Transcript
  • 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

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    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

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    4.9 Screening for curli positive clinical isolates and effect of nisin on curli expression 71

    4.10 In silico molecular docking analysis of nisin with Csg A and Csg B 75

    4.11 Cytotoxicity evaluation and anti-metastatic efficacy 78

    4.11.1 MTT based cytotoxicity assay 78

    4.11.2 Apoptosis assay 80

    4.11.3 Scratch/ In vitro Wound Healing Assay 82

    4.12 Hemolytic activity of nisin 84

    4.13 NET induction assay 85

    4.14 Effect of exogenous Catalase on Nisin induced ROS production and NET formation 85

    5. CHAPTER 5: DISCUSSION 88-103

    SUMMARY 100-103

    6. CHAPTER 6: REFERENCES 104-118

    PUBLICATIONS 119

    APPENDIX 120-126

  • vi

    LIST OF FIGURES

    Figure 1.1: Modules of non-ribosomal peptide synthetases (NRPSs)

    Figure 1.2 Crystal structures and reaction catalyzed by the core-domains of NRPSs

    Figure 1.3: Types of non-ribosomal peptide synthetases (NRPSs)

    Figure 2.1: Schematic of subtilin production in B. subtilis

    Figure 3.1: Structures of dehydro residues and lanthionine residues

    Figure 3.2: Structures of few type A, type B and type C lantibiotics

    Figure 3.3: A cartoon depicting the general life cycle of motile bacterial cells as they swarm on surfaces

    Figure 3.4 Static Bioreactor setup for slide assay of Biofilm

    Figure 3.5 Hex docking parameters for molecular docking analysis

    Figure 4.1: Purification and potency testing of lantibiotics

    Figure 4.2: Purity analysis with Tricine SDS-PAGE

    Figure 4.3: Activity evaluation of glycosylated nisin

    Figure 4.4: Effect of nisin on swarming of Proteus

    Figure 4.5: Effect of subtilin on swarming of Proteus

    Figure 4.6: Microtiter plate Biofilm assay

    Figure 4.7: Effect of subtilin and nisin on biofilm of E.coli and of subtilin on co- culture of P. mirabilis with E. coli

    Figure 4.8: Effect of partially fractionated subtilin on P. mirabilis Biofilm

    Figure 4.9: Slide assay for visual evaluation of Biofilm

    Figure 4.10: E. coli Biofilm Inhibition by Nisin

    Figure 4.11: Dislodgement of P. mirabilis preformed biofilms after nisin treatment

    Figure 4.12: Screening for pathogenic curli positive E. coli

    Figure 4.13: Overall comparative evaluation of the effect of nisin on curli expression in E. coli (2), (3) and (MTCC 119)

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    Figure 4.14: In silico Csg B- Nisin and Csg A-Csg B docking analysis by Hex molecular docking software

    Figure 4.15: Csg A-Nisin docking analysis and list of all interacting residues of Csg A, Csg B and Nisin

    Figure 4.16: MTT assay and DNA fragmentation analysis by Agarose gel electrophoresis

    Figure 4.17: Comet Assay

    Figure 4.18: Annexin V-Cy3 Apoptosis Detection

    Figure 4.19: Anti-metastatic activity by Scratch Assay

    Figure 4.20: (A) Fluorescent microscopic examination of Neutrophil extracellular trap (NET) induction by nisin

    (B) Induction of NET formation by nisin

    Figure 4.21: I. Quantitative NBT assay

    II. Comparative Fluorescent microscopic examination of Neutrophil extracellular trap (NET) induction by Nisin and LPS in presence and absence of exogenous catalase

    Graph 4.1: Monitoring the effect of subtilin and nisin on biofilms of P. mirabilis, P. aeruginosa and E. coli

    Graph 4.2: Quantitative evaluation of varying concentrations of nisin of P. mirabilis biofilm by slide assay

    Graph 4.3: Quantitative evaluation of biofilm inhibitory concentration of nisin

    Graph 4.4: Graphical representation of percent biofilm inhibitory potential of nisin on selected Gram negative bacteria

    Graph 4.5: Suppression of P. mirabilis and E. coli (2) Hemolysin after nisin treatment

    Graph 4.6: Significant reduction in rhamnolipid production in P. aeruginosa

    Graph 4.7: Cytotoxicity evaluation of nisin on tumor cell lines

    Graph 4.8: Hemolytic activity evaluation of nisin on isolated human RBCs

  • viii

    LIST OF TABLES

    Table 3.1: List of organisms used for screening of curli positive clinical isolates

    Table 4.1: Screening of curli positive clinical isolates

  • ix

    ABBREVIATIONS

    ABC ATP-binding cassette proteins

    AI Autoinducer

    AICD Activation induced cell death

    AIP Autoinducer peptide

    AMP Antimicrobial peptides

    ASL Acylhomoserine lactone

    ATCC American type cell culture

    CR Congo red

    DMEM Dulbeccos modified eagles medium

    DMSO Dimethyl sufoxide

    DNA Deoxyribonucleic acid

    FBS Fetal Bovine serum

    GI Gastrointestinal tract

    HBSS Hank's balanced salt solution

    HIC Hydrophobic interaction chromatography

    HSL Homoserine lactone

    IC50 Half maximal (50%) inhibitory concentration

    IL Interleukin

    LB Luria Bertani medium

    LPS Lipopolysaccharide

    MALDI-

    Tof

    Matrix-assisted laser desorption/ionization Time of flight Mass

    spectroscopy

    MH Mueller-Hinton medium

    MTCC Microbial type culture collection

    MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

    NADPH Nicotinamide adenine dinucleotide phosphate (reduced from)

    NBT Nitroblue tetrazolium

    NET Neutrophil extracellular traps

    NRPSs Non-ribosomal peptide synthetases

    PAGE Polyacrylamide Gel Electrophoresis

    PBMC Peripheral blood mononuclear cells

  • x

    PHA Phytohemagglutinin

    PI Propidium Iodide

    PMA Phorbol 12-myristate 13-acetate

    PMN Polymorphomononuclear neutrophils

    QS Quorum sensing

    ROS Reactive oxygen species

    RPMI-

    1640 Rosewell Park Memorial institute-1640 medium

    SDS Sodium Dodecyl Sulfate

    TNF Tumor necrosis factor

  • Introduction

    1

    GENERAL INTRODUCTION

    In this world of competitive fitness each living organism has to constantly compete

    throughout its existence. Bacteria are no exceptions to this rule. But, because of their

    higher reproductive rate, they can evolve faster and thus quickly adapt to the changing

    environment. The major selective pressure here is exerted by the available source of

    nutrition. Organisms which can best utilize the nutritional resource have an edge over

    other competitors. Eukaryotes cannot match the competitive quickness of bacteria;

    rather, they serve as soft targets to those bacteria which can survive on them. These

    bacteria could then either behave pathogenically or can co-operate with their host.

    Considering this overall scenario, all the multi-cellular eukaryotes seem to carry a

    disadvantage of slower reproductive and thereby evolution rate. Complexity of higher

    eukaryotes further adds up to this disadvantage and thus it might seem improbable for

    us to have any chance of eradicating deadly bacterial infections. But there is a positive

    aspect to this situation which seems to be more encouraging- what if we focus on

    those bacteria which seem to have a symbiotic relation with the host? These

    organisms might also encounter a crisis situation and will be losing their friendly

    environment in the event of deadly infection. Thus some of the self sustaining

    strategies employed by these microorganisms could beneficially serve the host

    organism to combat the bacterial pathogen.

    While living freely in the environment, bacteria create and stay in their own niche.

    Occasionally, to create such a microenvironment, several bacteria (both pathogenic

    and non-pathogenic) naturally produce variety of extracellular factors. These factors

    serve as an invitation to some mutualistic microorganisms and exclude other

    unwanted bacteria. Considering the latter, the exclusion factors either behave as

    repellents or might exterminate the competitor microorganisms from the local

    vicinity. Many of the bifidobacteria and other non-pathogenic bacteria are well known

    to produce a variety of such factors. Both Gram positive and negative bacteria

    produce extracellular signaling molecules involved in bacterial cell-cell

    communication (Hardman, Stewart et al. 1998). Some of these molecules are also

    reported to have antibacterial activity (Kleerebezem 2004; Heeb, Fletcher et al. 2011).

    Moreover, in course of evolution, bacteria have learnt to modulate such molecules in

  • Introduction

    2

    a way that the target microorganism finds it difficult to acquire resistance against

    these antibiotics of bacterial origin. Production of these signaling molecules/

    antimicrobial compounds/virulence factors is under a strict genetic control and

    frequently requires some specific environmental stimulus (Horswill, Stoodley et al.

