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January 2016 TARGETING MULTI-DRUG RESISTANT PATHOGENS WITH NOVEL Mohamed F. Mohamed Purdue University

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Recommended Citation Mohamed, Mohamed F., "TARGETING MULTI-DRUG RESISTANT PATHOGENS WITH NOVEL ANTIMICROBIAL PEPTIDES" (2016). Open Access Dissertations. 1223. https://docs.lib.purdue.edu/open_access_dissertations/1223

This document has been made available through Purdue e-Pubs, a service of the Purdue University Libraries. Please contact [email protected] for additional information. TARGETING MULTI-DRUG RESISTANT PATHOGENS WITH NOVEL ANTIMICROBIAL PEPTIDES by Mohamed Fathy Kamel Mohamed

A Dissertation Submitted to the Faculty of Purdue University In Partial Fulfillment of the Requirements for the degree of

Doctor of Philosophy

Department of Comparative Pathobiology West Lafayette, Indiana December 2016 ii

THE PURDUE UNIVERSITY GRADUATE SCHOOL STATEMENT OF DISSERTATION APPROVAL

Dr. Mohamed N. Seleem Department of Comparative Pathobiology Dr. Lynn F. Guptill-Yoran Department of Comparative Pathobiology Dr. Ramesh Vemulapalli Department of Comparative Pathobiology Dr. Yoon Yeo Industrial and Physical Pharmacy

Approved by: Dr. Suresh K. Mittal Head of the Departmental Graduate Program iii

To my big family (my parents, brothers and sisters) and my small family (Somaia and Salma) for their endless love, support and encouragement

iv

ACKNOWLEDGMENTS

First and foremost, I would like to thank Allah (God) for providing me the blessings and power to complete this work. I could never have done this without the faith I have in you. I would like to express my gratitude to my major advisor Dr. Mohamed Seleem for his guidance and support during my PhD. He encouraged me to be an independent researcher. He always pushed me hard to think outside the box and to work on new research avenues. He improved my poster and oral presentation skills. He also helps me to improve my writing and preparation of manuscripts. Aside from academia, he usually shares helpful advices about living abroad and how to enjoy our free times. I would also like to thank my committee members; Dr. Ramesh Vemulapalli, Dr. Lynn Guptill and Dr. Yoon Yeo for their feedback and suggestions. Dr. Ramesh Vemulapalli comments and suggestions after department seminars was so helpful to improve my research. Dr. Lynn Guptill provided me valuable comments on our project (Staphylococcus pseudintermedius). Dr. Yoon Yeo provided me valuable comments and technical support during our meetings on collaborative projects. I would like to express my gratitude to my professors in the department of bacteriology, immunology and mycology, Faculty of Veterinary Medicine, Beni-Suef University, Egypt. I would like to thank Dr. Fawzi R El-Seedy, Dr. Ismail Abd El- Hafeez Radwan Rahil, Dr. Walid Hamdi Hassan, Dr. Hala Hassan, Dr. Ahmed Abed, and Dr Amr Shehata for their help, encouragement and support. I thank all my past and current lab colleagues: Haroon Mohamed, Waleed Younis, Ahmed Hassan, Hassan Elsayed, Youssef Ahmed, Mostafa Abushahba, Maha Ahmed, Omar Ameen, Ahmed Omar, Ekramy Elsayed, Neha Debral, Manish Nebal, Anna Brezeden, Yihua Pei and Neha Argwal for their support and fun times. Special thanks to Haroon Mohamed for editing my manuscripts. Thanks to Barbara White for her support and guide during graduate school. v

To my parents and my brothers and sisters, I am speechless! I can barely find the words to express all the wisdom, love and support you've given me. I thank my wife Somaia and my daughter Salma for their love, patience, emotional support and sacrifices. Finally, I would like to thank the Egyptian Ministry of Higher Education and the Egyptian Cultural and Educational Bureau (ECEB) for financial support of my graduate studies. vi

TABLE OF CONTENTS

LIST OF TABLES ...... ix LIST OF FIGURES ...... xi LIST OF ABBREVIATIONS ...... xiv ABSTRACT ...... xvi CHAPTER 1. LITERATURE REVIEW ...... 1 1.1 Introduction ...... 1 1.2 Mechanism of action of antimicrobial peptides ...... 2 1.2.1 Cell membrane lysis ...... 2 1.2.2 Non-permeabilizing mechanism (targeting intracellular components) ...... 3 1.2.2.1 Inhibition of cell wall synthesis ...... 3 1.2.2.2 Inhibition of nucleic acid and synthesis: ...... 4 1.3 Advantages of Antimicrobial peptides ...... 5 1.3.1 Broad spectrum, rapid killing and synergism with ...... 5 1.3.2 Active against drug resistant pathogens and low emergence of bacterial resistance to antimicrobial peptides ...... 6 1.3.3 Anti-endotoxic activity ...... 7 1.3.4 Immune-modulatory properties ...... 8 1.3.5 Wound healing properties ...... 9 1.3.6 Efficacy of peptides on established biofilms ...... 9 1.4 Limitations of antimicrobial peptides ...... 10 1.4.1 Potential for host toxicity ...... 10 1.4.2 Susceptibility to proteolytic digestion ...... 12 1.4.3 Impaired antibacterial activity in the presence of physiologically relevant conditions (e.g., salts, acidic pH, plasma, and serum) ...... 13 1.4.4 Cost to manufacture ...... 13 1.5 Antimicrobial peptides in clinical trials ...... 14 1.6 References ...... 17 CHAPTER 2. TARGETING METHICILLIN-RESISTANT WITH SHORT SALT-RESISTANT SYNTHETIC PEPTIDES...... 33 vii

2.1 Abstract ...... 33 2.2 Introduction ...... 34 2.3 Materials and methods ...... 35 2.4 Results ...... 41 2.5 Discussion ...... 48 2.6 References ...... 52 CHAPTER 3. EFFICACY OF SHORT NOVEL ANTIMICROBIAL AND ANTI- INFLAMMATORY PEPTIDES IN A MOUSE MODEL OF METHICILLIN- RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) SKIN INFECTION ...... 66 3.1 Abstract ...... 66 3.2 Introduction ...... 66 3.3 Materials and methods ...... 67 3.4 Results and discussion ...... 68 3.5 References ...... 71 CHAPTER 4. ANTIBACTERIAL ACTIVITY OF NOVEL CATIONIC PEPTIDES AGAINST CLINICAL ISOLATES OF MULTI-DRUG RESISTANT STAPHYLOCOCCUS PSEUDINTERMEDIUS FROM INFECTED DOGS ...... 75 4.1 Abstract ...... 75 4.2 Introduction ...... 76 4.3 Materials and methods ...... 77 4.4 Results ...... 82 4.5 Discussion ...... 85 4.6 References ...... 90 CHAPTER 5. EVALUATION OF SHORT SYNTHETIC ANTIMICROBIAL PEPTIDES FOR TREATMENT OF DRUG-RESISTANT AND INTRACELLULAR STAPHYLOCOCCUS AUREUS ...... 104 5.1 Abstract ...... 104 5.2 Introduction ...... 105 5.3 Materials and methods ...... 106 5.4 Results and discussion ...... 112 5.5 Conclusion ...... 124 viii

5.6 Reference ...... 126 CHAPTER 6. A SHORT D-ENANTIOMERIC ANTIMICROBIAL PEPTIDE WITH POTENT IMMUNOMODULATORY AND ANTIBIOFILM ACTIVITY AGAINST MULTIDRUG-RESISTANT PSEUDOMONAS AERUGINOSA AND ACINETOBACTER BAUMANNII ...... 144 6.1 Abstract ...... 144 6.2 Introduction ...... 145 6.3 Materials and methods ...... 147 6.4 Results and discussion ...... 154 6.5 Conclusion ...... 169 6.6 References ...... 171 CHAPTER 7. TARGETING BIOFILMS AND PERSISTERS OF ESKAPE PATHOGENS WITH P14KANS, A KANAMYCIN PEPTIDE CONJUGATE ...... 196 7.1 Abstract ...... 196 7.2 Introduction ...... 197 7.3 Materials and methods ...... 198 7.4 Results and discussion ...... 204 7.5 Conclusion ...... 212 7.6 References ...... 214 VITA ...... 230 PUBLICATIONS ...... 234

ix

LIST OF TABLES

Table 1.1. Antimicrobial peptides (AMPs) in past and current clinical trials...... 29 Table 1.2. continued ...... 30 Table 2.1. Minimum inhibitory concentration (MIC) of peptides and antibiotics against clinical and drug-resistant Staphylococci strains ...... 55 Table 2.1.continued ...... 56 Table 2.1.continued ...... 57 Table 2.2. Amino acid sequence and physicochemical properties of peptides used in this study ...... 57 Table 2.3. Fractional inhibitory concentration (FIC) index for the combination of peptides together or with lysostaphin ...... 58 Table 4.1. Amino acid sequence and physicochemical properties of peptides used in this study ...... 93 Table 4.2. Minimum inhibitory concentration (MIC) of peptides against methicillin sensitive Staphylococcus pseudintermedius (MSSP) strains ...... 93 Table 4.2. continued ...... 94 Table 4.2. continued ...... 95 Table 4.3. Minimum inhibitory concentration (MIC) of peptides against methicillin resistant Staphylococcus pseudintermedius (MRSP) strains ...... 96 Table 4.4. The values of FIC index for the combination of peptides and antimicrobial compounds against Staphylococcus pseudintermedius (SP3) ...... 97 Table 4.5. Cytotoxicity and therapeutic index of peptides ...... 97 Table 5.1. Amino acid sequence and physicochemical properties of peptides used in this study ...... 130 Table 5.2. Minimum inhibitory concentration (MIC) of peptides and antibiotics against clinical and drug-resistant staphylococci isolates ...... 130 Table 5.2. continued ...... 131 Table 5.2. continued ...... 132 x

Table 5.3. Minimum inhibitory concentration (MIC) of peptides and antibiotics against clinical resistant Staphylococcus aureus (VRSA) isolates ...... 133 Table 5.4. Minimum inhibitory concentration (MIC) of peptides against Methicillin resistant Staphylococcus aureus (MRSA) USA300 in different media conditions ...... 134 Table 5.5. The fractional inhibitory concentration index range of peptides in combination with each other and with antibiotics against Staphylococcus aureus isolates ...... 134 Table 5.6. Resensitization of vancomycin resistant S. aureus (VRSA) to vancomycin, teicoplanin and oxacillin using a sub-inhibitory concentration (½×MIC) of WR12 or D- IK8 ...... 135 Table 6.1. Amino acid sequence and physicochemical properties of peptides used in this study ...... 176 Table 6.2. Minimum inhibitory concentration (MIC) (µM) of peptides against clinical and drug-resistant isolates of P. aeruginosa and A. baumannii ...... 177 Table 6.3. Minimum inhibitory concentration (MIC) (µM) of peptides against clinical isolates of colistin resistant P. aeruginosa isolated from cystic fibrosis patients...... 178 Table 6.4. MIC values of designed peptides in the presence of salts, pH 5.5 or pH 6.5, fetal bovine serum (FBS, 2%, 5% or 10%), human serum (2% or 5%), plasma from human (2% or 5%), against P. aeruginosa PAO1 ...... 179 Table 6.5. The fractional inhibitory concentration index range of peptide, D-RR4 in combination with antibiotics against bacterial isolates ...... 180 Table 6.6. Toxicity and therapeutic index of the designed peptides ...... 181 Table 7.1. Minimum inhibitory concentration (MIC) (µM) of compounds against clinical and drug-resistant bacterial isolates...... 219

xi

LIST OF FIGURES

Figure 1.1. Various models of membranolytic mechanism of action of antimicrobial peptides (AMPs)...... 31 Figure 1.2. Intracellular mechanism of action of antimicrobial peptides...... 32 Figure 2.1. Bacterial killing kinetics of peptides...... 59 Figure 2.2. Effect of culture conditions on killing kinetics of peptides...... 59 Figure 2.3. Synergistic killing of peptides with lysostaphin by time kill assay...... 60 Figure 2.4. Killing kinetics of MRSA USA300 exposed to peptides by measuring optical density...... 60 Figure 2.5. Permeabilization of the cytoplasmic membrane of MRSA USA300 as a function of peptide concentration...... 61 Figure 2.6. Interaction of peptides with plasmid DNA...... 62 Figure 2.7. The effect of peptides and antibiotics on established biofilms of S. aureus (A) and S. epidermidis (B)...... 63 Figure 2.8. The release of hemoglobin in the supernatant of human erythrocytes after treatment peptides...... 65 Figure 2.9. Cytotoxicity of peptides to HeLa cells...... 65 Figure 3.1. Efficacy of treatment of MRSA skin lesions with peptides and antibiotics. .. 73 Figure 3.2. The effect of peptides on cytokines (TNF-α, and IL-6) production in MRSA skin lesions...... 74 Figure 4.1. Bacterial killing kinetics of peptides against S. Pseudointermedius...... 98 Figure 4.2. Growth kinetics of SP3 exposed to 5X MIC of peptides...... 99 Figure 4.3. Permeabilization of the cytoplasmic membrane of SP3 as a function of peptide concentration...... 100 Figure 4.4. TEM micrographs of untreated and peptide treated S. pseudintermedius SP3...... 101 Figure 4.5. Cytotoxicity assay of peptides against mammalian cell lines...... 102 Figure 4.6. Drug resistance development profiles of S. pseudintermedius (SP6) exposed to peptides...... 103 xii

Figure 5.1. Permeabilization of the cytoplasmic membrane of MRSA USA300 as a function of peptide concentration...... 136 Figure 5.2. The kinetics of killing of peptides and antibiotics against logarithmic, persister cells and stationary phase of MRSA USA300...... 137 Figure 5.3. The effect of peptides on staphylococcal biofilms Peptides (WR12, D-IK8 and LL-37) and antibiotics (vancomycin & ) investigated against 24 hr (a&c) and 48 hr (b&d) old biofilms of S. aureus (a&b) and S. epidermidis (c&d)...... 138 Figure 5.4. Survival and permeabilization of the cytoplasmic membrane of VRS10 after treatment with 0.5 X MIC of WR12 and D-IK8...... 139 Figure 5.5. Sub-inhibitory concentration of WR12 and D-IK8 increase the uptake and binding of fluorescently labeled vancomycin (bodipy vancomycin)...... 140 Figure 5.6. Toxicity (a) and intracellular anti-staphylococcal activity (b) of peptides in human keratinocyte (HaCaT)...... 141 Figure 5.7. Internalization studies of WR12 inside macrophage cells...... 142 Figure 5.8. Efficacy of peptides and control antibiotics in bacterial load (a) and level of pro-inflammatory cytokines (b&d) in a murine model of MRSA skin infection...... 143 Figure 6.1. Helical wheel projections of the designed peptides ...... 182 Figure 6.2. CD analyses of the designed peptides ...... 183 Figure 6.3. Analytical HPLC spectra demonstrating proteolytic activity of trypsin and proteinase K on RR4 and D-RR4 after 0, 4 and 24 hours of incubation...... 184 Figure 6.4. The killing kinectics of peptides. Time-kill of peptides and antibiotics against logarithmic (A&B) and stationary phase (C&D) cultures of P. aeruginosa PAO1 (A&C) and A. baumannii ATCC BAA-1605 (B&D)...... 186 Figure 6.5. Permeabilization of bacterial membrane by peptides ...... 187 Figure 6.6. TEM micrographs of untreated and peptide-treated P. aeruginosa PAO1 .. 188 Figure 6.7. Drug-resistance development profile of P. aeruginosa PAO1 exposed to peptides ...... 189 Figure 6.8. Efficacy of peptides on established biofilms of P. aeruginosa PAO1 and A. baumannii ATCC BAA-1605 ...... 190 Figure 6.9. Intracellular antibacterial activity of peptides in murine macrophage...... 191 Figure 6.10. Anti-inflammatory effect and LPS binding activity of peptides ...... 192 xiii

Figure 6.11. Evaluation of toxicity and efficacy of peptides in Caenorhabditis elegans...... 193 Figure 7.1. The designed dual agents (P14KanS) composed of the , kanamycin and the antimicrobial peptide, P14LRR ...... 220 Figure 7.2. Mechanism of action of P14KanS...... 221 Figure 7.3. The killing kinectics of P14KanS and control antibiotics against logarithmic (A&B) and stationary phase (C&D) cultures of S. aureus ATCC 6538 (A&C) and S. epidermidis ATCC 35984 (B&D)...... 222 Figure 7.4. The killing kinectics of P14KanS and control antibiotics against logarithmic (A&B) and stationary phase (C&D) cultures of P. aeruginosa PAO1 (A&C) and A. baumannii ATCC BAA-1605 (B&D)...... 223 Figure 7.5. Efficacy of P14KanS on established biofilms of A) S. aureus ATCC 6538; B) S. epidermidis ATCC 35984; C) P. aeruginosa PAO1 and A. baumannii ATCC BAA- 1605...... 224 Figure 7.6. Toxicity of P14KanS against mammalian cells...... 226 Figure 7.7. Intracellular antibacterial activity of P14KanS in human keratinocytes (HaCaT)...... 226 Figure 7.8. Anti-inflammatory effect and LPS binding activity of P14KanS...... 227 Figure 7.9. Drug-resistance development profile of S. aureus...... 228 Figure 7.10. Efficacy of P14KanS in a Caenorhabditis elegans model of bacterial infection...... 229

xiv

LIST OF ABBREVIATIONS

AMPs Antimicrobial peptides CD Circular dichroism CF Cystic fibrosis CFU Colony-forming unit CLSI Clinical and Laboratory Standards Institute DMEM Dulbecco's Modified Eagle's medium ELISA -linked immunosorbent assay FBS Fetal bovine serum FIC The fractional inhibitory concentration index FITC Fluorescein isothiocyanate HaCaT Human keratinocyte cells HPLC High Performance Liquid Chromatography IL-6 Interleukin-6 LPS Lipopolysaccharides MHA Mueller-Hinton agar MHB Mueller-Hinton broth MIC Minimum inhibitory concentration MRSA Methicillin-resistant Staphylococcus aureus MRSE Methicillin-resistant Staphylococcus epidermidis. MRSP Methicillin-resistant Staphylococcus pseudintermedius MSA Mannitol salt agar MSSP Methicillin-susceptible Staphylococcus pseudintermedius NPN 1-N-phenylnaphthylamine OD Optical density PBS Phosphate buffered saline PI Propidium Iodide xv

TLR Toll-like receptor TNF-α Tumor necrosis factor-α TSA Trypticase soy agar TSB Trypticase soy broth VRSA Vancomycin-resistant Staphylococcus aureus

xvi

ABSTRACT

Author: Mohamed, Mohamed, FK. Ph.D. Institution: Purdue University Degree Received: December 2016 Title: Targeting Multi-Drug Resistant Pathogens with Novel Antimicrobial Peptides Major Professor: Dr. Mohamed N Seleem.

The emergence of antibiotic resistance is a notorious problem worldwide. In the United States alone, antibiotic-resistant bacteria infect at least two million people and kill around 23,000 patients each year. Half of these infections are attributed to the bacterial pathogen, methicillin resistant Staphylococcus aureus (MRSA) according to a report published in 2013 by the Centers for Disease Control and Prevention (CDC). Similarly, S. pseudintermedius is a leading cause of opportunistic infections in pet animals and has zoonotic potential. Furthermore, the marked increase in the incidence of infections due to extensively drug-resistant and pandrug-resistant isolates of Pseudomonas aeruginosa and Acinetobacter baumannii presents an alarming challenge that necessitates the development of novel therapeutic alternatives to traditional antibiotics in order to address this scourge. Currently, there is significant interest in antimicrobial peptides (AMPs) as novel therapeutic alternatives to conventional antibiotics. AMPs are major constituents of the host innate defense system for most organisms. AMPs are composed of a short sequence of amino acids (less than 50 residues), with a cationic charge and amphipathic character. AMPs possess several unique features that support their utility as antibacterial agents including possessing rapid bactericidal activity against a wide spectrum of pathogenic bacteria. Additionally, AMPs are typically not susceptible to the same mechanisms that confer resistance to traditional antibiotics. However, naturally-derived AMPs do possess limitations that have hindered their translation as clinically useful agents including, moderate antimicrobial activity, toxicity to host tissues, intolerance to physiological conditions, sensitivity to enzymatic degradation, and high manufacturing costs due to their complex design and long length of amino acids. Thus, there is a critical need for finding small peptides with strong antibacterial activity, salt tolerance and low toxicity. To address these issues, we screened a library of short synthetic peptides that were developed for xvii alternative therapeutic applications and identified four peptides, namely RR, RRIKA, WR12 and D-IK8, with potent antimicrobial activity against a panel of drug-resistant staphylococci including MRSA. In contrast to many natural AMPs; these peptides retained their activity in the presence of high salt concentrations. WR12 and D-IK8 were able to eradicate persister cells, MRSA in stationary growth phase, and showed significant clearance of intracellular MRSA in comparison to antibiotics of choice (vancomycin and linezolid). In addition to their antibacterial activity against MRSA, these peptides were found to possess more potent activity against clinical isolates of methicillin-resistant S. pseudintermedius (MRSP) isolated from infected dogs. Topical application of investigated peptides effectively reduced both the bacterial load and inflammatory cytokines in a murine model of MRSA skin lesions. Next, we designed modified derivatives of the RR peptide to enhance the peptide’s potency and spectrum of antimicrobial activity against Gram-negative pathogens (P. aeruginosa and A. baumannii). From the peptides designed in this study, RR4 and its D- enantiomer, D-RR4, were the most potent analogues with at least 32-fold improvement in antimicrobial activity observed. Interestingly, D-RR4 demonstrated potent activity against colistin-resistant, cystic fibrosis strains of P. aeruginosa indicating a potential therapeutic advantage of this peptide over several AMPs. Of note, D-RR4 was able to bind to LPS to reduce the endotoxin-induced proinflammatory cytokine response in macrophages. Furthermore, D-RR4 protected Caenorhabditis elegans worms from lethal infections of P. aeruginosa and A. baumannii and enhanced the activity of colistin in vivo against colistin- resistant P. aeruginosa. The ability of bacterial pathogens to form biofilms that are highly tolerant to antibiotics further aggravates the situation and can lead to recurring infections. To address this significant problem, we conjugated the antibiotic kanamycin with a novel antimicrobial peptide (P14LRR) to develop a kanamycin peptide conjugate (P14KanS). P14KanS was superior in disrupting adherent bacterial biofilms when compared to conventional antibiotics. Furthermore, P14KanS protected C. elegans from lethal infections of biofilm-forming Gram-positive and Gram-negative pathogens. In conclusion, this study supports the potential of investigated peptides and the kanamycin peptide conjugate for the treatment of multi-drug resistant bacterial infections. 1

CHAPTER 1. LITERATURE REVIEW

1.1 Introduction

The rapid development and spread of bacterial resistance to conventional antibiotics has become a serious global concern (1-4). Currently, members of ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), represent one of the biggest threats to health as a result of their multidrug resistance (5). In the United States alone, antibiotic-resistant bacteria infect at least two million people and kill around 23,000 patients each year (6). Half of these infections are attributed to the bacterial pathogen, methicillin resistant Staphylococcus aureus (MRSA) (6). Furthermore, the marked increase in the incidence of infections due to extensively drug-resistant and pandrug- resistant isolates of Pseudomonas aeruginosa and Acinetobacter baumannii (7-9) presents an alarming challenge that necessitates the development of novel therapeutic alternatives to traditional antibiotics in order to address this scourge. Antimicrobial peptides (AMPs) have shown significant promise in recent years as novel therapeutic alternative to treat infections caused by multidrug-resistant pathogens (10-16). AMPs are short sequences of amino acids (less than 50 residues), with a cationic charge and amphipathic character (15, 17). AMPs constitute the first line of innate immunity against infections (15, 16). Decreased production of AMPs in the host is associated with increased susceptibility to various infections (18-20). For example, decreased production of AMPs in the dermis is associated with increased susceptibility to various skin infection involving S. aureus in humans (21). Moreover, reduced expression of β- (hBD-2) in premature infants increases their susceptibility to pneumonia (19). In addition, mice deficient in the -related antimicrobial peptide (CRAMP) were susceptible to elevated persistence of P. aeruginosa in the lungs (18). AMPs possess several unique features that support their utility as antibacterial agents including possessing rapid bactericidal activity against a wide spectrum of pathogenic bacteria. Additionally, AMPs are typically not susceptible to the same mechanisms that confer resistance to traditional antibiotics. However, AMPs do possess limitations that have hindered their translation as clinically 2 useful agents including, toxicity to host tissues, intolerance to physiological conditions, sensitivity to enzymatic degradation, and high manufacturing costs due to their complex design and long length of amino acids. Here we discuss the different mechanisms of action of AMPs and their advantages over conventional antibiotics. Moreover, we discuss challenges to using AMPs as therapeutic agents and proposed solutions to overcome these limitations. We conclude by discussing AMPs that successfully reached clinical trials

1.2 Mechanism of action of antimicrobial peptides

The exact mechanism of action of AMPs has been controversial, until now (17, 22). Initially, it was believed that AMPs produce effects on bacterial function only by permeabilizing and destabilizing cell membranes (22). However, later studies demonstrated that some peptides penetrate the bacterial membrane without damaging it, and accumulate intracellularly to inhibit multiple internal targets including nucleic acid synthesis, protein synthesis, cell division and cell wall synthesis (22, 23). Below, we discuss the different modes of action of peptide antibiotics.

1.2.1 Cell membrane lysis

Most antimicrobial peptides belong to the class of membrane-lytic agents (22). Initially, these peptides interact with the negatively charged lipid group exposed on bacterial surfaces (lipopolysaccharides in Gram negative and lipo-teichoic acids in case of Gram positive) via electrostatic forces (17). This leads to destabilization of the anionic phospholipids groups to form pores and consequently killing of the microorganism due to leakage of intracellular contents (17, 22, 24). The most widely accepted models of membrane lysis by AMPs are the barrel-stave model, carpet model, and toroidal model for killing organisms (figure 1.1). In barrel-stave model, peptides position themselves for binding on the cell membranes, this leads to peptide aggregation and conversion to a bilayer membrane. So in this way the hydrophobic peptides align with the lipid core and hydrophilic peptides form an access pore in the interior part of membrane (figure 1.1A). The carpet model is described as a disruption of the membrane like "detergent action” by the binding of peptides to the outer surface (phospholipids) of cell membrane and forming a prolonged layer or carpet (figure 1.1B). 3

In the toroidal model, attached peptides start aggregation and force the lipid monolayer to bend incessantly through the pores. In this way the core is lined by both the inserted peptides and the lipid head groups (figure 1.1C) (17, 22, 24).

1.2.2 Non-permeabilizing mechanism (targeting intracellular components)

Recent studies suggest that membrane disruption is not the only mechanism of bacterial killing by AMPs. Otherwise, as presented in (figure 1.2), some peptides can penetrate membranes and induce killing by acting on one or more intracellular targets; such as inhibiting nucleic acids and protein synthesis or inhibition of cell wall synthesis and cell division (17, 22, 23).

1.2.2.1 Inhibition of cell wall synthesis

The cell wall is a vital bacterial component that preserves cell structure and protects the bacteria from osmotic lysis (25). As the cell wall is absent in mammalian cells and present only in prokaryotic cells, this structure was the target of many successful antibiotics such as penicillins and cephalosporins (25). Recent studies revealed that some antimicrobial peptides such as the fungal , plectasin, do not permeabilize the cytoplasmic membrane nor cause leakage of intracellular ions (K+) or ATP (26). Instead, the study found that plectasin kills bacteria by inhibiting the biosynthesis of cell wall by binding to a precursor of synthesis (lipid II) (26). This mechanism was found in other peptide antibiotics such as some (Lcn972), lantibiotics (mersacidin), and lipo-peptides (daptomycin) (25, 27). Moreover, , an antibacterial peptide commonly used as a food preservative, has several mechanisms; initially it binds to lipid II and then induces pore formation, leakage of cell contents, and death (28-30). Another study showed that nisin binds to lipids III and IV, inhibiting the biosynthesis of teichoic and lipoteichoic acids (31). Additionally, nisin and another AMP (Pep5) activate the function of an (N-acetylmuramoyl-L-alanine amidase) which triggers bacterial lysis, as demonstrated in Staphylococcus simulans (32). 4

1.2.2.2 Inhibition of nucleic acid and protein synthesis:

Some peptides translocate the bacterial cell membrane to reach intracellular macromolecules such as DNA, RNA and and inhibit their synthesis (22, 23). For example, buforin II, a short linear peptide consisting of 21 residues, is able to penetrate the bacterial membrane and accumulate in the cell without causing damage to the cell membrane (33). The proline hinge (Pro11) of the buforin peptide was responsible for this type of translocation as evidenced by its ability to translocate another non-cell permeable peptide inside cells (34). After internalization inside bacterial cells, buforin II showed high affinity for binding to DNA, RNA and interfering with their functions leading to cell death (33). This potent ability for binding nucleic acids could be interpreted that buforin II has sequence similarity to the N-terminal region of histone H2A (34). Other peptides from different families (such as , human neutrophil platelet -1 defensin and pleurocidin) inhibit nucleic acid and protein synthesis in bacteria as evidenced by blocking the uptake of radioactive precursors (thymidine, uridine and leucine) in the macro-molecular biosynthesis technique (22, 35, 36). Another group of peptides called proline-rich antimicrobial peptides (such as pyrrhocoricin, drosocin and apidaecin) have been discovered in insects. These peptides kill bacteria without a membrane lytic mechanism of action. Instead, they translocate inside bacterial cells by binding to a specific bacterial transporter (SbmA) and accumulate inside cells to bind the target molecule (37-42). Initially, it was shown that proline rich peptides bind with high affinity to a heat shock protein (DnaK) in bacterial lysates at low micromolar concentration (23). However, recent studies showed that DnaK is not the main target of these peptides as evidenced by the observation that the DnaK null mutant bacteria were more susceptible to proline peptides compared to wild-type strains, which excludes DnaK as the target (42). Moreover, the investigators found that proline-rich peptides are capable of binding strongly to the 70S ribosomal subunit in nanomolar concentrations thus inhibiting protein biosynthesis very efficiently (42). The mechanism of translation inhibition of the oncocin derivative (Onc 112) in complex with the 70S ribosomal subunit has been resolved recently by crystallography (38, 39). The authors showed that the peptide interacts with the exit tunnel of the ribosome and interferes with the binding of 5 aminoacyl-tRNA. This leads to destabilization of the initiation complex and inhibition of translation (38, 39). Indolicidin, a very short cationic peptide isolated from bovine neutrophils has been shown to inhibit DNA and RNA synthesis at lower concentrations (43-45). However, protein synthesis inhibition was achieved only at higher concentrations. Moreover, bacterial elongation and filamentation was observed in E. coli treated with indolicidin (43). Similar filamentation was seen in Salmonella Typhimurium treated with PR-39 (a proline– arginine-rich peptide) which also inhibited nucleic acid and protein synthesis (46, 47). Bacterial filamentation arises from defects in septum formation which could be result of inhibition of nucleic acid replication or inhibition of membrane proteins responsible for septum formation and cell division (22). A recent study showed that a hexapeptide ,WRWYCR, could induce filamentation and killing of S. Typhimurium in vitro and inside macrophages by inhibiting responsible for DNA repair leading to double-stranded DNA breaks and defects in chromosomal segregation (48). Microcins, a group of peptides isolated from bacteria, don’t cause membrane lysis, for example. Microcin C penetrate bacterial membranes and blocks translation by interfering with aminoacyl-tRNA synthetase function (49, 50). Another modified derivative, (Microcidin B17) inhibits the replication of DNA by targeting DNA gyrase (51). In conclusion, antimicrobial peptides have different mechanisms of action which mainly depend on the structure of the peptide and the amino acids incorporated in the structure. Moreover, some peptides have multiple mechanisms of bacterial killing which could explain why it is difficult for bacteria to develop resistance to antimicrobial peptides.

1.3 Advantages of Antimicrobial peptides

1.3.1 Broad spectrum, rapid killing and synergism with antibiotics.

Most antimicrobial peptides discovered to date have demonstrated potent activity against many Gram-positive and Gram-negative bacteria (15, 16, 22); some antimicrobial peptides also have antifungal (52, 53), antiviral (54, 55) (56) and antiparasitic activity (57). This broad-spectrum activity is very useful when treating poly-microbial infections, 6 especially in immunocompromised patients (such as HIV patients), where many infections are prevalent (58, 59). Moreover, in contrast to conventional antibiotics, antimicrobial peptides kill bacteria very rapidly, within minutes to a few hours, and they have potent bactericidal activity at concentrations similar to or above their MIC (15, 22). AMPs with fast bactericidal activity have several advantages including limiting spread of infection, improving treatment prognosis, limiting the emergence of resistance, and reducing duration of treatment (52). Furthermore, several peptides have been successful in combination with current antibiotics (60-63), as peptides cause weakening of the cell membrane to increase the access of antibiotics into bacterial cells to bind to their targets (63). The synergistic effect of this combination has several advantages. These advantages include minimizing the likelihood of emergence of bacterial drug-resistance to either agent, lowering the dose required for each antibacterial agent thus mitigating drug toxicity, and expanding the spectrum of pathogens that can be targeted (64). For example, the amphibian antimicrobial peptide, esculentin-1b (1–18) was also reported to cause permeabilization of bacterial membranes at subinhibitory concentrations leading to synergism with conventional antibiotics against Escherichia coli (65). The peptidomimetic OAK (oligo- acyl-lysyl), was able to resensitize erythromycin-resistant E. coli to erythromycin both in vitro and in an animal model of infection. The authors explained the mechanism of resensitization to be transient depolarization of the membrane potential at subinhibitory concentrations (66).

