Antimicrobial chromogranin A derived peptides and Staphylococcus aureus: from host pathogen ...

361 Pages·2013·9.05 MB·English

UNIVERSITÉ DE STRASBOURG

Ecole Doctorale Des Science De La Vie Et De La Santé (ED 414)
Inserm UMR 1121 Biomatériaux et Bioingénierie

THÈSE

Présentée par :

Rizwan ASLAM

Soutenue le : 15 Avril 2013

Pour obtenir le grade de : Docteur de l’université de Strasbourg
Discipline/ Spécialité : Aspects Moléculaire et Cellulaire de la Biologie

Les peptides antimicrobiens dérivés de la
chromogranine A et Staphylococcus aureus:
de l’analyse de l’interaction hôte-pathogène au
développement de revêtement de polymère antimicrobien

THÈSE dirigée par :
Mme METZ BOUTIGUE Marie Hélène Directrice de Recherche, Université de Strasbourg
M. PREVOST Gilles MCU-PH, Université de Strasbourg

RAPPORTEURS :
M. DRIDER Djamel Pr, Université de Lille-1
M. BERJEAUD Jean-Marc Pr, Université de Poitiers
M. BECHINGER Burkhard Pr, Université de Strasbourg

AUTRES MEMBRES DU JURY :
M. JACQUES Philippe Pr, Université de Lille-1

Acknowledgement
I humbly bow my head before God, the most beneficent and merciful, whose blessing
flourished my thoughts and thrived my ambitions. I could never have done this
without the faith I have in You, the Almighty. I would like to express my deep and
sincere gratitude to my supervisor, Dr. Marie Hélène METZ-BOUTIGUE, for making it
possible for me to work in such a prestigious scientific environment. She has been
very supportive, encouraging and kind. I owe a great deal of appreciation for her
valuable advice, constructive criticism and extensive discussions around my work. It
has been a great honor to have her as a supervisor.
I feel immense pleasure to gratefully acknowledge and to express a deep sense of
gratitude to my co-supervisor Dr. Gilles PRÉVOST for his creative suggestions,
motivation and exemplary guidance throughout the course of my doctoral research
and also during thesis writing.
I wish to express my cordial appreciation to my examiners, Prof. Bukhard Bechinger,
Prof. Djamel DRIDER, Prof. Jean-Marc BERJEAUD, and Prof. Philippe JACQUES for
the acceptance to be a referee. I feel immense pleasure to gratefully acknowledge
and to express a deep sense of gratitude to Prof. Pierre SCHAAF, Prof. Jean-Claude
VOEGEL, Dr. Francis SCHNEIDER, Dr. Fouzia BOULMEDAIS, Dr. Alain VAN
DORSSELAER, Dr. Jean-Marc STRUB, for their enthusiasm, creative suggestions
and motivation throughout the course of my doctoral research.
Moreover, I offer my profound gratitude to Prof. Youssef HAIKEL for his moral and
financial support. Words are lacking to express my gratitude for Higher Education
Commission of Pakistan and especially Dr. Atta ur Rehman, for providing me with a
scholarship during my study in France. The opportunity of studying in France has
really broadened my horizon and widened my perspectives in life.
My sincere thanks are also extended to Dr. Jean Francois CHICH and Dr. Peiman
SHOOSHTARIZADEH for their help in learning various techniques and to evaluate
results more precisely and also offer them my zealous thanks. I also benefited by
outstanding support of Bernard GUEROLD, for providing various synthetic peptides
for my experiments. My heartfelt thanks are also extended to Dr. Philippe LAVALLE
and Dr. Roxane FABRE for their expertise in confocal microscopy and offer my
zealous thanks to Dr. Gwenaelle CADO and Lydie SEON for preparation of
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multicoated PEM films. I would like to offer my gratitude to Dr. Benoît-Joseph
LAVENTIE, Daniel KELLER, Mira TAWK and Raymonde GIRARDOT for support to
complete flow cytometry analysis and providing bacterial strains and PMNs.
I sincerely acknowledge my colleagues especially Dr. Atiandou M. Cynthia, Charlotte
BACH, Martine RIVET, Cosette BETSCHA, Sophie HELLE, Merium SAHRAOUI, Dr.
Céline MARBAN, Ouria TAHAR, and Qiongue Hu for their support during my projects
and for facilitating all experimental or administrative issues.
I own my sincere appreciations to Sultan ALI, and Ghulam HUSSAIN for their
support, guidance and affection. I would also like to extend huge, warm thanks to my
friends Asghar Shabbir, Azeem Sultan, Sarfraz Shafiq, Muhammad Nauman Zahid,
Tariq Mahmood, Adnan Khan Niazi and Azhar Ayyaz Pirzadoo for their love and
sincerity. I am eternally grateful to my beloved mother, my brothers and my sister for
their unconditional love, fidelity, endurance and encouragement. I would like to
thanks to a person whose ambidextrous nature made me more enthusiastic. There
are so many others whom I may have inadvertently left out and I sincerely thank all of
them for their help.