    2007). For pathogenic microorganisms the host factors (hormones, surface receptors

    etc.) serve as the trigger (Hardman, Stewart et al. 1998) while for others, sheer

    presence of competitor bacteria in the vicinity can suffice. So, it seems likely that

    some factors secreted by the pathogenic bacteria to mark their presence within the

    host may trigger the production of some anti-pathogenic or pathogen attenuating

    compounds by the host-symbiont bacteria. Identification of extracellular signaling

    molecules of some symbiotic bacteria and testing their effect on the virulence of

    pathogenic microorganisms could thus be an interesting area of biochemical and

    pharmacological research. Furthermore, as many of these molecules are antibacterials

    of bacterial origin their fitness as antibiotics could be much better than the

    conventional antibiotics. At first instance this might seem to be the same old strategy

    of drug development but a deeper thought will reveal another aspect of this approach.

    Equipped with an extracellular signaling function, these antibiotics might also

    modulate the virulence of the pathogen by interfering with pathogens inherent

    signaling mechanism without actually killing it. The view might sound too optimistic

    but does have some physiological evidences (Heeb, Fletcher et al. 2011). Even some

    conventional antibiotics were found to modulate bacterial behavior but only when

    tested at their sub-inhibitory concentrations against selected bacteria (Yim, Huimi

    Wang et al. 2007; Fajardo and Martnez 2008).

    Logically, it does not seem probable that an organism as small as a bacterium will

    invest its energy into synthesis of any extracellular compound unless the compound

    has multiple functions. This could be especially true in case of Gram positive bacteria

    where these extracellular signaling molecules are essentially small proteins or

    peptides. Recent reports indicate that several such extracellular peptides have

    antimicrobial activity in addition to their signaling property (Gillor 2007). The

    success of these antimicrobial peptides (AMPs) could be understood from the fact that

    till date there are very few reports of development of resistance against them.

  • Introduction

    3

    Interestingly these are known to affect the target microorganism through more than

    one mechanism. Employment of such multiple strategies could be one of the reasons

    for their success (Hoffmann, Pag et al. 2002), making it extremely difficult for the

    microorganism at the receiving end to develop resistance. Although tremendously

    effective, their activity is mostly restricted to a group or a class of bacteria. Several

    other bacteria seem to escape from the activity of these highly efficient molecules.

    When reports document that the growth of some bacteria is not inhibited by an

    AMP it does not necessarily indicate inefficiency of that AMP. But this might hint

    towards its probable involvement in bringing about some beneficial behavioral

    modulation within the seemingly resistant bacterium. Thus without actually

    challenging the growth, some bacterial molecules could still prove effective and

    thereby maximize survival opportunities of the producer organisms.

    Turning our focus back to the host co-operators, i.e., the symbiotic bacteria and their

    extracellular molecules secreted in the event of host-pathogen insult, an additional

    activity could be assigned to these molecules. Sometimes, instead of directly affecting

    the pathogen, these may positively modulate the hosts inherent immune mechanisms

    for effective eradication of the pathogen (Bahrami, Macfarlane et al. 2011).

    Furthermore, as human immune mechanisms do employ several antimicrobial/

    immune reactive peptides of their own, involvement of bacterial AMPs in such

    mechanisms cannot be completely ruled out. Therefore analyzing the effects of AMPs

    produced by symbiont bacteria on the hosts immune system may reveal some

    interesting aspects of communication between the host and its symbiotic bacteria.

    1.1 Antimicrobial Peptides: a general overview

    Approximately in the past two and a half decades, hundreds of antimicrobial peptides

    have been isolated from a wide variety of plants, invertebrates, amphibians, and

    mammals, as well as from bacteria and fungi. The widespread occurrence of these

    antimicrobial peptides suggests their key role in host defence (Boman 1995)

    mechanisms. Several of these peptides exhibit a wide array of activities viz.

    bactericidal, fungicidal, antiviral and even antitumour effects have been observed in

    some cases. Despite their broad range of activities, many of these peptides have

    remarkably low cytotoxicity against normal mammalian cells. Hence, naturally

  • Introduction

    4

    occurring antimicrobial peptides may provide a feasible alternative to conventional

    antibiotics, which are becoming increasingly ineffective owing to the emergence of

    resistant bacterial strains (Davies 1994; Nikaido 1994; Swartz 1994).

    Diversity of AMPs with respect to their origins and activities demands investigation

    of some similar features for their proper categorization which could further prove

    beneficial for designing some improved synthetic analogs. A critical structural

    examination reveals some common trends. Despite very meagre sequence homology,

    almost every AMP is preferentially cationic and bears high proportion of hydrophobic

    amino acids. This composition of AMPs makes them attain an amphipathic structure

    which further suggests their involvement in membrane active microbicidal

    mechanisms. Although the membrane disrupting activity is believed to be employed

    by most of the AMPs there is still some degree of uncertainty about this belief.

    Therefore a simpler way of AMP classification could be on the basis of their synthesis

    mechanism used by the producer organism. Broadly therefore, all the AMPs can be

    categorized into two classes, non-ribosomally synthesized peptides, such as the

    gramicidins, polymyxins, bacitracins, glycopeptides, etc., and ribosomally

    synthesized (natural) peptides. The former are often drastically modified and are

    largely produced by bacteria, whereas the latter are produced by all species of life

    (including bacteria) as a major component of the natural host defense molecules of the

    producer species. Ribosomally synthesized AMPs of bacterial origin are often

    referred to as bacteriocins which are mostly produced by the gram positive bacteria. A

    brief discussion about these two categories of AMPs is given below:

    1.1.1 Non-ribosomally synthesized AMPs

    These are commonly produced by bacteria, fungi and streptomycetes and may contain

    two or more moieties derived from amino acids. Considering this definition, almost

    every antibiotic known currently would seem to have been synthesized by similar

    mechanisms. So, differing from this definition we would rather concentrate only on

    longer peptides, which essentially have some specific peptide synthesis strategy.

    Longer peptides falling in this class are synthesized by multienzyme complexes, the

    so-called non-ribosomal peptide synthetases (NRPSs) and they are said to operate

    through the thiotemplate multienzymic mechanism(Kleinkauf and von DHren

  • Introduction

    5

    1990; Finking and Marahiel 2004). NRPS enzymes comprise of repeating modules

    that are responsible for the sequential selection, activation, and condensation of

    precursor amino acids. A module is defined as a section of the NRPSs polypeptide

    chain that is responsible for the incorporation of one building block into the growing

    polypeptide chain. Thus selective activation of the amino acids by the protein

    modules decides the primary structure of the peptide. The modules can be further

    subdivided into catalytic domains. The concept of modules and activities of the

    catalytic domains are detailed in figure 1.1 and 1.2. Final tailoring-steps, including

    glycosylation and prenylation, serve to further decorate these non-ribosomal peptides

    (Wilkinson and Micklefield 2009).

    Figure 1.1 Coming from the gene, modules that are responsible for the incorporation of one amino acid can be identified on the protein level. Modules can be subdivided into domains that harbor the catalytic activities for substrate activation (A-domain), covalent loading (CP-domain), and peptide bond formation (C-domain). Modules lacking a C-domain are used to initiate non-ribosomal peptide synthesis, while those harboring a C-domain qualify for elongation. Figure incorporated from Finking and Marahiel 2004.

    The seemingly simple mechanism of NRPSs, however, involves some intricacies.

    Depending on the degree of complexity associated with the peptide synthesis, the

    NRPSs are classified into three types, Type A, B and C. The strategies employed by

    each of these NRPS types are as depicted in figure 1.3 with examples. For further

    details regarding these peptides one is advised to refer to better texts as it is out of the

    scope of our study.

  • Introduction

    6

    Figure 1.2 Crystal structures and reaction catalyzed by the core-domains of NRPSs. The arrows point to the most obvious features of the crystal structures. The A-domain is composed of a large N-terminal domain and a small C-terminal subunit. Ser-45 of the TycC3-PCP is the attachment site for the 4PP cofactor. The C-domain exhibits a C-terminal domain (black) and an N-terminal domain (dark gray). The N-face of the C-domain is shaded in light gray, and the C-face is composed of the remainder of the protein. The crystal structure of the Cy-domain is unknown but thought to be similar to the C-domain. Although it is viewed as an optional domain, it can replace the C-domain and has therefore been included in this figure. Figure incorporated from Finking and Marahiel 2004.