1.3.2 Active against drug resistant pathogens and low emergence of bacterial resistance to antimicrobial peptides Due to their unique mechanism of killing (22), antimicrobial peptides are not cross- resistant with traditional antibiotics (15). Additionally, most antimicrobial peptides are effective against multi-drug and pan-drug resistant strains (10, 63, 67, 68). Furthermore, bacteria are less prone to develop resistance to antimicrobial peptides compared to conventional antibiotics (11, 67, 69, 70). This most likely is due to the fact that peptides target the cell membrane or essential processes in the bacterial cell (i.e. are “dirty” drugs) 7

(22). Thus, mutations that confer resistance to peptides are less likely to occur as this would require alterations in the lipid profile of the bacterial cell membrane or multiple mutations in many essential targets which would affect the fitness of the microorganism. Previous reports indicated that after 7 passages at a subinhibitory concentration with pexiganan, bacteria failed to develop resistance (7). These data were confirmed in clinical trials as no significant resistance to pexiganan emerged among patients who received pexiganan. However, in the same study, bacterial resistance to ofloxacin emerged in some patients who received ofloxacin (2). Furthermore, several studies with different antimicrobial peptides that have conducted resistance studies for longer duration of time than pexiganan (20-30 passages), demonstrated either complete lack of resistance to the AMPs (8) (9, 10) or their AMPs displayed a lower propensity to select for resistant bacteria compared to conventional antibiotics (11).

1.3.3 Anti-endotoxic activity

A key virulence factor unique to Gram-negative bacterial pathogens is the presence of lipopolysaccharide (LPS). LPS is a principal structure of the outer membrane of Gram- negative bacteria. During bacterial death, cell replication, or exposure to certain antibiotics, LPS is released from bacteria leading to stimulation of proinflammatory cytokine release such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) from mononuclear phagocytes. Excessive LPS exposure leads to overproduction of these cytokines which can result in septic shock and death. Annually there are 18 million cases of septic shock globally with a mortality rate of ~30% (71, 72). Various antimicrobial peptides are able to bind to LPS and interfere with its binding to lipopolysaccharide binding protein (LBP) and consequently block the activation of toll- like receptor 4. This leads to inhibition of proinflammatory cytokine release and other mediators involved in septic shock (13, 73-76). For example, the human cathelicidin (LL- 37) inhibited the binding of LPS to CD14 on mononuclear phagocytes preventing the expression of TNF-α. In the murine model of endotoxin shock, LL-37 demonstrated potent inhibition of proinflammatory cytokines (77-80). Moreover, LL-37 prevents the LPS- induced apoptosis of endothelial cells by interfering with the assembly of the LPS receptor complex, consisting of CD14 and Toll like receptor 4 (TLR4) and phosphorylation of c- 8

Jun N-terminal kinase (JNK) (81).Therefore, by neutralizing the effect of LPS, AMPs prevent severe reactions such as sepsis and endotoxaemia which are responsible for many deaths worldwide (71, 72). This is particularly advantageous in the treatment of infections caused by Gram-negative bacteria, as most traditional antibiotics (particularly cell wall synthesis inhibitors) cause lysis and more release of LPS during treatment (82-84).

1.3.4 Immune-modulatory properties

Besides their potent antimicrobial activity, various AMPs have direct immune- modulatory effects that play a role in clearance of a bacterial infections (13, 76, 77, 85- 90). For example, LL-37 was found to play a role in activation of chemotaxis, modulation of cytokine release, regulation of immune cell differentiation and stimulation of dendritic cells functions (13, 76, 77, 85-90). LL-37, also, was found to be involved is the formation of the neutrophil extracellular traps (NETs). NETs are extracellular fibers primarily consisting of neutrophil DNA which bind pathogens. Human cathelicidin appears to be an initiator of NET formation as well as its defender, protecting from bacterial nucleases (80, 91). Biochemical features facilitating these activities are hydrophobicity and the cationicity, respectively. LL-37 have a chemotactic property as it induced migration of highly purified human neutrophils and CD4 T cells and human monocytes (79, 80, 92). Moreover, the innate defense regulator peptide-1 (IDR-1) has no direct antimicrobial activity in vitro, but it has been shown to protect mice from bacterial infection (93).IDR- 1 selectively increases the production of the anti-inflammatory cytokine (IL-10) in the blood and suppresses pro-inflammatory cytokines (TNF- and IL-6) at the site of infection, (93). Moreover, IDR-1 act as chemotactic by recruiting both monocytes and macrophages to the site of infection by increasing the production of two important chemokines (MCP-1 and RANTES) that play a role in protection against infection (93). Given the potent immunomodulatory properties of IDR-1, this agent has a therapeutic potential to be used to clear bacterial infections. Thus, the overall net result of the immunomodulatory functions of AMPs leads to the control of inflammatory responses in the affected host without altering or compromising the immune responses that are essential for the clearance of the bacterial infection. 9

1.3.5 Wound healing properties

Wound treatment is an economic burden globally (94, 95). Some antimicrobial peptides in addition to their immune-modulatory effect, have been shown to accelerate wound healing by promoting neovascularization and angiogenesis (96-99). Moreover, peptides stimulate the migration and proliferation of epithelial cells and keratinocytes into damaged tissues (97). Furthermore, peptides have been shown to stimulate the deposition of extracellular matrix to accelerate wound repair (12, 100, 101).

1.3.6 Efficacy of peptides on established biofilms

Biofilms are aggregated bacterial communities covered by polysaccharide matrix that protects bacteria from host immune defenses and hinders antibiotics from targeting deep- seated bacteria encased within the biofilm (104). Biofilm development has been linked to serious infections including pneumonia in cystic fibrosis patients and colonization of medical devices (105). Biofilms of A. baumannii permit this pathogen to survive on abiotic equipment in hospital settings. This facilitates contact with susceptible patients and has led to outbreaks of ventilator-associated pneumonia (106). Biofilms pose a serious medical challenge that are difficult to control. There is a critical need to find novel approaches to address this problem. Recently, several reports have presented AMPs that possess potent antibiofilm activity compared to conventional antibiotics. For example, de la Fuente-Nunez et al. reported a potent broad-spectrum antibiofilm peptide (peptide 1018), despite its weak antimicrobial activity against planktonic bacteria (107). The mechanism of antibiofilm activity of peptide 1018 was found to occur through binding and degradation of the stress related second messenger nucleotide, guanosine pentaphosphate (pppGpp) (107). The same group designed a series of more potent antibiofilm peptides that work by the same mechanism. The most potent antibiofilm peptides in the series were two D enantiomeric peptides (DJK-5 and DJK-6). These peptides were resistant to proteolysis and protected invertebrates from lethal P. aeruginosa infections (108). The synthetic peptides IDR-1018, DJK5, and DJK6 acted synergistically against several Gram-negative pathogens with one or more of the major conventional antibiotics ceftazidime, ciprofloxacin, imipenem and tobramycin, lowering their effective concentrations up to 64-fold (109). IDR-1018 also showed synergy with 10 the antiseptic agent chlorhexidine against multispecies oral biofilm (110)and DJK-6 enhanced the activity of the carbapenem imipenem against plasmid-mediated carbapenems-producing Klebsiella pneumoniae (111), highlighting how peptides can be used to repurpose antibiotics. Another group of researchers reported a de novo engineered cationic antimicrobial peptide (WLBU2) that reduced P. aeruginosa biofilm growth on CF airway epithelial cells and protected mice from lethal P. aeruginosa infections A specifically targeted AMPs, C16G2 can specifically recognize S. mutans and disrupt its biofilms (115). Therefore, this peptide selectively eliminates the plaque bacterium, S. mutans from the normal microflora in humans (115). Currently, C16G2 is under clinical trial phase II as mouthwash for the treatment of dental caries caused by S. mutans by C3 Jian, Inc. (ClinicalTrials.gov registration number NCT02254993).

1.4 Limitations of antimicrobial peptides

Despite the great potential of AMPs including their broad spectrum and rapid bactericidal activity, and low resistance mechanism, they still have several problems with application to clinical cases such as host toxicity, liability to , impaired activity in the presence of biological fluids (serum, plasma, high concentration of salts and acidic pH); and high cost of synthesis (15, 16, 22, 116). Below, we discuss these challenges and proposed solutions to overcome these limitations.

1.4.1 Potential for host toxicity

Since the mechanism of action of most antimicrobial peptides is complex and involves damaging the cell membrane, there is concern pertaining to toxicity to host cells/tissues (15, 16, 22). Indeed, peptides tested at relatively high concentrations required for bacterial killing tend to be non-selective and have high potential for blood cell hemolysis, mitochondrial toxicity and eukaryotic cell necrosis. Thus, all clinical trials up to date are limited to topical application for surface infections rather than parenteral and oral administration (116). However, it should be possible to develop synthetic AMPs which can overcome the above obstacles by designing improved short peptides and modifying the physicochemical features of AMPs (117-119). As for the toxicity of AMPs, high 11 hydrophobicity is related to increased hemolytic activity (119). Control of hydrophobicity and charge can reduce the toxicity of AMPs and closely link the improved selectivity to bacteria (119). For example, several analogs from brevinin-1EMa, a frog AMP, were designed by amino-acid substitution to monitor the effect of hydrophobicity. An analog peptide in which Leu was substituted with Ala in the hydrophobic side of the amphipathic helix showed decreased hydrophobicity, showing lower hemolytic activity against human blood cells and a higher therapeutic index than the parent molecule (120). A more recent study designed a series of analogues of the modified derivative, AR-23, by substitution of Ile with positively charged residues (Arg or Lys) (121). Substitution of Ile on the nonpolar face with positively charged Lys dramatically altered the hydrophobicity, amphipathicity, helicity and the hemolytic activity of the modified peptides. Of all the analogues, A(A1R, A8R, I17K), a peptide with Ala1-Arg, Ala8-Arg and Ile17-Lys substitutions, exhibited similar bactericidal activity and anti-biofilm activity to AR-23 but had much lower hemolytic activity and cytotoxicity against mammalian cells compared with AR-23 (121) . Likewise, the improvement of selectivity by design of molecular targeted AMPs, such as bacterium-selective AMPs, specifically targeted AMPs and environment-sensing AMPs, can be a solution for reducing toxicity (115, 122, 123).An example of specifically targeted AMPs is a fusion peptide, C16G2. Generally, a fusion peptide has two domains: one is a domain for selectively targeting bacteria and the other is a domain for bactericidal action. The two domains are connected with a short linker. The targeting domain consists of species-specific monoclonal antibodies or target bacterial-specific pheromones and delivers the AMP domain by binding to the bacterial surface (115, 122).The C16G2 peptide is conjugated to the targeting domain, which can specifically recognize Streptococcus mutans because it was made from a pheromone produced by S. mutans. Therefore, this peptide selectively eliminates the plaque bacterium S. mutans from the normal microflora in humans. Currently, C16G2 is under clinical trial phase II as mouthwash for the treatment of dental caries caused by S. mutans by C3 Jian, Inc. (ClinicalTrials.gov registration number NCT02254993) (123). Environment-sensing AMPs use the changes in the surrounding induced by pathogenic organisms. For example, the AAP2 peptide was designed to be activated at acidic pH but not at physiological pH 7.5. Under low pH conditions triggered by highly populated bacterium such as S. mutans, 12 this AMP can show maximum activity and successfully remove infected microorganisms (124). Recently, other techniques such as polymeric nanoencapsulation of peptides and liposomal formulations have been applied to solve stability and toxicity problems (125). Encapsulation of AMPs using nanoparticles such as nanospheres and nanovesicles could improve therapeutic activity and stability at the same time, which was demonstrated in the study of the P34 peptide encapsulated in phosphatidylcholine nanovesicles (126). In addition, as shown in several examples such as indolicidin and polymyxin E, their liposomal formulations reduced peptide toxicity and increased plasma half-life maintaining their original activity (127). Moreover, PEGylating peptides and usage of drug delivery systems have been newly introduced in this field (128). The PEGylated M33 peptide showed its increased resistance to peptidase and decreased toxicity, resulting in improving peptide activity (128).

1.4.2 Susceptibility to proteolytic digestion

Susceptibility of peptides to digestion by proteases serves as major impediment to the clinical application of AMPs (15). The linear structure of AMPs can be easily attacked by host proteases and peptidases (15, 129). The short half-lives of AMPs that may be caused by proteolysis make repetitive administration of AMPs quite problematic with high cost (15). Moreover, proteolytic activity of serum is strong, which can provide unfavorable circumstances to the administered AMPs into blood (15). That is why AMPs are usually used as topical treatment for skin infections associated with burns, diabetic wounds, the eyes and gum infections in practice. Thus, to overcome the limitation of application, the escaping strategy for degradation of AMPs is required. Moreover, peptides have high affinity to bind serum proteins leaving small unbound fraction in the circulation. Furthermore, peptides are rapidly cleared from circulation which results in short half-life in plasma and failure to access pathogens inside tissues to clear infections (130). These issues hindered the development of antimicrobial peptides in systemic applications (both oral and parenteral), and explain why antimicrobial peptides that reach the clinical phases are designed for topical applications (16) (54). (130). However, several strategies have been investigated to overcome these issues. The strategies employed include, 1) substitution of amino acids with their D isoform, 13

rendering the peptide resistant to the action of proteases (69, 108); 2) creation of chemical compounds that mimic the structure of antimicrobial peptides (peptoids (131-133) and peptidomimetics (134, 135)) which are more stable to proteolysis; and 3) packaging of peptides inside a carrier such as liposomes and nanoparticle results in increasing the peptide half-life in circulation (136-139).

1.4.3 Impaired antibacterial activity in the presence of physiologically relevant conditions (e.g., salts, acidic pH, plasma, and serum) A significant drawback of many antimicrobial peptides is their antibacterial activity is impaired in the presence of high concentration of salts; this severely limits their clinical utility (140, 141). For example, in the epithelial cell secretions of a CF patients, the NaCl concentration is approximately 120 mM (142); at this concentration β-defensins, lost its antibacterial activity. Recently, several studies reported that abnormally reduced pH in the airway surface liquid of CF patients (pH 6.8) inhibits the antimicrobial activity of host defense peptides and may impair the host’s defense against pathogens (143, 144). In addition to salts and acidity, serum and plasma can also impair the antibacterial activity of conventional antibiotics and peptides either via proteolytic digestion or binding to protein or lipid fractions (145). Several strategies have been investigated to overcome these issues, for example, De novo designing of tryptophan and arginine rich AMPs can improve antimicrobial activity under physiological conditions (e.g., salts, plasma, and serum) without negatively impacting host toxicity (24, 146, 147). In addition, substitution of tryptophan- and histidine-rich peptides with the bulky amino acid, naphthylalanine have shown to increase the serum stability and boost salt resistant of a short Trp-rich antimicrobial peptide, S1 (141).

1.4.4 Cost to manufacture

The cost of synthesizing antimicrobial peptides in bulk are very expensive compared to conventional antibiotics (15). A single gram of peptide (sufficient for an average daily dose orally or via intravenous injection) may cost $100- $600 to manufacture (15). This is considered one of the major hurdles for development of peptides for large scale 14 productions and testing in different clinical applications (15). Various strategies have been developed to decrease the cost of AMPs production. The strategies employed include, A) Size reduction and/or de novo synthesis of AMPs. For example, Won et al. tried to synthesize the shortest AMP analogs from Gaegurin 5, which showed good antimicrobial activity in their studies. They found that 11 residues are the marginal length showing good activity (148). B) Development of a suitable expression system is another strategy to decrease the cost, as seen in the example of plectasin, of which the fungal expression system was helpful to obtain a high yield of peptides (149). The recombinant human lactoferrin also has been expressed in transgenic cattle to provide the large-scale production of lactoferrin for pharmaceutical use (150). In addition, the improved solubility of AMPs using a fusion expression system that contains a solubility-enhancing moiety could increase efficiency in producing AMPs, leading to a reduction in cost Recent studies have shown that thioredoxin and small ubiquitin-related modifiers can be used for improving the solubility of peptides and finally producing high yields of peptides (151, 152).

1.5 Antimicrobial peptides in clinical trials

AMPs in current and past clinical trials have been reviewed here (Table 1.1). One of the AMPs that has advanced furthest in clinical trials is pexiganan for the treatment of diabetic foot ulcers (153). Pexiganan (22 amino acids peptide) is an analog of 2. Magainin was first discovered from the skin of the African clawed frog Xenopus laevis in 1987. Pexiganan showed excellent in vitro broad-spectrum bactericidal activity against large panel of clinically isolated bacteria. Topical application of 2% pexiganan cream showed therapeutic resolution equivalent to oral ofloxacin for treatment of mild infections of diabetic foot ulcers (153). Interestingly, no significant resistance to pexiganan emerged among patients who received pexiganan. However, bacterial resistance to ofloxacin emerged in some patients who received ofloxacin (153). Currently, pexiganan is undergoing phase 3 development as a topical agent for treatment of mild infections of diabetic foot ulcers (ClinicalTrials.gov registration numbers NCT01594762 and NCT01590758). Another compound that showed success in clinical trials is the 15 peptidomimetic, brilacidin. Brilacidin is a defensin-mimetic that targets the bacterial membrane, similar to AMPs. Brilacidin was capable of inhibiting growth of a collection of multidrug-resistant S. aureus isolates at a concentration of 2 μg/ml. Brilacidin also demonstrated clinical efficacy and safety in two phase II clinical trials for the treatment of acute bacterial skin and skin structure infections (ABSSSI). The FDA approved brilacidin to advance into Phase III clinical trials (123, 156, 157). Omiganan (MBI-226) is a synthetic analog (12 amino acid residue) of bovine indolicidin. It has several benefits such as it can be applied topically up to 5% concentration without toxicity and resistance (123, 158). Unfortunately, Phase III clinical trials for the prevention of infectious complications after blood vessel catheterization failed. This is because the study involving more than 1400 patients did not show statistically significant evidence of higher efficacy compared to standard treatment. Omiganan 1% gel, named Omigard, is now under another Phase III clinical trial for the prevention of catheter-related infections as topical treatment by Carrus Capital Corporation (123). In addition, Cutanea Life Sciences Inc. has developed a Phase II clinical trial for the treatment of rosacea (123). Iseganan (IB-367) is a synthetic derivative (18 amino acids) of pig protegrin. Iseganan forming a b-hairpin structure stabilized by two disulfide bonds. Iseganan demonstrated potent and broad-spectrum antibacterial and antifungal activity (159, 160). A Phase I clinical study demonstrated its safety and provided the first evidence of efficacy by the reduction of microorganisms by mouth treatment over ten days (159). Phase II clinical trials involving 134 patients indicated that IB-367 decreased the population of bacteria in infected lungs and helped in recovering pulmonary function in cystic fibrosis patients (160). Iseganan was intended to be developed as a local mouthwash to inhibit ulcerative oral mucositis for patients who are susceptible to infection with high risk. The company, IntraBiotics Pharmaceuticals, tried a Phase III clinical study with 502 patients receiving stomatotoxic chemotherapy. Unfortunately, this trial was not successful because of a dispensing error of a portion or the clinical supplies during the trial, resulting in consequent reduction of statistical power (161). In addition, another Phase III trial with 545 patients receiving radiotherapy for head-and-neck malignancies did not show any clinical improvement in ulcerative oral mucositis (162). As a result, the development of IB-367 was stopped for this particular indication (163). 16

A number of LL-37 related peptides have also shown the potential for therapeutic development], such as OP-145, which was developed by OctoPlus, and when the peptide was included in cream formulations for nasal application, these preparations were found to be efficacious in the eradication of MRSA carriage (164, 165).The completion of phase I/II clinical trials by OP-145 also showed that the peptide was safe and efficacious as a treatment for chronic otitis media (Table 1.1). This disease affects millions of people worldwide and is highly recalcitrant to treatment by conventional antibiotics, which is now known to be primarily due to bacterial biofilms (166). Derivatives of human lactoferrin have shown the potential for therapeutic development such as hLF(1-11), which was developed by AM Pharma, and in clinical trials the peptide was safely injected into neutropenic stem cell transplantation patients (167).Currently, hLF(1-11) in clinical trial II for the prevention of bacteremia and fungal infections in immunocompromised individuals (Table 1.1). Lytixar (LTX-109) is a synthetic, membrane-degrading peptide that has been developed by Lytix Biopharma (Oslo); this peptide has completed a Phase I/IIa clinical trial for nasally colonized MRSA. The anterior nares are the main reservoir for colonization by S. aureus and the nasal carriage of MRSA is an important risk factor for subsequent infection and transmission of this pathogen, which has led to intensive efforts to identify agents able to efficiently reduce MRSA colonization (168, 169).A significant effect on nasal decolonization of MRSA and MSSA was observed after only two days of LTX-109 treatment in subjects treated with 2% or 5% LTX-109, compared to the vehicle (168, 169). The success of pexiganin, brilacidin, hLF(1-11), OP-145 and LTX-109 demonstrates the promise that antimicrobial peptides have as potential topical agents for treatment of infections caused by multidrug-resistant pathogens.

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160. Kollef M, Pittet D, Sanchez Garcia M, Chastre J, Fagon JY, Bonten M, Hyzy R, Fleming TR, Fuchs H, Bellm L, Mercat A, Manez R, Martinez A, Eggimann P, Daguerre M, Luyt CE, Prevention of Pneumonia Study Trial G. 2006. A randomized double-blind trial of iseganan in prevention of ventilator-associated pneumonia. Am J Respir Crit Care Med 173:91-97. 161. Giles FJ, Miller CB, Hurd DD, Wingard JR, Fleming TR, Sonis ST, Bradford WZ, Pulliam JG, Anaissie EJ, Beveridge RA, Brunvand MM, Martin PJ, Investigators P-CT. 2003. A phase III, randomized, double-blind, placebo-controlled, multinational trial of iseganan for the prevention of oral mucositis in patients receiving stomatotoxic chemotherapy (PROMPT-CT trial). Leuk Lymphoma 44:1165-1172. 162. Trotti A, Garden A, Warde P, Symonds P, Langer C, Redman R, Pajak TF, Fleming TR, Henke M, Bourhis J, Rosenthal DI, Junor E, Cmelak A, Sheehan F, Pulliam J, Devitt-Risse P, Fuchs H, Chambers M, O'Sullivan B, Ang KK. 2004. A multinational, randomized phase III trial of iseganan HCl oral solution for reducing the severity of oral mucositis in patients receiving radiotherapy for head- and-neck malignancy. Int J Radiat Oncol Biol Phys 58:674-681. 163. Elad S, Epstein JB, Raber-Durlacher J, Donnelly P, Strahilevitz J. 2012. The antimicrobial effect of Iseganan HCl oral solution in patients receiving stomatotoxic chemotherapy: analysis from a multicenter, double-blind, placebo- controlled, randomized, phase III clinical trial. J Oral Pathol Med 41:229-234. 164. de Breij A, Riool M, Kwakman PH, de Boer L, Cordfunke RA, Drijfhout JW, Cohen O, Emanuel N, Zaat SA, Nibbering PH, Moriarty TF. 2016. Prevention of Staphylococcus aureus biomaterial-associated infections using a polymer-lipid coating containing the antimicrobial peptide OP-145. J Control Release 222:1-8. 165. Malanovic N, Leber R, Schmuck M, Kriechbaum M, Cordfunke RA, Drijfhout JW, de Breij A, Nibbering PH, Kolb D, Lohner K. 2015. Phospholipid-driven differences determine the action of the synthetic antimicrobial peptide OP-145 on Gram-positive bacterial and mammalian membrane model systems. Biochim Biophys Acta 1848:2437-2447. 166. Yang R, Sabharwal V, Okonkwo OS, Shlykova N, Tong R, Lin LY, Wang W, Guo S, Rosowski JJ, Pelton SI, Kohane DS. 2016. Treatment of otitis media by transtympanic delivery of antibiotics. Sci Transl Med 8:356ra120. 167. Dijkshoorn L, Brouwer CP, Bogaards SJ, Nemec A, van den Broek PJ, Nibbering PH. 2004. The synthetic N-terminal peptide of human lactoferrin, hLF(1-11), is highly effective against experimental infection caused by multidrug-resistant Acinetobacter baumannii. Antimicrob Agents Chemother 48:4919-4921. 168. Nilsson AC, Janson H, Wold H, Fugelli A, Andersson K, Hakangard C, Olsson P, Olsen WM. 2015. LTX-109 is a novel agent for nasal decolonization of methicillin-resistant and -sensitive Staphylococcus aureus. Antimicrob Agents Chemother 59:145-151. 169. Saravolatz LD, Pawlak J, Johnson L, Bonilla H, Saravolatz LD, 2nd, Fakih MG, Fugelli A, Olsen WM. 2012. In vitro activities of LTX-109, a synthetic antimicrobial peptide, against methicillin-resistant, vancomycin-intermediate, vancomycin-resistant, daptomycin-nonsusceptible, and linezolid-nonsusceptible Staphylococcus aureus. Antimicrob Agents Chemother 56:4478-4482. 29

Table 1.1. Antimicrobial peptides (AMPs) in past and current clinical trials.

Antimicrobial Description Indication Phase Clinical trial ID Company Peptides Topical cream for the treatment of Pexiganan An analogue of Dipexium Pharma diabetic foot 3 NCT00563394 (MSI-78) magainin. /MacroChem/Genaera infections and ulcers. Topical antibiotic for the treatment of nasal carriers Lytixar of methicillin- A (LTX- resistant 2 NCT01223222 Lytix Biopharma. peptidomimetic. 109) Staphylococcus NCT01158235 aureus (MRSA) and Gram- positive bacteria Mouthwash for the treatment of Ardea chemotherapy 3 Biosciences/national induced oral Cancer Institute. Iseganan A derivative of mucositis. (IB-367) protegrin 1. Mouthwash for the treatment of IntraBiotics ventilator- 3 NCT00118781 Pharmaceuticals. associated pneumonia. Intravenous administration for A derivative of hLF1–11 the treatment of 2 NCT00509938 AM Pharma. human lactoferrin. bacteraemia and fungal infections. Treatment for Novexatin A synthetic cyclic fungal infections of 2 NCT02343627 NovaBiotics (NP213), AMP. the toenail. Topical cream for the treatment of Mallinckrodt/Cutanea skin antisepsis, 3 NCT00231153 Life Sciences, Inc. prevention of catheter infections

Omiganan Topical cream for (MBI 226, An analogue of the treatment of MX-226, indolicidin. usual type vulvar CSL-001) intraepithelial neoplasia/moderate Cutanea Life 3 NCT00608959 to severe Sciences, Inc. inflammatory acne vulgaris/mild to moderate atopic dermatitis. 30

Table 1.2. continued

Synthetic 24- Ear drops for the OP-145 mer LL37 treatment of chronic 2 ISRCTN84220089 OctoPlus analogue otitis Intravenous administration for the treatment of acute bacterial skin A defensin Brilacidin, and skin structure mimetic (PMX- Infection caused by 3 NCT01211470 Cellceutix. (Arylamide 30063) Gram-positive oligomer). bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Specifically, Mouthwash for the targeted treatment of dental C16G2 antimicrobial caries caused by 2 NCT02254993 C3 Jian, Inc. peptide Streptococcus (STAMP) mutans.

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Figure 1.1. Various models of membranolytic mechanism of action of antimicrobial peptides (AMPs). AMPs associating with the bacterial membrane (top left). The red and blue parts of the peptide represents a hydrophilic and a hydrophobic surfaces, respectively. A) The barrel- stave model, B) The carpet model and C) The toroidal pore model. Reproduced with permission from Biochimica et Biophysica Acta (BBA) – Biomembranes, ref (19).

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Figure 1.2. Intracellular mechanism of action of antimicrobial peptides. Several peptides can penetrate membranes and induce killing by acting on one or more intracellular targets; such as inhibiting nucleic acids and protein synthesis, inhibition of cell wall synthesis and cell division. Reproduced with permission from Nature Reviews Microbiology, ref (12).

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CHAPTER 2. TARGETING METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS WITH SHORT SALT- RESISTANT SYNTHETIC PEPTIDES

(Mohamed, M.F., Hamed, M.I., Panitch, A., Seleem, M.N (2014) Targeting methicillin- resistant Staphylococcus aureus with short salt-resistant synthetic peptides. Antimicrobial agents and chemotherapy doi:10.1128/AAC.02578-14)

2.1 Abstract

The seriousness of microbial resistance combined with the lack of new antimicrobials have increased the interest in the development of antimicrobial peptides (AMPs) as novel therapeutics. In this study, we evaluated the antimicrobial activity of two short synthetic peptides, namely RRIKA and RR. These peptides exhibited potent antimicrobial activity against Staphylococcus aureus and their antimicrobial effects were significantly enhanced by addition of three amino acids in the C terminus, which consequently increased the amphipathicity, hydrophobicity and net charge. Moreover, RRIKA and RR demonstrated a significant and rapid bactericidal effect against clinical and drug-resistant Staphylococcus isolates including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), linezolid-resistant S. aureus and methicillin-resistant S. epidermidis. In contrast to many natural AMPs; RRIKA and RR retained their activity in the presence of physiological concentrations of NaCl and MgCl2. Both RRIKA and RR enhanced the killing of lysostaphin over 1000-fold and eradicated MRSA and VRSA isolates within 20 minutes. Furthermore, the peptides presented were superior in reducing adherent biofilms of S. aureus and S. epidermidis when compared to conventional antibiotics. Our findings indicate that the staphylocidal effects of our peptides were through permeabilization of the bacterial membrane, leading to leakage of cytoplasmic contents and cell death. Furthermore, peptides were not toxic to HeLa cells at 4 to 8 fold their antimicrobial concentrations. The potent and salt-insensitive antimicrobial activities of these peptides 34 present an attractive therapeutic candidate for treatment of multidrug-resistant S. aureus infections.

2.2 Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) infections have become a major public health concern in both hospital and community settings (1, 2). Since the late 1990s, community-associated MRSA (CA-MRSA) emerged as a principal cause of skin and soft- tissue epidemics throughout the world (3). It has been estimated that mortality rates from MRSA infections in US hospitals are higher than infections caused by HIV/AIDS and tuberculosis combined (4). Furthermore, there is a considerable medical challenge for treating MRSA infections due to the remarkable ability of S. aureus to develop resistance to multiple antibiotics, thus limiting the number of viable therapeutic options (2, 5). Therefore, there is an urgent need to develop novel antimicrobials with unique mechanisms of action to combat MRSA.

Antimicrobial peptides (AMPs) are key components of the host innate defense against infections in most creatures (6, 7). The potential therapeutic applications of AMPs are significant (8). They have rapid and broad spectrum antibacterial activity. In addition, it is extremely difficult for bacteria to evolve resistance against AMPs that disrupt the microbial membrane, as it would necessitate fundamental alterations in the lipid composition of the bacterial membranes. Though AMPs have many important antibacterial properties, several studies have exposed their potential limitations as therapeutic agents.

A significant number of natural AMPs are large in size with high host cytotoxicity and moderate antimicrobial activity. Moreover, their production cost is high (8, 9). In addition, many natural AMPs lose their antimicrobial activity in physiological salt concentrations (10, 11). These characteristics have substantially hindered their pharmaceutical development as new therapeutic agents. Thus, successful development of novel AMPs as future therapeutics requires identification of short AMPs demonstrating strong antimicrobial activity, salt tolerance and minimal toxicity to host tissues. 35

Previously, our group has developed short synthetic peptide templates for therapeutic applications against hyperplasia and inflammation (12-14). However, their antimicrobial activity was never explored. In the present study, we screened four synthetic peptides and identified two peptides, namely RRIKA and RR, with potent antimicrobial activity against a panel of clinical and drug resistant Staphylococci strains. In addition, we investigated the synergistic activity of these peptides in combination with clinically relevant antimicrobials and examined the peptides’ ability to disrupt staphylococcal biofilms. Moreover, we performed a series of experiments to explore the antibacterial mechanism of action of RRIKA and RR and examined the toxicity of these peptides toward mammalian cells.

2.3 Materials and methods

Bacterial strains and media

All Staphylococcus strains tested in this study are presented in (Table 2.1). Mueller- Hinton broth (MHB) and Mueller-Hinton agar (MHA) were purchased from Sigma- Aldrich, while trypticase soy broth (TSB) and trypticase soy agar (TSA) were purchased from Becton-Dickinson, Cockeysville, Md.

Reagents, peptides and antibiotics

Nisin (Sigma, N5764), melittin from honey bee venom (Sigma, M2272), magainin I (Sigma, M7152), vancomycin hydrochloride (Gold Biotechnology), linezolid (Selleck Chemicals), ampicillin sodium salt (IBI Scientific), recombinant lysostaphin (3000 U/ mg) from Staphylococcus simulans (Sigma, L9043) and calcein AM (Molecular Probes, Life Technologies) were all purchased from commercial vendors.

Peptide synthesis

Peptides RRIKA, RR, KAF, and FAK were synthesized on Knorr-amide resin (Synbiosci Corp.) using standard FMOC chemistry. Two different chemistries were used to couple each amino acid. The first coupling reagents were N-hydroxybenzotriazole (HoBt)/N, N’- diisopropylcarbodiimide (DIC) and the second coupling reagents were 2- (1Hbenzotriazole-1-yl)-1, 1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and 36 lutidine. For FITC labeled YARA, an aminohexanoic acid spacer was added to the N- terminus to serve as a spacer for the addition of FITC isomer 1 (Molecular Probes). The FITC isomer was solubilized in 12:7:5 pyridine/DMF/DCM and incubated with the deprotected peptide overnight. A ninhydrin test was used to check complete coupling of FITC to the peptide. Following synthesis, the peptide was cleaved from the resin with a trifluoroacetic acid-based cocktail, precipitated in ether, and recovered by centrifugation. The recovered peptide was dried in vacuo, resuspended in MilliQ purified water, and purified using an FPLC (ÄKTA Explorer, GE Healthcare) equipped with a 22/250 C18 prep-scale column (Grace Davidson). An acetonitrile gradient with a constant concentration of 0.1% trifluoroacetic acid was used to achieve purification. Desired molecular weight was confirmed by time-of-flight MALDI mass spectrometry using a 4800 Plus MALDI TOF/TOF™ Analyzer (Applied Biosystems).