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Table of Contents
Acknowledgements………………………………………………..………….. i
Table of contents………………………………………………………………..……….iii
Abbreviations………………………………………………………….………ix
List of Figures…………………………………………………………………….……..xii
List of Tables……………………………………………………………………………xiv
Part I: Introduction
1 Staphylococcus aureus ................................................................................. - 19 -
1.1 Virulence factors of the S. aureus ....................................................................... - 20 -
1.1.1 Surface proteins ............................................................................................... - 21 -
1.1.1.1 Cell wall and capsular factors ................................................................ - 22 -
1.1.1.2 Microbial Surface Components Recognizing Adhesive Matrix Molecules
(MSCRAMM) ......................................................................................................... - 22 -
1.1.1.2.1 Fibronectin binding proteins (A and B) ............................................. - 23 -
1.1.1.2.2 Protein A ............................................................................................ - 24 -
1.1.1.2.3 Collagen adhesion proteins ................................................................ - 24 -
1.1.1.2.4 Clumping factors (A and B) .............................................................. - 24 -
1.1.1.3 Secretable expanded repertoire adhesive molecules (SERAMs) ........... - 25 -
1.1.2 Secreted proteins ............................................................................................. - 26 -
1.1.2.1 Toxins .................................................................................................... - 26 -
1.1.2.1.1 Staphylococcal Enterotoxins ............................................................. - 26 -
1.1.2.1.2 Exfoliative toxins ............................................................................... - 28 -
1.1.2.1.3 Toxic shock syndrome toxins ............................................................ - 29 -
1.1.2.1.4 Staphylococcal Hemolysins ............................................................... - 31 -
1.1.2.1.4.1 Alpha hemolysin ......................................................................... - 31 -
1.1.2.1.4.2 Beta hemolysin ........................................................................... - 32 -
1.1.2.1.4.3 Gamma hemolysins .................................................................... - 32 -
1.1.2.1.4.4 Delta hemolysin .......................................................................... - 33 -
1.1.2.1.5 Panton valentine leucocidin ............................................................... - 33 -
1.1.2.1.6 Leukotoxin LukE/D ........................................................................... - 34 -
1.1.2.1.7 Other Bi-component cytolytic toxins ................................................ - 35 -
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1.1.2.2 Proteases of S. aureus ............................................................................ - 35 -
1.2 Host defences against S.aureus infection ............................................................ - 36 -
1.3 Immune evasion strategies of S. aureus .............................................................. - 39 -
1.3.1 Resistance to phagocytosis .............................................................................. - 40 -
1.3.2 Complement inactivation ................................................................................ - 40 -
1.3.3 Inhibition of neutrophil chemotaxis ................................................................ - 42 -
1.3.4 Leukocytes destruction by toxins .................................................................... - 43 -
1.3.5 Resistance to lysozyme ................................................................................... - 44 -
1.3.6 Survival of S. aureus in neutrophil phagosomes ............................................. - 44 -
1.3.7 Subversion of the humoral immune response ................................................. - 44 -
1.3.8 Resistance to killing by antimicrobial peptides ............................................... - 45 -
1.4 Resistance to chemotherapeutic agents ................................................................... - 45 -
2 Immune system ........................................................................................... - 47 -
2.1 Introduction ............................................................................................................. - 47 -
2.1.1 Innate immune system ..................................................................................... - 48 -
2.1.1.1 Components of innate immune system .................................................. - 48 -
2.1.1.1.1 Physical and chemical barriers .......................................................... - 49 -
2.1.1.1.2 Pattern recognition receptors (PRRs) ................................................ - 50 -
2.1.1.1.3 Cells of innate immune system .......................................................... - 51 -
2.1.1.1.3.1 Mast cells .................................................................................... - 51 -
2.1.1.1.3.2 Natural killer cells ...................................................................... - 52 -
2.1.1.1.3.3 Granulocytes (basophils and eosinophils) .................................. - 53 -
2.1.1.1.3.4 Phagocytic cells .......................................................................... - 54 -
2.1.1.1.3.4.1 Macrophages ........................................................................ - 55 -
2.1.1.1.3.4.2 Dendritic cells ...................................................................... - 55 -
2.1.1.1.3.4.3 Polymorphonuclear neutrophils ........................................... - 55 -
2.1.1.1.4 Complement system .......................................................................... - 57 -
2.1.1.1.5 Cytokines ........................................................................................... - 59 -
2.1.1.1.6 Antimicrobial peptides ...................................................................... - 61 -
2.1.2 Interrelation between immune, nervous and endocrine system. ..................... - 61 -
3 Antimicrobial peptides ................................................................................ - 64 -