  • Introduction

    7

    Figure 1.3 (A) Surfactin synthetase is a linear NRPS. Synthesis proceeds from module 1 to module 7 until the final product is cleaved off by the TE-domain of the last module. (B) The five modules of the gramicidin S-synthetase are used twice in succession and the TE-domain cyclodimerizes the two pentapeptides, which yields the homodimeric product. (C) NS is decorated with DHB and with two molecules of DHB-mOx by the second C-domain of VibF. Synthesis of DHB-mOx is realized by transfer of DHB to VibF, which condenses it with the heterocyclized Thr. Figure incorporated from Finking and Marahiel 2004.

    1.1.2 Ribosomally synthesized AMPs

    Almost every form of life throughout the phylogenetic tree produces this category of

    AMPs. Such conservation in spite of a huge genetic divergence from bacteria to

    human beings says all about the success of these AMP molecules. Similar activity but

    little sequence homology suggests that each peptide has evolved (probably

    convergently) to act optimally in the environment in which it is produced and against

    local microorganisms. All of these are produced via the conventional ribosome driven

    translational pathway and may undergo some posttranslational modifications.

    Irrespective of their origin and sequence many of these peptides tend to attain some

    similar active conformations like some form amphipathic -helices, some have

    cysteine knots, some predominantly have -sheets, some undergo unusual amino acid

    modifications whereas still others have an uncommon composition of regular amino

  • Introduction

    8

    acids. Therefore origin-wise distribution and functional modification of these peptides

    from higher eukaryotes to prokaryotes could be briefly summarized as follows:

    (i) Mammalian AMPs:

    Mammalian AMPs are essentially associated with the innate immune system. They

    are preferentially crowded at the mucosal surfaces and participate in the primary

    immune response. As is evident from the statement, epithelial cells and Neutrophils

    contribute to the highest extent in the production of these peptides. Additionally, cells

    like the Paneth cells of intestine, platelets etc. are also known to produce some AMPs

    locally (Ouellette and Bevins 2001). The most extensively studied mammalian AMPs

    are defensins. These can be categorized into two groups, classical (sometimes called

    -defensins) and -defensins. Both contain three pairs of disulfide linked cysteines

    and a high arginine content, but the location and connectivity of the cysteines are

    different between the two groups and there are also differences in other conserved

    amino acids. The classical defensins are present primarily within neutrophils and

    Paneth cells, and the -defensins are isolated from epithelial cells, neutrophils, and

    leukocytes. Research has also proved the existence of defensins in other eukaryotes

    viz. insects, plants, amphibians etc. Unlike defensins, - helical AMPs produced by

    mammals include cathelicidin found in lysosomes of macrophages and neutrophils.

    The best known human cathelicidin is a 37 amino acid peptide, LL37 (Yang, Chen et

    al. 2000). Furthermore, proteolytic degradation of some higher proteins is also

    documented to produce peptide fragments with antimicrobial activity. For example,

    an 11 amino acid peptide fragment released after peptic digestion of human

    lactoferrin, peptide fragments of H2A histone, bovine casein etc. are documented to

    have substantial antimicrobial activity (Lahov and Regelson 1996).

    (ii) Amphibian AMPs:

    Eukaryotic AMPs probably got more publicity after they were first identified and

    isolated in enormous amounts from the skin of Amphibian species. May be a relative

    ease in isolation gave them the recognition and studies on these AMPs started to

    gather some attention amongst researchers. After the isolation of bombinin from

    Bombina variegate in 1970 and later magainins from Xenopus spp., the researchers

    went on to isolate more than a dozen of AMPs from the Xenopus spp. alone.

  • Introduction

    9

    Moreover, later on some AMPs like the dermaseptins were found to possess good

    antifungal properties too. One surprising thing to note was that there exists very little

    sequence homology even within the AMPs isolated from same species. But as far as

    their charge and secondary structures are concerned most of them are either cationic,

    amphipathic alpha helical, e.g., magainins, dermaseptins and buforin II, or are

    cysteine disulfide loop peptides, e.g., ranalexins and the brevinins from Rana frog.

    Most widely studied amphibian AMPs are the 23 amino acid peptides, magainins,

    isolated from the skin of Xenopus laevis (Zasloff 1987). Magainins are expressed as

    pre-pro-proteins containing one copy of magainin1 and five copies of magainin2 that

    are further separated proteolytically (Terry, Poulter et al. 1988). A unique feature

    about an AMP, buforin II, isolated from Bufo bufo gargarizans stomach is that it is

    derived from proteolysis of H2A histone.

    (iii) Plant AMPs:

    Plant defense system also largely relies on their collection of antimicrobial peptides.

    The first isolated AMPs of plant origin were thionins, which not only have a broad

    range antibacterial and antifungal activity but has also proved to be toxic to various

    mammalian cell types. Plants are also reported to produce AMPs that are structurally

    similar to mammalian defensins and hence are aptly referred to as Plant defensins.

    As opposed to mammalian defensins plant defensins are more potent antifungal

    molecules. They are also demonstrated to have highly specific targets on the fungal

    microsomal membranes and hyphae. Even coconut water has been recently reported

    to have some unique AMPs (Santi M. Mandal 2009). Coconut water peptide Cn-

    AMP1 with high proportion of hydrophobic amino acids is the most active peptide

    within the collection of three AMPs isolated from the same source- the others are

    named Cn-AMP2 and 3 (Santi M. Mandal 2009).

    (iv) Insect AMPs:

    Insect body fluids and their external secretions are rich sources of this category of

    AMPs. Cecropins isolated from the hemolymph of a giant silkworm moth,

    Hyalophora cecropia (Steiner, Hultmark et al. 1981) are one of the most extensively

    investigated -helical insect AMPs. These are expressed as pre-pro-proteins and are

  • Introduction

    10

    further processed proteolytically to yield an active cecropin molecule (Gudmundsson,

    Lidholm et al. 1991). Cecropins have potent antibacterial activity, especially against

    Gram positive bacteria and display minimal cytotoxicity against insect and

    mammalian cells. Peptides similar to the Hyalophora cecropins were also found in

    many other insects. A molecule similar to insect cecropins, Cecropin P1 was isolated

    from pig intestine as well (Lee, Boman et al. 1989), suggesting that cecropins may be

    widespread throughout the animal kingdom. Apart from cercropins, bee venom is

    known to contain a cytotoxic AMP called melittin and even the royal jelly of

    honeybees contains a family of AMPs called Jelleines. Moreover, hemolymph of

    Drosophilla may contain at least seven AMPs out of which some are specifically

    expressed depending on the type of invading microorganism. Thus, it could be stated

    that within insects AMPs perform a major antiinfective role.

    (v) Bacterial AMPs:

    As opposed to higher eukaryotes, bacteria can produce their AMPs either ribosomally

    or Non-ribosomally as described above. Both Gram positive as well as Gram negative

    bacteria produce ribosomal AMPs, which could be cationic or neutral and may

    undergo some posttranslational modifications. These are commonly referred to as

    becteriocins and the producer organism is immune against its own bacteriocins. Some

    peptide bacteriocins, including the Escherichia coli 7-amino-acid peptide microcin

    C7, inhibit protein synthesis, and the Lactococcus peptide mersacidin, which inhibits

    peptidoglycan biosynthesis, have specific mechanisms which inhibit bacterial

    functions. However, most of these peptides, e.g., nisin and epidermidin, are thought to

    permeabilize target cell membranes. The speciality of bacteriocins is that many of

    these have some unusual, modified or uncommon composition of amino acids

    probably to escape from the proteolytic attack of some bacterial extracellular

    proteases.

    Targeted modification of some amino acid residues has lead to the emergence of an

    entirely new class of bacterial AMPs called lantibiotics. These are very stable AMPs,

    which is a property that is not usually associated with most other AMPs.