Antibacterial assays

Minimum inhibitory concentrations (MIC) of peptides and antibiotics were evaluated with the broth microdilution technique in MHB with an initial inoculum of 5×105 cells non- treated Polystyrene microtiter plates (CytoOne, CC7672-7596) in accordance with the Clinical and Laboratory Standards Institute (CLSI) (15). The MIC was interpreted as the lowest concentration of peptide or antibiotic that completely inhibited the visible growth of bacteria after 16 hr incubation of the plates at 37°C. Each agent was tested in triplicate in at least two independent experiments. The highest MIC value was reported.

Antimicrobial activity in the presence of salts

To investigate the activity of peptides in the presence of high salt concentrations, the MIC was again identified as described above except, fixed concentrations of NaCl or MgCl2 were added to each well of the microtiter plate. For further analysis of the effect of salinity on killing ability of peptides; logarithmic phase of MRSA USA300 and VRSA VRS13 at 5×105 colony forming units/ml (CFU/ml) were incubated with 2 × MIC of RRIKA and

RR, in MHB, with different concentrations of NaCl (0, 50, 100, 150 mM) or MgCl2 (0, 1, 2, 3 mM) for 4 hr. Bacteria were serially diluted and plated, in triplicate, on TSA plates. CFUs were counted after incubation of plates for 24 hr at 37°C. 37

Time-kill assay

An overnight culture of MRSA USA300 was diluted in fresh MHB and incubated until optical density (OD600) ≈1, was reached, then washed twice with phosphate buffered saline (PBS) diluted to ~5×105 CFU/ml in MHB. Peptides, vancomycin and linezolid were added at concentrations equivalent to 5 × MIC. Peptide diluent (sterile water) served as a negative control. Bacterial cell viability was monitored up to 24 hr. Samples were removed at specific intervals, serially diluted in PBS, and plated, in triplicate, on TSA plates. CFUs were counted after 24 hr incubation of plates at 37°C. To study the effect of serum and salinity on the killing kinetics of the peptides, a time-kill assay was performed as described above with the exception that bacteria and peptides at 5 × MIC were incubated in MHB alone or with 10% fetal bovine serum (FBS) or incubated in PBS (containing 137 mM NaCl).

Synergy with antimicrobials

The fractional inhibitory concentration (FIC) index was utilized to determine the relationship between antimicrobial agents. Peptide MICs against test microorganisms were determined in triplicate samples. Two-fold serial dilutions of antimicrobials (lysostaphin, vancomycin, linezolid and nisin) were tested in the presence of a fixed concentration of peptide, equals to ¼ × peptide MIC. It is worthy to note that none of the peptides killed the test microorganisms at their quarter MICs. The FIC index was calculated as follows: FIC = 0.25 + MIC (antibiotic in combination)/MIC (antibiotic alone); 0.25 is equal to MIC (peptide in combination)/MIC (peptide alone). An FIC index of ≤ 0.5 is considered to demonstrate synergy. Additive effect was defined as an FIC index of 1. Antagonism was defined as an FIC index of > 4(16). To further investigate the synergism of peptides with lysostaphin, the time-kill method of determining synergy was utilized as described elsewhere (17). MRSA USA300 and VRSA VRS13 were incubated with ½ × MIC of RRIKA or ½ × MIC of RR alone; or in combination with ¼ × MIC of lysostaphin (MIC of lysostaphin against MRSA USA300 and VRSA VRS13 were 0.04 and 0.08 µM, respectively). Samples were obtained at different time points, then diluted and plated on TSA plates. The plates were incubated for 24 hr at 37°C before CFU were determined. Synergy was defined as a 100-fold or 2- 38

log10 decrease in colony count by the combination of two agents together as compared to each agent tested alone. Additivity or indifference was defined as a 10-fold change in CFU and antagonism was defined as a 100-fold increase in CFU by the combined peptide treatment in comparison to the single treatment.

Bacterial membrane disruption activity (Bacteriolysis)

Cell lysis, as indicated by a decrease in OD595, was determined as described before (18). Briefly, MHB was inoculated with an overnight culture of MRSA USA300 and incubated aerobically at 37°C until an OD595≈0.6 was reached and then diluted to OD595≈0.2 in MHB (equivalent to ≈108 CFU/mL). 200 µL of diluted bacteria was added to all wells of a 96- well plate. RRIKA and RR were added in concentrations equivalent to 4 × MIC. Nisin at 4 × MIC was used as a positive control and untreated bacteria served as a negative control. MHB with the same concentrations of drugs served as blanks. Turbidity was monitored at defined intervals up to 10 hr in a Molecular Devices Vmax microplate reader at 595 nm absorption. The assay was carried out in triplicate samples for each treatment regimen.

Calcein leakage assay

Membrane permeabilization of S. aureus by peptides was monitored and quantified by the leakage of the preloaded fluorescent dye, calcein, as described before (19). MRSA USA300 was grown in MHB to logarithmic phase at 37°C. Cells were then harvested by centrifugation, washed twice with PBS, and then adjusted spectrophotometrically to an 9 OD600 of 1.0 (≈10 CFU/ml) in PBS containing 10% (vol/vol) MHB. Then MRSA cells were incubated with 3 µM calcein AM for 1 hr at 37°C. Calcein-loaded cells were harvested by centrifugation (3,000 × g, 10 min), suspended in PBS, and diluted to achieve a final inoculum of 107 CFU/ml. Aliquots of 100 µL were then added into a sterile black- wall 96-well plate. RRIKA and RR were added in concentrations equivalent to 5 × and 10 × MIC. Bacteria treated with peptide diluent (sterile water) and nisin (10 µg/mL) served as negative and positive controls, respectively. Calcein leakage was measured for 120 min using a fluorescence plate reader (FLx800 model BioTek® Instruments, Inc. Winooski, Vermont). Membrane permeabilization (%) was calculated as the absolute percent calcein leakage by peptides with respect to calcein-loaded with no-peptide treated cells. Experiments were done in triplicate and repeated independently twice. 39

DNA binding assay

The ability of RRIKA and RR to bind DNA was investigated by the electrophoretic mobility shift assay as has been previously described (20). In brief, increasing concentrations of RRIKA, RR and magainin were incubated with 250 ng of plasmid DNA (pUC19) in 30 µL binding buffer (10 mM Tris-HCl, 1 mM EDTA buffer, pH 8.0) at room temperature for 30 min. After incubation, the DNA was analyzed by 1% gel electrophoresis. DNA migration was visualized by ethidium bromide staining.

Quantification of activity against biofilms

Biofilm-forming clinical isolates of S. aureus (ATCC 6538) and S. epidermidis (ATCC 35984) were grown overnight in TSB. After incubation, cultures were diluted 1:100 in TSB supplemented with 1% glucose. Diluted bacteria were inoculated in either 24- or 96- well flat bottom cell culture plates (polystyrene) and incubated at 37°C for 24 hr. The culture medium was subsequently removed and wells were carefully washed with PBS three times to remove planktonic bacteria before re-filling wells with fresh MHB. Peptides and antibiotics were added at different concentrations and plates were incubated at 37°C for 24 hr. After the removal of medium at the end of incubation, wells were rinsed by submerging the entire plate in a tub containing tap water. Biofilms were stained with 0.1% (w/v) crystal violet for 30 min. After staining, the dye was removed and the wells washed with water. The plates were dried for at least 1 hr, prior to addition of ethanol (95%) to solubilize the dye bound to the biofilm. The optical densities (OD) of biofilms were measured at 595 nm absorbance by using a microplate reader (Bio-Tek Instruments Inc.). An inverted microscope (Vista Vision, VWR), with attached camera and 25X objective was used to photograph the biofilm in 24-well plates.

Hemolysis assay

Human red blood cells (RBCs) (Innovative Research “Novi, MI”) were pelleted by centrifugation at 2000 rpm for 5 min followed by washing three times with PBS. An 8% suspension (v/v) of RBCs was prepared in PBS and 50 L of the solution was transferred to a 96-well plate. Then, 50 L of different concentration of peptides in PBS were added to give a final suspension of 4% (v/v) of RBCs. PBS served as a negative control. 0.1 % 40 triton X-100 as well as 5 µM melittin served as a positive control. The plate was incubated at 37°C for 1 hr. The plate was subsequently centrifuged at 1000 rpm for 5 min at 4°C. 75 L aliquots of the supernatants in each well were carefully transferred to a new sterile 96-well plate. Finally, the hemolytic activity was evaluated by measuring the optical absorbance at OD405 with a microplate reader (Vmax, Molecular Devices). The hemolysis percentage was calculated based on the 100% release with 0.1 % triton X-100 or 5 µM melittin. Experiments were done in triplicates.

Cytotoxicity assay

The toxicity of RRIKA, RR and melittin against HeLa cells was carried out using the ® CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay kit from Promega (21). 4 Briefly, ~ 2 × 10 HeLa cells suspended in 200 µL of DMEM supplemented with 10%

FBS, were seeded in 96-well plates and incubated at 37°C in a 5% CO2 atmosphere for 24 hr. The HeLa cells were further incubated with different concentration of peptides for 24 hr. At the end of the incubation period, the culture media was discarded and the cells in each well were washed with PBS. 100 µL of cell culture media and 20 µL CellTiter 96®

AQueous assay reagent were added next (solution reagent contains a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium, inner salt; MTS] ). The plate was returned to the incubator for 2 hr to allow color development. The intensity of color was quantified at 490 nm using a 96-well microplate reader (Vmax, Molecular Devices). Results were expressed as the percentage mean absorbance by cells upon incubation with peptide with respect to incubation with the control (untreated wells).

Statistical analyses

The mean absorbance of crystal violet in the biofilm reduction was compared between the groups using the two-tailed Student t test (p ˂0.05). All statistical analyses were performed with Microsoft Excel’s statistical program.

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2.4 Results

Antimicrobial activity

The amino acid sequences and characteristics of the synthetic peptides used in this study are shown in Table 2.2. We screened the synthetic peptides against a panel of clinical and drug-resistant Staphylococcal strains (Table 2.1). RRIKA inhibited the growth of methicillin-sensitive S. aureus (MSSA), MRSA, VISA, VRSA, linezolid-resistant S. aureus, methicillin-resistant S. epidermidis and multidrug-resistant strains from concentrations ranging from 2 to 4 µM. RR inhibited the same isolates with MICs ranging from 8 to 32 µM. In contrast, KAF and FAK peptides were inactive against all strains tested up to 64 µM (data not shown).

Bacterial killing kinetics

Figure 2.1 presents the rate of microbial killing by RR, RRIKA, vancomycin and linezolid when MRSA USA300 was exposed to 5 × MIC of each treatment over a 24 hr incubation period at 37 ºC. Both RRIKA and RR exhibited a rapid bactericidal effect with 3-log10 reduction (99.9 % clearance) within 60 and 90 min, respectively. In addition, both peptides completely eliminated the inoculum (5×105 CFU/mL) within 120 and 180 min, respectively. In comparison, vancomycin achieved a 3-log10 bacterial reduction only after 24 hr; while linezolid (a protein synthesis inhibitor) exhibited a bacteriostatic effect producing only a single log bacterial reduction over the 24 hr incubation. The killing kinetics of RRIKA and RR in PBS (containing 137 mM NaCl) did not significantly change when compared to MHB (figure 2.2). In the presence of 10% FBS, RRIKA retained its bactericidal activity, though it required longer to kill the bacteria completely (6h), while RR lost its antibacterial activity at the same condition.

Antimicrobial activity in the presence of salts

The MIC of RRIKA and RR for MRSA USA300 was determined in the presence of NaCl or MgCl2. There was no increase in the MICs of RRIKA and only a one-fold increase in case of RR was observed in the presence of 100 mM NaCl. Moreover, the antimicrobial activity of RRIKA and RR was not impeded in the presence of MgCl2 at a concentration equivalent to reported physiological conditions (2 mM). To study the effect of salinity on 42 the killing ability of the peptides, CFU were counted after incubation of MRSA USA300 and VRSA VRS13 strains with RRIKA and RR (2 × MIC) in increasing concentrations of NaCl or MgCl2. There was no significant difference in the bactericidal activity of RRIKA in the presence of NaCl up to 150 mM. Similar patterns were observed for RR with the exception of one log10 increase in CFU observed at 150 mM NaCl; however, this peptide retained its bactericidal activity at this concentration (producing a 3-log10 reduction). Furthermore, physiological concentrations of MgCl2 (1, 2 and 3 mM) had no effect on the killing ability of both RRIKA and RR. Synergy with antimicrobial agents Both RRIKA and RR acted synergistically with lysostaphin against MSSA, MRSA and VRSA strains with a FIC index equal to 0.26 (Table 2.3). When both peptides were combined together, only one-fold decrease in each peptide’s MIC was observed (FIC index = 0.75). However, an additive effect was observed when each peptide was combined with vancomycin, linezolid or nisin against tested strains (data not shown). There were no antagonistic effects observed between the peptides and antimicrobials against all strains tested. Time-kill synergy studies revealed RRIKA and RR, at only ½ × MIC enhanced the killing of lysostaphin (at ¼ × MIC) more than 1000-fold and completely eradicated MRSA and VRSA isolates within 20 minutes (figure 2.3).

Bacterial membrane disruption activity (Bacteriolysis)

Since evidence of rapid cell lysis of treated cultures is indicative of membrane damaging activity of antimicrobial peptides (22, 23), we assessed whether the bactericidal activity of our peptides was associated with lysis of bacterial cells by measuring culture turbidity over time after treatment. We monitored the turbidity of MRSA cultures treated with 4 ×

MIC of peptides over a 10 hr period at OD595 via a microplate reader. As shown in figure

2.4 both RRIKA and RR caused a rapid decrease in OD595 even with a high inoculum concentration of 108 CFU/ml. Peptide RRIKA produced greater than 50% and 86% reduction in turbidity after 2.5 and 6 hr, respectively. RR produced a similar percentage of turbidity as RRIKA albeit at a slower rate (after 4.5 and 10 hr). Nisin at 4 × MIC, a polycyclic antibacterial peptide which is known as a membrane perturbing agent (24), 43 lysed cells at a slightly higher rate than RRIKA with more than 70% reduction in turbidity observed after 2.5 h. In contrast, wells treated with 4 × MIC of vancomycin (a cell wall biosynthesis inhibitor) had no effect on culture turbidity within the same time frame.

Permeabilization of S. aureus membrane (calcein leakage assay)

We monitored the effect of the peptides on MRSA membrane integrity by using the calcein leakage assay as described previously (19, 25). Damage of membrane integrity is indicated by calcein leakage from bacterial cells, leading to a reduction in fluorescence intensity. Figure 2.5 demonstrates that both peptides perturb the S. aureus cell membrane leading to leakage of preloaded calcein in both a concentration- and time-dependent manner. RRIKA was faster and more potent in comparison to RR in membrane perturbation. At 5 × MIC, RRIKA and RR caused more than 60% and 30% leakage respectively within 60 minutes. When the concentration of peptide was increased to 10 × MIC, a significant change in membrane damage was observed for both peptides. There was a more than 75% and 60% reduction in fluorescence intensity measured for RRIKA and RR respectively in the same time frame. Nisin at 5 × MIC resulted in more than 70% calcein leakage from cells, which is comparable to RRIKA at 10 × MIC. Vancomycin, as expected, had no effect on the bacterial cell membrane integrity even at high concentration (10 × MIC).

DNA binding properties

Since there was no obvious evidence that the antibacterial effect of AMPs was restricted to perforation of membranes, we explored the feasibility of other intracellular targets, such as DNA by assessing the DNA-binding properties of peptides (26, 27). As shown in figure 2.6, both peptides were able to bind the plasmid DNA and delay its electrophoretic migration into agarose gel, in a dose-dependent manner. At 8 µM concentration, of RRIKA peptide, a fraction of the plasmid DNA was still able to migrate into the gel as non-complexed DNA; however, at 16 µM, the majority of DNA remained in the gel’s wells. At higher concentrations, complete retardation of DNA migration was exhibited, implying that the DNA was aggregated by peptide. Similar patterns of migration were observed with the RR peptide except that free DNA was still seen at 16 µM concentration. 44

In contrast, magainin, a cationic antimicrobial peptide of amphibian skin, lacks this ability to bind DNA even at concentrations up to 80 µM.

Quantification of activity against biofilms

In order to evaluate the efficacy of RR and RRIKA against established biofilms, we measured the biofilm mass post-treatment by crystal violet staining biofilms formed by clinical isolates of S. aureus and S. epidermidis. As observed in figure 2.7A, both peptides significantly reduced S. aureus biofilms as compared to the antibiotics of choice. RRIKA and RR, at only 2 × MIC, dispersed more than 65% of mature biofilms (p < 0.01), while vancomycin and linezolid, at 16 × MIC, was only capable of reducing 40% of biofilm mass (p < 0.01). There was significant difference between peptide and antibiotics treated biofilms (p < 0.01).

With regards to S. epidermidis, both RR and RRIKA, at 32 and 64 × MIC, were able to reduce more than 50% of biofilm mass respectively (p < 0.01) (figure 2.7B). Vancomycin and linezolid, on the other hand, reduced only 9.5 and 10.7% of biofilm mass, respectively (p < 0.01) at concentrations equivalent to 256 × MIC. Also, we found statistical significance between peptide and antibiotics treated biofilms (p < 0.01).

Hemolysis assay and cell toxicity

We assessed the release of hemoglobin from human erythrocytes exposed to different concentrations of peptides. As depicted in figure 2.8, even at concentrations as high as 300 µΜ, RRIKA and RR exhibited minimal hemolysis against RBCs (maximum of 10% hemolysis observed). In contrast, melittin completely lysed RBCs (100% hemolysis) at a significantly lower concentration of 5 µM.

The cytotoxic effect of the peptides on HeLa cells was evaluated by the MTS assay. As depicted in figure 2.9, neither RRIKA nor RR was toxic to the mammalian cells tested up to concentrations of 32 µM and 64 µM, respectively. These values correlate to 8 and 4 ×

MIC for RRIKA and RR, respectively. The half maximal effective concentration (EC50) of RRIKA and RR against macrophage cell lines was 64 and 128 µM respectively. On the other hand, melittin showed high toxicity even at bacteriostatic concentration with EC50 less than 5 µM. 45

Discussion In this study, we evaluated the antimicrobial activity of two short synthetic peptides, and revealed their potential mechanisms of action. We observed that RRIKA and RR exhibited potent antibacterial activity against all tested S. aureus isolates. Moreover, our peptides demonstrated activity against multiple clinical isolates of MRSA, particularly MRSA USA300, a community-associated strain which is responsible for outbreaks of staphylococcal skin and soft-tissue infections (SSTI) in the United States (28). Similarly, the potent bactericidal activity was observed in other clinical MRSA isolates (USA100, USA200 and USA500) that are resistant to various antibiotic classes including macrolides, aminoglycosides, lincosamides, and fluoroquinolones. Additionally, we found that these peptides retained their activity against strains of S. aureus which have resistance to drugs of choice in treating MRSA infections, namely linezolid (MRSA NRS119) and vancomycin (VRSA strains). It is worthy to note that, the C terminus of the RR peptide was modified to contain three additional amino acids (isoleucine, lysine and alanine) which made the RRIKA peptide more amphipathic and hydrophobic and also increased its positive net charge. These modifications augmented the antibacterial activity of RRIKA which exhibited MICs two to eight folds lower than those of RR. The enhanced efficacy of RRIKA may reveal a functional structure activity relationship and provide a template for future peptide synthesis.

Time-kill kinetics revealed a major advantage of the synthetic peptides over tested antibiotics. While RRIKA and RR eliminated MRSA within 1-2 hr after treatment, vancomycin required 24 hr to reduce the bacterial CFU by 3-log10. Linezolid, on the other hand, exhibited only a bacteriostatic effect.

Growth kinetic measurements of MRSA exposed to peptides clearly demonstrated that RRIKA and RR peptides exhibited kinetic behavior similar to membrane lytic peptides such as the lantibiotic nisin (24) and amphibian magainin (23) but different from non-lytic peptides such as fungal plectasin (29) and amphibian buforin II (23). These data suggest that our peptides act by disrupting the integrity of the bacterial membrane. To validate this hypothesis, we studied the effect of peptides on MRSA membranes using a calcein leakage assay. Similar to the well-known membrane damaging peptide, nisin, both RRIKA and RR permeabilized S. aureus cells in a concentration and time-dependent manner. These 46 observations clearly validate that the mechanism of bacterial killing by our peptides is mediated by pore formation and disruption of the bacterial cell membrane, leading to leakage of cytoplasmic contents and ultimately cell lysis. However, recent evidence suggests that lysis of bacterial cells by pore formation is not the only mechanism of microbial killing of some AMPs and there are other possible intracellular targets, such as aggregation of DNA (26). To further explore this possibility, we examined the bacterial DNA-binding ability of the peptides by electrophoretic gel retardation. Our data indicated that both RRIKA and RR bind with bacterial DNA and alter its electrophoretic mobility in agarose gels. Although a detailed study on the interaction between peptides and nucleic acids in vivo is needed, the current results lead us to suggest that RRIKA and RR might inhibit the bacterial functions by binding to bacterial DNA after perturbing the cell membranes, resulting in an augmented effect by two different mechanisms. While RRIKA and RR are not toxic to mammalian cells at the levels used here, possible crossing of the peptides to the nuclear membrane and binding to eukaryotic DNA must be examined further.

Major limitations with the use of AMPs for systemic applications are their possible inactivation by serum or physiological concentrations of salts. Serum inactivate peptides either through cleavage by proteases or through binding with proteins or lipids (30) We observed that RRIKA but not RR retained its bactericidal activity, although at a slower rate, in the presence of 10% FBS. Furthermore, both RRIKA and RR retain their antibacterial activity when tested in increasing concentration of NaCl and MgCl2; in contrast to many well studied AMPs (such as LL-37, human β-defensin-1, gramicidins, bactenecins, and ) which showed substantially reduced antibacterial activities under the same conditions (11). Previously, Turner et al. reported that LL-37 and HNP- 1 demonstrated a 12-fold and 100-fold increase in the MIC of MRSA, respectively when 100 mM NaCl was added to the test medium (10). The ability to resist the effects of salt and serum provide a selective advantage for our peptides for potential therapeutics in physiological solutions.

After identifying that our peptides were capable of inhibiting bacterial growth alone, we wanted to explore their ability to be used in combination with other antimicrobials such 47 as lysostaphin. Lysostaphin is a zinc metalloenzyme that specifically cleaves the abundant pentaglycine cross-bridges of the staphylococcal cell wall. Several studies have reported the potential applications of lysostaphin in the treatment of staphylococcal infections, however, S. aureus has developed resistance against lysostaphin via different mechanisms (31). When we tested both RR and RRIKA in combination with lysostaphin, we observed a synergistic relationship demonstrated by complete eradication of MRSA and VRSA within 20 minutes (at very low concentrations). The observed synergistic effect may be due the cleaving of the cell wall peptidoglycan by lysostaphin, which allows more access of peptides to the bacterial membrane. The synergistic relationship observed between our peptides and lysostaphin is important as it has the advantage of reducing the emergence of bacterial resistance to both agents while also minimizing drug-associated toxicity (by lowering the therapeutic dose needed for each antimicrobial agent to effectively treat MRSA infection).

One of the difficult challenges facing current antibiotics is bacterial biofilms, which are vital in the pathogenesis of staphylococcal infections. Biofilms hinder the penetration of antimicrobials to access bacteria, leading to failure of treatments (32). Our data showed that both RR and RRIKA are capable of disrupting S. aureus and S. epidermidis biofilms more efficiently than drugs of choice (vancomycin and linezolid). Most bacteria living in biofilms are either slow-growing or non-growing dormant cells that are difficult to treat with antibiotics that normally inhibit macromolecular synthesis in growing cells (33). However our peptides target mainly the microbial cell membrane, a characteristic that is not only in dividing organisms, but also in quiescent cells or stationary phase bacteria (34).

Cell toxicity is one of the major limitations in the development of antibacterials, particularly if the target of action is the cell membrane (7). We observed minimal hemolysis (less than 10%) with our peptides even with high concentrations (300 µΜ). On the other hand, 5 µM of melittin completely lysed human RBCs (100% hemolysis). Furthermore, RRIKA and RR were not toxic to HeLa cells at 8 and 4 × MIC, respectively. 48

2.5 Discussion

In this study, we evaluated the antimicrobial activity of two short synthetic peptides, and revealed their potential mechanisms of action. We observed that RRIKA and RR exhibited potent antibacterial activity against all tested S. aureus isolates. Moreover, our peptides demonstrated activity against multiple clinical isolates of MRSA, particularly MRSA USA300, a community-associated strain which is responsible for outbreaks of staphylococcal skin and soft-tissue infections (SSTI) in the United States (28). Similarly, the potent bactericidal activity was observed in other clinical MRSA isolates (USA100, USA200 and USA500) that are resistant to various antibiotic classes including macrolides, aminoglycosides, lincosamides, and fluoroquinolones. Additionally, we found that these peptides retained their activity against strains of S. aureus which have resistance to drugs of choice in treating MRSA infections, namely linezolid (MRSA NRS119) and vancomycin (VRSA strains). It is worthy to note that, the C terminus of the RR peptide was modified to contain three additional amino acids (isoleucine, lysine and alanine) which made the RRIKA peptide more amphipathic and hydrophobic and also increased its positive net charge. These modifications augmented the antibacterial activity of RRIKA which exhibited MICs two to eight folds lower than those of RR. The enhanced efficacy of RRIKA may reveal a functional structure activity relationship and provide a template for future peptide synthesis.

Time-kill kinetics revealed a major advantage of the synthetic peptides over tested antibiotics. While RRIKA and RR eliminated MRSA within 1-2 hr after treatment, vancomycin required 24 hr to reduce the bacterial CFU by 3-log10. Linezolid, on the other hand, exhibited only a bacteriostatic effect.

Growth kinetic measurements of MRSA exposed to peptides clearly demonstrated that RRIKA and RR peptides exhibited kinetic behavior similar to membrane lytic peptides such as the lantibiotic nisin (24) and amphibian magainin (23) but different from non-lytic peptides such as fungal plectasin (29) and amphibian buforin II (23). These data suggest that our peptides act by disrupting the integrity of the bacterial membrane. To validate this hypothesis, we studied the effect of peptides on MRSA membranes using a calcein leakage assay. Similar to the well-known membrane damaging peptide, nisin, both RRIKA and 49

RR permeabilized S. aureus cells in a concentration and time-dependent manner. These observations clearly validate that the mechanism of bacterial killing by our peptides is mediated by pore formation and disruption of the bacterial cell membrane, leading to leakage of cytoplasmic contents and ultimately cell lysis. However, recent evidence suggests that lysis of bacterial cells by pore formation is not the only mechanism of microbial killing of some AMPs and there are other possible intracellular targets, such as aggregation of DNA (26). To further explore this possibility, we examined the bacterial DNA-binding ability of the peptides by electrophoretic gel retardation. Our data indicated that both RRIKA and RR bind with bacterial DNA and alter its electrophoretic mobility in agarose gels. Although a detailed study on the interaction between peptides and nucleic acids in vivo is needed, the current results lead us to suggest that RRIKA and RR might inhibit the bacterial functions by binding to bacterial DNA after perturbing the cell membranes, resulting in an augmented effect by two different mechanisms. While RRIKA and RR are not toxic to mammalian cells at the levels used here, possible crossing of the peptides to the nuclear membrane and binding to eukaryotic DNA must be examined further.

Major limitations with the use of AMPs for systemic applications are their possible inactivation by serum or physiological concentrations of salts. Serum inactivate peptides either through cleavage by proteases or through binding with proteins or lipids (30) We observed that RRIKA but not RR retained its bactericidal activity, although at a slower rate, in the presence of 10% FBS. Furthermore, both RRIKA and RR retain their antibacterial activity when tested in increasing concentration of NaCl and MgCl2; in contrast to many well studied AMPs (such as LL-37, human β-defensin-1, gramicidins, bactenecins, and magainins) which showed substantially reduced antibacterial activities under the same conditions (11). Previously, Turner et al. reported that LL-37 and HNP- 1 demonstrated a 12-fold and 100-fold increase in the MIC of MRSA, respectively when 100 mM NaCl was added to the test medium (10). The ability to resist the effects of salt and serum provide a selective advantage for our peptides for potential therapeutics in physiological solutions. 50

After identifying that our peptides were capable of inhibiting bacterial growth alone, we wanted to explore their ability to be used in combination with other antimicrobials such as lysostaphin. Lysostaphin is a zinc metalloenzyme that specifically cleaves the abundant pentaglycine cross-bridges of the staphylococcal cell wall. Several studies have reported the potential applications of lysostaphin in the treatment of staphylococcal infections, however, S. aureus has developed resistance against lysostaphin via different mechanisms (31). When we tested both RR and RRIKA in combination with lysostaphin, we observed a synergistic relationship demonstrated by complete eradication of MRSA and VRSA within 20 minutes (at very low concentrations). The observed synergistic effect may be due the cleaving of the cell wall peptidoglycan by lysostaphin, which allows more access of peptides to the bacterial membrane. The synergistic relationship observed between our peptides and lysostaphin is important as it has the advantage of reducing the emergence of bacterial resistance to both agents while also minimizing drug-associated toxicity (by lowering the therapeutic dose needed for each antimicrobial agent to effectively treat MRSA infection).

One of the difficult challenges facing current antibiotics is bacterial biofilms, which are vital in the pathogenesis of staphylococcal infections. Biofilms hinder the penetration of antimicrobials to access bacteria, leading to failure of treatments (32). Our data showed that both RR and RRIKA are capable of disrupting S. aureus and S. epidermidis biofilms more efficiently than drugs of choice (vancomycin and linezolid). Most bacteria living in biofilms are either slow-growing or non-growing dormant cells that are difficult to treat with antibiotics that normally inhibit macromolecular synthesis in growing cells (33). However our peptides target mainly the microbial cell membrane, a characteristic that is not only in dividing organisms, but also in quiescent cells or stationary phase bacteria (34).

Cell toxicity is one of the major limitations in the development of antibacterials, particularly if the target of action is the cell membrane (7). We observed minimal hemolysis (less than 10%) with our peptides even with high concentrations (300 µΜ). On the other hand, 5 µM of melittin completely lysed human RBCs (100% hemolysis). Furthermore, RRIKA and RR were not toxic to HeLa cells at 8 and 4 × MIC, respectively. In conclusion, our findings reveal the potent bactericidal action of peptides RRIKA and 51

RR against MRSA. The mechanism of action of the peptides is mainly due to bacterial lysis as a consequence of bacterial membrane disruption and possibly by binding of the peptides to bacterial DNA, which interferes with necessary cellular functions vital for microbial survival. Such effects are extremely challenging for pathogens to overcome by developing resistance; unlike current antibiotics, which usually inhibit metabolic pathways that can lead to bacterial resistance (8). To date, issues such as poor pharmacokinetics have limited the potential systemic applications of therapeutic AMPs (7, 8, 30). Therefore, AMPs which have advanced into preclinical or clinical trials, are indicated for topical treatment of bacterial skin infections (7, 8, 30) such as Pexiganan (Access Pharmaceuticals, Inc.) for curing diabetic foot ulcers (35). S. aureus is the most frequently isolated microorganism in diabetic foot infections, and those caused by MRSA are associated with the worse outcomes and more frequent amputations (36-41). Additionally, bacterial biofilms appear to play an important role in increasing the difficulty of treating these ulcers (40). Furthermore, it has been proven recently that the clinical severity of S. aureus skin infection is driven by the inflammatory response to the bacteria, rather than bacterial burden (42-44). Taken together, the characteristics of the presented peptides with combined bactericidal, anti-biofilm and anti-inflammatory effect may offer an effective way for treating Staphylococcal skin infection. Therefore, these results support the potential for further study and development of RRIKA and RR as topical therapeutics particularly in an era of emerging drug resistance.

