DHA modified tat peptide as an effective antimicrobial agent

DHA MODIFIED TAT PEPTIDE AS AN EFFECTIVE ANTIMICROBIAL AGENT AJITHA SUNDARESAN (B.Tech, Chemical Engineering) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2011 ACKNOWLEDGEMENT First and foremost, I would like to thank both my supervisors, Dr Yen Wah Tong and Dr Yi Yan Yang, for their constant support and encouragement even during the tough phases of my Master’s program I am extremely grateful for their invaluable guidance and inputs through the course of this project I would also like to sincerely thank Dr Yang Chuan and Dr Nikken Wiradharma for the long discussions and continuous guidance and assistance, which helped me to shape my ideas and understand my research better A special thanks to Mr Luo Jingnan for providing valuable suggestions on improving this work Next, I would like to thank the Department of Chemical and Biomolecular Engineering (National University of Singapore, Singapore) and Youth Research Program (Institute of Bioengineering and Nanotechnology (IBN), Singapore) for facilitating this research I also thank IBN for funding this project In addition, I would like to express my heartfelt gratitude to all my lab members at both NUS ChBE and IBN, for their kind support Last but not the least, I would like to thank my parents and my friends for their constant encouragement and understanding I TABLE OF CONTENTS ACKNOWLEDGEMENT I TABLE OF CONTENTS II SUMMARY V LIST OF TABLES AND FIGURES VII NOMENCLATURE IX INTRODUCTION 1.1 BACKGROUND 1.2 OBJECTIVE LITERATURE REVIEW 2.1 BACTERIAL AND FUNGAL INFECTIONS – AN OVERVIEW 2.1.1 Tuberculosis 2.1.2 Meningitis 2.1.3 Pneumonia 2.2 MORPHOLOGY AND STRUCTURE OF PATHOGENS – A PERSPECTIVE 2.2.1 The Bacterial Cell 2.2.1.1 Morphology of the Gram-negative bacteria 2.2.1.2 Morphology of the Gram-positive bacteria 2.2.2 The Fungal Cell 2.2.3 Comparison of lipids in prokaryotic and eukaryotic membranes – A means of selective activity 2.3 CHALLENGES FACING THE CONTROL OF INFECTIOUS DISEASES 2.4 ANTIMICROBIAL AGENTS AND THEIR MODES OF ACTION 2.4.1 Antibiotics 2.4.1.1 β-lactams 2.4.1.2 Glycopeptide antibiotics 2.4.1.3 Macrolides 2.4.1.4 Tetracyclines 2.4.2 Alternate strategies for treatment of infectious diseases 2.5 ANTIMICROBIAL PEPTIDES 2.5.1 Natural Antimicrobial Peptides 2.5.2 Mechanism of action of Antimicrobial peptides 2.5.2.1 The carpet model 2.5.2.2 The Barrel-Stave model 5 6 8 10 10 11 11 13 13 14 14 15 15 15 16 17 19 19 20 II 2.5.2.3 The Toroidal Pore model 2.5.2.4 Alternate methods of action 2.5.3 Synthetic Antimicrobial peptides 2.5.4 Factors affecting antimicrobial activity of peptides 2.5.4.1 Cationic charge 2.5.4.2 Secondary structure 2.5.4.3 Hydrophobicity 2.5.5 Fatty acid conjugation of Antimicrobial peptides DESIGN AND CHARACTERIZATION OF SYNTHETIC AMPHIPHILIC PEPTIDES – DHA-G3R6TAT-NH2 AND BA-G3R6TAT-NH2 3.1 AMPHIPHILIC PEPTIDES DESIGN AND BACKGROUND 3.2 MATERIALS AND METHODS 3.2.1 Materials 3.2.2 Methods 3.2.2.1 Solution phase synthesis of DHA-G3R6TAT-NH2 and BA-G3R6TAT-NH2 3.2.2.2 Purification by precipitation and dialysis 3.2.2.3 Matrix assisted Laser desorption and Ionization-Time of Flight (MALDI-TOF) 3.2.2.4 Proton Nuclear Magnetic Resonance (1H-NMR) 3.2.2.5 Dynamic light scattering 3.2.2.6 Critical Micelle concentration measurement 3.3 RESULTS AND DISCUSSION 3.3.1 MALDI-TOF 3.3.2 1H-NMR 3.3.3 Dynamic light scattering 3.3.4 Critical Micelle concentration measurement IN VITRO STUDY OF THE BIOLOGICAL ACTIVITY OF SYNTHETIC AMPHIPHILIC PEPTIDES – DHA-G3R6TAT-NH2 AND BA-G3R6TAT-NH2 4.1 MATERIALS AND METHODS 4.1.1 Materials 4.1.2 Methods 4.1.2.1 Preparation of Tryptic Soy Broth (TSB) medium 4.1.2.2 Preparation of Yeast Mould broth 4.1.2.3 Inoculation of