  • Introduction

    11

    1.2 Quorum Sensing

    Bacteria being simple unicellular organisms, their involvement in cell-cell

    communication was overlooked for quite a long time. The preliminary evidences on

    bacterial communication were obtained from the studies conducted on a

    bioluminescent bacterium Vibrio fischeri in 1970(Nealson, Platt et al. 1970). The

    bacterium resides symbiotically in the light organ of the luminescent Hawaiian Squid,

    Euprymna Scolopes, and is responsible for the squids bioluminescence. It was

    observed that this microorganism is luminescent only at high cell densities (1010-1011

    cells/ml) while fails to do so at lower population size. Subsequent research in the field

    demonstrated the involvement of a signaling molecule whose critical threshold levels

    when achieved cause activation of the genes responsible for bioluminescence. In

    several instances certain bacterial extracellular molecules were considered to provide

    the bacterial population a means to determine its numerical size or density, as is

    described above. So, in 1990s a term quorum-sensing (QS) was introduced to

    describe a process of intercellular bacterial communication through diffusible

    signaling molecules, required to sense minimal functional bacterial population for

    particular gene expression (Fuqua, Winans et al. 1994). Therefore, in simple terms,

    bacteria are capable of monitoring their own population density and use this form of

    communication to coordinate expression of particular genes. Although widely

    accepted, the term quorum sensing in its strictest sense does not always describe

    appropriate situations where bacteria employ diffusible signaling molecules. For

    example, accumulation or loss of a signaling molecule will also depend upon the local

    environmental conditions. Thus, it is also possible for a single bacterial cell to switch

    from the non-quorate to the quorate state depending on its local environment, as

    has been observed for Staphylococcus aureus trapped within an endosome in

    endothelial cells (Qazi, Counil et al. 2001). Considering the same, may be diffusion

    sensing or compartment sensing are more appropriate terms since the signal

    molecule is supplying information with respect to the local environment rather than

    cell population density per se (Redfield 2002; Winzer, Hardie et al. 2002). However

    still, as routinely practiced, the terms diffusion sensing and quorum sensing will be

    treated synonymously all throughout this text, with proper description of the

    environmental conditions wherever required.

  • Introduction

    12

    Bacteria as they grow release a wide variety of small molecules including secondary

    metabolites such as antibiotics and siderophores (iron chelators), metabolic end

    products and cell-to-cell signaling molecules which function as pheromones and are

    sometimes termed autoinducers. Both Gram positive and Gram negative bacteria

    produce such signaling molecules. The fact that many of these molecules are

    associated with activation of communal responses within bacterial population like

    swarming initiation, biofilm formation, biosurfactant production, expression of

    virulence factors etc. lead to extensive research in this field. In the past decade

    researchers have contributed exhaustively for isolation, purification and identification

    of several signaling molecules as well as for elucidation of their mode of action.

    Based on the type of signaling molecules involved, broadly there can be two

    mechanisms associated with bacterial cell-cell signaling, viz., Peptide-mediated QS

    and Non-peptide QS. The former is preferentially operative in Gram positive bacteria

    while the latter is prevalent in Gram negative bacteria. After a brief description of

    Non-peptide QS, particular attention will be given to the Peptide-mediated QS

    mechanisms.

    1.2.1 Non-peptide QS mechanisms

    Quorum sensing mechanism in Gram negative bacteria largely depends upon low

    molecular weight hydrophobic molecules viz. Homoserine lactones (HSL), Alkyl

    quinolones, Furanosyl diesters (AI-2), Fatty acid methyl esters etc. HSL or more

    specifically, Acyl homoserine lactone i.e. AHL-mediated quorum sensing is employed

    by diverse Gram negative proteobacteria belonging to , and subdivisions, but no

    AHL-producing Gram positive bacteria have so far been identified (Withers, Swift et

    al. 2001; Cmara, Williams et al. 2002; Chhabra, Philipp et al. 2005). In fact the first

    identified QS molecule was 3-oxo-C6-HSL (3-oxohexanoylhomoserine lactone), an

    autoinducer responsible for the bioluminescence of V. fischeri (Eberhard, Burlingame

    et al. 1981). Thereafter, a variety of cellular responses of Gram negative bacteria were

    found to be controlled by certain specific HSLs. For example, serine-protease and

    metalloprotease production in Aeromonas hydrophila is regulated by N-butanoyl-

    HSL, carbapenem synthesis by Erwinia carotovora is regulated by N-(3-oxo-C6)-

    HSL, and virulence determinants in Pseudomonas aeruginosa are activated by N-(3-

  • Introduction

    13

    oxododecanoyl)-HSL (Miller and Bassler 2001; Whitehead, Barnard et al. 2001). In

    general, most Gram negative bacteria are said to rely on a highly conserved LuxI

    LuxR (luminescence genes of V. fischeri) system of QS. This involves a transcription

    regulatory receptor protein-homologous to Lux R- and its respective ligand ie the

    signal molecule (N-AHL) which is synthesized by luxI homologues (Gray and Garey

    2001). Whenever the threshold levels of appropriate AHL are achieved, respective

    receptor protein binds the AHL and the expression of target gene(s) is altered.

    1.2.2 Peptide mediated QS mechanisms

    As opposed to Gram negative bacteria, Gram positive organisms exclusively utilize

    comparatively heavier molecules like peptides for their cell-cell communication. The

    QS in these organisms is said to be controlled by a two component signal transduction

    mechanism consisting of a histidine kinase and a response-regulating protein that

    triggers signal transduction by using phosphorylation to convey information (Miller

    and Bassler 2001). The signal peptides are essentially synthesized ribosomally and

    may or may not undergo any post-translational modifications. Mature signal peptides

    are further actively pumped out of the cell mostly via an ABC (ATP-binding cassette)

    exporter protein. Once the threshold levels are reached, the peptide binds to the

    extracellular domain of its cognate histidine kinase receptor leading to

    autophosphorylation of a specific histidine residue of its cytosolic domain. The

    phosphate group is then transferred to a conserved aspartate residue of a cytoplasmic,

    DNA-binding, two component system response regulator. The phosphorylated form

    of the response regulator further modulates expression of quorum sensingregulated

    genes (Kleerebezem, Quadri et al. 1997; Bassler 1999). These signal peptides are

    sometimes also referred to as autoinducer peptides (AIP) as they frequently positively

    autoregulate their own synthesis (Shpakov 2009). Although this is a basic template

    employed in every Gram positive bacterial signaling system, it could be significantly

    varied in some cases. For example, in Bacillus subtilis, phr signaling system that

    involves Phr-pentapeptides responsible for competence and sporulation. Right from

    its synthesis this peptide undergoes several proteolytic modification steps including

    during its extracellular transport and final extracellular processing to generate mature

    peptapeptide (ARNQT) (Stephenson, Mueller et al. 2003). Instead of interacting with

  • Introduction

    14

    any surface receptor protein this pentapeptide uniquely makes a direct entry into its

    target cell through an Opp oligopeptide permease system. Once inside, the peptide

    inactivates phosphatase thereby maintaining the active form of DNA-binding

    response regulator phosphorylated (Perego and Brannigan 2001). However, this is one

    of the extreme examples of the deviation from the basic model of QS popular within

    Gram positive bacteria.

    Several signaling peptides of Gram positive bacteria are reported to have substantial

    antimicrobial activity (Gillor 2007). Such dual function peptides serve as an effective

    weapon for the producer organism to eliminate competitive microorganisms and self

    sustenance. Perhaps the most widely studied bacteriocin belonging to this sub-group

    of signaling peptides is nisin produced by Lactococcus lactis (Sturme, Kleerebezem et

    al. 2002). It serves as an autoinducer pheromone peptide in L.lactis (Kleerebezem

    2004) while exercises its bacteriocidal function on other non-self Gram positive

    bacteria but is relatively ineffective against Gram negative microorganisms unless

    under some modified conditions. Although there are several suspected modes of

    action for antibacterial activity of nisin, there is a long standing belief that it kills the

    target organism through membrane pore formation. Another peptide with similar

    properties produced by Bacillus subtilis is subtilin which along with nisin is included

    into a special class of stable AMPs called Lantibiotics. Despite preferential

    involvement of lantibiotics in bacterial signaling some non-lantibiotic unmodified

    AMPs also serve as signaling molecules, for example, carnobacteriocin peptides of

    Carnobacterium piscicola LV17B and Plantaracin A of L. plantarum C11 (Quadri

    2002).

    Peptide-mediated QS although more prevalent, is not the only cell-cell

    communication mechanism operative within Gram positive bacteria. -butyrolactone

    in Steptomyces spp. and Autoinducer-2 (AI-2) dependent mechanisms are the two

    additional documented mechanisms. Presently, AI-2 mediated is the only known QS

    mechanism shared by both Gram positive and Gram negative organisms. Existence of

    such common signaling mechanism suggests the possibility of interspecies

    communication in bacteria. AI-2 signal has been shown to activate an ABC

    transporter protein Lsr in S. typhimurium (Taga, Miller et al. 2003). Furthermore, such

  • Introduction

    15

    findings might be extrapolated to associate the participation of AI-2 mechanisms in

    expression of some signaling AMPs.