52

2.6 References

1. Naimi TS, LeDell KH, Como-Sabetti K, Borchardt SM, Boxrud DJ, Etienne J, Johnson SK, Vandenesch F, Fridkin S, O'Boyle C, Danila RN, Lynfield R. 2003. Comparison of community- and health care-associated methicillin-resistant Staphylococcus aureus infection. Jama 290:2976-2984. 2. Tsuji BT, Rybak MJ, Cheung CM, Amjad M, Kaatz GW. 2007. Community- and health care-associated methicillin-resistant Staphylococcus aureus: a comparison of molecular epidemiology and antimicrobial activities of various agents. Diagnostic microbiology and infectious disease 58:41-47. 3. Stryjewski ME, Chambers HF. 2008. Skin and soft-tissue infections caused by community-acquired methicillin-resistant Staphylococcus aureus. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 46 Suppl 5:S368-377. 4. Boucher HW, Corey GR. 2008. Epidemiology of methicillin-resistant Staphylococcus aureus. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 46 Suppl 5:S344-349. 5. Chambers HF, Deleo FR. 2009. Waves of resistance: Staphylococcus aureus in the antibiotic era. Nature reviews. Microbiology 7:629-641. 6. Boman HG. 1995. Peptide antibiotics and their role in innate immunity. Annu Rev Immunol 13:61-92. 7. Zasloff M. 2002. Antimicrobial peptides of multicellular organisms. Nature 415:389-395. 8. Hancock RE, Sahl HG. 2006. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nature biotechnology 24:1551-1557. 9. Silva ON, Mulder KC, Barbosa AE, Otero-Gonzalez AJ, Lopez-Abarrategui C, Rezende TM, Dias SC, Franco OL. 2011. Exploring the pharmacological potential of promiscuous host-defense peptides: from natural screenings to biotechnological applications. Frontiers in microbiology 2:232. 10. Turner J, Cho Y, Dinh NN, Waring AJ, Lehrer RI. 1998. Activities of LL-37, a cathelin-associated antimicrobial peptide of human neutrophils. Antimicrobial agents and chemotherapy 42:2206-2214. 11. Chu HL, Yu HY, Yip BS, Chih YH, Liang CW, Cheng HT, Cheng JW. 2013. Boosting salt resistance of short antimicrobial peptides. Antimicrobial agents and chemotherapy 57:4050-4052. 12. Lopes LB, Flynn C, Komalavilas P, Panitch A, Brophy CM, Seal BL. 2009. Inhibition of HSP27 phosphorylation by a cell-permeant MAPKAP Kinase 2 inhibitor. Biochemical and biophysical research communications 382:535-539. 13. Lopes LB, Brophy CM, Flynn CR, Yi Z, Bowen BP, Smoke C, Seal B, Panitch A, Komalavilas P. 2010. A novel cell permeant peptide inhibitor of MAPKAP kinase II inhibits intimal hyperplasia in a human saphenous vein organ culture model. J Vasc Surg 52:1596-1607. 14. Ward B, Seal BL, Brophy CM, Panitch A. 2009. Design of a bioactive cell- penetrating peptide: when a transduction domain does more than transduce. Journal of peptide science : an official publication of the European Peptide Society 15:668-674. 53

15. CLSI. 2007. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard M7-A7. CLSI, Wayne, PA. 16. Yan H, Hancock RE. 2001. Synergistic interactions between mammalian antimicrobial defense peptides. Antimicrobial agents and chemotherapy 45:1558- 1560. 17. White RL, Burgess DS, Manduru M, Bosso JA. 1996. Comparison of three different in vitro methods of detecting synergy: time-kill, checkerboard, and E test. Antimicrobial agents and chemotherapy 40:1914-1918. 18. Oliva B, Miller K, Caggiano N, O'Neill AJ, Cuny GD, Hoemann MZ, Hauske JR, Chopra I. 2003. Biological Properties of Novel Antistaphylococcal Quinoline- Indole Agents. Antimicrobial agents and chemotherapy 47:458-466. 19. Xiong YQ, Mukhopadhyay K, Yeaman MR, Adler-Moore J, Bayer AS. 2005. Functional interrelationships between cell membrane and cell wall in antimicrobial peptide-mediated killing of Staphylococcus aureus. Antimicrobial agents and chemotherapy 49:3114-3121. 20. Yan J, Wang K, Dang W, Chen R, Xie J, Zhang B, Song J, Wang R. 2013. Two hits are better than one: membrane-active and DNA binding-related double-action mechanism of NK-18, a novel antimicrobial peptide derived from mammalian NK-lysin. Antimicrob Agents Chemother 57:220-228. 21. Mosmann T. 1983. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55-63. 22. Boman HG, Agerberth B, Boman A. 1993. Mechanisms of action on Escherichia coli of cecropin P1 and PR-39, two antibacterial peptides from pig intestine. Infection and immunity 61:2978-2984. 23. Park CB, Kim HS, Kim SC. 1998. Mechanism of action of the antimicrobial peptide buforin II: buforin II kills microorganisms by penetrating the cell membrane and inhibiting cellular functions. Biochemical and biophysical research communications 244:253-257. 24. Ruhr E, Sahl HG. 1985. Mode of action of the peptide antibiotic nisin and influence on the membrane potential of whole cells and on cytoplasmic and artificial membrane vesicles. Antimicrobial agents and chemotherapy 27:841-845. 25. Koo SP, Bayer AS, Yeaman MR. 2001. Diversity in antistaphylococcal mechanisms among membrane-targeting antimicrobial peptides. Infection and immunity 69:4916-4922. 26. Brogden KA. 2005. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Micro 3:238-250. 27. Park CB, Kim HS, Kim SC. 1998. Mechanism of Action of the Antimicrobial Peptide Buforin II: Buforin II Kills Microorganisms by Penetrating the Cell Membrane and Inhibiting Cellular Functions. Biochemical and biophysical research communications 244:253-257. 28. King MD, Humphrey BJ, Wang YF, Kourbatova EV, Ray SM, Blumberg HM. 2006. Emergence of community-acquired methicillin-resistant Staphylococcus aureus USA 300 clone as the predominant cause of skin and soft-tissue infections. Ann Intern Med 144:309-317.

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29. Schneider T, Kruse T, Wimmer R, Wiedemann I, Sass V, Pag U, Jansen A, Nielsen AK, Mygind PH, Raventós DS, Neve S, Ravn B, Bonvin AMJJ, De Maria L, Andersen AS, Gammelgaard LK, Sahl H-G, Kristensen H-H. 2010. Plectasin, a Fungal Defensin, Targets the Bacterial Cell Wall Precursor Lipid II. Science 328:1168-1172. 30. Yeung AT, Gellatly SL, Hancock RE. 2011. Multifunctional cationic host defence peptides and their clinical applications. Cellular and molecular life sciences : CMLS 68:2161-2176. 31. Kumar JK. 2008. Lysostaphin: an antistaphylococcal agent. Applied microbiology and biotechnology 80:555-561. 32. Mah TF, O'Toole GA. 2001. Mechanisms of biofilm resistance to antimicrobial agents. Trends in microbiology 9:34-39. 33. Hurdle JG, O'Neill AJ, Chopra I, Lee RE. 2011. Targeting bacterial membrane function: an underexploited mechanism for treating persistent infections. Nature reviews. Microbiology 9:62-75. 34. Yeaman MR, Yount NY. 2003. Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55:27-55. 35. Lipsky BA, Holroyd KJ, Zasloff M. 2008. Topical versus systemic antimicrobial therapy for treating mildly infected diabetic foot ulcers: a randomized, controlled, double-blinded, multicenter trial of pexiganan cream. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 47:1537- 1545. 36. Dang CN, Prasad YD, Boulton AJ, Jude EB. 2003. Methicillin-resistant Staphylococcus aureus in the diabetic foot clinic: a worsening problem. Diabetic medicine : a journal of the British Diabetic Association 20:159-161. 37. Lipsky BA, Stoutenburgh U. 2005. Daptomycin for treating infected diabetic foot ulcers: evidence from a randomized, controlled trial comparing daptomycin with vancomycin or semi-synthetic penicillins for complicated skin and skin-structure infections. The Journal of antimicrobial chemotherapy 55:240-245. 38. Tentolouris N, Jude EB, Smirnof I, Knowles EA, Boulton AJ. 1999. Methicillin- resistant Staphylococcus aureus: an increasing problem in a diabetic foot clinic. Diabetic medicine : a journal of the British Diabetic Association 16:767-771. 39. Fejfarova V, Jirkovska A, Skibova J, Petkov V. 2002. [Pathogen resistance and other risk factors in the frequency of lower limb amputations in patients with the diabetic foot syndrome]. Vnitrni lekarstvi 48:302-306. 40. Lipsky BA, Berendt AR, Cornia PB, Pile JC, Peters EJ, Armstrong DG, Deery HG, Embil JM, Joseph WS, Karchmer AW, Pinzur MS, Senneville E, Infectious Diseases Society of A. 2012. 2012 Infectious Diseases Society of America clinical practice guideline for the diagnosis and treatment of diabetic foot infections. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America 54:e132-173. 41. Sotto A, Lina G, Richard JL, Combescure C, Bourg G, Vidal L, Jourdan N, Etienne J, Lavigne JP. 2008. Virulence potential of Staphylococcus aureus strains isolated from diabetic foot ulcers: a new paradigm. Diabetes Care 31:2318-2324. 55

42. Montgomery CP, Daniels MD, Zhao F, Spellberg B, Chong AS, Daum RS. 2013. Local inflammation exacerbates the severity of Staphylococcus aureus skin infection. PloS one 8:e69508. 43. Pirofski LA, Casadevall A. 2008. The damage-response framework of microbial pathogenesis and infectious diseases. Adv Exp Med Biol 635:135-146. 44. Casadevall A, Pirofski LA. 2003. The damage-response framework of microbial pathogenesis. Nature reviews. Microbiology 1:17-24.

Table 2.1. Minimum inhibitory concentration (MIC) of peptides and antibiotics against clinical and drug-resistant Staphylococci strains

MIC(µM) Strain Type Strain ID Origin Phenotypic Peptide Peptide L V N A Properties RRIKA RR Quality control ATCC - and biofilm 2 16 2 0.5 1 ≤0.25 6538 forming strain Methicillin Resistant to RN4220 United States 4 8 1 0.5 nd ≤0.25 sensitive mupirocin Staphylococcu United Resistant to NRS72 4 32 4 0.5 2 >32 s aureus Kingdom penicillin (MSSA) United NRS77 - 4 16 2 0.5 2 ≤0.25 Kingdom NRS846 - - 4 16 4 0.5 2 ≤0.25 NRS860 - - 4 16 1 0.5 2 ≤0.25 Resistant to United States ciprofloxacin, USA100 2 16 4 0.5 nd >32 (Ohio) clindamycin, erythromycin

Resistant to Methicillin ciprofloxacin, United States resistant clindamycin, USA200 (North 4 8 4 0.5 nd >32 Staphylococcu erythromycin, Carolina) s aureus gentamicin, (MRSA) and methicillin

Resistant to United States erythromycin, USA300 4 16 4 0.5 1 >32 (Mississippi) methicillin, and tetracycline United States Resistant to USA400 (North methicillin and 2 8 4 0.5 nd >32 Dakota) tetracycline

56

Table 2.2.continued United States Resistant to USA500 4 8 4 1 nd >32 (Connecticut) ciprofloxacin, Resistant to United States USA700 erythromycin 2 8 4 1 nd >32 (Louisiana) and methicillin United States Resistant to USA800 2 8 4 0.5 nd >32 (Washington) methicillin Resistant to USA100 United States erythromycin 4 8 4 0.5 nd >32 0 (Vermont) and methicillin USA110 United States Resistant to 2 8 4 1 nd >32 0 (Alaska) methicillin United States Resistant to NRS194 (North 2 8 4 1 nd >32 methicillin Dakota) Resistant to NRS108 France 4 8 4 0.5 nd >32 gentamicin United States NRS119 Resistant to 12 (Massachusett 4 16 0.5 nd >32 (LinR) linezolid 8 s) ATCC United States Resistant to 4 16 4 0.5 nd >32 43300 (Kansas) methicillin ATCC Lisbon, Multidrug- 4 16 4 0.5 nd >32 BAA-44 Portugal resistant strain Resistant to erythromycin NRS70 Japan 4 16 2 0.5 2 >32 and spectinomycin Resistant to United NRS71 tetracycline 4 16 2 0.5 2 >32 Kingdom and methicillin Resistant to NRS100 United States tetracycline 4 16 2 0.5 2 >32 and methicillin United States Resistant to NRS123 (North tetracycline 4 16 2 0.5 1 >32 Dakota) and methicillin Resistant to aminoglycoside s and tetracycline NRS1 Japan 2 16 2 8 nd >32 Glycopeptide- Vancomycin intermediate intermediate Staphylococcus Staphylococcu aureus s aureus Glycopeptide- (VISA) United States intermediate NRS19 4 8 2 4 nd >32 (Illinois) Staphylococcus aureus Glycopeptide- intermediate NRS37 France 2 8 2 8 nd >32 Staphylococcus aureus Vancomycin Resistant Resistant to >1 Staphylococcu VRS1 United States 4 32 1 1 >32 vancomycin 28 s aureus (VRSA) 57

Table 2.3.continued Resistant to VRS2 United States 4 16 1 16 1 >32 vancomycin Resistant to VRS3a United States 4 16 1 128 2 >32 vancomycin Resistant to VRS4 United States 4 32 2 128 nd >32 vancomycin Resistant to VRS5 United States 4 32 2 128 nd >32 vancomycin Resistant to >1 VRS6 United States 4 32 1 nd 2 vancomycin 28 Resistant to VRS7 United States 4 16 2 128 nd >32 vancomycin Resistant to >1 VRS8 United States 4 16 1 nd 32 vancomycin 28 Resistant to >1 VRS9 United States 4 16 1 nd >32 vancomycin 28 Resistant to >1 VRS10 United States 4 16 4 nd >32 vancomycin 28 Resistant to >1 VRS11b United States 4 32 1 nd >32 vancomycin 28 Resistant to >1 VRS12 United States 4 32 2 nd >32 vancomycin 28 Resistant to >1 VRS13 United States 4 16 1 1 16 vancomycin 28 Prototype biofilm producer, S. epidermidis NRS101 United States 2 8 2 0.5 nd >32 Resistant to methicillin and gentamicin L: linezolid, V: vancomycin, N: nisin, A: ampicillin, nd: not determined

Table 2.4. Amino acid sequence and physicochemical properties of peptides used in this study

% of amino Molecular Peptide Amino acid sequence Length Charge acids that are weight hydrophobic

RR WLRRIKAWLRR 11 1553.9 + 5 54 %

RRIKA WLRRIKAWLRRIKA 14 1866.3 + 6 57 %

KAF KAFAKLAARKA 11 1174.4 + 4 63 %

FAK FAKLAARLYRKA 12 1407.7 + 4 58 % 58

Table 2.5. Fractional inhibitory concentration (FIC) index for the combination of peptides together or with lysostaphin FIC indexa

MSSA MRSA VRSA Compound (RN4220) (USA300) (VRS13)

RRIKA RR RRIKA RR RRIKA RR

RRIKA - 0.75 - 0.75 - 0.75

RR 0.75 - 0.75 - 0.75 -

Lysostaphin 0.26 0.26 0.26 0.26 0.26 0.26 a The FIC index was determined in the presence of a fixed concentration of peptide, equivalent to ¼ ×MIC. 59

Figure 2.1. Bacterial killing kinetics of peptides.

RRIKA, RR, vancomycin and linezolid at 5X MIC against MRSA USA300 in MHB (Mueller Hinton broth). Samples treated with peptide diluent (sterile water) were used as a control. The results are given as means ± SD (n = 3; data without error bars indicate that the SD is too small to be seen)

(A ) R R IK A (B ) R R

1 0 1 0

8 8

L

L

m

m /

6 C o n tro l / 6 C o n tro l

U

U F

F R R IK A in M H B R R in M H B C

C 4 4 0

0 R R IK A in P B S 1

1 R R in P B S

g

g o o R R IK A + 1 0 % F B S R R + 1 0 % F B S

2 L 2 L

0 0 0 2 4 6 8 1 0 1 2 1 4 0 2 4 6 8 1 0 1 2 1 4 T im e (h r ) T im e (h r )

Figure 2.2. Effect of culture conditions on killing kinetics of peptides.

RRIKA (A), RR (B) at 5X MIC against MRSA USA300. Abbreviations, MHB “Mueller Hinton broth”, PBS “phosphate buffered saline” or MHB+10% FBS “fetal bovine serum”. Untreated samples were used as a control was. The killing curves were identical (overlapping in the figure) for RRIKA in MHB and in PBS.

60

(A ) M R S A " U S A 3 0 0 " (B ) V R S A " V R S 1 3 " 1 0 1 0 C o n tro l 8

R R IK A 0 .5 X M IC 8 C o n tro l L

L R R IK A 0 .5 X M IC

m R R 0 .5 X M IC

/ m

6 /

U 6 U

F L y s o s ta p h in 0 .2 5 X M IC R R 0 .5 X M IC

F

C C

0 L y s o s ta p h in 0 .2 5 X M IC 0 1 4 R R IK A 0 .5 X M IC + 1 4

g R R IK A 0 .5 X M IC +

L y s o s ta p h in 0 .2 5 X M IC g

o

o L

L L y s o s ta p h in 0 .2 5 X M IC 2 R R 0 .5 X M IC + L y s o s ta p h in 2 R R 0 .5 X M IC + 0 .2 5 X M IC L y s o s ta p h in 0 .2 5 X M IC 0 0 0 1 0 0 2 0 0 3 0 0 4 0 0 0 1 0 0 2 0 0 3 0 0 4 0 0 T im e (m in u te s ) T im e (m in u te s ) Figure 2.3. Synergistic killing of peptides with lysostaphin by time kill assay.

MRSA USA300 (A) and VRSA VRS13 (B) were incubated with 0.5X MIC of RRIKA or RR alone or in combination with 0.25X MIC of lysostaphin. Samples were obtained at different time points, then plated and counted. Control was untreated samples. The killing curves were identical (overlapping in the figure) for RRIKA and RR in combination with lysostaphin. The results are given as means ± SD (n = 3; data without error bars indicate that the SD is too small to be seen).

Figure 2.4. Killing kinetics of MRSA USA300 exposed to peptides by measuring optical density.

4X MIC of RRIKA, RR, nisin, vancomycin and sterile water (control) as observed by measuring OD595 by microplate reader over time. Results are representative of two separate experiments, each was done in triplicate. Error bars represent standard deviation values (data without error bars indicate that the SD is too small to be seen). 61

Figure 2.5. Permeabilization of the cytoplasmic membrane of MRSA USA300 as a function of peptide concentration.

Permeabilization indicated by percent of calcein leakage for 60 min exposure. The results are given as means ± SD (n = 3; data without error bars indicate that the SD is too small to be seen).

62

Figure 2.6. Interaction of peptides with plasmid DNA.

Binding was assayed by measuring inhibition of migration by plasmid DNA. Different concentrations of peptides were incubated with 250 ng for 1 hr at room temperature prior to electrophoresis on a 1.0% agarose gel. The numbers above the lanes represent the concentration (in µM) of magainin, RRIKA or RR. Lane C, control consisting of plasmid DNA only.

63

Figure 2.7. The effect of peptides and antibiotics on established biofilms of S. aureus (A) and S. epidermidis (B).

Bacteria were incubated at 37°C in glucose supplemented TSB medium for 24 h to allow biofilm formation. Then wells were rinsed with PBS and the medium was switched to MHB containing different concentrations of peptides and antibiotics. Following incubation for 24 h, wells were washed again and left for air dry. Surface associated biofilm was visualized either unstained by inverted microscope 25X objectives and an attached camera (middle) or the adherent biofilm stained by crystal violet and the wells imaged with digital camera (above), then the dye was extracted with ethanol, measured at 595 nm absorbance and presented as percentage of biofilm reduction compared to untreated wells “control” (below). All experiments were done in triplicate for statistical significance. ”. One asterisk (*) indicates statistically different than the positive control (p < 0.01). Two asterisks (**) indicates statistically different than the antibiotic treated wells (p < 0.01). 64

Figure 2.7 continued 65

Figure 2.8. The release of hemoglobin in the supernatant of human erythrocytes after treatment peptides.

Increasing amounts of RRIKA and RR was incubated with human erythrocytes for one hour and OD was measured at 415 nm. Melittin (5 µM) and 0.1 % of triton X-100 served as positive controls. Phosphate buffered saline (PBS) served as a negative control.

1 2 0 M e littin

1 0 0 R R IK A

) R R %

( 8 0

s C o n tro l

l l

e 6 0

c

e v

i 4 0 L

2 0

0 l o M M 8 4 2 6 8 4 2 r 2 6 3 1 t   1 n 0 5 1 o in C n t ti t t li li e e M M C o n c e n tra tio n (M ) Figure 2.9. Cytotoxicity of peptides to HeLa cells.

Cytotoxicity assay showing the percent mean absorbance at 490 nm after incubating HeLa cells with RRIKA and RR and melittin at different concentrations. Diluent was used as a control. Cell viability was measured by MTS assay. Results are expressed as means from three measurements ± standard deviation. 66

CHAPTER 3. EFFICACY OF SHORT NOVEL ANTIMICROBIAL AND ANTI- INFLAMMATORY PEPTIDES IN A MOUSE MODEL OF METHICILLIN- RESISTANT STAPHYLOCOCCUS AUREUS (MRSA) SKIN INFECTION

(Mohamed, M.F., Seleem, M.N (2014) Efficacy of short novel antimicrobial and anti- inflammatory peptides in a mouse model of methicillin-resistant Staphylococcus aureus (MRSA) skin infection. Drug Design, Development and Therapy 2014:8; 1979–1983.)

3.1 Abstract

The therapeutic efficacy of two novel short antimicrobial and anti-inflammatory peptides (RR and RRIKA) was evaluated in a mouse-model of staphylococcal skin infection. RR (2%) and RRIKA (2%) significantly reduced the bacterial counts and the levels of pro- inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), in methicillin-resistant Staphylococcus aureus (MRSA) USA300-0114 skin lesions. Furthermore, the combined therapy of RRIKA (1%) and lysostaphin (0.5%) had significantly higher antistaphylococcal and anti-inflammatory activity compared to monotherapy. This study supports the potential use of these peptides for topical treatment of MRSA skin infections.

3.2 Introduction

Bacterial infections, especially those caused by Staphylococcus aureus, are the most significant complication encountered in the management of wounds. (1) Furthermore, multidrug resistant S. aureus strains and their secreted toxins are responsible for interfering with the wound healing process and creating portals of entry for systemic complications in affected patients. (2) With the increasing incidence of staphylococcal resistance to topical antimicrobials such as mupirocin and fusidic acid, (3-5) there is a pressing need to develop novel antimicrobials and new approaches to circumvent this burgeoning problem. Recently, there has been increased interest in the development of antimicrobial peptides (AMPs) as novel therapeutics due to their high potency, broad spectrum of activity, and reduced potential for resistance development. (6, 7) In addition 67 to the potent bactericidal activity of AMPs, the recognized anti-inflammatory response of certain AMPs should be an advantage in the treatment of S. aureus skin infections. (8) In a recent study, we described two novel short peptides, RR (WLRRIKAWLRR) and RRIKA (WLRRIKAWLRRIKA), with potent bactericidal activity in vitro against multiple clinical isolates of MRSA (6). In particular, the peptides were active against the highly virulent MRSA USA300, a community-associated strain responsible for outbreaks of staphylococcal skin and soft-tissue infections (SSTIs) in the United States. (9) Moreover, RR and RRIKA were superior in reducing adherent biofilms of both S. aureus and S. epidermidis when compared to conventional antibiotics. Furthermore, both RR and RRIKA enhanced the antistaphylococcal activity of lysostaphin in vitro more than 1,000- fold. (6) Although lysostaphin demonstrated potent efficacy against MRSA infections in different animal models, (10-12) its therapeutic potential was hampered by the emergence of bacterial resistance. (10, 13) In the light of our previous results, showing enhancement of the antimicrobial effectiveness of lysostaphin against MRSA when combined with AMPs in vitro, (6) we moved forward with an in vivo experiment in a mouse model of MRSA skin infection.

3.3 Materials and methods

Bacterial isolate: Community-acquired methicillin-resistant S. aureus (CA-MRSA) strain NRS384 (MRSA USA300-0114 isolated from a wound from a patient in Mississippi, USA. The strain is resistant to erythromycin and tetracycline; positive for mec (subtype IV); pvl+-; MLST sequence type (ST) 8-; eGenomic spa type 1, eGenomic spa repeats YHGFMBQBLO; Ridom spa type t008; agr grp I. Peptides and antibacterial agents: RR and RRIKA were synthesized by GenScript (Piscataway, NJ). Recombinant lysostaphin was purchased from AMBI PRODUCTS LLC. (Lawrence, NY). Linezolid and fusidic acid were purchased from Chem-Impex International, Inc. (Wood Dale, IL).

68

Mice infection: Female BALB/c mice (6-8 weeks old) were obtained from Harlan Laboratories, Indianapolis, IN. All procedures were approved by the Purdue University Animal Care and Use Committee (PACUC). The murine model of MRSA skin infection has been described before. (14, 15) Eight groups of mice (n=5) were inoculated with 40 µl of MRSA USA300 (3×107) colony forming unit (CFU) intradermally. Forty-eight hours after infection and formation of an open wound, 6 groups were treated topically with either 2% fusidic acid, 2% RR, 2% RRIKA, 1% RRIKA, 0.5% lysostaphin or 1% RRIKA plus 0.5% lysostaphin formulated in 20 mg petroleum jelly. One group received vehicle only (petroleum jelly) and the last group was treated orally with linezolid (25 mg/kg). All groups were treated twice a day for 3 days. Twenty-four hours after the last treatment, the area around the wound was lightly swabbed with 70% ethanol and the wound was excised for bacterial counting after homogenization. Cytokines detection: Enzyme-linked immunosorbent assay (ELISA) development kits for detection of cytokines were purchased from R&D Systems, Inc. (Minneapolis, MN). Skin lesions (around 1 squared cm) were centrifuged at high speed for 10 minutes. The supernatant were removed and kept in -20 freezer till the time of assay. The supernatant was examined for cytokine producion: tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) using ELISA as described before. (16) Cytokine levels were expressed as percent change relative to negative control. Statistical analyses: Data were presented as mean ± SD. Statistical analyses were assessed by GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA). P values were calculated by the two-tailed Student t test. P values of ˂ 0.05 were considered significant.

3.4 Results and discussion

As shown in Figure 3.1, all treatments significantly reduced the mean bacterial counts compared with the control group (P ≤ 0.01). Among groups treated with monotherapy, the group treated with 2% RRIKA had the highest reduction in CFU (2.08 ± 0.20 log10), 69

followed by 2% fusidic acid (1.94 ± 0.36 log10), 2% RR (1.83 ± 0.30 log10), and linezolid

(1.74 ± 0.53 log10). Groups treated with 0.5 % lysostaphin or 1% RRIKA achieved a significant reduction of 1.79 ± 0.56 log10 and 1.08 ± 0.36 log10, respectively, when compared with the control group (P ≤ 0.01) Figure 3.1. The combination therapy (0.5% lysostaphin with 1% RRIKA) was significantly more effective than treatment with RRIKA or lysostaphin alone and achieved statistically significant bacterial reduction of

2.65 ± 0.44 log10 compared to the negative control (P ≤ 0.01). The bacterial reduction in the combined therapy was also statistically significant compared to monotherapy (P ≤

0.05) or control antibiotics (P ≤ 0.05). Although, there is 0.25 log10 difference between treatments of RRIKA (2%) and RR (2%), this is not statistically significant (P = 0.15). The reason for that is not clear. However, it may be explained as peptides have concentration dependent killing activity and at higher concentration (2%) they may behave the same (17, 18). Previously, RR and RRIKA showed potent immune-modulatory activities in vitro through inhibition of mitogen-activated protein kinase (MK2). (19, 20) In this study, topical treatment with RRIKA and RR significantly reduced TNF-α and IL-6 production in MRSA skin lesions. As shown in Figure 3.2 (A & B), 2% RR and 2% RRIKA reduced the TNF-α level by 49% and 56%, respectively, while the IL-6 level was reduced by 29% and 60%, respectively. Treatment with 0.5% lysostaphin and 2% fusidic acid caused 39% and 29% reduction of the TNF-α level, respectively, and 13% and 25% reduction of the IL-6 level, respectively, which is in agreement with previous findings. (21, 22) There was a synergistic anti-inflammatory response observed when 0.5% lysostaphin was combined with 1% RRIKA. The combined therapy significantly reduced TNF-α and IL-6 levels by 62% and 67%, respectively. In this study, topical application of RR and RRIKA was shown to be very effective in reducing the bacterial load in MRSA skin lesions. Moreover, peptides reduced the release of TNF-α and IL-6 which might benefit the healing of infected wounds. (26-30) In addition, the combination of RRIKA with lysostaphin is significantly more effective in the treatment of MRSA skin lesions than treatment with either peptide alone. This combination therapy is also expected to overcome some of the limitations associated with lysostaphin monotherapy through hindering the emergence of bacterial resistance and lowering the required therapeutic dose. In conclusions, our 70 findings in MRSA skin lesions should significantly impact and inform efforts to use a combination of anti-inflammatory and AMP therapies as novel topical treatment options for multidrug-resistant pathogens.

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3.5 References

1. Sen CK, Gordillo GM, Roy S, Kirsner R, Lambert L, Hunt TK, Gottrup F, Gurtner GC, Longaker MT. 2009. Human skin wounds: a major and snowballing threat to public health and the economy. Wound Repair Regen 17:763-771. 2. Guo S, Dipietro LA. 2010. Factors affecting wound healing. J Dent Res 89:219- 229. 3. Dobie D, Gray J. 2004. Fusidic acid resistance in Staphylococcus aureus. Arch Dis Child 89:74-77. 4. Kresken M, Hafner D, Schmitz FJ, Wichelhaus TA, Paul-Ehrlich-Society for C. 2004. Prevalence of mupirocin resistance in clinical isolates of Staphylococcus aureus and Staphylococcus epidermidis: results of the Antimicrobial Resistance Surveillance Study of the Paul-Ehrlich-Society for Chemotherapy, 2001. Int J Antimicrob Agents 23:577-581. 5. Chambers HF, Deleo FR. 2009. Waves of resistance: Staphylococcus aureus in the antibiotic era. Nat Rev Microbiol 7:629-641. 6. Mohamed MF, Hamed MI, Panitch A, Seleem MN. 2014. Targeting Methicillin- Resistant Staphylococcus aureus with Short Salt-Resistant Synthetic Peptides. Antimicrob Agents Chemother 58:4113-4122. 7. Hancock RE, Sahl HG. 2006. Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies. Nat Biotechnol 24:1551-1557. 8. Montgomery CP, Daniels MD, Zhao F, Spellberg B, Chong AS, Daum RS. 2013. Local inflammation exacerbates the severity of Staphylococcus aureus skin infection. PLoS One 8:e69508. 9. King MD, Humphrey BJ, Wang YF, Kourbatova EV, Ray SM, Blumberg HM. 2006. Emergence of community-acquired methicillin-resistant Staphylococcus aureus USA 300 clone as the predominant cause of skin and soft-tissue infections. Ann Intern Med 144:309-317. 10. Kumar JK. 2008. Lysostaphin: an antistaphylococcal agent. Appl Microbiol Biotechnol 80:555-561. 11. Desbois AP, Gemmell CG, Coote PJ. 2010. In vivo efficacy of the antimicrobial peptide ranalexin in combination with the lysostaphin against wound and systemic meticillin-resistant Staphylococcus aureus (MRSA) infections. International Journal of Antimicrobial Agents 35:559-565. 12. Kokai-Kun JF, Walsh SM, Chanturiya T, Mond JJ. 2003. Lysostaphin cream eradicates Staphylococcus aureus nasal colonization in a cotton rat model. Antimicrobial Agents and Chemotherapy 47:1589-1597. 13. Kusuma C, Jadanova A, Chanturiya T, Kokai-Kun JF. 2007. Lysostaphin-resistant variants of Staphylococcus aureus demonstrate reduced fitness in vitro and in vivo. Antimicrobial Agents and Chemotherapy 51:475-482. 14. Cho JS, Zussman J, Donegan NP, Ramos RI, Garcia NC, Uslan DZ, Iwakura Y, Simon SI, Cheung AL, Modlin RL, Kim J, Miller LS. 2011. Noninvasive in vivo imaging to evaluate immune responses and antimicrobial therapy against Staphylococcus aureus and USA300 MRSA skin infections. J Invest Dermatol 131:907-915. 72

15. Cho JS, Pietras EM, Garcia NC, Ramos RI, Farzam DM, Monroe HR, Magorien JE, Blauvelt A, Kolls JK, Cheung AL, Cheng G, Modlin RL, Miller LS. 2010. IL- 17 is essential for host defense against cutaneous Staphylococcus aureus infection in mice. J Clin Invest 120:1762-1773. 16. De Vry CG, Valdez M, Lazarov M, Muhr E, Buelow R, Fong T, Iyer S. 2005. Topical application of a novel immunomodulatory peptide, RDP58, reduces skin inflammation in the phorbol ester-induced dermatitis model. J Invest Dermatol 125:473-481. 17. Lohner K, Blondelle SE. 2005. Molecular mechanisms of membrane perturbation by antimicrobial peptides and the use of biophysical studies in the design of novel peptide antibiotics. Comb Chem High Throughput Screen 8:241-256. 18. Yeaman MR, Yount NY. 2003. Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55:27-55. 19. Ward B, Seal BL, Brophy CM, Panitch A. 2009. Design of a bioactive cell- penetrating peptide: when a transduction domain does more than transduce. J Pept Sci 15:668-674. 20. Lopes LB, Brophy CM, Flynn CR, Yi Z, Bowen BP, Smoke C, Seal B, Panitch A, Komalavilas P. 2010. A novel cell permeant peptide inhibitor of MAPKAP kinase II inhibits intimal hyperplasia in a human saphenous vein organ culture model. J Vasc Surg 52:1596-1607. 21. Schmelcher M, Powell AM, Becker SC, Camp MJ, Donovan DM. 2012. Chimeric phage lysins act synergistically with lysostaphin to kill mastitis-causing Staphylococcus aureus in murine mammary glands. Appl Environ Microbiol 78:2297-2305. 22. Rubin BK, Tamaoki J. 2005. Antibiotics as anti-inflammatory and immunomodulatory agents. Birkhä user, Basel ; Boston. 23. Miller LS, Cho JS. 2011. Immunity against Staphylococcus aureus cutaneous infections. Nat Rev Immunol 11:505-518. 24. Cho JS, Pietras EM, Garcia NC, Ramos RI, Farzam DM, Monroe HR, Magorien JE, Blauvelt A, Kolls JK, Cheung AL, Cheng GH, Modlin RL, Miller LS. 2010. IL-17 is essential for host defense against cutaneous Staphylococcus aureus infection in mice. Journal of Clinical Investigation 120:1762-1773. 25. Cho JS, Guo Y, Ramos RI, Hebroni F, Plaisier SB, Xuan CY, Granick JL, Matsushima H, Takashima A, Iwakura Y, Cheung AL, Cheng GH, Lee DJ, Simon SI, Miller LS. 2012. Neutrophil-derived IL-1 beta Is Sufficient for Abscess Formation in Immunity against Staphylococcus aureus in Mice. Plos Pathogens 8. 26. Jialal I, Miguelino E, Griffen SC, Devaraj S. 2007. Concomitant reduction of low- density lipoprotein-cholesterol and biomarkers of inflammation with low-dose simvastatin therapy in patients with type 1 diabetes. J Clin Endocrinol Metab 92:3136-3140. 27. Wallace HJ, Stacey MC. 1998. Levels of tumor necrosis factor-alpha (TNF-alpha) and soluble TNF receptors in chronic venous leg ulcers--correlations to healing status. J Invest Dermatol 110:292-296. 28. Cowin AJ, Hatzirodos N, Rigden J, Fitridge R, Belford DA. 2006. Etanercept decreases tumor necrosis factor-alpha activity in chronic wound fluid. Wound Repair Regen 14:421-426. 73

29. Donath MY. 2014. Targeting inflammation in the treatment of type 2 diabetes: time to start. Nat Rev Drug Discov 13:465-476. 30. Khanna S, Biswas S, Shang Y, Collard E, Azad A, Kauh C, Bhasker V, Gordillo GM, Sen CK, Roy S. 2010. Macrophage dysfunction impairs resolution of inflammation in the wounds of diabetic mice. PLoS One 5:e9539.