bacteria and fungi 4.1.2.4 Minimum Inhibitory Concentration Assay 4.1.2.5 Colony Formation Assay 42 20 21 21 22 22 23 24 24 27 27 28 28 29 29 31 32 32 33 33 34 34 36 37 38 40 40 40 41 41 41 41 42 III 4.1.2.6 Field Emission Scanning Electron Microscopy (FE-SEM) 4.1.2.7 Haemolysis Assay 4.2 RESULTS AND DISCUSSION 4.2.1 Minimum Inhibitory Concentration Assay 4.2.2 Colony Formation Assay 4.2.3 Field Emission Scanning Electron Microscopy (FE-SEM) 4.2.4 Haemolysis Assay CONCLUSIONS AND FUTURE WORK 5.1 CONCLUSIONS 5.2 FUTURE WORK AND RECOMMENDATIONS REFERENCES 43 44 44 44 52 53 57 58 58 60 61 IV SUMMARY Indiscriminate use of conventional antibiotics has led to the development of resistance by various strains of bacteria and fungi against majority of drugs currently in use Hence the need to develop alternative strategies for combating these microbial infections is highly pertinent and relevant to the field of medicinal therapy and healthcare development Antimicrobial peptides (AMPs) are one such promising candidate, owing to their broad spectrum of activity and unique mechanisms of action, which renders it difficult for the pathogen to develop resistance against them Ideally, such antimicrobial peptides should selectively target microbial membranes without affecting the host mammalian cells This study aims to develop a new synthetic antimicrobial amphiphilic peptide to target different bacteria and fungi without causing appreciable cytotoxicity The first part of the thesis deals with the design and chemical synthesis of a polyunsaturated fatty acid conjugated peptide Several characterization techniques were used to confirm the successful conjugation of the fatty acid to the peptide and to understand its functional attributes A control amphiphilic peptide which differs only in the degree of unsaturation of the fatty acid was similarly synthesized and characterized The two peptides were also found to self-assemble in solution forming nanoparticles The next part of the thesis describes the in vitro studies done to test the biological activity of the synthetic amphiphilic peptides The minimum inhibition concentration assay was performed against pathogenic organisms like Staphylococcus aureus and Candida albicans The cytotoxicity of the two amphiphilic peptides was also tested by means of the haemolysis assay against rat red blood cells The conjugation of the V polyunsaturated fatty acid to the peptide improved its antimicrobial activity without compromising on its haemolytic activity In conclusion, the polyunsaturated fatty acid-peptide conjugate could be used as a potential therapeutic to combat microbial infections VI LIST OF TABLES AND FIGURES TABLES Table 1: Estimated Tuberculosis incidence, prevalence and mortality region wise for the year 2009 Table 2: Particle size determination of nanoparticles formed by DHA-G3R6TATNH2 and BA-G3R6TAT-NH2 FIGURES Figure 1: Chemical structures of (a) G3R6TAT-NH2, (b) DHA-G3R6TAT-NH2, (c) BA-G3R6TAT-NH2 Figure 2: MALDI-TOF spectrum of the (a) unconjugated peptide (G3R6TAT-NH2) 2667 Da, (b) docosahexaenoic acid conjugated peptide (DHA-G3R6TATNH2) – 2978 Da and (c) behenic acid conjugated peptide (BA-G3R6TATNH2) – 2990 Da Figure 3: Figure 4: Plot of I338/I334 vs logarithm of concentration of DHA-G3R6TAT-NH2 and BA-G3R6TAT-NH2 in PBS buffer Figure 5: Minimum inhibitory concentration determination of G3R6TAT-NH2 (250 µg/ml), DHA-G3R6TAT-NH2 (31.2 µg/ml) and BA-G3R6TAT-NH2 (250 µg/ml) against Staphylococcus aureus Figure 6: Minimum inhibitory concentration determination of G3R6TAT-NH2 (15.6 µg/ml), DHA-G3R6TAT-NH2 (15.6 µg/ml) and BA-G3R6TAT-NH2 (62.5 µg/ml) against Bacillus subtilis Figure 7: Minimum inhibitory concentration determination of G3R6TAT-NH2 (>500 µg/ml), DHA-G3R6TAT-NH2 (500 µg/ml) and BA-G3R6TAT-NH2 (>500 µg/ml) against Escherichia coli Figure 8: Minimum inhibitory concentration determination of G3R6TAT-NH2 (>500 µg/ml), DHA-G3R6TAT-NH2 (>500 µg/ml) and BA-G3R6TAT-NH2 (>500 µg/ml) against Pseudomonas aeruginosa Figure 9: Minimum inhibitory concentration determination of G3R6TAT-NH2 (62.5 µg/ml), DHA-G3R6TAT-NH2 (62.5 µg/ml) and BA-G3R6TAT-NH2 (62.5 µg/ml) against Candida albicans Figure 10: Concentration dependant killing efficiency of DHA-G3R6TAT-NH2 against (a) S.aureus (b) C.albicans H-NMR of DHA-G3R6TAT-NH2 and BA-G3R6TAT-NH2 show successful conjugation of DHA and BA to G3R6TAT-NH2 respectively VII Figure 11: FE-SEM images of B.Subtilis (a) without and (b) with treatment with of DHA-G3R6TAT-NH2 at a lethal dose of 50 µg/ml for hour Figure 12: FE-SEM images of S.aureus (a) without and (b) with treatment with DHA-G3R6TAT-NH2 at a lethal dose of 100 µg/ml for hour Figure 13: FE-SEM images of C.albicans (a) without and (b) with treatment with DHA-G3R6TAT-NH2 at a lethal dose of 200 µg/ml for hour Figure 14: Plot of % Haemolysis vs concentration of G3R6TAT-NH2, DHA-G3R6TATNH2 and BA-G3R6TAT-NH2 VIII NOMENCLATURE ABBREVIATIONS: AMP – Antimicrobial Peptide BA – Behenic acid BA-G3R6TAT-NH2 – Behenic acid TAT peptide conjugate CL - Cardiolipin CMC – Critical Micelle Concentration CHCA – α-Cyano Hydroxy Cinammic acid d-DMSO – Deuterated dimethyl sulfoxide d-CHCl3 – Deuterated chloroform DCC – Dicyclohexyl carbodiimide DCM – Dichloromethane DIW - De-ionized water DMF – Dimethylformamide DHA – Docosahexaenoic acid DHA-G3R6TAT-NH2 – Docosahexaenoic acid TAT peptide conjugate FE-SEM – Field Emission Scanning Electron Microscope G3R6TAT-NH2 – TAT peptide sequence HIV – Human Immunodeficiency Virus H- NMR – Proton Nuclear Magnetic Resonance LPS – Lipopolysaccharide MALDI-TOF – Matrix assisted Laser Desorption Ionization – Time of Flight MDR – Multi Drug resistance MIC – Minimum Inhibitory Concentration NHS – N-Hydroxy Succinimide O.D – Optical Density PBS – Phosphate Buffer Saline IX a b Figure 11: FE-SEM images of B.subtilis (a) without and (b) with treatment with of DHA-G3R6TAT-NH2 at a lethal dose of 50 µg/ml for hour 54 a b Figure 12: FE-SEM images of S.aureus (a) without and (b) with treatment with DHA-G3R6TAT-NH2 at a lethal dose of 100 µg/ml for hour 55 a b Figure 13: FE-SEM images of C.albicans (a) without and (b) with treatment with DHA-G3R6TAT-NH2 at a lethal dose of 200 µg/ml for hour 56 4.3.4 Haemolysis assay The haemolysis assay was performed to investigate the selective activity of the DHA conjugated peptide towards microbial membranes The unconjugated peptide and the DHA-peptide conjugate were found to possess negligible haemolytic activity at even the highest concentration tested (which was much higher than their MIC levels) while the BA-peptide conjugate was slightly haemolytic at the highest concentration tested For example, DHA-G3R6TAT-NH2 doesn’t exhibit haemolysis even at a high concentration of 500 µg/ml which is much higher than its MIC against B.subtilis (15.6 µg/ml), S.aureus (31.25 µg/ml) and C albicans (62 µg/ml) This thereby indicates that DHA-G3R6TAT-NH2 can be used a prospective antimicrobial agent without appreciable cytotoxicity effects 100 Comparison of haemolytic activity % Haemolysis 80 60 DHA-G3R6TAT-NH2 40 G3R6TAT-NH2 BA-G3R6TAT-NH2 20 -50 -20 50 100 150 200 250 300 350 400 450 500 550 Concentration in µg/ml Figure 14: Plot of % Haemolysis vs concentration of G3R6TAT-NH2, DHA-G3R6TAT-NH2 and BAG3R6TAT-NH2 57 CONCLUSIONS AND FUTURE WORK 5.1 Conclusions Research in the field of infectious diseases therapy and drug discovery has been focussed on combating multi-drug resistance and developing