    No matter which form of QS signals are used by the bacteria, the fact that co-

    ordinated group behaviour can provide a survival benefit, has been extensively

    utilized by bacteria. The pathogenic/opportunistic microorganisms are reported to

    manifest symptomatic diseases within the host organism through such QS strategies.

    Recently, therefore much of the therapeutic research has concentrated around

    discovery of certain QS attenuating molecules. Such anti-QS molecules may not cause

    any selective pressure on the growth of target bacterium thereby reducing the

    frequency of acquiring resistance against them. This is the basic rationale behind

    designing an alternative therapeutic approach for treatment of some resistant bacterial

    pathogens. Our entire existing knowledge of bacterial cell-cell communication could

    thus be utilized for designing quorum disrupting molecules. Structural information of

    QS molecules has given a better insight for chemical synthesis of some inhibitory

    analogues. However, exploiting naturally synthesized quorum quenching molecules

    could be a still better strategy. Changing the present approach of research could be

    beneficial for identification of such naturally produced anti-QS molecules. For

    example, the studies conducted on QS molecules till date has focused mainly on

    intraspecies autoregulatory mechanisms of signaling. Even after identification of

    some bacteriocins as QS signals, studies on their autoinduction received extra

    attention instead of recognizing the fact that these same molecules are also sensed as

    the death signals by certain target microorganisms (Gillor 2007). Thus implication

    of bacterial molecules for our benefit may serve as a more rewarding approach.

    1.3 Immune system: an AMP perspective

    Innate immune system in higher eukaryotes has been frequently found to recruit

    AMPs as the first line of defense and to provide effective barrier against constant

    microbial assault. Although most epithelial cells are preferentially engaged in the

    synthesis and secretion of AMPs, it is the cells of immune system that essentially

    require their assistance to elicit a proper immune response. Induction of -defensins in

    various epithelia including human GI and respiratory tract and skin depends upon

    some bacterial stimuli like LPS as well as on some host cytokines like IL-1 and

  • Introduction

    16

    TNF. In mammals, IL-1 can be liberated from monocytes, macrophages, dendritic

    cells, or from injured epithelial cells (Murphy, Robert et al. 2000). Not only can these

    AMPs act as direct effectors but can also alert the adaptive immune system to elicit an

    antigen specific immune response (Risso 2000). For example, the -defensins of the

    human neutrophil directly attract human peripheral blood T cells that express

    CD4/CD45RA (naive) and CD8 antigens; the -defensins also attract immature

    dendritic cells both in vitro and after injection under the skin of mammals (Chertov,

    Yang et al. 2000). Such chemoattractant property is associated with almost every

    AMP and at times this could be very selective for a particular category of cell (Yang,

    Chen et al. 2000; Yang, Chen et al. 2000). For example, an inducible defensin HBD-2

    selectively attracts the memory subset of peripheral T cells along with immature

    dendritic cells (Yang, Chen et al. 2000). This leads to a highly sophisticated immune

    response ensuring the memory of pathogen specific antigen by the host T cells.

    Therefore, AMPs are very aptly referred to as host defense peptides because they

    seem to take part in almost every step of host defense against the pathogen (Zasloff

    2002).

    Recent data suggest that commensals also provide protection by chronically

    stimulating epithelial surfaces to express host antimicrobial peptides at levels that kill

    microbial pathogens (Boman 2000). These commnensal microorganisms, for

    example, Fusobacterium nucleatum in mouth and Lactobacillus in gut, are relatively

    resistant to hosts endogenous AMPs (Zasloff 2002). In the gingival epithelium, F.

    nucleatum stimulates the inducible defensin HBD-2, whereas P. gingivalis, the

    anaerobe that destroys gum tissue, does not, behaving as a silent invader

    (Krisanaprakornkit, Kimball et al. 2000). Interestingly however, it was demonstrated

    recently that F. nucleatum also produces a bacteriocin that mainly inhibits

    Lactobacilli, thus eliminating its chance of colonizing at the oral surfaces (Testa, Ruiz

    de Valladares et al. 2003). This important observation directs towards two vital

    possibilities, firstly, F. nucleatum bacteriocin production might be under the control

    of some host, pathogen or competitor microorganism derived signals so as to prevent

    Lactobacillus colonization in mouth but ensuring the same in gut. Secondly, F.

    nucleatum bacteriocin might serve as an actual chemical stimulus for the synthesis of

  • Introduction

    17

    inducible defensin HBD-2. Furthermore as bacteriocin production is an energetically

    exhausting process it must not be constitutive but inducible, either by host signals or

    by some pathogen/competitor microorganism derived factors. The latter situation

    seems to be more probable as these symbiotic bacteria colonizing on epithelial

    surfaces might encounter the pathogen first, thereby inducing their bacteriocin

    production which might further stimulate production of host defense peptides. In

    either of the situation the symbiotic bacterium seems to assist its host to effectively

    eradicate bacterial pathogen may be through its bacteriocin that might directly or

    indirectly stimulate host defense responses. There are ample evidences of host

    pathogen interactions or pathogenic lipopeptide dependent induction of host defense

    responses, so the possibility of adoption of similar induction strategies by commensal

    derived peptides cannot be ruled out (Infante-Duarte, Horton et al. 2000; Kreth,

    Merritt et al. 2009).

    Thus it seems logical to concentrate on bacterial AMPs especially those derived from

    symbiotic or non-pathogenic bacteria, which might stimulate host immune responses

    against pathogens. As most of the AMPs are unstable and prone to degradation, the

    first objective of the study was to select and choose relatively stable AMPs for the

    analysis. Lantibiotics therefore became an obvious choice.

  • Aims and Objectives

    18

    AIMS AND OBJECTIVES

    A widespread distribution of peptide antibiotics in all forms of life and the fact that

    there are comparatively minor evidences of development of bacterial resistance

    against them has gathered extensive research attention. However we decided to focus

    mainly on their signaling aspect rather than their antimicrobial activity. The

    modulation of gene expression and associated changes in the community behavior of

    certain bacterial species by peptide bacteriocins gave a positive indication towards

    their involvement in cell-cell communication. Moreover prominent dependence of

    immune system in higher eukaryotes on AMPs makes them an attractive and potential

    candidate for drug development. Therefore the present study is an attempt to

    accumulate further evidences about the therapeutic potential of selected AMPs by

    recording the response of animal and microbial cells against them in vitro. The

    proposed objectives for the work were as under:

    1. Selection and purification of antimicrobial peptides.

    2. Determining the antimicrobial spectrum of selected peptides.

    3. Screening and sorting of selected peptides on the basis of their activities.

    4. Unfolding the peptides and checking the alteration in their potency, if any, on

    refolding.

    5. Chemical modification of peptides and reassessment of their potency.

  • Literature Survey

    19

    LITERATURE SURVEY

    Antibacterial peptides have long been looked upon as the potential targets for

    therapeutic purpose, the reason being the vast repertoire that could be achieved using

    these biomolecules (McCafferty, Cudic et al. 1999; Reddy, Yedery et al. 2004). These

    not only kill the microorganism directly but might also modulate the host immune

    system to effectively deal with the invader microorganism (Reddy, Yedery et al.

    2004). Some of these peptides also have a unique quorum sensing activity with which

    one can send the message across in the microorganisms own language. With this

    spectrum of activities the antimicrobial peptides might appear as the ideal targets for

    future pharmaceutical research but the greatest setback here is the stability of these

    peptides. To some extent, the answer to this query is given by the microorganisms

    themselves. A class of stable antimicrobial peptides known as Lantibiotics, is

    synthesized by some Gram positive bacteria to restrict the growth of other competitive

    Gram positive bacteria. There has been a lot of development in the subject of

    lantibiotics in the last few decades, due to their inherent stability as well as potent

    microbicidal activity. There are also evidences of these being useful against multidrug

    resistant pathogens (Severina, Severin et al. 1998).

    Lantibiotics are a special class of ribosomally synthesized AMPs that undergo

    extensive posttranslational modification to achieve remarkable stability. They have an

    unusual amino acid called lanthionine and/or methyl lanthionine which stabilizes the

    peptide and even protects it from the protease attack (Sahl and Bierbaum 1998).

    Lanthionine and/or methyl lanthionine residues are synthesized when the dehydrated

    serine and/or threonine residues in the prepeptide are stereo and regiospecifically

    linked via a thioether bridge to a nearby cysteine residue (Rink, Wierenga et al. 2007;

    Willey and van der Donk 2007). Special enzymes are recruited for this

    posttranslational modification of the lantibiotic prepeptide (Sahl and Bierbaum 1998;

    Bonelli, Wiedemann et al. 2006; Christianson 2006; Rink, Wierenga et al. 2007).