Figure 3.1. Efficacy of treatment of MRSA skin lesions with peptides and antibiotics.

Mice were treated topically with RRIKA (2%), RR (2%), fusidic acid (2%), linezolid (25 mg/kg), RRIKA (1%), lysostaphin (0.5%), combined therapy of RRIKA (1%) and lysostaphin (0.5 %) and petroleum jelly (negative control) twice daily for 3 days. The two tailed Student t test, was used to determine statistical significance (a P value of < 0.05 was considered significant) (* P ≤ 0.05) (** P ≤ 0.01). 74

Figure 3.2. The effect of peptides on cytokines (TNF-α, and IL-6) production in MRSA skin lesions.

Tissue homogenate supernatants were examined for cytokine production using ELISA. Cytokine levels were expressed as percent change relative to negative control. Data are presented as mean ± SD from duplicates consisting of four mice per group. Statistical analysis was calculated by the two-tailed Student t test. P values of ˂ 0.05 were considered significant (*).

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CHAPTER 4. ANTIBACTERIAL ACTIVITY OF NOVEL CATIONIC PEPTIDES AGAINST CLINICAL ISOLATES OF MULTI-DRUG RESISTANT STAPHYLOCOCCUS PSEUDINTERMEDIUS FROM INFECTED DOGS

(Mohamed, M.F., Hammac G.K., Guptill, L. and Seleem, M.N (2014) Antibacterial activity of novel cationic peptides against clinical isolates of multi-drug resistant Staphylococcus pseudintermedius from infected dogs. PLoS ONE 9(12): e116259. doi: 10.1371/journal.pone.0116259)

4.1 Abstract

Staphylococcus pseudintermedius is a major cause of skin and soft tissue infections in companion animals and has zoonotic potential. Additionally, methicillin-resistant S. pseudintermedius (MRSP) has emerged with resistance to virtually all classes of antimicrobials. Thus, novel treatment options with new modes of action are required. Here, we investigated the antimicrobial activity of six synthetic short peptides against clinical isolates of methicillin-susceptible and MRSP isolated from infected dogs. All six peptides demonstrated potent anti-staphylococcal activity regardless of existing resistance phenotype. The most effective peptides were RRIKA (with modified C terminus to increase amphipathicity and hydrophobicity) and WR-12 (α-helical peptide consisting exclusively of arginine and tryptophan) with minimum inhibitory concentration50 (MIC50) of 1 µM and MIC90 of 2 µM. RR (short anti-inflammatory peptide) and IK8 “D isoform” demonstrated good antimicrobial activity with MIC50 of 4 µM and MIC90 of 8 µM. Penetratin and (KFF)3K (two cell penetrating peptides) were the least effective with MIC50 of 8µM and MIC90 of 16 µM. Killing kinetics revealed a major advantage of peptides over conventional antibiotics, demonstrating potent bactericidal activity within minutes. Studies with propidium iodide and transmission electron microscopy revealed that peptides damaged the bacterial membrane leading to leakage of cytoplasmic contents and consequently, cell death. A potent synergistic increase in the antibacterial effect of the cell penetrating peptide (KFF)3K was noticed when combined with other peptides and 76 with antibiotics. In addition, all peptides displayed synergistic interactions when combined together. Furthermore, peptides demonstrated good therapeutic indices with minimal toxicity toward mammalian cells. Resistance to peptides did not evolve after 10 passages of S. pseudintermedius at sub-inhibitory concentration. However, the MICs of amikacin and ciprofloxacin increased 32 and 8 fold, respectively; under similar conditions. Taken together, these results support designing of peptide-based therapeutics for combating MRSP infections, particularly for topical application.

4.2 Introduction

Methicillin-susceptible Staphylococcus pseudintermedius (MSSP) and methicillin- resistant S. pseudintermedius (MRSP) are a leading cause of skin and ear infections and post-operative wound infections in dogs and cats (1, 2). S. pseudintermedius isolates can also cause infections in humans as apparent zoonotic transfer from dogs has been reported (2-5). Similar to methicillin-resistant Staphylococcus aureus (MRSA), MRSP is a nosocomial pathogen that can colonize personnel in veterinary hospitals (1, 6). Recent studies reported that MRSP from Europe and North America emerged resistance to virtually all classes of antimicrobial agents used in veterinary medicine (7). Such dissemination of multidrug resistant staphylococci among dogs raises concern due to the few therapeutic options available for treatment (8). Therefore, there is an urgent need for novel antimicrobial compounds with new mechanisms of action. Antimicrobial peptides (AMPs) serve as an alternative novel therapeutic approach against microbial infections. AMPs constitute the first line of defense against invading pathogens in most multicellular organisms. They have been discovered from a broad range of organisms, from microorganisms to plants and from insects to mammals (9). AMPs are generally between 12 and 50 amino acids in length with a cationic charge and contains up to 50% hydrophobic amino acids. They have the ability to form an amphipathic secondary structure that allows the peptides to partition into the bacterial membrane lipid bilayer (10). The mechanism of action of AMPs involves binding to the negatively charged anionic phospholipids on lipopolysaccharide (LPS) of Gram-negative bacteria or to the teichoic 77 acids of Gram-positive bacteria. Once there are sufficient aggregated peptides, they destabilize the lipid head groups, to produce pores by one of various potential mechanisms (barrel-stave, carpet or toroidal-pore) leading to leakage of cytoplasmic contents and bacterial cell death (9, 11). However, membrane disruption is not the only proven mechanism of bacterial killing by AMPs. Instead, peptides can traverse bacterial membranes and induce killing through inhibition of specific macromolecular synthesis pathways (11). Several studies have reported the potency of AMPs in combating S. aureus infections (12, 13); however, to our knowledge, there are limited data about their activity and potential use against S. pseudintermedius (14, 15). In the present study, we investigated the antimicrobial activity of six synthetic short peptides against clinical isolates of MSSP and MRSP from infected dogs. Moreover, we performed a series of experiments to explore their antibacterial mechanism of action. Finally, we examined their toxicity toward mammalian cells and assessed the ability of S. pseudintermedius to develop resistance to peptides.

4.3 Materials and methods

Peptides, antibiotics and reagents

Peptides (RRIKA, RR, WR-12, IK8 “D isoform”, (KFF)3K and penetratin) were synthesized by GenScript (Piscataway, NJ) using solid-phase 9-fluorenylmethoxy carbonyl (Fmoc) chemistry and purified to a purity of 98% using reverse-phase high- performance liquid chromatography (HPLC). Peptide mass was confirmed by mass spectrometry (Table 4.1). Nisin (Sigma, N5764), melittin from honey bee venom (Sigma, M2272), ampicillin sodium salt (IBI Scientific), ciprofloxacin (Sigma), amikacin hydrate (Sigma) and propidium iodide (Molecular Probes, Life Technologies) were all purchased from commercial vendors. Mueller-Hinton broth (MHB) and Mueller-Hinton agar (MHA) were purchased from Sigma-Aldrich, while trypticase soy broth (TSB) and trypticase soy agar (TSA) were purchased from Becton-Dickinson, Cockeysville, Md.

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Bacterial isolates

Forty isolates of S. pseudintermedius (30 MSSP and 10 MRSP) identified at the Indiana Animal Disease Diagnostic laboratory from specimens collected from dogs admitted to the small animal teaching hospital at Purdue University were included in the study (Tables 4.2 and 4.3). Clinical specimens were inoculated onto 5% sheep blood agar and incubated at 35°C for 18-24 hours. White, beta-hemolytic colonies with morphology suggestive of Staphylococcus sp. were used to inoculate a coagulase tube test and were identified to the species level using the Vitek 2 (BioMérieux). Vitek identification of S. pseudintermedius was confirmed by a positive coagulase test. Antimicrobial susceptibility was determined by broth microdilution using the SensiTitre (Trek). Isolates demonstrating resistance to oxacillin, a surrogate for methicillin, with a MIC greater than or equal to 0.5 μg/mL were screened for mecA by PCR as previously described (16). A mecA positive result was assigned to samples with a visible 310 bp band on a 1.5% agarose gel.

Antibacterial assays

The broth microdilution technique was used to determine the minimum inhibitory concentrations (MIC) of peptides and antibiotics in MHB in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines (17). Peptides were dissolved in sterile distilled water containing 0.02% acetic acid. MIC assays were carried out with an initial bacterial inoculum of 5×105 colony forming unit (CFU/ml). Peptides and antibiotics were added to Polystyrene 96 well plates (CytoOne, CC7672-7596) at desired concentrations and plates were incubated for 24 hr at 37 ºC. MIC was defined as the lowest concentration of peptide which inhibited the visible growth of bacteria. MIC was done in triplicates and the highest value was reported.

Time kill assay

One strain of MRSP (SP3) was grown overnight in MHB, then diluted in fresh MHB and incubated for 2 hr until logarithmic growth phase was achieved. Then bacteria were centrifuged and washed twice with phosphate buffered saline (PBS) and adjusted to ~5×105 CFU/ml in MHB. Peptides and antibiotic (amikacin) at 5X MIC were added in triplicates to adjusted bacteria, and incubated aerobically at 37 ºC. Aliquots at specified 79 time points were taken, serially diluted in PBS and plated in triplicate on MHA. CFUs were counted after incubation of plates for 24 hr at 37 ºC.

Growth Kinetics

The turbidity of bacterial culture after exposure to peptides was measured as described previously (13). Briefly, logarithmic growth of SP 3 was diluted in MHB to reach OD600 ≈ 0.25. One mL of diluted bacteria was added to a plastic cuvette (light path 1 cm). Peptides and antibiotics were added to cuvettes at a concentration equivalent to 5 X MIC as determined in antibacterial assays above. Cuvettes were incubated aerobically at 37 ºC. At specific time points, OD was measured at 600 nm wavelength by spectrophotometer. Cuvettes containing MHB without bacteria and with the same concentrations of peptide or antibiotic served as blanks. Nisin at 5 X MIC and untreated bacteria were used as positive and negative controls, respectively. The assay was repeated twice and the average was reported.

Bacterial membrane permeability

Bacterial membrane damage was assessed by fluorescence change of propidium iodide dye as described previously (18). Briefly, SP3 was grown in MHB to logarithmic phase of growth then diluted to OD600 ≈ 0.25. Propidium iodide was mixed with bacteria to a final concentration of 10 µM. Aliquots of 200 µL were then loaded into black wall 96 microplates and drugs were added at 5 X and 10 X MIC in triplicate. Fluorescence was monitored for 30 minutes at excitation and emission wavelengths of 585 and 620 nm, respectively, using a fluorescence plate reader (FLx800 model BioTek® Instruments, Inc. Winooski, Vermont). The percentage of membrane permeabilization was calculated as the percent of fluorescent intensity of peptide-treated samples with respect to fluorescence intensity of propidium iodide-loaded, peptide untreated samples.

Transmission electron microscopy

SP3 was grown to OD600 0.2. Bacterial cells were harvested by centrifugation, resuspended in PBS, and treated with peptides at 10 X MIC for 30 minutes. RRIKA and penetratin were also tested at lower concentration 1 X MIC to study the concentration- dependent effect of peptides. After treatment with peptides, the bacterial pellets were fixed 80 with 2.5% buffered glutaraldehyde for 1 hour. After fixation, cells were treated with 1% buffered osmium tetroxide for 1 hour, stained en bloc with 1% uranyl acetate, dehydrated in a graded series of ethanol, and embedded in white resin. The buffer used was 0.1M sodium cacodylate, pH 7.4. Ultrathin sections were prepared on Formvar-coated grids and stained with 1% uranyl acetate and lead citrate. Microscopy was performed with a Philips CM-100 microscope under standard operating conditions.

Synergism studies

The synergistic effect between peptides and antimicrobial agents was determined by the combination assay as described previously (19). Peptide MICs against SP3 were determined in triplicate. Two-fold serial dilutions of antimicrobials (antibiotic and peptides) were tested in the presence of a fixed concentration of peptide of concern (equal to ¼ × peptide MIC, which did not inhibit the growth of bacteria alone). The fractional inhibitory concentration (FIC) index was calculated as follows: FIC of drug A = MIC of drug A in combination/MIC of drug A alone, FIC of drug B = MIC of drug B in combination/MIC of drug B alone, and FIC index = FIC of drug A + FIC of drug B. An FIC index of ≤ 0.5 is considered to demonstrate synergy. Additive was defined as an FIC index of 1. Antagonism was defined as an FIC index of > 4.

Cytotoxicity analysis

Peptides were assayed at different concentrations against macrophage-like cell line (J774A.1) and human keratinocyte (HaCat) to determine the potential toxic effect in vitro. Briefly, J774A.1 and HaCat cells were seeded at a density of 1.5 x 104 per well in a tissue culture 96-well plate (CytoOne, CC7682-7596) in DMEM media supplemented with 10% fetal bovine serum (FBS), and incubated at 37°C in a 5% CO2 atmosphere for 24 hours. The cells were treated with peptides at concentrations range from 8 to 256 µM for 24 hours. Untreated cells were used as a negative control. Melittin 5 µM and DMSO 10% were used as a positive control for J774A.1 and HaCat cells, respectively. After incubation, the cells were washed three times with PBS and the media in each well were replaced with 100 µL of DMEM media and 20 µL of assay reagent, MTS 3-(4,5-dimethylthiazol-2-yl)- 5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Promega, Madison, WI, USA). The cells were incubated further for 4 h at 37 ºC. Corrected absorbance 81 readings (actual absorbance readings for each treatment subtracted from background absorbance) were taken using a kinetic ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA). Cell viability was expressed as percentage of absorbance in comparison to negative control, untreated cells. Therapeutic index of the peptides, was calculated as the ratio of mammalian IC50 (the concentration of peptide which inhibit 50% of mammalian cells) to geometric mean of the MIC of peptides against S. pseudintermedius. When there was no detectable cytotoxicity at 256 µM, we used a more than sign “>” to calculate the therapeutic index. Larger values of the therapeutic index indicate greater cell selectivity of peptides. (20)

Multi-step resistance selection

To assess the ability of S. pseudintermedius to develop resistance to the peptides and antibiotics we used amikacin and ciprofloxacin-susceptible strain (SP6) in a multi-step resistance selection experiment (Table 4.2). SP6 strain was exposed for ten passages over a period of ten days to various concentrations of the peptides (RRIKA and IK8 “D isoform”) and antibiotics (amikacin and ciprofloxacin). For each subsequent passage, the inoculum for the MIC determination was adjusted to a final density of approximately 5 × 5 1 10 CFU/mL using the bacterial cells growing at sub-inhibitory concentration ( /2 X MIC). Bacteria were incubated for 16 hr and the MIC value was recorded for each passage. Results were expressed as the ratio of MIC value after each passage compared to the initial MIC value.

Statistical analyses

Cytotoxicity data were subjected to statistical analysis using GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA). Cytotoxicity data were analyzed using one-way ANOVA, with post hoc Tukey’s multiple comparisons test. P-values of ˂ 0.01 were considered significant.

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4.4 Results

Antimicrobial activity

The amino acid sequences and characteristics of the synthetic peptides used in this study are shown in Table 4.1. All synthetic peptides demonstrated potent inhibitory activity. As shown in Table 4.2, the most effective peptides were RRIKA and WR-12 with MIC50 and

MIC90 (the MICs at which 50% and 90% of the isolates were inhibited, respectively) were found to be 1 µM and 2 µM, respectively. RR and IK8 “D isoform” demonstrated good antimicrobial activity with MIC50 of 4 µM and MIC90 of 8 µM. Penetratin and (KFF)3K were the least effective with MIC50 of 8 µM and MIC90 of 16 µM.

The MICs for MRSP were not significantly different from those for the nonresistant strains. Interestingly, some peptides (such as Penetratin and (KFF)3K) demonstrated lower

MICs values for methicillin-resistant (MIC50 and MIC90 equal to 4 and 8 µM, respectively) compared to methicillin sensitive strains (Table 4.3).

Time kill assay

To determine the killing kinetics of peptides, we exposed a MRSP strain (SP3) to 5 X MIC of peptides and control antibiotic (amikacin). As depicted in Figure 4.1, all peptides demonstrated rapid bactericidal activity. RRIKA, RR and (KFF)3K were the most rapid in killing as viable MRSP were not detectable after 10 minutes. Penetratin, IK8 “D isoform” and WR-12 achieved more than 5 log10 reduction within 25, 45 and 60 minutes respectively. In contrast, amikacin achieved less than one log10 reduction within 3 hours and demonstrated bactericidal activity only after 12 hr (data not shown).

Growth kinetics

As a primary objective to understand the mechanism of action of peptides against S. pseudintermedius, we monitored the turbidity of SP3 culture exposed to 5 X MIC of peptides over time by a spectrophotometer. As shown in Figure 4.2, all peptides reduced the optical density of S. pseudintermedius suspensions, and the degree of reduction over time varied among peptides. Results for RRIKA, RR and (KFF)3K were similar in rate and percentage to the rapidly acting membrane-damaging peptide, nisin (21). These peptides produced greater than 50% and 95% reductions in turbidity after 60 and 120 83 minutes, respectively. IK8 “D isoform” and WR-12 achieved a similar percentage of turbidity reduction as nisin albeit at a slower rate (after 3 and 7 hours). However, the culture turbidity of SP exposed to penetratin remained constant for 2 hours, and turbidity was reduced by less than 50% after 7 hours. In contrast, a DNA synthesis inhibitor, ciprofloxacin, and a protein synthesis inhibitor, amikacin, did not reduce the culture turbidity within the same time frame, indicating a different mechanism of action.

Membrane permeability

In order to observe the permeability effect of peptides on MRSP membrane, we monitored the fluorescent intensity of bacterial culture mixed with propidium iodide (PI) after exposure to peptides. As shown in Figure 4.3, fluorescence intensity remained stable during incubation of untreated bacteria in the presence of PI. This result confirmed the integrity of the bacterial membrane under control conditions. When exposed to peptides, the cells became permeable to PI and fluorescence increased within 2 minutes, suggesting very rapid membrane disruption activity. The permeabilization effect was concentration and time-dependent. At 5X MIC; nisin, RRIKA, RR and IK8 “D isoform” were faster and more potent at membrane perturbation in comparison to (KFF)3K, WR-12 and penetratin (Figure 4.3A). However, when the concentration of peptides was increased to 10 X MIC, similar pattern was observed for all peptides, suggesting dose dependent activity (Figure 4.3B). In contrast, ciprofloxacin, as expected, had no effect on membrane permeabilization, even at a higher concentration.

Transmission electron microscopy

To explore the possible alternative mechanism of action of peptides on S. pseudintermedius, transmission electron microscopy (TEM) was done on thin sections of MRSP (SP3) that had been exposed to peptides for 30 min (Figure 4.4). TEM micrographs of control S. pseudintermedius in dividing cells (Figure 4.4A) and single cells (Figure 4.4B) displayed round cocci with intact cell membranes and well-defined cell wall. The nucleoid region showed a non-homogenous electron density. However, after exposure to peptides, the cytoplasm showed a more homogenous electron density (Figures 4.4C, D and E). At a 1X MIC of RRIKA, multiple rounded double-layered mesosome-like structures were noticed as well as rounded bodies in the cytoplasm with similar electron 84 density as that of the septal cell wall layer (Figures 4.4C and D). At 10 X MIC of RRIKA, pore formation and lysed cells were observed (Figure 4.4E&F). RR (at 10 X MIC) showed the most damaging effect on the cell wall, including alteration in cell wall thickness and cell wall rupture (Figure 4.4G). (KFF)3K caused deviated septa and cytoplasmic inclusions. (Figure 4.4H). Cell wall disintegration and pore formation could be seen in IK8 “D isoform” treated bacteria (Figure 4.4I). Furthermore, some large non-membrane- enclosed bodies with similar electron density to that of the cell wall murein layer were seen in S. pseudintermedius cells at a 10 X MIC of WR-12 (Figure 4.4K). Penetratin at 1 X MIC showed the lowest damaging effect (or didn’t show clear damaging effect) as most cells had normal appearance (data not shown) with the exception of small percentage of cells that displayed a disintegrated membrane separated from the cell wall (Figure 4.4L). However, at 10 X MIC, large pores were seen (Figure 4.4M). Taken together, these data demonstrate the membrane-damaging effect of the investigated peptides.

Synergism studies

As shown in Table 4.4, all peptides displayed synergistic interactions when combined together. The FIC index varied from 0.37 to 0.5. When peptides were combined with antibiotics; only (KFF)3K displayed potent synergism with amikacin and gentamicin with FIC index equal to 0.37 and 0.26, respectively. However, RRIKA, RR, WR-12, IK-8 and penetratin showed an additive effect with amikacin and gentamicin. Notably, there were no antagonistic effects observed between peptides and antibiotics.

Cytotoxicity studies

To evaluate the safety of the peptides toward mammalian cells, we assessed the cytotoxic effect of different concentration of peptides on macrophage-like cell line (J774A.1) and human keratinocyte (HaCat) by the MTS assay. RR, IK8 “D isoform”, (KFF)3K and penetratin peptides were not toxic to mammalian cells at concentrations up to 64µM (Figure 4.5). RRIKA and WR-12, were not toxic up to 32 µM, however they exhibited toxicity at 64 µM (P <0.01). In contrast, melittin peptide was toxic to J774A.1at 5 µM (P <0.01); this concentration is equal to the MIC of the melittin (Figure 4.5). 85

Table 4.5 shows the therapeutic index of the peptides. All peptides displayed higher cell selectivity with therapeutic index of RRIKA, RR, WR-12, IK8 “D isoform”, (KFF)3K and penetratin equal to 55.6, 31, 85.3, >51.2, 33 and >29.2, respectively, in case of macrophage-like cell line and 111.2, 62, 85.3, >51.2, >33 and >29.2, respectively, in case of human keratinocyte suggesting a favorable drug safety profile. On the other hand, melittin exhibited a very low therapeutic index of 1 (TI=1) in case of macrophage-like cell line.

Multi-step resistance selection

In order to investigate if the physical perturbation and disruption of microbial membranes by peptides can adequately overcome conventional mechanisms of drug resistance, SP6 isolate exposed to sub-inhibitory concentrations of IK8 “D isoform” and RRIKA were subcultured for ten serial passages to determine if a shift in the MIC of each peptide tested would be observed against MSSP. Two classes of clinically used antibiotics including the protein synthesis inhibitor, amikacin and DNA synthesis inhibitor, ciprofloxacin were included as controls in this study. As shown in Figure 4.6, MSSP was not able to develop resistance to the peptides despite the repeated exposure with sub-lethal doses of peptides. However, treatment of SP6 with amikacin induced a shift in the MIC as early as passage 4. There was a 32 fold increase in the MIC against amikacin by passage 10. Ciprofloxacin mediated a slightly delayed onset of drug resistance, with doubling of MIC occurring later at passage 6 which subsequently increased to 8-fold the original MIC value by passage 10.

4.5 Discussion

The rapid emergence of bacterial resistance among S. pseudintermedius isolates and the dearth of effective antimicrobials call for alternative strategies to battle infections caused by this pathogen. One approach which warrants attention as a potential novel therapy for S. pseudintermedius infections is antimicrobial peptides (AMPs). There are very few reports describing the antimicrobial activity of naturally-derived AMPs against S. pseudintermedius (14, 15). Additionally, there are several drawbacks of naturally-derived AMPs, including high toxicity and increased cost of production due to the complexity of 86 their design and their large size, which will limit their translational clinical applications (3, 4). Given the significant problem posed by the rapid emergence of multidrug-resistant S. pseudintermedius and the potential benefits of using AMPs as an alternative therapy, more effort and attention needs to be focused on developing potent, short, and less toxic AMPs to overcome their therapeutic limitations. In the present study, six synthetic peptides were investigated for their ability to inhibit clinical isolates of MSSP and MRSP (Table 4.1). These peptides were chosen due to their short sequence (8-16 amino acids), their simple composition, and their cationic charge. The AMPs, RR and RRIKA, are derivatives of an anti-inflammatory cell-penetrating peptide (13, 22, 23). Previously, the efficacy of these two peptides against human MRSA clinical isolates were shown in vitro and in a mouse model of MRSA skin infection (13, 23). In light of our previous success in targeting MRSA (13, 23), we tested these two peptides against MSSP and MRSP. RR exhibited potent antimicrobial activity against clinical isolates of S. pseudintermedius with

MIC50 and MIC90 of 4 µM and 8 µM, respectively. Furthermore, addition of three amino acids (I, K and A) in the C terminus of RR significantly enhanced the antimicrobial activity fourfold against S. pseudintermedius. This enhancement of activity is probably due to the enhanced amphipathicity, hydrophobicity, and net charge of the newly derived peptide RRIKA. RRIKA exhibited antimicrobial activity against clinical isolates of S. pseudintermedius with MIC50 and MIC90 of 1 µM and 2 µM, respectively. Additionally, further advantage of using these two peptides has been demonstrated by their potent activity in physiological concentrations of NaCl and MgCl2, in contrast to most natural AMPs (13, 24). Previous studies demonstrated the activity of WR-12 and IK8 “D isoform” peptides against S. aureus (25, 26). However, their activity against S. pseudintermedius was not investigated. WR-12 is a broad-spectrum AMP and consists exclusively of arginine and tryptophan, arranged to form idealized amphipathic helices (25). In our study, WR-12 demonstrated potent antimicrobial activity against MSSP and MRSP with MIC50 and

MIC90 of 1 µM and 2 µM, respectively. Peptide IK8 “D isoform” in a previous study showed potent antimicrobial activities against MRSA (26). Additionally, the D-amino acids substitution of this peptide prevented proteolytic degradation by mammalian or microbial proteases, which makes it an excellent candidate for in vivo applications (26). 87

Peptide IK8 “D isoform” in our study exhibited MIC50 and MIC90 of 4 µM and 8 µM, respectively, against MSSP and MRSP. The antimicrobial activities of the cell-penetrating peptides (CPPs) have been widely explored due to their low toxicity and their potential clinical applications (27-31). We sought to investigate the antimicrobial activity of two CPPs, penetratin and (KFF)3K, that share the same characteristic of AMPs, including basic amino acids composition and cationic charge. The CPPs showed antimicrobial activity against MSSP with MIC50 and

MIC90 of 8 µM and 16 µM, respectively. Furthermore, they showed improved activity against MRSP with MIC50 and MIC90 of 4 µM and 8 µM, respectively. We assessed the bacterial killing kinetics of the peptides in comparison with the aminoglycoside antibiotic, amikacin. The peptides showed total and rapid elimination of S. pseudintermedius within minutes, while amikacin exhibited a much slower killing kinetic with only 3 log10 reduction after 12 hours incubation (Figure 4.1). This fast bacterial elimination by the AMPs represents a unique advantage of the peptides over conventional antibiotics that should lead to better treatment outcomes. Understanding the mechanism of antimicrobial activity of these AMPs on S. pseudintermedius is an essential step for enhancing their activity, reducing their toxicity, and furthering their development as a potential therapeutic option. Hence, we explored S. pseudintermedius membrane-perturbing abilities of these peptides. Growth kinetics study of S. pseudintermedius incubated with the peptides and control antibiotics revealed a membrane-damaging activity and a lytic mechanism of action similar to the lytic peptide nisin (Figure 4.2). The membrane-damaging activity of the peptides was further confirmed by the ability of propidium iodide (PI) to enter damaged S. pseudintermedius and intercalate with bacterial DNA. The fluorescence intensity of PI increased significantly after 2 minutes of addition of the peptides, clearly demonstrating their fast permeabilization characteristic (Figure 4.3). Additionally, transmission electron microscopy of S. pseudintermedius that had been exposed to the peptides for 30 minutes showed the presence of mesosome structure and damaged membranes (Figure 4.4). The observed membrane-damaging effect is similar to those seen with well-characterized AMPs such as defensins and bactenecins (32, 33). 88

The checkerboard assay was utilized to ascertain whether these peptides have potential to be combined with each other or with antibiotics against S. pseudintermedius (34). Although peptides that work by the same mechanism of action are less likely to exhibit synergistic activity, our peptides were found to exhibit a synergistic relationship with each other against S. pseudintermedius (Table 4.4). This finding suggests that, although these peptides share a common mechanism of action by damaging the bacterial membrane, they may use different pathways such as barrel-stave, carpet or toroidal-pore to permeabilize the bacterial membrane (9, 11). Collectively, these results provide valuable insight into potential combination therapy that could reduce the dose of each peptide and minimize potential host toxicity and emergence of resistant pathogens. With the exception of

(KFF)3K, none of the peptides showed synergistic activity with antibiotics. This result is probably due to their rapid bactericidal activity that masked the activity of antibiotics (19,

35, 36). However, (KFF)3K is the only peptide that displayed potent synergism with the antibiotics, gentamicin and amikacin. That synergism could be explained by their ability to permeabilize the membrane, at low concentration, without killing the bacteria, which will enhance entrance of antibiotics inside bacterial cells. A significant challenge facing AMPs which has limited their use in therapeutic applications is associated cytotoxicity to host tissues (37). However, our peptides demonstrated high therapeutic profiles, and there was no toxicity observed on mammalian cells (J774A.1 and HaCat) at concentrations several times higher than their bactericidal concentrations (Table 4.5 and Figure 4.5). After confirming the potent bactericidal activity of the peptides and their mechanism of action against S. pseudintermedius, we next turned our attention to assessing the likelihood that S. pseudintermedius would quickly develop resistance to these peptides. Repeated subculturing of S. pseudintermedius, in the presence of sub-inhibitory concentration, in a multi-step resistance selection for ten serial passages did not result in the development of bacterial resistance to the peptides. On the other hand, amikacin- and ciprofloxacin-resistant mutants of S. pseudintermedius readily emerged. The low frequency of emerging bacterial resistance to AMPs is attributed to the complexity of their mechanism of action and the extremely difficult changes required by the outer membrane to create a mutant, which cannot be acquired by simple bacterial mutation (10, 38). 89

We have successfully demonstrated the potential usage of AMPs against MSSP and MRSP clinical isolates. The AMPs were superior to antibiotics, demonstrating potent and rapid bactericidal activity against multidrug resistant S. pseudintermedius and low frequency of resistance. Moreover, AMPs such as RR and RRIKA (23) have been shown to possess potent anti-inflammatory properties and antibiofilm properties and to enhance wound healing. These are essential qualities for efficient treatment of skin infections caused by S. pseudintermedius. Taken together, the characteristics of these AMPs as antimicrobial agents may offer a significant improvement over current approaches and have a strong potential to be used for treatment of infections caused by multidrug resistant S. pseudintermedius.

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4.6 References

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14. Fazakerley J, Crossley J, McEwan N, Carter S, Nuttall T. 2010. In vitro antimicrobial efficacy of beta-defensin 3 against Staphylococcus pseudintermedius isolates from healthy and atopic canine skin. Vet Dermatol 21:463-468. 15. Santoro D, Maddox CW. 2014. Canine antimicrobial peptides are effective against resistant bacteria and yeasts. Vet Dermatol 25:35-e12. 16. Vannuffel P, Gigi J, Ezzedine H, Vandercam B, Delmee M, Wauters G, Gala JL. 1995. Specific detection of methicillin-resistant Staphylococcus species by multiplex PCR. J Clin Microbiol 33:2864-2867. 17. CLSI. 2007. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard M7-A7. CLSI, Wayne, PA. 18. Nagant C, Pitts B, Nazmi K, Vandenbranden M, Bolscher JG, Stewart PS, Dehaye JP. 2012. Identification of peptides derived from the human antimicrobial peptide LL-37 active against biofilms formed by Pseudomonas aeruginosa using a library of truncated fragments. Antimicrob Agents Chemother 56:5698-5708. 19. Zhang L, Benz R, Hancock RE. 1999. Influence of proline residues on the antibacterial and synergistic activities of alpha-helical peptides. Biochemistry 38:8102-8111. 20. Bahnsen JS, Franzyk H, Sandberg-Schaal A, Nielsen HM. 2013. Antimicrobial and cell-penetrating properties of penetratin analogs: effect of sequence and secondary structure. Biochim Biophys Acta 1828:223-232. 21. Ruhr E, Sahl HG. 1985. Mode of action of the peptide antibiotic nisin and influence on the membrane potential of whole cells and on cytoplasmic and artificial membrane vesicles. Antimicrobial Agents and Chemotherapy 27:841- 845. 22. Ward B, Seal BL, Brophy CM, Panitch A. 2009. Design of a bioactive cell- penetrating peptide: when a transduction domain does more than transduce. J Pept Sci 15:668-674. 23. Mohamed MF, Seleem MN. 2014. Efficacy of short novel antimicrobial and anti- inflammatory peptides in a mouse model of methicillin-resistant Staphylococcus aureus (MRSA) skin infection. Drug Des Devel Ther 8:1979-1983. 24. Chu HL, Yu HY, Yip BS, Chih YH, Liang CW, Cheng HT, Cheng JW. 2013. Boosting salt resistance of short antimicrobial peptides. Antimicrob Agents Chemother 57:4050-4052. 25. Deslouches B, Steckbeck JD, Craigo JK, Doi Y, Mietzner TA, Montelaro RC. 2013. Rational design of engineered cationic antimicrobial peptides consisting exclusively of arginine and tryptophan, and their activity against multidrug- resistant pathogens. Antimicrob Agents Chemother 57:2511-2521. 26. Ong ZY, Cheng J, Huang Y, Xu K, Ji Z, Fan W, Yang YY. 2014. Effect of stereochemistry, chain length and sequence pattern on antimicrobial properties of short synthetic beta-sheet forming peptide amphiphiles. Biomaterials 35:1315- 1325. 27. Splith K, Neundorf I. 2011. Antimicrobial peptides with cell-penetrating peptide properties and vice versa. Eur Biophys J 40:387-397.