alternative medicine Antimicrobial peptides are a unique class of compounds which hold great promise as an alternative to conventional antibiotics This study aims to design a potential therapeutic antimicrobial peptide drug to target different types of bacteria and fungi A hydrophilic peptide (G3R6TAT-NH2), capable of crossing the blood brain barrier was conjugated to a hydrophobic long chain polyunsaturated fatty acid (docosahexaenoic acid) to form a peptide amphiphile, which is necessary for antimicrobial action To study the effect of presence of double bonds in the fatty acid of the amphiphilic peptide, a similar saturated fatty acid (behenic acid) of the same carbon chain length as DHA was conjugated to G3R6TAT-NH2 and characterized MALDI-TOF spectrum of both the conjugates DHA-G3R6TAT-NH2 and BA-G3R6TAT-NH2 confirmed the successful conjugation of the peptide to the respective fatty acids 1HNMR was used to further substantiate the formation of the two fatty acid-peptide conjugates Pyrene was used as a probe to determine the critical micelle concentration of DHAG3R6TAT-NH2 and BA-G3R6TAT-NH2 The DHA conjugated peptide was found to have a significantly higher critical micelle concentration than the BA conjugated peptide, indicating that DHA-G3R6TAT-NH2 existed as free molecules in all the biological experiments performed in this study 58 The addition of fatty acid to the peptide was done to improve the antimicrobial activity of the parent peptide Several studies have been reported in literature where fatty acid conjugation has been used as a technique to improve antimicrobial activity of peptides Most of these studies till now have used only saturated fatty acid conjugated peptides to compare the antimicrobial activity [57],[70] This work however involved the novel use of a polyunsaturated fatty acid to compare the effect of unsaturation on the antimicrobial activity of fatty acid conjugated peptides The minimum inhibitory concentration assay indicated that the conjugation of DHA to G3R6TAT-NH2 significantly increased its activity against S.aureus alone while maintaining similar activity against B.subtilis Similarly, the presence of six double bonds of DHA was found to be crucial in the activity of DHA conjugated peptide against Gram-positive bacteria as BA-G3R6TAT-NH2 displayed a much higher MIC value in comparison with DHA-G3R6TAT-NH2 However, both the amphiphilic peptides exhibited similar activity against C albicans All the three peptides G3R6TAT-NH2, DHA-G3R6TAT-NH2 and BA-G3R6TAT-NH2 failed to inhibit the growth of Gram-negative bacteria The results from the haemolysis assay indicated the potential of DHA-G3R6TAT-NH2 to be used as a therapeutic as it was found to be non-haemolytic even at concentrations much higher than its MIC values, while BA-G3R6TAT-NH2 was found to be mildly haemolytic at higher concentrations The ability of DHA-G3R6TAT-NH2 to function as a strong antibiotic was further proved by the colony formation assay which indicated that 99.9% of S.aureus and C.albicans 59 were killed at concentration levels of MIC and above FE-SEM images also showed the damage of bacterial and yeast cells due to the action of DHA-G3R6TAT-NH2 Hence, the conjugate DHA-G3R6TAT-NH2 can be used as a therapeutic drug to treat infections caused by Gram-positive bacteria and fungi without appreciable cytotoxicity effects 5.2 Future work and recommendations This work was aimed at developing a prospective alternate form of medicine to treat bacterial and fungal infections The in vitro studies indicated the ability of the developed synthetic drug to target pathogenic organisms like S.aureus and C.albicans However, it needs to be 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