    Some additional modifications include production of D-amino acids within

    lantibiotics that may suggest towards their probable involvement in some neuronal

    processes similar to neuropeptides. This supposition is strengthened by recent

  • Literature Survey

    20

    identification of lantionine binding sites in some neurologically important proteins of

    mammalian CNS (Hensley, Christov et al. 2010). Two very well known and widely

    studied lantibiotics are nisin and subtilin. Nisin is produced by Lactococcus lactis and

    is the best characterized lantibiotic (Chan, Bycroft et al. 1989; Rink, Wierenga et al.

    2007). It is widely used in food industry as an antibacterial agent (Delves-Broughton,

    Blackburn et al. 1996; Delves-Broughton 2005). Inspite of its routine use from the

    day it was first analyzed by Gross & Morell in the early 1970s, till date there are very

    few reports of microbial resistance against it (Breidt, Crowley et al. 1995; Davies,

    Falahee et al. 1996). Similar is the story of its structural cousin, subtilin (Chan,

    Bycroft et al. 1992) produced by Bacillus subtilis but surprisingly, it is not as

    commonly applied as nisin.

    Another important biochemical function of lantibiotics that has been recently

    uncovered is their unique quorum sensing activity. Nisin and subtilin specifically

    were found to act as peptide pheromones which can control their own biosynthesis

    (Kleerebezem and Quadri 2001; Kleerebezem 2004). Later on, however the process

    was found to be commonly utilized by every lantibiotic producing Gram positive

    bacterium. Taking nisin and subtilin as the model, it is elucidated that there is an

    entire gene cluster specially dedicated for lantibiotic synthesis, producer organisms

    self immunity and autoregulation of lantibiotic production as depicted in figure 2.1

    (Quadri 2002; Kleerebezem 2004; Gillor 2007; Willey and van der Donk 2007;

    Shpakov 2009). Such an arrangement of genes indicates the importance of lantibiotics

    for the producer organism, for example, an intact lantibiotic locus is extremely

    essential for the virulence and intracellular survival of Streptococcus pyogenes within

    murine macrophages (Phelps and Neely 2007). There are reports which suggest that

    some Streptomycete lantibiotics are indirectly involved in triggering cellular

    differentiation for aerial hyphea formation in Streptomycete (Kodani, Lodato et al.

    2005; Willey, Willems et al. 2006). Swarming is also a kind of morphological

    differentiation observed in several bacteria (Kim, Killam et al. 2003). Therefore, there

    is a possibility that lantibiotics like nisin and subtilin may pronounce some effect on

    swarming of their respective producers and/or other Gram positive bacteria.

    Additionally, lantibiotics are unable to kill Gram negative bacteria, except under

  • Literature Survey

    21

    specialized conditions (Olasupo, Fitzgerald et al. 2003) and presently there are no

    evidences about their effect on the swarming behavior of Gram negative bacteria

    indicating negligence of research towards this aspect. Furthermore, swarming

    behavior involves QS and is linked with biofilm formation, disease manifestation and

    expression of virulence factors in pathogenic bacteria (Rozalski, Sidorczyk et al.

    1997; Eberl, Molin et al. 1999; Parsek and Singh 2003).

    Biofilms are specialized microbial communities that frequently harbor genetically

    divergent microorganisms (Kolenbrander 2000). The transformation of free

    swimming planktonic bacteria into adherent multicellular microbial community

    requires specific morphological differentiation and associated modulation of gene

    expression (Kolenbrander 2000; Stoodley, Sauer et al. 2002; Waite, Paccanaro et al.

    2006). This entire transformation is under strict QS control (Irie and Parsek 2008;

    Senadheera and Cvitkovitch 2008) and is essential for the bacterium to adopt

    according to changing surrounding environment (O'Toole, Kaplan et al. 2000). A

    bacterium within the biofilm or in a swarming mode is highly resistant to antibiotics

    as well as to host defense mechanisms (Kim and Surette 2003). Colonizing host target

    tissues after switching on the biofilm mode thus seems to be advantageous to the

    bacterium (Parsek and Singh 2003; Waite, Paccanaro et al. 2006), particularly to a

    pathogen, thereby making current treatment strategies ineffective. Therefore, recently

    there were some extensive studies targeting towards discovery of molecules capable

    of modulating QS mechanisms required for biofilm and/or swarming switchover

    (Wang, Lai et al. 2006).

    Several recent reports suggest quorum quenching strategies might serve as highly

    efficient, emerging alternative therapeutic approach for treatment of deadly bacterial

    infections (Rasmussen and Givskov 2006; Martin, Hoven et al. 2008; Raina, De Vizio

    et al. 2009). There are evidences which indicate high biofilm inhibitory potential of

    lantibiotic nisin on some specific bacteria viz. Oenococcus oeni and Listeria

    monocytogenes (Bower, McGuire et al. 1995; Bower, Daeschel et al. 1998; Nel,

    Bauer et al. 2002). Moreover, nisin has also demonstrated certain degree of

    immunomodulatory efficacy in vivo, in mice and fish models, as well as in vitro (de

  • Literature Survey

    22

    Pablo, Gaforio et al. 1999; Villamil, Figueras et al. 2003), however there are no direct

    evidences of human immune system modulation.

    Figure 2.1 Presubtilin is the product of the spaS gene; it is modified and transported by the membrane-associated subtilin synthetase complex (Kiesau, Eikmanns et al. 1997), which consists of SpaB, SpaC, and SpaT. After export, the leader is cleaved by any one of three exoproteases (subtilisin, WprA, Vpr) (Corvey, Stein et al. 2003). Mature subtilin serves as ligand for the sensor kinase SpaK, which when activated phosphorylates SpaR, a positive regulator of spaS and the biosynthetic and immunity operons. Stationary-phase control of spaS transcription is regulated indirectly through H control of spaRK; other genes in the cluster are dependent on A for transcription. H itself is subject to transcriptional control by the transition state regulator AbrB (Stein, Borchert et al. 2002). Incorporated from (Willey and van der Donk 2007).

    Interestingly, no lantibiotic tested thus far has been shown to have an exceptional

    cytotoxic effect on the eukaryotic cells (Maher and McClean 2006). With such

    diverse and multi-dimensional characters lantibiotics become attractive targets for

    pharmaceutical research and drug development.

  • Material and Methods

    23

    Materials

    Chemicals, Research Kits and Reagents

    Acetic acid Galcial Merck Chemicals Ltd.

    Acetone Merck Chemicals Ltd.

    Acetonitrile Merck Chemicals Ltd.

    Acrylamine Merck Chemicals Ltd.

    Agarose Low EEO Merck Chemicals Ltd.

    Ammonium Acetate Merck Chemicals Ltd.

    Ammonium Chloride Merck Chemicals Ltd.

    Ammonium persulphate Merck Chemicals Ltd.

    Annexin V-Cy3 Apoptosis detection Kit BioVision, CA, USA.

    Bnromophenol Blue Himedia, India

    Boric Acid Merck Chemicals Ltd.

    Butanol Merck Chemicals Ltd.

    Calcium Chloride Himedia, India

    Catalase Himedia, India

    Citric Acid mono hydrate Merck Chemicals Ltd.

    Congo Red Himedia, India

    Coomassie Brilliant Blue R-250 Himedia, India

    Crystal Violet Himedia, India

    Dimethyl sufoxide

    Biogene, Reagents, Inc,

    USA

    DNase I Himedia, India

    Ethanol Merck Chemicals Ltd.

    Ethylenediaminetetraacetic acid Himedia, India

    Ferric Chloride Himedia, India

    Formaldehyde Merck Chemicals Ltd.

    GH polypropylene Membrane Pall Corporation, USA

    Glucose Merck Chemicals Ltd.

  • Material and Methods

    24

    Gluteraldehyde Himedia, India

    Glycerol Merck Chemicals Ltd.

    Hanks Balance Salt Solution Invitrogen, USA

    HEA HyperCel Mixed-mode Chromatography

    Resin Pall Corporation, USA

    Hydrochloric acid Merck Chemicals Ltd.

    Hydrogen peroxide Merck Chemicals Ltd.

    Lactose Merck Chemicals Ltd.

    Lipopolysaccharide Fluka, Germany

    Magnesium Suphate Himedia, India

    Manganese Chloride Himedia, India

    Mercaptoethanol SRL, India

    Methanol Merck Chemicals Ltd.

