to Mr. Vilas Gedam, Mr. Pravin Attarkar and above all to Mr. Rakesh Gedam, non-teaching staff of Hislop College for their untiring support all throughout the course of my work. Thanks are also due to Mrs. Seema Bhange, Librarian, for making all the important literature available.

    Last but not the least, although I know my thanks are too less for their efforts, still I would like to thank my Parents and my elder Brother, who actually made me what I am today and for constantly inspiring me to stand tall against all odds. But it would be unjust if I do not make a special mention of someone who has completely changed my attitude towards life and my research work. The special person is none other than my wife, Mrs. Pradnya Mahatme-Begde to whom I am thankful, especially because she listened to all my rubbish and still did not mention even a bit of it in front of anyone and also for her extreme care and unconditional love for me.

    I believe God has blessed me with such wonderful people, so, I would like to thank the Almighty for being so kind to me and for being there with me all throughout in the form of each and everyone mentioned above.

  • iii

    TABLE OF CONTENTS

    Acknowledgement i-ii

    Table of Contents iii-v

    List of Figures vi-vii

    List of Tables viii

    Abbreviations ix-x

    1. CHAPTER 1: INTRODUCTION 1-17

    GENERAL INTRODUCTION 1

    1.1 Antimicrobial Peptides: a general overview 3

    1.1.1 Non-ribosomally synthesized AMPs 4

    1.1.2 Ribosomally synthesized AMPs 7

    1.2 Quorum Sensing 11

    1.2.1 Non-peptide QS mechanisms 12

    1.2.2 Peptide mediated QS mechanisms 13

    1.3 Immune system: an AMP perspective 15

    2. CHAPTER 2: AIMS AND OBJECTIVES 18-22 LITERATURE SURVEY 19

    3. CHAPTER 3: MATERIAL AND METHODS 23-51

    MATERIALS 23-25

    Chemicals, Research Kits and Reagents 23

    Culture Media 25

    METHODOLOGY 26-51

    3.1 Selection, Isolation and Modification of AMPs 26

    3.1.1 Introduction 26

    3.1.2 Media and Culture conditions 29

    3.1.3 Isolation and purification 29

  • iv

    3.1.4 Alternate Culture media and Subtilin isolation procedure 30

    3.1.5 Tricine-SDS-PAGE of purified subtilin and nisin 32

    3.1.6 Chemical Glycosylation of commercial nisin 33

    3.2 Effect of subtilin and nisin on bacteria 34

    3.2.1 Introduction 34

    3.2.2 Microorganisms, media and growth conditions 36

    3.2.3 Nisin and subtilin stock solutions 36

    3.2.4 Swarming Motility Assay 37

    3.2.5 Biofilm Assay 37

    3.2.6 Virulence factor analysis 40

    3.3 In vitro Response of animal cells to subtilin and nisin 45

    3.3.1 Introduction 45

    3.3.2 Effect on Tumor cells 46

    3.3.3 Effect on Human Immune System 47

    3.3.4 In vitro Hemolytic activity of Nisin on Human RBCs 49

    3.3.5 Neutrophil extracellular trap (NET) induction assay 49

    3.3.6 Quantitation of Neutrophil NADPH oxidase 50

    3.3.7 Effect of exogenous Catalase on Nisin 50 induced ROS production and NET formation

    4. CHAPTER 4: OBSERVATION AND RESULTS 52-87

    4.1 Isolation and purification of Nisin and Subtilin 52

    4.2 Tricine SDS-PAGE 52

    4.3 Chemical Glycosylation of nisin 55

    4.4 Effect of nisin and subtilin on swarming of Proteus 55

    4.6 Biofilm assay 59

    4.6.1 Microtiter plate assay 59

    4.6.2 Slide assay 63

    4.7 Effect of nisin on P. mirabilis and E. coli Hemolysin 70

    4.8 Effect of nisin on Rhamnolipid production of P. aeruginosa 70

  • v

    4.9 Screening for curli positi

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