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28. Kuriakose J, Hernandez-Gordillo V, Nepal M, Brezden A, Pozzi V, Seleem MN, Chmielewski J. 2013. Targeting intracellular pathogenic bacteria with unnatural proline-rich peptides: coupling antibacterial activity with macrophage penetration. Angew Chem Int Ed Engl 52:9664-9667. 29. Bond PJ, Khalid S. 2010. Antimicrobial and cell-penetrating peptides: structure, assembly and mechanisms of membrane lysis via atomistic and coarse-grained molecular dynamics simulations. Protein Pept Lett 17:1313-1327. 30. Henriques ST, Melo MN, Castanho MA. 2006. Cell-penetrating peptides and antimicrobial peptides: how different are they? Biochem J 399:1-7. 31. Wadhwani P, Reichert J, Burck J, Ulrich AS. 2012. Antimicrobial and cell- penetrating peptides induce lipid vesicle fusion by folding and aggregation. Eur Biophys J 41:177-187. 32. Shimoda M, Ohki K, Shimamoto Y, Kohashi O. 1995. Morphology of defensin- treated Staphylococcus aureus. Infect Immun 63:2886-2891. 33. Friedrich CL, Moyles D, Beveridge TJ, Hancock RE. 2000. Antibacterial action of structurally diverse cationic peptides on gram-positive bacteria. Antimicrob Agents Chemother 44:2086-2092. 34. Mohammad H, Mayhoub AS, Cushman M, Seleem MN. 2014. Anti-biofilm activity and synergism of novel thiazole compounds with glycopeptide antibiotics against multidrug-resistant Staphylococci. J Antibiot (Tokyo) doi:10.1038/ja.2014.142. 35. Piers KL, Hancock RE. 1994. The interaction of a recombinant cecropin/melittin hybrid peptide with the outer membrane of Pseudomonas aeruginosa. Mol Microbiol 12:951-958. 36. Fidai S, Farmer SW, Hancock RE. 1997. Interaction of cationic peptides with bacterial membranes. Methods Mol Biol 78:187-204. 37. Zasloff M. 2002. Antimicrobial peptides of multicellular organisms. Nature 415:389-395. 38. Yeaman MR, Yount NY. 2003. Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55:27-55.

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Table 4.1. Amino acid sequence and physicochemical properties of peptides used in this study Peptide Molecular Hydrophobic amino Amino acid sequencea Length Charge designation weight acids

RR WLRRIKAWLRR 11 1553.9 + 5 54 %

RRIKA WLRRIKAWLRRIKA 14 1866.3 + 6 57 %

WR-12 RWWRWWRRWWRR 12 2072.4 + 6 50 %

IK8 “D irikirik 8 1040.28 + 4 50 % isoform” 1

(KFF)3K KFFKFFKFFK 10 1413.7 + 4 60 %

penetratin RQIKIWFQNRRMKWKK 16 2246.7 + 7 37 %

1D-amino acid substitution aSmall underlined residues represent D-amino acids

Table 4.2. Minimum inhibitory concentration (MIC) of peptides against methicillin sensitive Staphylococcus pseudintermedius (MSSP) strains

MIC (µM)

K

3

12 “D “D

Isolates Origin - Resistance phenotype

RR

KFF)

WR

IK8

RRIKA

isoform” ( Penetratin SP1 urine 1 4 2 4 4 8 PEN, AMP PEN, AMP, CLIN, ENRO, ERYTH, SP2 urine 2 8 2 8 16 16 GEN,MARBO, TMP-SMX SP4 urine 2 4 4 8 16 16 PEN, AMP, TMP-SMX SP6 ear 2 4 1 4 16 8 - AMK, SP7 ear 1 4 2 4 8 8 PEN,AMP,CLIN,ENR,ERM,GEN,MARB, TMP-SMX

94

Table 4.3. continued SP10 urine 1 4 1 4 8 8 PEN,AMP,CLIN, ERM, TMP-SMX SP11 hair 1 4 1 4 8 8 PEN, AMP,CHL,CLIN, 0. 0. 0. 0. SP12 urine 8 4 PEN,AMP,AMK,ENR,GEN,MARB, TMP-SMX 5 5 5 5 skin 0. SP13 1 4 4 8 8 PEN,AMP abscess 5 SP14 ear 2 4 4 4 8 8 PEN,CHL,CLIN,ERM, SP15 urine 1 4 2 8 8 8 - abdomi nal SP16 swab 2 8 1 8 8 8 - (surger y) skin 3 PEN, AMP, SP17 1 8 2 8 8 abscess 2 AMK,CLIN,ENR,ERM,GEN,MARB, TMP-SMX SP18 urine 1 4 2 8 8 8 PEN synovia l SP19 fluid(sti 1 4 1 4 8 8 PEN,AMP,CEF fle swab) 1 1 SP20 urine 1 4 1 8 PEN,AMP,DOX 6 6 SP21 urine 1 4 1 4 8 8 - bladder 1 SP22 1 4 1 8 8 - swab 6 SP23 wound 1 4 1 4 8 8 PEN,AMP, 1 SP24 abscess 1 4 2 8 8 PEN 6 conjunc 1 AMK,BAC,CHL,ERM,NEO,OXY, TOB, TMP- SP26 1 4 1 4 4 tiva 6 SMX

95

Table 4.4. continued epiderm al collarett PEN,AMP,AMK,CHL,CLIN,ENR,ERM,GEN,M SP27 1 4 4 8 8 8 es ARB, TMP-SMX ( abdo men) tooth SP30 root 1 4 1 4 8 8 PEN,AMK,CLIN,ERM,GEN, TMP-SMX abscess SP31 abscess 1 4 1 4 4 8 - 0. SP32 abscess 1 4 4 4 8 PEN,AMP,AMK,ERM,CLIN,GEN, TMP-SMX 5 conjunc 0. SP33 1 4 4 8 8 BAC,CIP,ERM,MOX,NEO,OXY, TMP-SMX tiva 5 SP34 urine 2 4 2 8 8 8 - SP35 urine 2 4 1 4 8 8 - swab at SP36 bone 2 8 1 8 8 8 PEN,AMP,CLIN,ENR,ERM,MARB, TMP-SMX plating 0. SP37 urine 4 2 4 8 8 PEN and AMP 5

MIC50 1 4 1 4 8 8 1 1 MIC90 2 8 2 8 6 6 Abbreviation: PEN: penicillin, AMP, ampicillin, AMK: amikacin, CEF: cefpodxime, CLIN: clindamycin, GEN: gentamicin, CHL: chloramphenicol, ENR: enrofloxacin, MARB: marbofloxacin, ERM: erythromycin, BAC: bacitracin, NEO: neomycin, TOB: tobramycin, CIP: ciprofloxacin, OXY: Oxytetracyclin, TMP-SMX: trimethoprime sulphamethoxazole.

96

Table 4.5. Minimum inhibitory concentration (MIC) of peptides against methicillin resistant Staphylococcus pseudintermedius (MRSP) strains Abbreviation:OXA: oxacillin, AMK: amikacin, CEF: cefpodxime, CLIN: clindamycin, GEN: gentamycin, CHL: chloramphenicol, ENR: enrofloxacin, MARB: marbofloxacin, ERM: erythromycin, BAC: bacitracin, NEO: neomycin, TOB: tobramycin, CIP:

MIC (µM)

K 3

Isolat Resistance phenotype

12 “D “D Origin -

es RR

WR

IK8

RRIKA

isoform” (KFF) Penetratin orthope SP3 dic 1 4 0.5 4 8 4 CEF,ERM,CLIN ,IMI, ,OXA,TIC implant AMK,CEF,CHL,CLIN,ENR,ERM,GEN,IMI,M SP5 urine 1 4 1 4 4 8 ARB,OXA,TIC, TMP-SMX SP8 urine 1 4 4 4 4 4 CEF,ERM,CLIN ,IMI,OXA,TIC,CHL AMK,CEF,CHL,CLIN,ENR,ERM,GEN,IMI,M SP9 1 4 0.5 4 8 8 skin ARB,OXA,TIC, TMP-SMX AMK,CEF,CHL,CLIN,ENR,ERM,GEN,IMI,M SP25 urine 1 2 0.5 2 4 4 ARB,OXA,TIC, TMP-SMX AMK,

SP28 1 4 2 4 8 8 CEF,CHL,CLIN,ENR,ERM,GEN,IMI,MARB, urine OXA,TIC, TMP-SMX CEF,CHL,CLIN,ENR,ERM,GEN,IMI,MARB, SP38 implant 0.5 2 0.5 4 2 4 OXA,TIC, TMP-SMX AMK, SP39 ear 0.5 0.5 0.5 2 0.5 2 CEF,ENR,ERM,GEN,IMI,MARB,OXA,TIC SP40 ear 2 4 2 4 8 8 CEF,ENR,ERM,CLIN ,IMI,MARB,OXA,TIC AMK, Sp41 urine 1 4 1 4 8 8 CEF,CHL,CLIN,ENR,ERM,GEN,IMI,MARB, OXA,TIC, TMP-SMX

MIC50 1 4 0.5 4 4 4

MIC90 1 4 2 4 8 8 ciprofloxacin, OXY: Oxytetracyclin, TMP-SMX: trimethoprime sulphamethoxazole, TIC: ticarcillin, IMI: imipenem.

97

Table 4.6. The values of FIC index for the combination of peptides and antimicrobial compounds against Staphylococcus pseudintermedius (SP3)

FIC indexa Compound IK8 “D RRIKA RR WR-12 Penetratin (KFF)3K isoform” RRIKA - 0.5 0.5 0.5 0.37 0.37

RR 0.5 - 0.5 0.5 0.5 0.37

WR-12 0.37 0.5 - 0.5 0.5 0.5

IK8 “D 0.5 0.5 0.5 - 0.75 0.5 isoform” Penetratin 0.5 0.37 0.5 0.5 - 0.37

(KFF)3K 0.37 0.37 0.5 0.5 0.37 -

Amikacin 1 0.75 1 0.75 0.75 0.37

Gentamicin 1 0.75 0.75 1 1 0.26 a The FIC index was determined in the presence of a constant amount of peptide, equal to one -quarter of the peptide MIC.

Table 4.7. Cytotoxicity and therapeutic index of peptides

a b c GM MIC (µM) IC50 (µM) TI Peptide S. pseudintermedius J774A.1/HaCaT J774A.1/HaCaT RRIKA 1.15 64/128 55.6/111.2 RR 4.12 128/256 31/62 WR-12 1.5 128/128 85.3/85.3 IK8 “D isoform” 5 >256/>256 >51.2/>51.2 Penetratin 8.76 >256/>256 >29.2/>29.2

(KFF)3K 7.75 256/>256 33/>33 Melittin 2 2/ND 1/ND a GM, geometric mean of the MICs of the peptides against S. pseudintermedius strains. b IC50 (Inhibitory Concentration 50), peptide concentration that inhibits 50 % of macrophage cell line (J774A.1) or human keratinocyte (HaCaT). c TI ( Therapeutic index), the ratio of the IC50 over the geometric mean MIC value. ND: not determined. 98

8

C o n tr o l 6 R R IK A

L R R

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F C

0 K F F 3 K 1

g 2 IK 8 " D iso fo rm "

o L P e n e tr a tin

0 A m ik a c in 0 1 0 2 0 3 0 4 0 5 0 6 0 1 2 0 1 5 0 1 8 0

T im e (m in ) Figure 4.1. Bacterial killing kinetics of peptides against S. Pseudointermedius.

SP 3 exposed to peptides (RRIKA, RR, WR-12, KFF3K, IK8 “D isoform” and penetratin) and antibiotic (amikacin) at 5X MIC in MHB (Mueller Hinton broth). Samples treated with peptide diluent (sterile water plus 0.2% acetic acid) were used as a control. The killing curves were identical (overlapping in the figure) for RRIKA, RR and KFF3K. The results are given as means ± SD (n = 3; data without error bars indicate that the SD is too small to be seen).

99

0 .8 C o n tro l

N is in 0

0 R R IK A

6 0 .6

D R R

O

t W R -1 2 a

0 .4

y t

i IK 8 "D iso fo rm "

d i

b P enetratin r

u 0 .2

T K F F 3 K

A m ik a c in 0 .0 C ip ro flo x a c in 0 2 4 6 8 T im e (h r ) Figure 4.2. Growth kinetics of SP3 exposed to 5X MIC of peptides.

SP3 exposed to 5X MIC of peptides, antibiotics and sterile water (control) and OD600 was measured by spectrophotometer over time. Results are representative of two separate experiments, each done in triplicate. Error bars represent standard deviation values.

100

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% (

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s R R IK A

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Figure 4.3. Permeabilization of the cytoplasmic membrane of SP3 as a function of peptide concentration. Peptides at 5X MIC (A) and 10 X MIC (B) incubated with bacteria and leakage was indicated by percent of propidium iodide fluorescence. Each experiment was done in triplicate, and the values represent means ± standard deviations.

101

Figure 4.4. TEM micrographs of untreated and peptide treated S. pseudintermedius SP3.

Control bacteria either dividing cells (A) or single celled (B), the cells are round and intact, with a well-defined cell membrane. The intracellular DNA region exhibits a highly inhomogeneous electron density. C&D: 1X MIC RRIKA; E&F: 10X MIC RRIKA, G: 10X MIC RR; H: 10X MIC KFF3K; I: 10X MIC, IK8 “D isoform”; K: 10X MIC WR-12; L: 1X MIC penetratin; M: 10X MIC penetratin. 102

Figure 4.5. Cytotoxicity assay of peptides against mammalian cell lines.

Cytotoxicity assay showing the percent mean absorbance at 490 nm after incubating macrophage cell line (J774.1) (A &B) and human keratinocyte (HaCaT) (C&D) with peptides at different concentrations. Melittin and DMSO served as positive control in J774.1 and HaCat cells, respectively. Water (peptide diluent) served as negative control. Cell viability was measured by MTS assay. Results are expressed as means from three measurements ± standard deviation. Data were analyzed using one-way ANOVA, with post hoc Tukey’s multiple comparisons test to determine statistical significance. Two asterisks (**) means statistical different (p< 0.01) from the negative control while (ns) means there was no statistical significance from the negative control. 103

3 2

C I R R IK A

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n

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n 1 6

a A m ik a c in

h

c

d

l 8

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0 0 2 4 6 8 1 0 N u m b e rs o f p a s s a g e s

Figure 4.6. Drug resistance development profiles of S. pseudintermedius (SP6) exposed to peptides.

S. pseudintermedius (SP6) was exposed to ½ X MIC concentrations of peptides (IK8 “D isoform” and RRIKA) and antibiotics (amikacin and ciprofloxacin) for 10 passages.

104

CHAPTER 5. EVALUATION OF SHORT SYNTHETIC ANTIMICROBIAL PEPTIDES FOR TREATMENT OF DRUG- RESISTANT AND INTRACELLULAR STAPHYLOCOCCUS AUREUS

(Mohamed, M.F., Ahmed Abdelkhalek and Seleem, M.N (2016) Evaluation of short synthetic antimicrobial peptides for treatment of drug-resistant and intracellular Staphylococcus aureus. Scientific reports, Scientific Reports 6, Article number: 29707 (2016) doi:10.1038/srep29707)

5.1 Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) infections present a serious challenge because of the emergence of resistance to numerous conventional antibiotics. Due to their unique mode of action, antimicrobial peptides are novel alternatives to traditional antibiotics for tackling the issue of bacterial multidrug resistance. Herein, we investigated the antibacterial activity of two short novel peptides (WR12, a 12 residue peptide composed exclusively of arginine and tryptophan, and D-IK8, an eight residue β- sheet peptide) against multidrug resistant staphylococci. In vitro, both peptides exhibited good antibacterial activity against MRSA, vancomycin-resistant S. aureus, linezolid-resistant S. aureus, and methicillin-resistant S. epidermidis. WR12 and D-IK8 were able to eradicate persisters, MRSA in stationary growth phase, and showed significant clearance of intracellular MRSA in comparison to both vancomycin and linezolid. In vivo, topical WR12 and D-IK8 significantly reduced both the bacterial load and the levels of the pro-inflammatory cytokines including tumor necrosis factor-α (TNF- α) and interleukin-6 (IL-6) in MRSA-infected skin lesions. Moreover, both peptides disrupted established in vitro biofilms of S. aureus and S. epidermidis significantly more so than traditional antimicrobials tested. Taken together, these results support the potential of WR12 and D-IK8 to be used as a topical antimicrobial agent for the treatment of staphylococcal skin infections. 105

5.2 Introduction

The rapid development and spread of bacterial resistance to conventional antibiotics, particularly those associated with staphylococcal infections, has become a serious global concern. Nearly 11,000 people die each year from a methicillin-resistant Staphylococcus aureus (MRSA)-related infection alone in the United States; this figure represents nearly half of all fatalities caused by antibiotic-resistant bacteria pathogens (1, 2). Staphylococcus aureus is the pathogen most frequently isolated from human skin and wound infections (2, 3). Staphylococcal biofilms and toxins can evade the host immune system, leading to recurring/chronic infections, prolonging inflammation, and hindering the process of wound healing (4, 5). Furthermore, the emergence of MRSA strains exhibiting resistance to topical drugs of choice, including mupirocin and fusidic acid, is a significant public health challenge that requires novel therapeutic alternatives (6, 7). Antimicrobial peptides (AMPs) have shown significant promise in recent years as novel therapeutic agents to treat infections caused by multidrug-resistant pathogens (1). AMPs are a major component of the human skin’s innate immunity and a decrease in the production of AMPs in the dermis is associated with increased susceptibility to skin infection with S. aureus in humans(8). In addition to possessing potent antibacterial activity, AMPs have several unique advantages over traditional antibiotics. These advantages include AMPs possess a broad spectrum of activity, low potential for resistance development, ability to neutralize virulence factors released by pathogens, and ability to modulate the host immune response(1). However, there are several limitations for utilizing naturally-derived AMPs, particularly for treatment of invasive infections. These limitations include host toxicity, degradation by proteases, extensive serum binding, loss of antimicrobial activity in the presence of physiological concentration of salts, and high cost of production due to their complex design. These limitations need to be addressed, and new avenues need to be pursued in order to transform AMPs into novel therapeutic agents capable of being used clinically. One avenue gaining momentum is the utilization of AMPs as topical antibacterial agents. Multiple AMPs have already reached various stages of clinical trials for the treatment and prevention of bacterial infections(1). In the present study, the in vitro and in vivo antibacterial activity of two unique AMPs, 106

WR12 and IK8 all D (D-IK8), was investigated against multidrug-resistant S. aureus. These two peptides have several advantages, including potent antibacterial activity, high selectivity, and short and simple sequences (8-12 amino acids). Their simplified sequences should facilitate their rapid production, decrease their cost of synthesis, and accelerate their translational clinical applications (9, 10). WR12 (RWWRWWRRWWRR) is a de novo designed short peptide composed of 12 amino acids with broad-spectrum antibacterial activity. It is composed exclusively of arginine and tryptophan and is designed to form standard amphipathic helices with six cationic charges and 50% hydrophobicity(9). Peptide D-IK8 (irikirik) is a short synthetic β-sheet forming peptide composed only of eight amino acids with four cationic charges and 50% hydrophobicity. Importantly, substitution of the L-amino acids of IK8 with the D-isoform provides resistance to enzymatic degradation by animal and bacterial proteases (10). Both WR12 and D-IK8 kill bacteria by disrupting bacterial membranes, leading to leakage of intracellular contents and, consequently, bacterial death. This mechanism explains the low emergence of bacterial resistance observed after serial passage with sub-inhibitory concentrations of peptides (9, 10). The aim of this study was to investigate the spectrum of antibacterial activity of the designed peptides against a collection of important multidrug-resistant strains of staphylococci isolated from clinical settings, to assess their ability to kill persisters and MRSA in stationary phase of growth, to explore their antibiofilm activity and their ability to clear intracellular infections, to investigate their ability to be used in combination with conventional antibiotics, and to assess their efficacy and immune-modulatory effect in a murine model of MRSA skin infection.

5.3 Materials and methods

Bacterial isolates, peptides and reagents

Methods were carried out in accordance with approved guidelines. Peptides, WR12, D isoform of IK8 (“here we refer to it as “D-IK8”), LL-37 and pexiganan (Table 5.1) were synthesized by GenScript (Piscataway, NJ) using solid-phase 9-fluorenylmethoxy carbonyl (Fmoc) chemistry and purified to a purity of 95% using reverse-phase high- 107 performance liquid chromatography (HPLC). Peptide mass was confirmed by mass spectrometry (supplementary materials). Clinical isolates of Staphylococci are presented in Table 5.2 and 5.3. Antibiotics were purchased from commercial vendors, vancomycin hydrochloride (Gold Biotechnology, St. Louis, MO), linezolid (Selleck Chemicals, Houston, TX), clindamycin (TCI chemicals, Portland, OR), erythromycin, gentamicin were purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum (FBS) was purchased from Sigma-Aldrich (St. Louis, MO). Dulbecco's Modified Eagle's medium (DMEM) was purchased from Life technologies. MTS reagent was purchased from Promega (Madison, WI). Mueller-Hinton broth (MHB) was purchased from Sigma- Aldrich, while Trypticase soy broth (TSB) and Trypticase soy agar (TSA) were purchased from Becton-Dickinson, Cockeysville, MD. Mannitol salt agar (MSA) was purchased from Hardy Diagnostics (Santa Maria, CA). Enzyme-linked immunosorbent assay (ELISA) development kits for cytokines detection were purchased from R&D Systems, Inc. (Minneapolis, MN).

Antibacterial assays

The minimum inhibitory concentrations (MIC) of peptides and antibiotics were determined by the broth microdilution technique according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (56). MIC assays were carried out with an initial bacterial inoculum of 5×105 colony forming units (CFU/ml) in MHB. Peptides and antibiotics were added to polystyrene 96-well plates at desired concentrations. MHB were supplemented with 50 mg/liter Ca2 in case of daptomycin. MIC was defined as the lowest concentration of peptide or antibiotic which inhibited the visible growth of bacteria.

Antibacterial assays in presence of salts:

To investigate the activity of peptides in the presence of high salt concentrations, WR12, D-IK-8 and pexiganan were tested against MRSA USA300 in a cation-adjusted MHB or in MHB with added concentrations of NaCl (150 mM) or MgCl2 (2 mM). The MIC was subsequently identified, as described before and according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (56) 108

To determine the effect of protease digestion on the antimicrobial activity of the peptides, each peptide was first incubated with trypsin at a molar ratio of 500: 1 (peptide: enzyme) in the digestion buffer (50 mM Tris–HCl, pH 7.4) at 37°C for 4 hours. After incubation, the digestion mixture was heated at 80°C for 10 min to halt the enzyme reaction. After these treatments, the procedures conducted were the same as the MIC assay described above.

Membrane permeabilization assay (Calcein leakage assay)

Membrane permeabilization of S. aureus by peptides was monitored and quantified by the leakage of the preloaded fluorescent dye, calcein, as described before (57). MRSA USA300 and VRS10 was grown in MHB to logarithmic phase at 37°C. Cells were then harvested by centrifugation, washed twice with PBS, and then adjusted 9 spectrophotometrically to an OD600 of 1.0 (≈10 CFU/ml) in PBS containing 10% (vol/vol) MHB. Then MRSA cells were incubated with 3 µM calcein AM for 1 hr at 37°C. Calcein- loaded cells were harvested by centrifugation (3,000 × g, 10 min), suspended in PBS, and diluted to achieve a final inoculum of 107 CFU/ml. Aliquots of 100 µL were then added into a sterile black-wall 96-well plate. In case of MRSA USA300, WR12 and D-IK8 were added in concentrations equivalent to 5 × and 10 × MIC. In case of VRS10, WR12 and D-IK8 were added in concentrations equivalent to 0.5 × MIC and incubated for 1 hr. Bacteria treated with peptide diluent (sterile water) served as negative controls. Calcein leakage was measured for 120 min using a fluorescence plate reader (FLx800 model BioTek® Instruments, Inc. Winooski, Vermont). Membrane permeabilization (%) was calculated as the absolute percent calcein leakage by peptides with respect to calcein- loaded with no-peptide treated cells. Experiments were done in triplicate and repeated independently twice.

Time-kill assay

MRSA USA300 was grown overnight in MHB, then diluted in fresh MHB and incubated aerobically at 37 ºC until bacteria reached logarithmic phase of growth (OD600 = 0.2). Then bacteria were diluted to 5.6 ×106 CFU/ml in MHB. Peptides (at 5 × and 10 × MIC) and antibiotics at 10 × MIC were added to diluted bacteria, and incubated aerobically at 37 ºC in a shaking incubator at 250 r.p.m. Aliquots at specified time points were taken, 109 serially diluted in phosphate-buffered saline (PBS) and plated, in triplicate, on TSA. CFUs were counted after incubation of plates for 24 hours at 37 ºC. The kinetics of killing against stationary phase bacteria was done as described previously (24, 27, 28). MRSA USA300 was grown in MHB with aeration at 250 r.p.m. at 37 ºC overnight. Then bacteria were exposed to peptides and antibiotics at 10 × MIC. Aliquots were taken and plated as described above. Formation of persister cells was done as described before (24, 27, 28). Briefly, MRSA USA300 was grown overnight in MHB, then diluted in fresh MHB and incubated until cells reached logarithmic phase of growth. Bacteria were then exposed to10 × MIC ciprofloxacin for 6 hours. Peptides and antibiotics, at 10 × MIC, were added to bacteria after 6 hours from ciprofloxacin treatment. Bacteria were incubated aerobically at 37ºC in a shaking incubator at 250 r.p.m. Aliquots at specified time points were taken and counted as described above.

Efficacy of peptides on Staphylococcus biofilms

The efficacy of peptides to disrupt biofilms was conducted as described before (57). Briefly, isolates of S. aureus (ATCC 6538) and S. epidermidis (ATCC 35984) grown overnight were diluted 1:100 in TSB + 1% glucose and incubated in 96-well plates at 37°C for 24 hours or 48 hours. After removing media, wells were rinsed with PBS to remove planktonic bacteria before re-filling wells with fresh MHB. Peptides and antibiotics were added at desired concentrations and plates were incubated at 37°C for 24 hours. After incubation, wells were washed and biofilms were stained with 0.5% (w/v) crystal violet for 30 minutes. The dye was solubilized with ethanol (95%) and the optical density (OD) of biofilms was measured.

Combination therapy analysis The synergistic effect between peptides and antibiotics was assessed by the combination assay as described previously (57). Two-fold serial dilutions of antimicrobials (WR12, D- IK8, and antibiotics) were tested in the presence of a fixed concentration of peptide equal to ¼ × peptide MIC, which did not inhibit the growth of bacteria alone. The fractional inhibitory concentration (FIC) index was calculated as follows: FIC of drug A = MIC of drug A in combination/MIC of drug A alone, FIC of drug B = MIC of drug B in combination/MIC of drug B alone, and FIC index = FIC of drug A + FIC of drug B. An 110

FIC index of ≤ 0.5 was classified as synergism. Additive was defined as an FIC index of 1. Antagonism was defined as an FIC index of > 4.

Re-sensitization of VRSA to vancomycin, teicoplanin and oxacillin in the presence of subinhibitory concentration of peptides

Resensitization of VRSA strains to antibiotics (vancomycin, teicoplanin and oxacillin) was done as described previously (58). Briefly, ½ × MIC of WR12 and D-IK8was incubated with VRSA (bacterial inoculum of 5×105 colony forming unit (CFU/ml)) in MHB at room temperature for 60 minutes. After incubation, peptide-treated bacteria were added in 96-well plates. Antibiotics, at a concentration equal to their MIC, were added to the first row and diluted. Bacteria treated with ½ × MIC of WR12 and D-IK8served as the negative control as we didn’t observe bacterial inhibition at this concentration. The plate was incubated for 24 hours at 37 ºC and the MIC was recorded.

Sub-inhibitory concentration of WR12 and D-IK8 increase the uptake and binding of bodipy vancomycin

We investigated the binding and association of fluorescently labeled vancomycin (bodipy vancomycin) with VRSA. Briefly, VRS10 was incubated with sub-inhibitory concentration of WR12 and D-IK8 (0.5 X MIC) for one hour and then treated with bodipy vancomycin (16 µg/ml) for 30 minutes. Samples treated with bodipy vancomycin only served as a control. After incubation, bacteria were centrifuged at 9,000×g for 5 min, washed four times with PBS, and resuspended in a small volume of PBS. Bacterial pellets were fixed with 4% paraformaldehyde, and visualized under 40X oil objective of Nikon A1R multi-photon inverted confocal microscope.

Toxicity of peptides on human keratinocytes

Peptides were assayed for potential in vitro toxicity against human keratinocytes (HaCaT) as described before(59). Briefly, cells were seeded at a density of 1.5 × 104 per well in a tissue culture 96-well plate (CytoOne, CC7682-7596) in DMEM supplemented with 10% fetal bovine serum (FBS), and incubated at 37°C in a 5% CO2 atmosphere for 24 hours. The cells were treated with compounds at different concentrations for 24 hours. After incubation, the cells were washed and incubated with 100 µL of DMEM media containing 111

20 µL of MTS reagent for 4 hours at 37 ºC. Corrected absorbance readings were taken using an ELISA microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Intracellular antibacterial efficacy of peptides in human keratinocytes

Infection of human keratinocytes (HaCaT) was done as described previously (46). Briefly, HaCaT cells were seeded and incubated as described above. Following incubation, the cells were infected either with methicillin-sensitive S. aureus ATCC 6538 or methicillin resistant S. aureus USA300, (at a multiplicity of infection 50:1) in DMEM + 10% FBS for 2.5 hours. After infection, the wells were washed with 200 µl media with lysostaphin (10 µg/ml) and further incubated for 30 minutes with lysostaphin to kill any remaining extracellular bacteria. Drugs were diluted in DMEM + 10% FBS to the desired concentrations (4 × MIC) and wells were treated with 100 µl of DMEM +10% FBS containing drugs for 24 hours. Medium alone was used as a negative control. After incubation, the media were aspirated and washed twice with PBS to remove any residual drugs. HaCaT cells were lifted with trypsin. Then 100 µl of PBS with 0.01% triton X was added in each well to lyse HaCaT cells. Subsequently, bacteria were diluted and plated on TSA plates. Plates were incubated at 37 ºC for 24 hours. After incubation, bacteria were counted and analyzed. Experiments were repeated twice independently and the average was reported.

Confocal Microscopy: Uptake of WR12-FITC in mammalian cells

J774A.1 cells were seeded at a density of 1.5 x 105 cells/well in a 4-well Lab-Tek chambered slides in DMEM media supplemented with 10% fetal bovine serum (FBS), and incubated at 37°C in a 5% CO2 atmosphere for 20 hours. The media were aspirated and the cells were washed 1X with 400 µL PBS. Following incubation, the cells were washed once with DMEM media. Then the cells were incubated with WR12-FITC (10 º µM) for 3 hours at 37 C and 5% CO2. The cells were washed 3X with PBS and visualized under 60X oil objective of Nikon A1R multi-photon inverted confocal microscope.

Efficacy of peptides in mice model of MRSA skin infection The animal care and all experiments were approved and performed in accordance with the guidelines approved by Purdue University Animal Care and Use Committee (PACUC). 112

Female BALB/c mice (6-8 weeks old) were obtained from Harlan Laboratories, Indianapolis, IN. All procedures were approved by the Purdue University Animal Care and Use Committee (PACUC) (protocol no: 1207000676). The murine model of MRSA skin infection was done as described previously (47, 48). Briefly, the posterior upper backs of mice were shaved and mice were injected intradermal by 40 µl of MRSA USA300 (3 × 107 CFU/40 µl) in sterile phosphate-buffered saline (PBS) using a 27-gauge insulin syringe. Mice were randomly divided into five groups and each group contained five animals. Forty-eight hours after infection and formation of open wound, groups of mice were treated topically either with 2% fusidic acid, 2% WR12 or 2% D-IK8 formulated in 20 mg petroleum jelly. One group received vehicle only (petroleum jelly) and the last group was treated orally with linezolid (25 mg/kg). All groups were treated twice a day for 3 days. Twenty-four hours after the last treatment, mice were euthanized, and the skin lesion was removed and homogenized in 1 ml tryptic soy broth. Samples were diluted, plated in mannitol salt agar in triplicate, and incubated aerobically at 37°C. After 24 hours incubation, the colony forming units (CFU) were counted. Cytokine detection of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in skin lesions was done using ELISA as described before and according to manufacturer instructions (47). Cytokine levels were expressed as percent change relative to negative control.

Statistical analyses

Statistical analyses were performed using GraphPad Prism 6.0 software (GraphPad Software, La Jolla, CA, USA). Comparison between two groups were analyzed using two- tailed unpaired Student t-tests. Comparison between three groups or more were analyzed using one-way ANOVA, with post hoc Tukey’s multiple comparisons test. P-values of ˂ 0.05 were considered significant.

5.4 Results and discussion

Antimicrobial activity

We explored the antibacterial activity of WR12 and D-IK8 against multidrug-resistant strains of staphylococci, including methicillin-sensitive S. aureus (MSSA), MRSA, 113 vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), linezolid-resistant S. aureus and methicillin-resistant S. epidermidis (MRSE) (Table 5.2 and 5.3). WR12 exhibited strong antibacterial activity against MRSA, inhibiting 50% of the strains (MIC50) at a concentration of 2 µM and inhibiting 90% of the strains (MIC90) at a concentration of 8 µM. The MIC50 and MIC90 of D-IK8 against MRSA strains were found to be 8 and 16 µM, respectively. Moreover, WR12 and D-IK8, demonstrated good activity against multiple clinical isolates of MRSA, particularly MRSA USA300, a community-associated strain responsible for major outbreaks of staphylococcal skin and soft-tissue infections (SSTI) (3). Similarly, potent antibacterial activity of these peptides was observed against other important clinical MRSA isolates (USA500, USA200, and USA100) that exhibit resistance to various antibiotic classes, including fluoroquinolones, macrolides, lincosamides, and aminoglycosides.