    MTT Himedia, India

    N, N' methylene Bisacrylamide Merck Chemicals Ltd.

    Nisin Himedia, India

    Nitroblue Tetrazolium

    Biogene, Reagents, Inc,

    USA

    Orcinol Himedia, India

    Osmium tertroxide SRL, India

    Petroleum ether Merck Chemicals Ltd.

    Phorbol 12-myristate 13-acetate MP Biochemicals, France

    Phosphate Buffered saline

    Biogene, Reagents, Inc,

    USA

    Phytohemagglutinin Invitrogen, USA

    Propidium Iodide MP Biochemicals, France

    Proteinase K Sigma-Aldrich, USA

    Rhamnose Merck Chemicals Ltd.

    Silver Nitrate Merck Chemicals Ltd.

    Sodium Acetate Merck Chemicals Ltd.

    Sodium Carbonate Merck Chemicals Ltd.

  • Material and Methods

    25

    Sodium Chloride Merck Chemicals Ltd.

    Sodium cynoborohydride Himedia, India

    Sodium dodecyl sulphate Merck Chemicals Ltd.

    Sodium Hydroxide SRL, India

    Sodium Phosphate dibasic Merck Chemicals Ltd.

    Sodium Phosphate monobasic Merck Chemicals Ltd.

    Sodium pyruvate Himedia, India

    Sodium Thiosulphate SRL, India

    Sulfuric acid Merck Chemicals Ltd.

    Tannic acid Himedia, India

    Tetramethylethylenediamine Merck Chemicals Ltd.

    tri Sodium Citrate dehydrate Merck Chemicals Ltd.

    Tricine Merck Chemicals Ltd.

    Trifluoro acetic Merck Chemicals Ltd.

    Tris Base Merck Chemicals Ltd.

    Tris HCl Merck Chemicals Ltd.

    Triton X-100 Merck Chemicals Ltd.

    Trypan Blue MP Biochemicals, France

    Zinc Chloride Merck Chemicals Ltd.

    Culture Media

    Brain Heart Infusion Broth Himedia, India

    CAS Amino Acids Himedia, India

    DMEM Medium Invitrogen, USA

    Fetal Bovine Serum Invitrogen, USA

    Luria Bertani (LB) broth Himedia, India

    Mueller Hinton (MH) broth Himedia, India

    Nutrient broth Himedia, India

    RPMI-1640 Medium Invitrogen, USA

  • Material and Methods

    26

    3.1 Selection, Isolation and Modification of AMPs.

    3.1.1 Introduction

    Selection of a source for purification of AMPs could have been a simple process due

    to their ubiquitous distribution throughout the phylogenetic tree. But the major hurdle

    in such a random selection of source and thereby an AMP molecule was associated

    with substantial instability of these molecules. Hassle free isolation, purification and

    activity evaluation of antimicrobial peptides demands the selection of some stable

    AMPs. This necessitated choosing a special class of stable AMPs synthesized by

    Gram positive bacteria called Lantibiotics.

    Lantibiotics are named so because of the presence, in their structures, of lanthionine

    residues (figure 3.1). Production of lanthionine residues involves a sequential cascade

    of posttranslational events, cross linking the target amino acids as well as sometimes

    creating some D-isomers of other amino acids. Such overall modification results into

    a mature and functional lantibiotic molecule (Sahl and Bierbaum 1998). The presence

    of these unusual residues, their influence on structure and activity of the peptides and

    their unique biosynthetic pathways have made lantibiotics attractive targets for

    research. Lantibiotics can be classified into types A, B and C according to their

    structural features (figure 3.2). Type A lantibiotics are linear, cationic peptides which

    include nisin, subtilin and sublancin. Type B such as cinnamycin are less cationic or

    neutral, while type C lantibiotics like mersacidin are small and neutral (Bonelli,

    Wiedemann et al. 2006).

  • Material and Methods

    27

    Figure 3.1 Structures of dehydro residues and lanthionine residues. The unusual dehydro residues and lanthionine residues observed in lantibiotics are formed as a result of post-translational modifications of naturally occurring amino acids.

  • Material and Methods

    28

    Figure 3.2 Structures of few type A, type B and type C lantibiotics. Lantibiotics are classified into type A, type B and type C based on their structural features. Nisin and subtilin belong to type A (nisin-like) lantibiotics. Cinnamycin is a type B (duramycin-like) lantibiotic, and mersacidin is a type C lantibiotic.

    Two well-studied type A lantibiotics are nisin and subtilin produced by Lactococcus

    lactis ATCC 11454 and Bacillus subtilis ATCC 6633, respectively. Nisin and subtilin

    are structural analogs exhibiting 60% identity in their amino acid residues. Nisin is a

    peptide of 34 amino acids while subtilin has 32 amino acids. Both peptides possess

    three dehydro residues - two Dha and one Dhb, and five lanthionine rings (figure 3.2).

    The lanthionine rings are named alphabetically with ring A at the amino terminus.

    Both nisin and subtilin are autoinducer pheromone peptides (Sturme, Kleerebezem et

    al. 2002; Kleerebezem 2004; Kleerebezem 2004; Kleerebezem, Bongers et al. 2004).

    Type A

    Type BType C

    Subtilin

    Nisin

  • Material and Methods

    29

    Under normal culture conditions the producer organisms accumulate these peptides to

    a threshold level and the production maximizes in the late log phase. Therefore,

    ability of nisin or subtilin production can be enhanced, when the respective producer

    organism is stimulated with its autoinducer peptide.

    3.1.2 Media and Culture conditions

    Lactococcus lactis (MTCC # 440/ATCC # 11454) and Bacillus subtilis (MTCC #

    441/ATCC # 6633) purchased from IMTECH, Chandigarh, India were grown and

    maintained on the media recommended by the supplier. The L. lactis growth media-

    Brain Heart Infusion Broth (Himedia # M210) was prepared and sterilized according

    to the manufacturers instructions. The organism was revived, checked for purity on

    BHI agar and a single isolated colony was inoculated in sterile BHI broth and was

    allowed to grow for 24 hrs at 37C. This was used as a starter culture for nisin

    production. Similarly, B. subtilis starter culture was prepared in Nutrient broth

    (Himedia # M002) and used for subtilin production.

    100 ml of BHI/Nutrient broth in 250 ml conical flask was inoculated with 100 l of

    the respective starter culture and the organisms were allowed to grow for 72 hrs at

    37C with continuous shaking at 100 rpm.

    3.1.3 Isolation and purification

    Nisin and subtilin were purified from the respective cultures as per the patented

    procedure of Dimick et. al. (Dimick, Alderton et al. 1947). Briefly, after the specified

    incubation period, the entire culture was acidified with HCl and the pH adjusted to

    2.5. Acidified culture was then extracted with half the volume of butanol with

    vigorous agitation. To the resulting butanol phase, half the volume of petroleum ether

    was added and the mixture thus obtained was extracted thrice with 1/3rd volume of 1%

    acetic acid. All the three aqueous fractions obtained were pooled and crude nisin or

    subtilin was precipitated by addition of sodium chloride to reach final concentration

    of 6 g %. The precipitate was washed thoroughly with 95% (v/v) ethanol and the dried

    precipitate was resuspended in sufficient quantity of distilled water. The pH of this

    solution was adjusted to 4.6 by NaOH and the impurity was precipitated by adjusting

    the salt concentration to 0.4 g % with NaCl. The process was repeated thrice and the

  • Material and Methods

    30

    last solid residue obtained was discarded while all the resulting aqueous fractions

    were pooled. Finally pure nisin or subtilin was precipitated from pooled aqueous

    fractions by adjusting the NaCl concentration to 10%. The resulting final precipitate

    of nisin or subtilin was washed twice with distilled water and finally with 95%

    ethanol, dried in vacuum and weighed to calculate the final yield.

    3.1.4 Alternate Culture media and Subtilin isolation procedure

    Subtilin producing B. subtilis strain was grown in modified M9 medium (Banerjee

    and Hansen 1988) with sodium pyruvate as sole carbon source at 30C as described

    by Parisot et. al. (Parisot, Carey et al. 2008). The isolation procedure was modified to

    devise a convenient and simple method. The culture was incubated for 72 hrs under

    continuous shaking conditions at 100 rpm to maintain good aeration and the culture

    medium was centrifuged at 9000 rpm for 15 min to collect the supernatant. The clear

    supernatant was filtered through 0.2 m GH polypro membrane filter (Pall

    Corporation, USA) under vacuum and immediately subjected to acetone precipitation.