3.1 Introduction ............................................................................................................. - 64 -
3.2 Structure of antimicrobial peptides ......................................................................... - 66 -
3.2.1 Chemical properties of the peptides ................................................................ - 66 -
3.2.2 Primary structure of AMPs ............................................................................. - 67 -
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3.2.3 Secondary structure of AMPs ......................................................................... - 68 -
3.2.4 Posttranslational modifications ....................................................................... - 68 -
3.3 Classification of antimicrobial peptides .................................................................. - 69 -
3.3.1 Cationic AMPs rich in specific amino acids ................................................... - 70 -
3.3.2 Cationic AMPs lacking cysteine ..................................................................... - 70 -
3.3.3 Anionic AMPs ................................................................................................. - 70 -
3.3.4 Cationic/anionic AMPs having disulfide bond ............................................... - 71 -
3.3.5 Cationic/anionic peptides derived from the large proteins .............................. - 71 -
3.4 Mechanism of action of antimicrobial peptides ...................................................... - 72 -
3.4.1 Interaction with membranes ............................................................................ - 72 -
3.4.1.1 Carpet like mechanism ........................................................................... - 72 -
3.4.1.2 Barrel stave model ................................................................................. - 73 -
3.4.1.3 Toroidal model ....................................................................................... - 73 -
3.4.2 Interaction with intracellular components ....................................................... - 74 -
3.5 Biological activities of antimicrobial peptides ........................................................ - 75 -
3.5.1 Direct antimicrobial activity ............................................................................ - 75 -
3.5.2 Immune modulation by AMPs ........................................................................ - 76 -
3.5.2.1 Recruitment of innate immune cells ...................................................... - 77 -
3.5.2.2 Endotoxin binding .................................................................................. - 77 -
3.5.2.3 AMPs induce cellular proliferation and differentiation ......................... - 77 -
3.5.2.4 Adaptive immune cells activation .......................................................... - 78 -
3.6 Antimicrobial peptide avoidance mechanisms adopted by microorganisms .......... - 78 -
4 Granins ...................................................................................................... - 80 -
4.1 Adrenal glands ......................................................................................................... - 80 -
4.2 Chromaffin cells ...................................................................................................... - 80 -
4.3 Granin family .......................................................................................................... - 81 -
4.3.1 Biological activities of the granin derived peptides ........................................ - 82 -
4.3.2 Chromogranin A .............................................................................................. - 84 -
4.3.2.1 Biological activities of the CgA derived peptides ................................. - 85 -
4.3.2.1.1 Catestatin ........................................................................................... - 88 -
4.3.2.1.2 Cateslytin ........................................................................................... - 89 -
4.3.3 Chromogranin B .............................................................................................. - 90 -
5 AMPS conjugated polyelectrolyte films ........................................................ - 93 -
5.1 Introduction to polyelectrolyte complexes .............................................................. - 93 -
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5.2 Application of polyelectrolyte complexes in the biomedical field ......................... - 93 -
5.3 Polyelectrolyte multilayered films .......................................................................... - 93 -
5.3.1 History of multilayered films .......................................................................... - 94 -
5.3.2 Methods to develop polyelectrolyte multilayer films ..................................... - 95 -
5.3.2.1 The soaking method (dipping method) .................................................. - 96 -
5.3.2.2 The centrifugation method (Spin coating method) ................................ - 96 -
5.3.2.3 Spray coating method ............................................................................ - 97 -
5.4 Application of polyelectrolyte multilayered films in the field of biomaterials ....... - 98 -
5.4.1 Biomaterials .................................................................................................... - 98 -
5.4.2 Cell adhesion to the PEM films....................................................................... - 99 -
5.4.2.1 Adhesion to films based on synthetic polyelectrolytes .......................... - 99 -
5.4.2.2 Adhesion of the films based on biopolymers (polysaccharides and
polypeptides) ......................................................................................................... - 100 -
5.4.3 Insertion of active molecules in a film .......................................................... - 100 -
5.4.3.1 Adsorption and absorption of molecules ............................................. - 100 -
5.4.3.2 Insertion of active molecules by coupling of the molecules ................ - 101 -
5.4.4 Antibacterial PEM films ................................................................................ - 101 -
5.4.4.1 Passive antibacterial PEM films .......................................................... - 101 -