WR12 and D-IK8 also showed potent antibacterial activity against all tested VRSA isolates that were resistant to vancomycin and teicoplanin, with MIC values ranging from 4-8 and 8-16 µM, respectively (Table 5.3). It is important to note that 85% of the VRSA strains we examined were isolated from wounds; thus the studied AMPs have potential to be used as a topical antimicrobial agent for the treatment of multidrug-resistant staphylococcal skin and wound infections (11).

The superior antibacterial activity of WR12 over D-IK8 may be attributed to the higher cationic charge and the increased length of amino acids of WR12 (12 residues) compared to D-IK8 (8 residues) (Table 5.1). Indeed, WR12 permeabilized the staphylococcal membrane more potently than D-IK8. As demonstrated in figure 5.1, at 5 × MIC, WR12 and D-IK8 caused more than 95% and 76% leakage of preloaded calcein dye within 60 minutes, respectively.

In contrast to WR12 and D-IK8, we detected very low activity and high level of resistance in staphylococci strains against the natural derived human AMP cathelicidin (LL-37) that protects human skin from bacterial infections (12). LL-37 showed activity only against two strains of staphylococci (S. aureus ATCC 6538 and S. epidermidis ATCC 35984 “NRS 101”) with a MIC value equal to 16 µM. The MIC values of LL-37 against all other strains were more than 128 µM. Our results correlate with previous reports that have found 114 high levels of resistance to LL-37 in clinical MRSA strains compared with MSSA bacterial isolates(13). The emergence of resistance among clinical isolates to natural peptides, such as LL-37, could be one of the factors that has contributed to their global epidemic(14).

We evaluated the activity of pexiganan against four isolates of staphylococci represent different antibiotic susceptibility phenotypes: MSSA, MRSA, MRSE, and VRSA. Pexiganan is an analog of the magainin peptides isolated from the skin of the African clawed frog. Pexiganan is the first AMPs that has advanced furthest in clinical trials for the treatment of diabetic foot ulcers. Pexiganan demonstrated comparable activity to D- IK8 and less activity than WR12 against MSSA, MRSA and VRSA (MIC of pexiganan is 16, 16, 32 µM against MSSA, MRSA and VRSA, respectively). However, pexiganan demonstrated enhanced activity than WR12 and D-IK8 against MRSE (MIC, 1 µM). Ge et al reported that the MIC50 and MIC90 of pexiganan against large panel of S. aureus isolates were 8 and 16 µg/ml, respectively (15). A more recent study reported that pexiganan MIC values among MSSA and MRSA strains isolated from diabetic foot infections ranged from 16 to 32 µg/ml(16). It is important to note that pexiganan have 22- amino-acid residues in contrast to WR12 (12 residues) and D-IK8 (8 residues). This ensures a relatively lower cost of production of WR12 and D-IK8 compared to pexiganan.

Antimicrobial activity in physiological concentrations of salts

One major limitation with the clinical translation of AMPs is their potential inactivation by salts present in the human body. Therefore, we tested the MIC of peptides against MRSA USA300 in cation-adjusted Mueller Hinton medium and in regular MHB, for comparison (Table 5.4). We did not find a significant difference in the MIC value of the peptides in the two conditions (there is no difference in the MIC of WR12 and only one fold increase of D-IK8 and pexiganan in the presence of cation-adjusted Mueller Hinton medium). To ensure that these peptides remain active in the presence of higher concentrations of cations, we tested the peptides in higher concentration of salts. As demonstrated in supplementary Table 5.4, there was no difference in the MIC of WR12 in the presence of 150 mM NaCl. However, there was a two-fold increase in the MIC observed for D-IK8 and pexiganan at the same conditions. In the presence of a 115

physiological concentration of MgCl2 (2 mM), we observed a one-fold increase in the MIC of WR12 and a two-fold increase in the MIC of D-IK8 or no change in the MIC of pexiganan, compared to regular MHB (Table 5.4). The superior salt stability of WR12 compared to D-IK8 and pexiganan is attributed to its amino acid composition. WR12 is composed of tryptophan and arginine residues which are known to improve antimicrobial activity under challenging salt conditions (9, 17, 18). In contrast, many well-studied AMPs (such as LL-37, human β-defensin-1, gramicidins, bactenecins, and magainins) demonstrated substantially reduced antibacterial activities under the same conditions (19). Previously, Turner et al. reported that LL-37 and HNP-1 demonstrated a 12-fold and 100- fold increase in the MIC of MRSA, respectively, when 100 mM NaCl was added to the test medium (20). The ability to resist the effects of salt provide a selective advantage for WR12 peptide for potential therapeutics in physiological solutions.

Bacterial killing kinetics

After confirming that the AMPs possessed excellent antimicrobial activity against multidrug-resistant staphylococcal clinical isolates, we next assessed the killing kinetics of these AMPs. Both peptides showed concentration-dependent killing of MRSA USA300. WR12 showed fast bactericidal activity and was capable of completely eliminating a high starting inoculum of MRSA USA300 (5.6 x 106 CFU/ml) within 30 minutes and 240 minutes at 10 × and 5 × MIC, respectively (figure 5.2a). D-IK8 showed slower bactericidal activity with complete clearance of MRSA within 90 minutes and 180 minutes at 10 × MIC and 5 × MIC, respectively. The rapid bactericidal activity of WR12, when compared to D-IK8, is mainly attributed to its ability to more rapidly permeabilize the staphylococcal membrane. In contrast to AMPs, conventional antibiotics demonstrated slower killing kinetics at 10 × MIC. As shown in figure 5.2b, vancomycin only produced a 2.5-log reduction after 12 hours of exposure and required 24 hours to completely eliminate MRSA USA300. It is worth noting that frequent clinical failure in MRSA patients receiving vancomycin treatment has been linked to the poor bactericidal activity of this drug (21). Ciprofloxacin showed bactericidal activity by reducing the starting inoculum of MRSA

USA300 3-log within six hours; however, complete clearance was not achieved, even after 24 hours of exposure. Linezolid, as expected, showed a bacteriostatic effect reducing 116

MRSA USA300 starting inoculum by 1.3-log after 24 hours of treatment. AMPs with fast bactericidal activity have several advantages over their counterparts, conventional antibiotics, including limiting spread of infection, improving outcome of the disease, reducing the potential emergence of bacterial resistance, and reducing duration of treatment (22).

Efficacy of peptides on persister cells and stationary phases of MRSA

Persister cells are phenotypic variants of the normal bacterial population. They are extremely tolerant to antimicrobials and contribute to chronic and latent infections (23-25) (26). To assess the ability of AMPs to eradicate persister cells, MRSA was treated with ciprofloxacin in order to produce persister cells. When treated with ciprofloxacin, MRSA USA300 (in exponential growth phase) produces a biphasic killing pattern that results in surviving persister cells (figure 5.2c). The subsequent addition of conventional antimicrobials such as vancomycin or linezolid had minimal impact in reducing the number of persisters, which is in agreement with previous studies (24, 27, 28). However, treatment with WR12 and D-IK8 resulted in complete eradication of persister cells after 2 and 24 hours, respectively (figure 5.2c).

The ability of both WR12 and D-IK8 to kill MRSA persisters led us to next assess their impact on stationary-phase S. aureus which is known to be tolerant to many antimicrobial agents (24, 27, 28). Treatment of stationary-phase MRSA USA300 with WR12 and D- IK8 resulted in complete eradication of MRSA after 2 and 6 hours, respectively (figure 5.2d). With the exception of vancomycin, conventional antibiotics (ciprofloxacin and linezolid) did not have any effect on stationary-phase MRSA. Vancomycin had minimal impact in reducing the number of stationary-phase MRSA by only 2.4-log after 48 hours of exposure. The superior activity of our AMPs, when compared to conventional antibiotics, against persisters and stationary phase MRSA could be explained by their unique antimicrobial mechanism of action. Many antibiotics require growing and metabolically active bacteria to exert their antimicrobial effects and inhibit intracellular bacterial targets including nucleic acids (ciprofloxacin) or protein (linezolid) or cell wall synthesis (vancomycin) (29). In contrast, the positive charge present in WR12 and D- IK8serves as a point of attraction with cell membranes and consequently targeted- 117 disruption of bacterial membrane and leakage of intracellular contents (9, 10). This unique mechanism of action of AMPs does not require cells to be metabolically active and is not impaired by the dormant and quiescent state of bacteria (stationary and persister cells) (29). The results garnered lend valuable insight into using AMPs as a possible future therapeutic option for the treatment of persistent bacterial infections.

Efficacy of peptides on Staphylococcus biofilms

Biofilm formation is one of the major virulence factors of S. aureus (30). The polysaccharide matrix of biofilm protects bacteria from host immune defenses and hinders the ability of antibiotics to target deep-seated bacteria residing within the biofilm(31). Furthermore, biofilms act as an infectious niche with sustained release of bacteria inside the host, which leads to chronic infection, relapses, life-threatening bloodstream infections, and treatment failure (30). Given the serious challenges associated with staphylococcal biofilms and their role in promoting recurring infections in the host, we next moved to assess whether our peptides are capable of disrupting mature biofilms (formed after 24 and 48 hours) of both S. aureus and S. epidermidis. As shown in figure 5.3a, both peptides (at 4 × MIC) significantly disrupted the 24 hour mature biofilms of S. aureus, reducing biofilm mass by 50%. Vancomycin and linezolid were required at a higher concentration (16 × MIC) to reduce the same percentage of biofilm mass (p < 0.05) (figure 5.3a). Although some conventional antibiotics might be capable of disrupting 24 hour-mature bacterial biofilm, most antibiotics are not effective against 48 hour-mature biofilms due to the dormant state of growth of the bacterial cells present within the mature-biofilms(32). To examine whether the potential therapeutic application of WR12 and DIK-8 could be expanded beyond just inhibition of 24 hour biofilm, the ability of both peptides to disrupt 48 hours-mature staphylococcal biofilm was tested. As expected, we observed that the mature 48 hour S. aureus biofilms were resistant to antibiotics with no significant reduction in the biofilm mass observed, even at very high concentrations (64 × MIC) (figure 5.3b). Interestingly, WR12 and DIK-8 (at 4 × MIC) showed more than 50% reduction of biofilm mass (p < 0.05) (figure 5.3b).

Next, we evaluated the ability of our peptides to disrupt established biofilms of S. epidermidis ATCC 35984 (NRS 101), a clinical high slime producer strain isolated from 118 septicemic patients with a colonized intravascular catheter (33). This strain is a multidrug- resistant strain, exhibiting resistance to methicillin, erythromycin, kanamycin, gentamicin, clindamycin, and trimethoprim. The great thickness of the slime matrix of S. epidermidis biofilms makes it extremely resilient to antibiotic penetration (34). Hence, the 24 hour- mature S. epidermidis biofilms were less susceptible to vancomycin and linezolid, even at 64 × MIC, showing only 30-35% biofilm inhibition. The 48 hour-mature biofilms were not susceptible to the effect of both antibiotics action, even at a very high concentration (256 × MIC). WR12 and DIK-8 (at 8 × MIC) significantly reduced biofilm mass by more than 70% and 50% in 24 and 48 hour-mature biofilms, respectively (p < 0.05) (figures 5.3c&d). The studied peptides proved to be far superior to antibiotics against biofilm due to their amphipathic nature and their high cationic charge, which may facilitate their penetration through the extracellular biofilm matrix. Furthermore, we included the known biofilm inhibitor, LL-37, as a control (35). In our studies, LL-37 disrupted both biofilms of staphylococci regardless of the maturation stage of the biofilm. Interestingly, WR12 and D-IK8showed improved activity compared to LL-37, particularly in 48 hour mature biofilms of both species of staphylococci (figure 5.3). This may be related to the more potent antimicrobial activity and amphipathicity of the designed peptides compared to LL- 37.

Combination therapy analysis

The potent antimicrobial activity of the AMPs indicated that they have the potential to be used alone for treatment of skin infections caused by S. aureus. Although the use of a single agent to treat skin infections caused by S. aureus is the most commonly used practice in clinical settings, combination therapy has several advantages. These advantages include minimizing the likelihood of emergence of bacterial drug-resistance, lowering the doses required for each antibacterial agent thus mitigating drug toxicity, and expanding the spectrum of pathogens that can be targeted (36). Furthermore, several topical treatments currently used for treatment of skin infections involve a combination of more than one antibiotic, such as Polysporin (bacitracin, polymyxin B sulfate, and gramicidin) and Neosporin (bacitracin, neomycin, and polymyxin B sulfate) (37). Thus, identifying AMPs to pair with conventional antibiotics used for treatment of S. aureus 119 infections has good potential to expand available treatment options. Keeping the above points in mind, we were curious to assess the synergistic action of WR12 and D-IK8in combination with each other and with conventional antibiotics against four staphylococcal isolates. The isolates were chosen to represent different antibiotic susceptibility phenotypes: MSSA, MRSA, MRSE, and VRSA.

As presented in Table 5.5, WR12 and D-IK8 displayed synergistic activity when combined against MSSA, MRSA, and MRSE but not against VRSA. The FIC indices against all isolates except VRSA varied from 0.27 to 0.38. When our peptides were combined with antibiotics, WR12 proved to be superior to D-IK8, as it displayed potent synergism with most topical antibiotics (fusidic acid and mupirocin) and systemic antibiotics (daptomycin, teicoplanin, vancomycin, linezolid, ciprofloxacin, meropenem and oxacillin) against most tested strains with FIC indices ranging from 0.26 to 0.5. D- IK8 demonstrated synergism with fusidic acid and daptomycin in all four strains tested with FIC indices ranging from 0.26 to 0.5. D-IK8 also showed synergism with teicoplanin in two strains and with oxacillin, vancomycin, meropenem, and linezolid in one strain. Remarkably, we found that both WR12 and D-IK8 showed potent synergism with vancomycin against a VRSA isolate (with a low FIC index of 0.27)). Since vancomycin is considered to have nephrotoxicity at higher concentrations, decreasing the therapeutic doses required to treat staphylococcal infection is beneficial, as it will reduce the adverse side effects in affected patients (36). Notably, there were no antagonistic interactions recorded between the peptides and antimicrobials. The synergistic interaction between peptides and antibiotics could be a result of the membrane permeabilization action of peptides, leading to more penetration of antibiotics inside bacterial cells and augmented killing13,14. The potent and broad synergism observed for WR12 compared to D-IK8 is attributed to its rapid and more potent membrane permeabilization (figure 5.1).

Re-sensitization of VRSA to vancomycin, teicoplanin, and oxacillin in the presence of a sub-inhibitory concentration of peptides

As a strong synergistic relationship was observed with the peptides and vancomycin against the VRSA10 strain, we hypothesized that these peptides could be used to re- sensitize vancomycin and teicoplanin-resistant S. aureus (VRSA) strains to the effect of 120 vancomycin, teicoplanin and oxacillin. To assess this, we incubated VRSA strains with a subinhibitory concentration of the peptides for one hour. Afterward, the broth microdilution assay was used to determine the sensitivity of VRSA strains to antibiotics. Both peptides demonstrated the ability to re-sensitize VRSA strains to the effect of vancomycin, teicoplanin, and oxacillin. Pretreatment with subinhibitory concentrations of WR12 resulted in an 8- to 256-fold reduction in the MICs of vancomycin, teicoplanin, and oxacillin in the four VRSA strains tested. Pretreatment of VRSA strains with sub- inhibitory concentrations of D-IK8 resulted in a 2- to 256-fold reduction in the MICs of vancomycin, teicoplanin, and oxacillin. This study confirmed that, in addition to being used as antimicrobial agents alone or in combination with antibiotics in the treatment of staphylococcal infections, the peptides have the potential to suppress resistance of VRSA to conventional antibiotics. Clearly, further studies are needed to understand the mechanism of re-sensitization and their potential clinical applications. To explore the mechanism of re-sensitization, we monitored the leakage of preloaded calcein dye after exposure of VRSA to ½ × MIC of peptides for one hour. As demonstrated in supplementary figure 5.4, WR12 and D-IK-8 demonstrated leakage of calcein dye without affecting the survival of the VRSA strain (figure 5.4). This demonstrates that at subinhibitory concentrations, the peptides permeabilized the membrane potentially leading to increased access of antibiotics to their target. To confirm this hypothesis, VRSA (VRS10) was incubated with a sub-inhibitory concentration (½ × MIC) of WR12 and D- IK8 for one hour. Then treated with fluorescently labeled vancomycin (bodipy vancomycin) for 30 minutes. Bacterial pellets were fixed with 4% paraformaldehyde, and visualized under confocal microscope. As demonstrated in figure 5.5, pre-treatment of VRSA with WR12 and D-IK8 led to increased uptake and binding of fluorescently labeled vancomycin in contrast to non-peptide pre-treated samples. The fluorescence was maximized at the cell division septa and the cell wall of VRSA. Several studies reported that membrane acting antimicrobials re-sensitize resistant bacteria to antibiotics by similar mechanisms. For example, the protein-lipid complex from human milk, HAMLET (human alpha-lactalbumin made lethal to tumor cells) was able to resensitize MRSA to methicillin and VISA to vancomycin by depolarization of the bacterial membrane and dissipation of the proton motive force leading to more access and increased cell-associated 121 binding of antibiotics (38, 39). Moreover, Minahk et al reported that sub-lethal concentrations of enterocin synergize with antibiotics by dissipation of the proton motive force leading to inhibition of bacterial efflux systems and more accumulation of antibiotics intracellularly (40). The amphibian antimicrobial peptide, esculentin-1b (1– 18) was also reported to cause permeabilization of bacterial membranes at subinhibitory concentrations leading to synergism with conventional antibiotics against E. coli (41). The peptidomimetic OAK (oligo-acyl-lysyl), was able to resensitize erythromycin-resistant Escherichia coli to erythromycin both in vitro and in an animal model of infection. The authors explained the mechanism of resensitization to be transient depolarization of the membrane potential at subinhibitory concentrations (42).

Toxicity of peptides on human keratinocytes

One of the major limitations for advancement of AMPs for clinical applications is toxicity to host tissues(1). Here, we evaluated the cytotoxic effect of IDK-8 and WK12 on human keratinocytes (HaCaT cells) using the MTS assay. The half maximal effective concentrations (EC50) of WR12 and D-IK8were 128 and >256 µM, respectively (figure

5.6a). These values correlate to 64- and >32-fold of the MIC50 for WR12 and D-IK8, respectively. These results suggest that these AMPs have a favorable drug safety profile. The low toxicity of peptides against mammalian cells compared to their potent antimicrobial activity suggests their selective actions against the negatively charged bacterial membranes compared to the zwitterionic mammalian membranes.

Intracellular antibacterial efficacy of peptides in human keratinocytes

Although S. aureus is not considered a typical intracellular pathogen, it can invade and thrive inside mammalian host cells. Moreover, treatment with conventional antimicrobials during the S. aureus intracellular invasion phase is a daunting task because most antimicrobials are unable to access intracellular replicative niches and achieve the optimum therapeutic concentrations within the infected cells(43). Accordingly, treatment with conventional drugs of choice (such as vancomycin and aminoglycosides) is often associated with high clinical failures that exceed 40% in intracellular MRSA infections due to poor intracellular penetration of these drugs (44, 45). Since intracellular persistence of S. aureus constitutes a potent virulence component for various skin diseases such as 122 impetigo and folliculitis (46), we chose to assess the ability of AMPs to kill invasive intracellular MRSA infections. Due to the fact that MRSA and MSSA infect, reside, and replicate inside human keratinocytes, it was important to test the activity of WR12 and D- IK8against MRSA and MSSA infected human keratinocytes. As depicted in figure 5.6b, both peptides showed significant reduction of intracellular MRSA and MSSA at 4 × MIC. D-IK8displayed the most potent activity with significant reduction of 96% ± 1.15 and 91.08% ±14.94 of intracellular MRSA and MSSA, respectively at 4 × MIC. WR12 at 4 × MIC demonstrated significant reductions of 40.98% ± 8.03 and 45% ± 6.23 of intracellular MRSA and MSSA, respectively. In contrast, antibiotics showed either low reduction (linezolid, 30-35%) or nonsignificant reduction (vancomycin) against intracellular staphylococci at 4 × MIC (figure 5.6b). Taken together, these results show that AMPs exhibits potent intracellular anti-staphylococcal efficacy in infected keratinocytes. These peptides, with their potent intracellular activity, could be useful in treating certain chronic exacerbating skin diseases such as Darier’s disease (keratosis follicularis) where S. aureus persists inside keratinocytes, leading to recurrent infections and treatment failure(46). Interestingly, D-IK8was more efficient than WR12 against MRSA and MSSA in infected keratinocytes compared to results obtained in pure culture. We hypothesized that the weak intracellular anti-staphylococcal activity of WR12 may be due to lack of intracellular penetration or due to reduced stability inside mammalian cells. To differentiate between the two possibilities, we conducted two experiments, (confocal study and stability study). Confocal studies of WR12-FITC demonstrated that this peptide penetrates and accumulates inside mammalian cells (figure 5.7). However, when WR12 was treated with trypsin for 4 hours, its anti-staphylococcal activity was abolished compared to D-IK8 (Table 5.4). This supports our hypothesis that the weak intracellular anti-staphylococcal activity of WR12 may be due to the reduced stability of this peptide intracellularly.

Efficacy of peptides in mice model of MRSA skin infection In light of our successful in vitro experiments, we moved forward with an in vivo experiment with a murine model of MRSA skin infection (47, 48). Briefly, groups of mice (n=5) were injected with a highly virulent community-acquired MRSA strain USA300- 0114. This clinical strain was isolated from a skin and soft-tissue outbreak in a state prison in Mississippi, USA(49). Twenty-four hours after the intradermal injection, mice 123 developed an abscess at the injection site. Within 48 hours of infection, the abscess further developed into an open wound. Open wounds were treated topically with either 2% peptides, 2% fusidic acid, or vehicle alone (petroleum jelly). An additional group was treated orally with 25 mg/kg linezolid. All groups of mice were treated twice daily for three days. As presented in figure 5.8a, all treatments significantly reduced the mean bacterial counts of MRSA in wounds compared to the control group (P ≤ 0.05). WR12 and D-IK8showed 1.71 ± 0.07 and 1.78 ± 0.07 log reduction of MRSA USA300, respectively. The group treated with fusidic acid produced a 1.94 ± 0.352 log reduction in bacterial count. The group treated with oral linezolid generated a 1.678 ± 0.38 log reduction in bacterial load. These results reveal that our peptides are very effective in reducing the bacterial load in MRSA skin lesions. The clinical severity of skin infections caused by S. aureus is driven by excess production of host pro-inflammatory cytokines more so than by bacterial burden. In addition, excessive host inflammation delays the wound healing process and leads to more scar formation(50) (51). We hypothesized that therapeutics with combined antibacterial and anti-inflammatory properties should be superior to traditional antibiotics for treatment of S. aureus skin infections. To confirm, we investigated the immune-modulatory effect of our peptides by measuring the levels of pro-inflammatory cytokines produced normally during infection, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) (52). As shown in figures 5.8b and 5.8c, both peptides produced a significant reduction in TNF- α and IL-6 levels compared to the untreated control and antibiotic-treated groups. WR12 and D-IK8reduced the TNF-α level by 50% and 44%, respectively, while the IL-6 level was reduced by 48% and 42%, respectively. However, treatment with 2% fusidic acid reduced TNF-α and IL-6 levels by only 29% and 25%, respectively, which is in agreement with previous findings(53). Linezolid, on the other hand, did not show any significant reduction in the levels of cytokines when compared to the control group. The combined antimicrobial and immunomodulatory effects of the AMPs should confer an added advantage in the treatment of S. aureus skin infection and might help in promoting epithelization and accelerating wound healing processes. Some AMPs have reached preclinical and clinical phases for topical treatment of bacterial infection. One of the AMPs that has advanced furthest in clinical trials is pexiganan for curing diabetic foot 124 ulcers (54). Topical application of 2% pexiganan cream showed therapeutic resolution equivalent to oral ofloxacin for treatment of mild infections of diabetic foot ulcers (54). Interestingly, no significant resistance to pexiganan emerged among patients who received pexiganan. However, bacterial resistance to ofloxacin emerged in some patients who received ofloxacin (54). Currently, pexiganan is undergoing phase 3 development as a topical agent for treatment of mild infections of diabetic foot ulcers (ClinicalTrials.gov registration numbers NCT01594762 and NCT01590758). Another compound that showed success in clinical trials is the peptidomimetic, brilacidin. Brilacidin is a defensin-mimetic that targets the bacterial membrane, similar to AMPs. The MIC90 of brilacidin against a collection of multidrug-resistant S. aureus isolates was 2 μg/ml. Brilacidin demonstrated clinical efficacy and safety on two studies of phase II clinical trials for the treatment of acute bacterial skin and skin structure infections (ABSSSI). The FDA approved brilacidin to advance into Phase III clinical trials (55). Lytixar (LTX-109) is a synthetic, membrane- degrading peptide that has been developed by Lytix Biopharma (Oslo); this peptide has completed a Phase I/IIa clinical trial for nasally colonized MRSA. A significant effect on nasal decolonization of MRSA and MSSA was observed after only two days of LTX-109 treatment in subjects treated with 2% or 5% LTX-109, compared to the vehicle. The success of pexiganin, brilacidin, and LTX-109 demonstrates the promise that antimicrobial peptides have as potential therapeutic agents for treatment of multidrug- resistant pathogens.

5.5 Conclusion

We have successfully demonstrated the potential utility of WR12 and D-IK8against MRSA and VRSA clinical isolates. WR12 and D-IK8were superior to antibiotics, demonstrating potent and rapid eradication of persister cells and MRSA cells in stationary phase of growth. Moreover, WR12 and D-IK8 disrupted the mature biofilms of staphylococci and were able to kill intracellular staphylococci at a more significant rate than antibiotics of choice. Additionally, WR12 and D-IK8augmented the antibacterial action of topical and systemic antibiotics, which will increase the clinical uses of these antibiotics and decrease their adverse effects. Finally, WR12 and D-IK8 significantly 125 reduced the count of MRSA in skin lesions and displayed potent immunomodulatory effects. Collectively, the qualities of WR12 and D-IK8 presented here have the potential to be used for different clinical applications, including resilient MRSA infections.

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5.6 Reference

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31. Mah TF, O'Toole GA. 2001. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol 9:34-39. 32. Wolcott RD, Rumbaugh KP, James G, Schultz G, Phillips P, Yang Q, Watters C, Stewart PS, Dowd SE. 2010. Biofilm maturity studies indicate sharp debridement opens a time- dependent therapeutic window. J Wound Care 19:320-328. 33. Christensen GD, Bisno AL, Parisi JT, McLaughlin B, Hester MG, Luther RW. 1982. Nosocomial septicemia due to multiply antibiotic-resistant Staphylococcus epidermidis. Ann Intern Med 96:1-10. 34. Singh R, Ray P, Das A, Sharma M. 2010. Penetration of antibiotics through Staphylococcus aureus and Staphylococcus epidermidis biofilms. J Antimicrob Chemother 65:1955-1958. 35. Dean SN, Bishop BM, van Hoek ML. 2011. Natural and synthetic cathelicidin peptides with anti-microbial and anti-biofilm activity against Staphylococcus aureus. BMC Microbiol 11:114. 36. Nguyen HM, Graber CJ. 2010. Limitations of antibiotic options for invasive infections caused by methicillin-resistant Staphylococcus aureus: is combination therapy the answer? Journal of Antimicrobial Chemotherapy 65:24-36. 37. Mohammad H, Cushman M, Seleem MN. 2015. Antibacterial Evaluation of Synthetic Thiazole Compounds In Vitro and In Vivo in a Methicillin-Resistant Staphylococcus aureus (MRSA) Skin Infection Mouse Model. PLoS One 10:e0142321. 38. Marks LR, Clementi EA, Hakansson AP. 2012. The human milk protein-lipid complex HAMLET sensitizes bacterial pathogens to traditional antimicrobial agents. PLoS One 7:e43514. 39. Marks LR, Clementi EA, Hakansson AP. 2013. Sensitization of Staphylococcus aureus to methicillin and other antibiotics in vitro and in vivo in the presence of HAMLET. PLoS One 8:e63158. 40. Minahk CJ, Dupuy F, Morero RD. 2004. Enhancement of antibiotic activity by sub-lethal concentrations of enterocin CRL35. J Antimicrob Chemother 53:240- 246. 41. Marcellini L, Borro M, Gentile G, Rinaldi AC, Stella L, Aimola P, Barra D, Mangoni ML. 2009. Esculentin-1b(1-18)--a membrane-active antimicrobial peptide that synergizes with antibiotics and modifies the expression level of a limited number of proteins in Escherichia coli. FEBS J 276:5647-5664. 42. Goldberg K, Sarig H, Zaknoon F, Epand RF, Epand RM, Mor A. 2013. Sensitization of gram-negative bacteria by targeting the membrane potential. FASEB J 27:3818-3826. 43. Ellington JK, Harris M, Hudson MC, Vishin S, Webb LX, Sherertz R. 2006. Intracellular Staphylococcus aureus and antibiotic resistance: implications for treatment of staphylococcal osteomyelitis. J Orthop Res 24:87-93. 44. Cruciani M, Gatti G, Lazzarini L, Furlan G, Broccali G, Malena M, Franchini C, Concia E. 1996. Penetration of vancomycin into human lung tissue. Journal of Antimicrobial Chemotherapy 38:865-869. 45. Van Bambeke F, Barcia-Macay M, Lemaire S, Tulkens PM. 2006. Cellular pharmacodynamics and pharmacokinetics of antibiotics: current views and perspectives. Curr Opin Drug Discov Devel 9:218-230. 129

46. von Eiff C, Becker K, Metze D, Lubritz G, Hockmann J, Schwarz T, Peters G. 2001. Intracellular persistence of Staphylococcus aureus small-colony variants within keratinocytes: a cause for antibiotic treatment failure in a patient with darier's disease. Clin Infect Dis 32:1643-1647. 47. Mohamed MF, Seleem MN. 2014. Efficacy of short novel antimicrobial and anti- inflammatory peptides in a mouse model of methicillin-resistant Staphylococcus aureus (MRSA) skin infection. Drug Des Devel Ther 8:1979-1983. 48. Thangamani S, Younis W, Seleem MN. 2015. Repurposing ebselen for treatment of multidrug-resistant staphylococcal infections. Sci Rep 5:11596. 49. Tenover FC, Goering RV. 2009. Methicillin-resistant Staphylococcus aureus strain USA300: origin and epidemiology. Journal of Antimicrobial Chemotherapy 64:441-446. 50. Eming SA, Krieg T, Davidson JM. 2007. Inflammation in wound repair: molecular and cellular mechanisms. J Invest Dermatol 127:514-525. 51. Montgomery CP, Daniels MD, Zhao F, Spellberg B, Chong AS, Daum RS. 2013. Local inflammation exacerbates the severity of Staphylococcus aureus skin infection. PLoS One 8:e69508. 52. Miller LS, Cho JS. 2011. Immunity against Staphylococcus aureus cutaneous infections. Nat Rev Immunol 11:505-518. 53. Rubin BK, Tamaoki J. 2005. Antibiotics as anti-inflammatory and immunomodulatory agents. Birkhä user, Basel ; Boston. 54. Lipsky BA, Holroyd KJ, Zasloff M. 2008. Topical versus systemic antimicrobial therapy for treating mildly infected diabetic foot ulcers: a randomized, controlled, double-blinded, multicenter trial of pexiganan cream. Clin Infect Dis 47:1537- 1545. 55. Vuong C, Yeh AJ, Cheung GY, Otto M. 2015. Investigational drugs to treat methicillin-resistant Staphylococcus aureus. Expert Opin Investig Drugs doi:10.1517/13543784.2016.1109077:1-21. 56. CLSI. 2007. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; approved standard M7-A7. CLSI, Wayne, PA. 57. Mohamed MF, Hamed MI, Panitch A, Seleem MN. 2014. Targeting Methicillin- Resistant Staphylococcus aureus with Short Salt-Resistant Synthetic Peptides. Antimicrob Agents Chemother 58:4113-4122. 58. Mohammad H, Mayhoub AS, Cushman M, Seleem MN. 2015. Anti-biofilm activity and synergism of novel thiazole compounds with glycopeptide antibiotics against multidrug-resistant staphylococci. J Antibiot (Tokyo) 68:259-266. 59. Mohamed MF, Hammac GK, Guptill L, Seleem MN. 2014. Antibacterial activity of novel cationic peptides against clinical isolates of multi-drug resistant Staphylococcus pseudintermedius from infected dogs. PLoS One 9:e116259.