    To the organism free spent medium, acetone was added to get the final concentration

    of 35% and crude subtilin was allowed to precipitate overnight at 0C. The precipitate

    thus obtained was collected by centrifugation at 15000 rpm, washed with acetone and

    allowed to dry in vacuum. The crude subtilin thus obtained was further purified by

    hydrophobic interaction chromatography. To enhance subtilin production, structurally

    similar commercial nisin was used as a stimulant in some cultures at sub-inhibitory

    concentrations.

    (i) Hydrophobic interaction chromatography (HIC)

    A glass column of 10 cm length and 1.5 cm diameter was packed evenly with HEA

    HyperCel Mixed-mode Chromatography Resin (Pall Corporation, USA).The column

    was equilibrated with 7 volumes of equilibrating/wash buffer comprising of 0.05 M

    Tris-Cl, pH 8.0 and the flow rate was adjusted to 0.5 ml/min. Crude subtilin was

    suspended in distilled water, acidified with glacial acetic acid and was equilibrated

    with 0.05 M Tris-Cl, pH 8.0. The equilibrated subtilin sample was placed on ice and

    slowly 1.5-2 ml loaded onto the column. The column was then washed with 3

    volumes of wash buffer. The less hydrophobic molecules were washed off the column

  • Material and Methods

    31

    in this step. Subtilin, a more hydrophobic peptide, was eluted with 0.2M

    Phosphate/0.1M Citrate elution Buffer with varying pH (2.6 7.0) and fractions were

    collected every minute in microcentrifuge tubes. The elution profile of subtilin was

    observed at 254 nm, 214 nm and 280 nm to monitor the absorbances of dehydro

    residues, peptide bond, and aromatic residues, respectively and all the collected

    fractions were tested for antimicrobial activity using halo assays.

    Alternatively, the acetone precipitated crude subtilin was also subjected for a partial

    purification process. The process involved concentration of 5 fold diluted saturated

    crude subtilin solution by passing it through 1 kDa and 3 kDa cutoff columns. The

    resulting supernatants and vacuum concentrated filtrates from the two columns were

    tested for their activities. The presence of subtilin in these fractions was analyzed by

    MALDI-Tof analysis.

    (ii) Spore outgrowth inhibition assay (halo assay)

    The antimicrobial activity of the lantibiotics subtilin (purified by both the methods)

    and nisin (both purified and commercial) was assessed by their ability to inhibit the

    outgrowth of B. cereus T spores (Michener 1955; Campbell and Sniff 1959; Liu and

    Hansen 1993; Paik, Chakicherla et al. 1998; Gut, Prouty et al. 2008). The spores were

    prepared for the assay by heat shocking the spores. 250 mg of lyophilized B. cereus

    spores was suspended in 20 ml of sterile distilled H2O. The spores were thoroughly

    dispersed in a glass homogenizer. The homogenized spores were then heat shocked

    for 2 h at 65C. The spore pellet was collected after centrifugation for 10 min at 4,000

    rpm. The spores were resuspended in 50 ml of 10% ethanol prepared in sterile water

    and stored at RT.

    The halo assay was performed on Mueller Hinton (MH) broth (Himedia # M391)

    solidified by 1.0 g % agarose. Tubes containing sterile MH agarose were melted and

    maintained at 40-45C before inoculation of B. cereus spore suspension. The plates

    were poured immediately after inoculating 50 l spore suspension and the medium

    was allowed to solidify at RT. Wells were punctured in solid medium under sterile

    conditions. 10 l of each fraction to be tested for antimicrobial activity was

  • Material and Methods

    32

    transferred to each well. Fractions with antimicrobial activity showed clear halos

    representing zones of growth inhibition amidst a lawn of bacterial cells.

    3.1.5 Tricine-SDS-PAGE of purified subtilin and nisin

    The subtilin and nisin fractions purified by conventional purification methods as well

    as by HIC were run on a Tricine-SDS-PAGE (Schagger 2006) to assess their purity.

    The Tricine-SDS-PAGE gel comprised of a separating gel, a spacer gel and a stacking

    gel. 14 cm x 16 cm gel plates were assembled with spacers of 1 mm thickness, and the

    edges and the bottom of the assembly were sealed with 1% molten agarose (in

    Distilled water). 150 l of 10% APS and 30 l of TEMED were added to 10 ml of the

    separating gel solution and this was immediately poured into the plate assembly. The

    solution was overlaid with isobutanol and allowed to polymerize for 45-60 min. After

    polymerization was complete, the overlay was poured off and the top of the gel

    washed with distilled H2O several times. The spacer gel was then allowed to

    polymerize on top of the separating gel. This was followed by polymerization of the

    stacking gel which occurred with the comb in place. The comb and clamps were then

    removed from the assembly, and the gel assembly was placed into the electrophoretic

    apparatus. Cathode buffer (0.1 M Tris at pH 8.25, 0.1 M Tricine, 0.1% SDS) was

    added to the upper chamber of the electrophoresis apparatus, while the lower reservoir

    was filled with anode buffer (0.2 M Tris, pH 8.9). 10 l of the sample was diluted

    with an equal volume of 2X gel loading buffer. The ultralow range marker (SIGMA-

    Aldrich) specially designed for use in the Tricine SDS-PAGE was also loaded on to

    the gel according to the manufacturers instructions. The samples were boiled for 3

    min before being loaded on the gel. The gel was electrophoresed at a constant voltage

    of 100 mV until the bromophenol blue tracking dye had just migrated into the

    separating gel. The gel was then electrophoresed at 15 mV until the tracking dye had

    migrated to the bottom of the gel (~17-19 hr). The gel was fixed and stained either

    using the silver staining protocol or with simple Coomassie Brilliant Blue R 250 stain

    as detailed by Schagger (Schagger 2006).

  • Material and Methods

    33

    3.1.6 Chemical Glycosylation of commercial nisin

    A saturated nisin (Himedia # RM5360) solution (~ 3.0 mM) in 0.1M sodium acetate

    was mixed with 1M lactose in 0.1M sodium acetate to get a final concentration of 100

    mM lactose. To this reaction mixture 5M sodium cynoborohydride was added as a

    reducing agent to give a final concentration of 1M. The resulting reaction mixture was

    incubated for 72 hrs at 37C (Hermanson 2008). After incubation the mixture was 5

    times diluted with distilled water and was subjected to centrifugal fractionation first

    through 3 kDa followed by 1 kDa cutoff column. The fractionation was done to

    remove all the chemical impurities from the modified peptides. The extent of

    glycosylation was tested by subjecting the peptides to Tricine SDS-PAGE.

    An alternative protocol for peptide modification with shorter span of incubation was

    also tried. Here the saturated solution of nisin was prepared in 20% DMSO. This nisin

    solution was almost 3 times more concentrated than that used in the procedure above.

    1M lactose used for glycosylation was also prepared in 30% glacial acetic acid in

    DMSO. These solutions were then mixed in the same proportion with sodium

    cynoborohydride as the previous procedure and incubated at 60-80C for 1-2 hrs at

    RT (Hermanson 2008). The resulting peptides were fractionated and separated by

    Tricine SDS-PAGE.

    The fractionated and purified glycosylated nisin peptides were also tested for their

    potency by performing halo assay on selected bacteria.

  • Material and Methods

    34

    3.2 Effect of subtilin and nisin on bacteria

    3.2.1 Introduction

    Bacterial quorum sensing provides an important growth advantage to bacteria by

    enhancing access to nutrients or environmental niches, it enables bacteria to mount

    defensive response against competing organisms, and also optimizes the ability of the

    cell to differentiate into morphological forms better adapted to survival in a hostile

    environment (Miller and Bassler 2001). Current understanding of QS is that each

    organism in a bacterial population constitutively synthesizes a signal molecule at low

    levels. This signal accumulates at a threshold concentration sufficient for induction of

    biological activity at a cell density that is appropriate for the induced activity to occur

    efficiently (Swift, Rowe et al. 2008). QS is thus an example of multicellular behavior

    in prokaryotes and regulates diverse physiological processes including

    bioluminescence, swarming, antibiotic biosynthesis, plasmid conjugal transfer and the

    production of virulence determinants in animal, fish and plant pathogens (Hardman,

    Stewart et al. 1998). QS-dependent co-ordinated group behavior often leads to the

    development of a complex microbial community known as biofilm. Biofilms are also

    important as environmental reservoirs for pathogens, and the biofilm growth mode

    may provide organisms with s


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