5.4.4.2 Active antibacterial PEM films ............................................................ - 102 -
Part II: Materials and methods
1 Preparation and purification of biological materials ...................................... - 103 -
1.1 Preparation of Leucocidin E-D ............................................................................. - 103 -
1.2 Gel electrophoresis and coomassie blue staining of the gel .................................. - 103 -
1.3 RP-HPLC of Leucocidins E-D .............................................................................. - 104 -
1.4 Isolation of polymorphonuclear neutrophils ......................................................... - 104 -
1.5 Preparation of secretions after PMNs stimulation by Leucocidin E-D ................. - 105 -
1.5.1 Purification of PMNs secretions by RP-HPLC ............................................. - 105 -
1.5.2 Desalting of the PMNs secretions ................................................................. - 105 -
1.6 Synthesis of peptides ............................................................................................. - 106 -
1.6.1 Purification of synthetic peptides by RP-HPLC ........................................... - 106 -
1.7 Isolation and characterization of staphylococcal strains ....................................... - 106 -
2 Preparation and characterization of material for polymer development.......... - 108 -
2.1 Preparation of modified synthetic antimicrobial peptide (CTL-Cys) for insertion in
biomaterials ................................................................................................................... - 108 -
2.1.1 Synthesis of rhodaminated peptides .............................................................. - 109 -
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2.2 Preparation of modified HA. ................................................................................. - 109 -
2.2.1 Preparation of HA-CTL-Cys ......................................................................... - 109 -
2.2.2 Preparation of HAFITC .................................................................................... - 111 -
2.2.3 Preparation of HAFITC-CTL ........................................................................... - 112 -
2.3 Gel filtration chromatography ............................................................................... - 112 -
2.4 PEM films buildup ................................................................................................ - 113 -
2.4.1 Polysaccharide solutions ............................................................................... - 113 -
2.4.2 Film buildup .................................................................................................. - 113 -
2.5 Physico-chemical characterization of the polymer films ...................................... - 114 -
2.5.1 Quartz Crystal Microbalance ........................................................................ - 114 -
2.5.2 Atomic Force Microscopy (AFM) ................................................................ - 114 -
2.5.3 Confocal Laser Scanning Microscopy (CLSM) ............................................ - 114 -
3 Methods of proteomic analysis ..................................................................... - 115 -
3.1 Sequence characterization by Edman sequencing ................................................. - 115 -
3.2 Western blot analysis of the PMNs secretion ........................................................ - 115 -
3.3 Matrix assisted laser desorption ionization time-of-flight Mass spectrometry (Maldi-
TOF) .............................................................................................................................. - 116 -
3.4 Nano LC-MS/MS Mass spectrometry analysis ..................................................... - 117 -
4 Methods to characterize biological activities ................................................. - 118 -
4.1 Flow-cytometry measurements: ............................................................................ - 118 -
4.2 Analysis of antimicrobial activity ......................................................................... - 119 -
4.2.1 Antibacterial test ........................................................................................... - 119 -
4.2.1.1 Kinetics of bacterial growth inhibition ................................................ - 120 -
4.2.2 Antifungal activity ......................................................................................... - 120 -
4.2.2.1 Kinetics of fungal growth inhibition .................................................... - 121 -
4.3 Confocal laser scanning microscopy(CLSM) ...................................................... - 121 -
4.4 Degradation analysis of synthetic peptides by S. aureus strains ........................... - 122 -
4.4.1 Purification of bacterial supernatants by RP-HPLC ..................................... - 122 -
4.5 PSG degradation by the V8 protease ..................................................................... - 122 -
4.6 Human gingival fibroblasts viability assays .......................................................... - 123 -
Part III: Results and discussion
Manuscript 1………………………………………………………………...….- 124-
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Activation of neutrophils by the two-component leukotoxin LukE/D from
Staphylococcus aureus: a proteomic analysis of the secretions
Manuscript 2……………………………………………………………………- 156-
Cateslytin a chromogranin A derived peptide is active against Staphylococcus
aureus and resistant to degradation by its proteases
Manuscript 3…………………………………………………………………….-176-
Staphylococcus aureus Glu-C protease subverts innate immunity by degradation of
Chromogranins and production of new antifungal peptides
Manuscript 4
Self-defensive biomaterial coating against bacteria and yeasts: polysaccharide
multilayer film with embedded antimicrobial peptide
Conclusion and Perspectives……………………………………………………….-191-
References………………………………………………………………………………-193-
Annexes
Chromogranin A-derived peptides are involved in innate immunity
The Natural Antimicrobial Chromogranins/Secretogranins- Derived Peptides –
Production, Lytic Activity and Processing by Bacterial Proteases
Proteomic analysis of activated PMNs secretion, by Staphylococcus aureus
leukotoxins LukE/D
Summary

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