development and optimization of topical antimicrobial agents based on human antimicrobial peptides

131 Pages·2016·2.79 MB·English

by  Giovanni Sanni

D
EVELOPMENT AND OPTIMIZATION
OF TOPICAL ANTIMICROBIAL
AGENTS BASED ON HUMAN

ANTIMICROBIAL PEPTIDES

Katia Pane

Dottorato in Scienze Biotecnologiche 28° ciclo
Indirizzo Biotecnologie Industriali e Molecolari
Università di Napoli Federico II

Dottorato in Scienze Biotecnologiche 28° ciclo
Indirizzo Biotecnologie Industriali e Molecolari
Università di Napoli Federico II

D
EVELOPMENT AND OPTIMIZATION
OF TOPICAL ANTIMICROBIAL
AGENTS BASED ON HUMAN

ANTIMICROBIAL PEPTIDES

Katia Pane

Dottoranda: Katia Pane

Relatore: Prof. Alberto Di Donato

Coordinatore: Prof. Giovanni Sannia

A chi mi è così vicino da lontano

INDEX

ABSTRACT pag. 1
RIASSUNTO pag. 2
Chapter 1-INTRODUCTION
1.1 Antiobiotic resistance pag. 7
1.2 General characteristics of host defence peptides pag. 7
1.3 Mechanism of action pag. 9
1.4 Biological roles of HDPs pag. 11
1.4.1 Examples of direct antimicrobial activity pag. 12
1.4.2 Examples of immune modulatory activity pag. 13
1.5 AMPs advantages as novel anti-infective agents pag. 15
1.6 Biotechnological application of AMPs pag. 15
1.7 Proteins carrier of cryptic AMPs pag. 16
1.8 A novel tool to identify new cryptic AMPs pag. 18
1.9 AMPs chemical and biological production methods pag. 18
1.10 Aims pag. 19
Chapter 2-MATERIALS AND METHODS
2.1 Materials
pag. 20
2.2 General procedures
pag. 20
2.3 Peptides
pag. 20
2.4 Bacterial growth culture media
pag. 20
2.5 Antibiotics
pag. 21
2.6 Construction of the expression vectors
pag. 21
2.7 Expression of recombinant proteins
pag. 21
2.8 Purification of fusion proteins
pag. 21
2.9 Acid cleavage of Asp-Pro and Asp Cys peptide bond
pag. 22
2.10 Purification of recombinant peptides
pag. 22
2.11 HPLC
pag. 22
2.12 PyMPO fluorophore conjugation to (C)GKY20 peptide
pag. 25
2.13 Antibacterial and antibiofilm assay
pag. 27
2.14 Bacterial strains used for antimicrobial susceptibility test
pag. 27
2.15 Blood cell isolation and lactate dehydrogenase (LDH) assay
pag. 27
2.16 Detection of cytokines/chemokines
pag. 27
2.17 MTT assay
pag. 27
2.18 Ellman assay
pag. 28
Chapter 3-RESULTS AND DISCUSSION
3.1 Development of a novel fusion system for recombinant AMPs production
pag. 29
3.2 Production of GKY20 with a N-terminal cysteine residue pag. 33
3.2.1 Selective labelling of (C)GKY20 pag. 36
3.3 Antimicrobial activity of thrombin derived peptides pag. 39
3.4 Expression of further potential human AMPs pag. 41
3.5 Purification of recombinant pepsinogen A-3 derived peptides pag. 43

pag. 47
3.5.1 Antimicrobial activity
3.6. Purification of recombinant β Fibrinogen derived peptides pag. 50
3.6.1 Antimicrobial activity pag. 55
3.7 Functional characterization of potential new human AMPs pag. 57
3.7.1 Antimicrobial activity pag. 57
3.7.2 Antibiofilm activity pag. 59
3.7.3 Immunomodulatory activity and ability to enhance the innate immune system pag. 63
Chapter 4-CONCLUSIONS pag. 69
REFERENCES
pag. 71
APPENDICES
pag. 78