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 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)
vii
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
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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
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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
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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
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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).
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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.
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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