130

Table 5.1. Amino acid sequence and physicochemical properties of peptides used in this study Peptide Molecular Hydrophobic Amino acid sequencea Length Charge designation weight amino acids

WR-12 RWWRWWRRWWRR 12 2072.4 + 6 50 %

D-IK8 irikirik 8 1040.28 + 4 50 %

Pexiganan GIGKFLKKAKKFGKAFVKILKK 22 2478.163 +9 45% aSmall underlined residues represent D-amino acids

Table 5.2. Minimum inhibitory concentration (MIC) of peptides and antibiotics against clinical and drug-resistant staphylococci isolates

MIC (µM)

Strain

Strain ID

Typea

37

- IK8

-

LL D

WR12

Linezolid

Kanamycin

Vancomycin

Erythromycin Ciprofloxacin Trimethoprim

< < ATCC 6538 8 16 2 0.5 4 1 4 0.25 0.25

RN4220 < < 8 >128 1 0.5 2 1 2 0.25 0.25 (NRS107) MSSA < < NRS77 8 >128 2 0.5 1 1 2 0.25 0.25

< NRS846 16 >128 4 0.5 >128 4 2 16 0.25

NRS860 4 16 >128 1 0.5 >128 4 16 2 USA100 MRSA 2 8 >128 4 0.5 >128 32 8 1 (NRS382)

USA200

4 8 >128 4 0.5 32 >128 >128 >128 (NRS383)

131

Table 5.3. continued USA300-0114 4 16 >128 2 0.5 >128 >128 4 2 (NRS384)

USA400 < < 8 >128 4 0.5 1 4 2 0.25 0.25 (NRS123)

USA500 8 16 >128 4 1 >128 >128 32 >128 (NRS385)

USA700 2 8 >128 4 1 8 >128 0.5 2 (NRS386)

USA800 < < 2 16 >128 4 0.5 1 2 (NRS387) 0.25 0.25

USA1000 2 8 >128 4 0.5 2 4 2 4 (NRS483)

USA1100 < 8 >128 4 1 1 0.5 2 1 0.25 (NRS484) < < NRS194 8 >128 4 1 1 2 2 0.25 0.25

NRS108 1 8 >128 4 0.5 32 >128 4 >128

NRS119 < 4 16 >128 128 0.5 >128 >128 >128 (LinR) 0.25

< ATCC 43300 4 >128 4 0.5 >128 >128 2 2 0.25

ATCC BAA- 8 >128 4 0.5 >128 >128 8 1 44 4

< NRS70 4 >128 2 0.5 >128 32 2 1 0.25

NRS71 4 8 >128 2 0.5 >128 >128 >128 4 < NRS100 16 >128 2 0.5 >128 4 2 8 0.25

< < NRS123 8 >128 2 0.5 1 4 2 0.25 0.25

132

Table 5.4. continued

NRS1 1 8 >128 2 8 >128 >128 16 2 VISA NRS19 1 8 >128 2 4 >128 >128 >128 >128

NRS37 1 8 >128 2 8 >128 >128 32 2 < MRSE NRS101 4 4 16 2 0.5 >128 >128 >128 0.25

a (MSSA) : methicillin sensitive Staphylococcus aureus; (MRSA): methicillin resistant Staphylococcus aureus; (VISA):vancomycin intermediate Staphylococcus aureus and (MRSE): methicillin resistant Staphylococcus epidermidis

133

Table 5.5. Minimum inhibitory concentration (MIC) of peptides and antibiotics against clinical vancomycin resistant Staphylococcus aureus (VRSA) isolates

MIC(µM)

Strain ID nin

37

- IK8

-

LL

WR12 D

Oxacillin

Kanamycin

Teicopla Vancomycin

VRS4 4 8 >128 32 64 >128 16

VRS5 8 16 >128 32 4 >128 8

VRS10 8 16 >128 >128 >128 >128 >128

VRS11a 4 16 >128 >128 >128 >128 >128

VRS11b 4 16 >128 >128 >128 >128 >128

VRS12 8 16 >128 >128 >128 >128 >128

VRS13 4 16 >128 >128 >128 >128 >128

134

Table 5.6. Minimum inhibitory concentration (MIC) of peptides against Methicillin resistant Staphylococcus aureus (MRSA) USA300 in different media conditions MIC (µM) Cation adjusted NaCl MgCl Trypsin MHB 2 MHB 150 mM 2 mM 1:500 WR12 4 4 4 8 > 64 D-IK8 16 32 64 64 16 Pexiganan 16 32 64 16 nd nd: not determined

Table 5.7. The fractional inhibitory concentration index range of peptides in combination with each other and with antibiotics against Staphylococcus aureus isolates

∑FICIa

IK8

-

strain

WR12 D

Oxacillin

Linezolid

Mupirocin

Teicoplanin

Meropenem

Fusidicacid

Daptomycin

Vancomycin Ciprofloxacin MSSA WR12 - 0.27 0.31 0.28 0.31 nd 0.26 0.28 0.50 0.28 0.27

ATCC D-IK8 0.31 - 0.31 0.50 0.31 0.31 0.75 1.25 1.25 0.50 0.38 6538

MRSA WR12 - 0.31 0.38 0.27 0.28 0.27 0.27 0.31 0.26 0.31 0.31 USA300 D-IK8 0.38 - 0.50 0.75 0.75 0.75 0.75 1.25 1.25 1.25 0.31 MRSE WR-12 - 0.38 0.38 0.38 0.26 0.31 0.38 0.50 0.75 0.75 0.28

NRS101 D-IK8 0.31 - 0.38 0.75 0.26 0.50 1.25 0.50 0.75 1.25 0.75

VRSA WR12 - 1.25 0.50 0.75 0.26 0.50 0.27 1.25 0.75 0.38 0.75

(VRS10) D-IK8 0.75 - 0.50 0.75 0.26 0.75 0.27 1.25 1.25 1.25 1.25 a ∑FICI, fractional inhibitory concentration index. FIC index was interpreted as follows: An FIC index of ≤ 0.5 is considered to demonstrate synergy. Additive was defined as an FIC index of 1. Antagonism was defined as an FIC index of > 4. nd, not determined. (MSSA): methicillin sensitive Staphylococcus aureus; (MRSA): methicillin resistant Staphylococcus aureus; (VRSA): vancomycin resistant Staphylococcus aureus and (MRSE) methicillin resistant Staphylococcus epidermidis

135

Table 5.8. Resensitization of vancomycin resistant S. aureus (VRSA) to vancomycin, teicoplanin and oxacillin using a sub-inhibitory concentration (½×MIC) of WR12 or D-IK8 Fold of re-sensitizationa

strain Vancomycin Teicoplanin Oxacillin WR12 8 32 128

No sensitization VRS4 D-IK8 2 32 effect.

WR12 256 256 256 VRS10 D-IK8 256 256 256

WR12 128 8 256 VRS12 No sensitization D-IK8 64 256 effect.

WR12 64 16 256 VRS13 D-IK8 128 32 256 a Fold of re-sensitization: it is the ratio of the MIC of antibiotic alone divided by the MIC of antibiotic after re-sensitization with (½×MIC) of peptides (WR12 or D-IK8).

136

Figure 5.1. Permeabilization of the cytoplasmic membrane of MRSA USA300 as a function of peptide concentration. Permeabilization is indicated by percent of calcein leakage for 60 min exposure. The results are given as means ± SD (n = 3; data without error bars indicate that the SD is too small to be seen). Permeabilization of the cytoplasmic membrane of MRSA USA300 as a function of peptide concentration 137

a - L o g a rith m ic p h a s e o f M R S A U S A 3 0 0 b - L o g a rith m ic p h a s e o f M R S A U S A 3 0 0

1 0 1 0 9

9 C o n tro l )

8 ) l l 8

C o n tro l V a n 1 0 X m

7 m / / 7

L in 1 0 X U

6 W R 1 2 5 X U 6 F

F C ip 1 0 X C

5 W R 1 2 1 0 X C (

( 5

0

4 0 4 1

D -IK 8 5 X 1 g

3 D -IK 8 1 0 X g 3

o o

L 2 L 2 1 1 0 0 0 6 0 1 2 0 1 8 0 2 4 0 0 6 1 2 1 8 2 4 T im e (m in ) T im e (h r )

c - P e rs is te r c e lls o f M R S A U S A 3 0 0 d - S ta tio n a ry p h a s e o f M R S A U S A 3 0 0

C ip 1 0 X 1 1 S e c o n d C IP th e n W R 1 2 1 0 X 1 0 1 0 a n tib io tic 9

C IP th e n D -IK 8 1 0 X C o n tro l )

) 9 a d d e d l l 8

8 C IP th e n L in 1 0 X W R 1 2 1 0 X

m

m / / 7

7 U U C IP th e n V a n 1 0 X

6 D -IK 8 1 0 X F

F 6

C C

( 5

( V a n 1 0 X

5 0

0 4 1

1 4 L in 1 0 X g

g 3 3 o

o C ip 1 0 X L L 2 2 1 1 0 0 0 6 1 2 1 8 2 4 3 0 0 6 1 2 1 8 2 4 3 0 3 6 4 2 4 8 T im e (h r ) T im e (h r )

Figure 5.2. The kinetics of killing of peptides and antibiotics against logarithmic, persister cells and stationary phase of MRSA USA300.

(a) Logarithmic phase of MRSA USA300 exposed to peptides (WR12, D-IK8) at 5X and 10X MIC or (b) antibiotics at 10X MIC. (c) The kinetics of killing of persister cells and (d) stationary phase of MRSA USA300 exposed to peptides (WR12, D-IK8) and antibiotics at 10X MIC. In figure 1c, CIP means treatment of MRSA with ciprofloxacin at 10X MIC for 6 hr then the surviving persisters were exposed to peptides or antibiotics at 10X MIC as the arrow pointed. Untreated samples served as a control. Abbreviations, Van, vancomycin; Lin, linezolid; Cip, ciprofloxacin. The results are given as means ± SD (no=3); data without error bars indicate that the SD is too small to be seen.

138

a - S . a u re u s b io film (2 4 h r) b - S . a u re u s b io film (4 8 h r)

e 1 2 0 1 2 0

g a t 1 0 0

n 1 0 0

e c r 8 0 8 0

e * p

* s 6 0 * 6 0 s * *

a * * # * # m

4 0 4 0

m

l i

f 2 0 2 0

o i

B 0 0 l C C l C C o IC IC IC I I o IC IC IC I I tr M M tr M M M M M M M M n n X o X X X X X o X X X X 4 4 4 6 6 4 4 4 4 4 C 1 1 C 6 6 2 8 7 2 8 7 1 3 d n 1 3 d n IK - li i IK - li i R - c R - L c L o y o y W D L z W D L z e m e m n o n o i c i c L n L n a a V V

c - S . e p id e r m id is b io film (2 4 h r) d - S . e p id e r m id is b io film (4 8 h r)

e 1 2 0 1 2 0

g

a t

n 1 0 0 1 0 0

e c r 8 0 * 8 0 *

e *

p

s 6 0 6 0 s * #

a * * #

* # m

4 0 4 0

* #

m

l i

f 2 0 2 0

o i

B 0 0 l C C l C C o IC IC IC I I o IC IC IC I I tr M M tr M M M M M M M M n n X o X X X X X o X X X X 8 8 8 4 4 8 8 8 6 6 C 6 6 C 5 5 2 8 7 2 8 7 2 2 1 3 d n 1 3 K - i i K - n R -I l c R -I id i L o y L l c W D L z W D L o y e m z e m in o c in o L n c a L n V a V Figure 5.3. The effect of peptides on staphylococcal biofilms Peptides (WR12, D- IK8 and LL-37) and antibiotics (vancomycin & linezolid) investigated against 24 hr (a&c) and 48 hr (b&d) old biofilms of S. aureus (a&b) and S. epidermidis (c&d). The adherent biofilm stained by crystal violet, then the dye was extracted with ethanol, measured at 595 or 490 nm absorbance and presented as percentage of biofilm reduction compared to untreated wells “control”. All experiments were done in triplicate for statistical significance. The two tailed Student t test, was used to determine statistical significance between two groups. One asterisk (*) indicates statistically different than control (p < 0.05). Symbol (#) indicates statistically different than the antibiotic treated wells (p < 0.05). 139

) W R 1 2

% (

6 0 1 0

n

o i

t 5 0

L

a 8 z

o

i l

g i 4 0

1

(

) b

0

b a e 6

C

a

e v

r r 3 0 F

s

m u

U r

)

c e

4 / (

m p

2 0

L e

n 2

a 1 0

r b

m 0 0

e M 0 .0 0 .5 1 .0

C o n c e n tr a tio n (X M IC )

) D -IK 8

% (

6 0 1 0

n

o i

t 5 0

L

a 8 z

o

i l

g i 4 0

1

(

) b

0

b a e 6

C

a

e v

r r 3 0 F

s

m u

U r

)

c e

4 / (

m p

2 0

L e

n 2

a 1 0

r b

m 0 0

e M 0 .0 0 .5 1 .0

C o n c e n tr a tio n (X M IC ) Figure 5.4. Survival and permeabilization of the cytoplasmic membrane of VRS10 after treatment with 0.5 X MIC of WR12 and D-IK8.

Membrane permeabilization is indicated by percent of calcein leakage after 60 min exposure to peptides. Bacterial count of VRS10 is presented as columns. The results are given as means ± SD (n = 3; data without error bars indicate that the SD is too small to be seen).

140

Figure 5.5. Sub-inhibitory concentration of WR12 and D-IK8 increase the uptake and binding of fluorescently labeled vancomycin (bodipy vancomycin).

VRSA (VRS10) was incubated with sub-inhibitory concentration of WR12 and D-IK8 for one hour or left untreated. Then treated with bodipy vancomycin for 30 minutes. Bacterial pellets were fixed with 4% paraformaldehyde, and visualized under 40X oil objective of Nikon A1R multi-photon inverted confocal microscope. DIC, differential interference contrast.

141 a 1 2 0

E C 5 0 > 2 5 6

) 1 0 0

% (

D -IK 8

8 0

s l

l W R 1 2

e 6 0 E C 5 0 = 1 2 8

c

e 4 0

v

i L 2 0

0 1 2 4 8 1 6 3 2 6 4 1 2 8 2 5 6

C o n c e n tr a tio n ( M ) b

M R S A U S A 3 0 0 M S S A A T C C 6 5 3 8

** **

e

e

g

g a

1 0 0 a 1 0 0 t

t ** **

n

n

e

e

c

c r r *

* * e

e *

p

p

l

l a

a 5 0 5 0

i

i

r

r

e

e

t

t c

c *

a

a

B B * 0 0 l l n d in d 8 o i i 2 8 o i 2 c l r c l 1 K tr 1 K t o I y o -I y R - n z R n z m D o m e D o e W o W o n C in C c i c L n L n a a V V Figure 5.6. Toxicity (a) and intracellular anti-staphylococcal activity (b) of peptides in human keratinocyte (HaCaT).

Cytotoxicity assay showing the percent mean absorbance at 490 nm after incubating human keratinocyte (HaCaT) with peptides (WR12 and D-IK8) at different concentrations. Sterile water (peptide diluent) served as negative control. Cell viability was measured by MTS assay. EC50 is the half maximal effective concentration which equal 128 µM for WR12 and >256 µM for D-IK8. Results are expressed as means from three measurements ± standard deviation. (b) The effect of (WR12 and D-IK8) and antibiotics (vancomycin & linezolid) to kill MRSA USA300 (left panel) and MSSA ATCC 6538 (right panel) inside HaCaT cells after treatment with 4 X MIC for 24 hr in DMEM +10 % FBS. Statistical analysis was calculated using one-way ANOVA, with post hoc Tukey’s multiple comparisons test. P values of ˂ 0.05 were considered significant. One asterisk (*) indicates significance from control negative. Two asterisk (**) indicates significance from control antibiotics. 142

Figure 5.7. Internalization studies of WR12 inside macrophage cells.

Confocal images demonstrate cell penetration of WR12-FITC (10 µM) inside macrophage cells. Lower panel is a higher magnification of upper panel. 143

Figure 5.8. Efficacy of peptides and control antibiotics in bacterial load (a) and level of pro-inflammatory cytokines (b&d) in a murine model of MRSA skin infection. Mouse was injected with the highly virulent MRSA USA300. After injection, mouse developed an abscess at the local site of injection over the back which further developed into an open wound within 48 hr from infection. Two days after the start of infection, mice were treated twice daily for 3 days either topically with fusidic acid (2%), D-IK8 (2%), WR12 (2%) formulated in petroleum jelly; or orally with linezolid (25 mg/kg). Petroleum jelly alone served as negative control. Figure (a) show the average log count of MRSA after treatment. Figure (b&c) show the effect of peptides on production of anti- inflammatory cytokines (TNF-α and IL-6) in MRSA skin lesions. Tissue homogenate supernatants were examined for cytokine production using ELISA. Cytokine levels were expressed as percent change relative to negative control. The two tailed Student t test, was used to determine statistical significance between two groups (a P value of < 0.05 was considered significant). One asterisk (*) indicates significance from control negative. Symbol (#) indicates statistically different than the antibiotic treated mice (p < 0.05). 144

CHAPTER 6. A SHORT D-ENANTIOMERIC ANTIMICROBIAL PEPTIDE WITH POTENT IMMUNOMODULATORY AND ANTIBIOFILM ACTIVITY AGAINST MULTIDRUG- RESISTANT PSEUDOMONAS AERUGINOSA AND ACINETOBACTER BAUMANNII

(Mohamed, M.F., Brezden A. Mohammed H, Chmielewski J, and Seleem, M.N (2016). A short D-enantiomeric antimicrobial peptide with potent immunomodulatory and antibiofilm activity against multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii. Submitted to Antimicrobial agents and chemotherapy, under review)

6.1 Abstract

The emergence of resistance of Pseudomonas aeruginosa and Acinetobacter baumannii to critical antibiotics, including colistin and carbapenems, represents a serious medical threat. Antimicrobial peptides (AMPs) represent a promising therapeutic alternative for treatment of bacterial infections. Herein, the antimicrobial activity of a short α-helical peptide, RR, was markedly improved through systematic modifications made to this peptide’s primary structure. From the peptides designed in this study, RR4 and its D- enantiomer, D-RR4, were the most potent analogues with at least 32-fold improvement in antimicrobial activity observed. The MIC50 of D-RR4 was 2 and 4 µM against multidrug- resistant strains of A. baumannii and P. aeruginosa, respectively. Interestingly, D-RR4 demonstrated potent activity against colistin-resistant, cystic fibrosis strains of P. aeruginosa indicating a potential therapeutic advantage of this peptide over several AMPs. In contrast to many natural AMPs, D-RR4 retained its antimicrobial activity under challenging physiological conditions (high salts, serum, and acidic pH). Furthermore, D- RR4 was superior in disrupting adherent biofilms of A. baumannii and P. aeruginosa when compared to conventional antibiotics. Remarkably, D-RR4 demonstrated strong synergy with colistin, meropenem and cefotaxime, even in the case of meropenem and 145 cefotaxime-resistant isolates. Of note, D-RR4 was able to bind to LPS to reduce the endotoxin-induced proinflammatory cytokine response in macrophages. Finally, D-RR4 protected Caenorhabditis elegans from lethal infections of P. aeruginosa and A. baumannii and enhanced the activity of colistin in vivo against colistin-resistant P. aeruginosa. The present study confirms D-RR4 possesses potent antimicrobial, antibiofilm and immunomodulatory activity that warrants further investigation.

6.2 Introduction

The emergence and spread of Pseudomonas aeruginosa and Acinetobacter baumannii strains exhibiting resistance to numerous antibiotics represents a serious global medical threat (1-3). P. aeruginosa has been linked to serious human illnesses including postoperative and burn wound infections, urinary tract infections, chronic leg ulcers, otitis and keratitis (4). Cystic fibrosis (CF) patients are particularly susceptible to pseudomonas infections that lead to progressive loss of lung function and premature death (5, 6). A. baumannii, like P. aeruginosa, has been associated with burn wound infections in addition to invasive syndromes such as ventilator-associated pneumonia and urinary tract infections (7, 8). Compounding the situation further, extensively drug-resistant and pandrug-resistant isolates of P. aeruginosa and A. baumannii have been increasingly reported worldwide (9-11). The ability of these resistant pathogens to form biofilms that are highly tolerant to antibiotics further aggravates the situation ultimately leading to persistent, recurring infections (5, 6). Furthermore, the emergence of resistance of P. aeruginosa and A. baumannii strains to antibacterials deemed agents of last resort, including colistin and carbapenems, presents an alarming challenge; this necessitates the development of novel therapeutic alternatives to traditional antibiotics in order to address this scourge (12-14). Currently, there is significant interest in antimicrobial peptides (AMPs) as novel therapeutic alternatives to conventional antibiotics. AMPs are major constituents of the host innate defense system for most organisms. AMPs possess several unique features that support their utility as antibacterial agents including possessing rapid bactericidal activity against a wide spectrum of pathogenic bacteria. Additionally, AMPs are typically not 146 susceptible to the same mechanisms that confer resistance to traditional antibiotics (15). However, naturally-derived AMPs do possess limitations that have hindered their translation as clinically useful agents including, moderate antimicrobial activity, toxicity to host tissues, intolerance to physiological conditions, sensitivity to enzymatic degradation, and high manufacturing costs due to their complex design and long length of amino acids (16-18). Furthermore, recent studies have reported that high salt and abnormally reduced pH in the airway surface liquid of CF patients inhibits the antimicrobial activity of host defense peptides including LL-37 and β-defensins leading to P. aeruginosa associated pulmonary infections in CF patients (18-20). Thus, successful translation of AMPs into the clinic requires that limitations due to inactivation by proteases and physiological conditions be overcome. We have recently identified a small synthetic peptide, RR (12 amino acids), with potent efficacy against methicillin-resistant Staphylococcus aureus both in vitro and in a murine staphylococcal skin infection model (21, 22). However, the antimicrobial activity of RR against Gram-negative pathogens is very weak. In the present study, we designed modified derivatives of RR to enhance the peptide’s potency and spectrum of antimicrobial activity while also reducing toxicity to eukaryotic cells. The analogues of RR were subsequently examined against a collection of important multidrug-resistant strains of P. aeruginosa and A. baumannii. The stability of the newly constructed derivatives to proteolytic degradation and inactivation by salts and serum was confirmed. Additionally, the mechanism of action and immune-modulatory activity of the new peptides was explored. Furthermore, the peptides’ ability to eradicate preformed biofilm and to clear an intracellular infection was examined. Finally, the antibacterial activity of the most promising analogue, D-RR4, was investigated in vivo in a Caenorhabditis elegans model of bacterial infection. Taken altogether, our findings indicate that D-RR4 is a promising AMP that warrants further investigation as an alternative therapeutic agent to traditional antibiotics.

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6.3 Materials and methods

Bacterial isolates, peptides and reagents

Clinical isolates of P. aeruginosa and A. baumannii are presented in Tables 1 and 2. Peptides were synthesized using solid-phase 9-fluorenylmethoxy carbonyl (Fmoc) chemistry (GenScript, Piscataway, NJ). Peptides were purified to a purity of 95% using reverse-phase high-performance liquid chromatography (HPLC). The correct peptide mass was confirmed via mass spectrometry. Melittin was purchased from Sigma-Aldrich (St. Louis, MO). Antibiotics were purchased from commercial vendors; proteinase K and Mueller-Hinton broth (MHB) were purchased from Sigma-Aldrich (St. Louis, MO). Trypsin was purchased from VWR (Arlington Heights, IL). The ENLITEN™ ATP Assay System and MTS reagent were purchased from Promega (Madison, WI, USA). Enzyme- linked immunosorbent assay (ELISA) development kits for cytokines detection were purchased from R&D Systems, Inc. (Minneapolis, MN).

Determination of minimum inhibitory concentration (MIC) values

The broth microdilution technique was utilized to assess the minimum inhibitory concentrations of designed peptides according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (23). P. aeruginosa and A. baumannii isolates were diluted to 5×105 colony forming unit (CFU/ml) in MHB. The designed peptides were added to 96-well plates at specified concentrations and subsequently serially diluted. The MIC was defined as the smallest concentration of peptide that prevented visual bacterial growth. MIC was done in triplicate and was repeated twice independently.

Circular dichroism spectroscopy (CD)

Data were recorded on a Jasco circular dichroism spectropolarimeter (Model J810) at 25 °C using a 1 mm path length quartz cell. The spectra were averaged over three scans taken from 250 to 190 nm, with a data pitch and bandwidth of 1 nm, at a scan rate of 20 nm/min. All peptides were analyzed at 50 µM concentration in deionized water, 30 mM sodium dodecyl sulfate (SDS), or 30% trifluoroethanol (TFE) in water (mimicking a membrane environment) as described previously (24, 25).

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Resistance to proteolytic digestion by mammalian and bacterial proteases

To examine the ability of peptides to resist proteolytic digestion by mammalian and bacterial proteases, RR4 and D-RR4 were incubated with trypsin or proteinase K at a molar ratio of 500: 1 (peptide: enzyme), respectively, in digestion buffer (50 mM Tris– HCl, pH 7.4) at 37 °C for 4 hours. After incubation, the digestion mixture was heated at 80°C for 10 minutes to halt the enzymatic reaction. After treatment, the MIC of the peptides was examined using the broth microdilution assay described above. Experiments were performed using triplicate samples for each peptide tested. Next, the stability of peptides to protease degradation was examined using HPLC as described previously (26).

Resistance to salts, plasma and serum

The activity of the designed peptides in different environmental conditions including salts, serum and plasma, was evaluated using the broth microdilution assay explained above. P. aeruginosa PAO1 was treated with peptides under specific physiological conditions as noted here: 100 mM NaCl, 150 mM NaCl, 2% human serum, 5% human serum, 2% FBS, 5% FBS, 10% FBS, 2% human plasma, and 5% human plasma. The MIC was also determined at pH 5.5 and pH 6.5.

Time-kill assay against logarithmic and stationary phase bacteria

P. aeruginosa PAO1 and A. baumannii ATCC BAA-1605 were grown in MHB with aeration at 250 rpm at 37 ºC overnight. Bacteria were subsequently diluted in fresh MHB and incubated aerobically at 37 ºC until the suspension reached logarithmic phase of 5 growth (OD600 = 0.2). Next, bacteria were diluted to 5×10 CFU/ml in MHB. Peptides (at 5 × and 10 × MIC) and antibiotics (at 5 × MIC) were added to diluted bacteria, and incubated aerobically at 37 ºC in a shaking incubator at 250 rpm. Aliquots at specified time points were taken, serially diluted in phosphate-buffered saline (PBS) and plated in triplicate on tryptic soy agar (TSA). CFUs were counted after incubation of plates for 24 hours at 37 ºC. In order to determine the kinetics of killing against stationary phase bacteria, P. aeruginosa PAO1 and A. baumannii ATCC BAA-1605 were grown in MHB with aeration at 250 rpm at 37 ºC overnight. Bacteria were subsequently exposed to peptides and antibiotics at 10 × MIC. Aliquots were taken and plated as described above. 149

Mechanism of action studies a) Outer membrane permeabilization assay

Permeation of the bacterial outer membrane by peptides was assessed using the non-polar fluorescent dye 1-N-phenylnaphthylamine (NPN) as described elsewhere (27). P. aeruginosa PAO1 in logarithmic growth phase was centrifuged for 10 minutes at 4000 × g, washed, and adjusted to OD600 of 0.5. A small aliquot (100 µl) of bacteria was added to black wall 96-well plates. NPN was mixed with bacteria at a final concentration of 10 μM. Peptides were added, in triplicates, at specified concentrations. Plates were monitored using a microplate reader (Molecular Devices, Sunnyvale, CA) at excitation and emission wavelengths of 350 nm and 420 nm, respectively. Data was corrected for background fluorescence and plotted as percentage ± standard deviation. This experiment was done in triplicate and was repeated twice independently. b) Inner (cytoplasmic) membrane permeabilization assay

Damage of the bacterial inner (cytoplasmic) membrane after exposure to peptides was evaluated using the fluorescent propidium iodide dye, as described elsewhere (28). P. aeruginosa PAO1, in logarithmic growth phase, was centrifuged for 10 min at 4000 × g, washed, and adjusted to OD600 of 0.2. An aliquot (100 µl) of bacterial suspension was added to black 96-well plates. Propidium iodide dye was added at a final concentration of 10 μM. Peptides were added, in triplicates, at specific concentrations. Bacteria treated with peptide diluent (distilled water) served as a negative control. Plates were read using a microplate reader at excitation and emission wavelengths of 585 nm and 620 nm, respectively. Data was corrected for background fluorescence and plotted as percentage ± standard deviation. This experiment was done in triplicate and was repeated twice independently. c) ATP leakage assay.

ATP leakage from bacteria treated with peptides was detected using a luminescence assay according to the manufacturer’s instructions. P. aeruginosa PAO1 was grown to logarithmic growth phase, centrifuged for 10 minutes at 4000 × g, washed, and adjusted to an OD600 of 0.2. An aliquot (100 µl) of bacterial suspension was added to white 96-well 150 plates. Peptides were added, in duplicate, at specific concentrations and incubated for 5 minutes at room temperature. Luciferase buffer (100 μl) was added to each well after incubation and luminescence was measured using a microplate reader d) Transmission electron microscopy

P. aeruginosa PAO1 was grown in MHB with aeration at 250 rpm at 37 ºC overnight. Bacteria were subsequently diluted in fresh MHB and incubated aerobically at 37 ºC until the suspension reached logarithmic phase of growth. Next, bacteria were harvested by centrifugation, resuspended in PBS and adjusted to an OD600 ~0.2. The bacterial suspension was exposed to peptides, at 1 × and 10 × MIC, for 60 minutes. After treatment, bacteria were centrifuged and the supernatant was discarded. Bacterial pellets were fixed and examined with a Philips CM-100 microscope as described previously (29).

Multi-step resistance selection

To evaluate the ability of P. aeruginosa PAO1 to acquire resistance to the D-RR4 peptide and control antibiotics, a multi-step resistance selection experiment was employed. P. aeruginosa PAO1 was serially passaged in the presence of a sub-inhibitory concentrations of D-RR4 and three different classes of antibiotics, tobramycin, rifampicin, and ciprofloxacin for ten serial passages. The MIC was monitored daily using the broth microdilution assay. Results were presented as the change in MIC measurement observed after each passage in comparison to the initial MIC value.

Combination studies with antibiotics

The synergistic effect between peptides and antibiotics was performed as described previously with the following modification; we tested a fixed concentration of peptide, 0.25 X MIC, which did not inhibit the growth of bacteria alone (21, 29-31). Synergism was done in triplicate and was repeated twice independently. Toxicity of peptides a) Toxicity of peptides against mammalian cell lines

Peptides were assayed for potential in vitro toxicity against human keratinocytes (HaCaT) and murine macrophages (J774A.1) as described before (29) with the following modifications. Cells were treated with peptides at various concentrations for 24 hours. 151

The therapeutic index of the peptides was calculated as the ratio of mammalian EC50 (the concentration of peptide which inhibits growth of 50% of mammalian cells) to the MIC50 of peptides against bacteria. When there was no detectable cytotoxicity at 256 µM, we used the higher value (512 µM) to calculate the therapeutic index. Larger values of the therapeutic index indicate greater cell selectivity of peptides (32) . b) Hemolysis assay

Peptides were assayed for hemolytic activity against human red blood cells as described previously (21).

Efficacy of peptides on bacterial biofilms

To examine the peptides’ ability to disrupt bacterial biofilms, the microtiter dish biofilm formation assay was employed (33). Briefly, overnight cultures of P. aeruginosa PAO1 and A. baumannii ATCC BAA-1605 were diluted 1:100 in M63 minimal medium supplemented with magnesium sulfate, glucose and casamino acids. Bacterial suspensions were incubated in 96-well plates at 37 °C for 24 hours. After removing media, wells were rinsed with PBS and fresh MHB was added. D-RR4 peptide and selected antibiotics were added at concentrations range from 1X to 32X their respective MICs and plates were incubated at 37 °C for 24 hours. After incubation, wells were washed and stained with 0.5% (w/v) crystal violet for 30 min. The dye was solubilized with ethanol (95%) and the biofilm mass was quantified at OD595. This experiment was done in triplicate and was repeated three times independently.

Intracellular antibacterial efficacy of peptides in murine macrophages

In order to assess the peptides’ ability to kill intracellular bacteria, murine macrophages (J774A.1) were infected, as described in previous studies (34-37). Briefly, J774A.1 was seeded and incubated as described above for the toxicity assessment. Following incubation, cells were infected with either P. aeruginosa PAO1 or A. baumannii ATCC 19606 (multiplicity of infection 100:1) in DMEM + 10% FBS for two hours. After infection, the wells were washed with 200 µl media containing gentamicin (50 µg/ml) and further incubated for 30 minutes with gentamicin to kill any remaining extracellular bacteria. Drugs were diluted in DMEM + 10% FBS (to a concentration equal to 8 × MIC) 152 and added to appropriate wells. Medium alone was used as a negative control. After incubation, the media was aspirated and washed twice with PBS to remove any residual test agent. Next, 100 µl of PBS containing 0.01% triton X-100 was added to lyse J774 cells. Subsequently, bacteria were diluted and plated on TSA plates. Plates were incubated at 37ºC for 16 hours. After incubation, bacteria were enumerated and analyzed. Experiments done in triplicate and were repeated twice independently and the average percent reduction of bacterial growth for each treatment regimen has been reported.

LPS studies a) Binding of peptides to lipopolysaccharide, LPS (LAL assay) To determine if the peptides could bind to LPS, a Limulus amoebocyte lysate (LAL) kit (GenScript, USA Inc.) was utilized as described in previous studies (38). Peptides were dissolved in pyrogen-free water and serially diluted in the same solvent. The peptides were then incubated with one endotoxin unit (EU) of LPS at 37 °C for 30 minutes in order to permit the peptides to bind LPS. The reaction mixture (50 μl) was added to 50 μl of LAL reagent, and further incubated for 10 min. Next, 100 μl of LAL chromogenic substrate was added. The mixture was incubated at 37 °C for 6 minutes. Samples were measured at OD545 using a microplate reader. Colistin was used as a positive control. The binding of peptides with LPS was expressed as percent change relative to the untreated control. b) Anti-inflammatory effect of peptides on LPS-stimulated macrophages

To assess the anti-inflammatory effect of our peptides on LPS stimulated macrophages, J774A.1 cells were seeded and incubated as described above (for toxicity assessment). Next, the cells were stimulated with LPS (150 ng/ml final concentration) in the presence of different concentration of peptides (RR and D-RR4). Cells that were stimulated with LPS alone and untreated cells served as controls. Cells were incubated for six h at 37°C and supernatants from each treatment were collected and stored at -20°C until use. Cytokine detection of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in supernatants was performed using ELISA as described previously, per the manufacturer’s instructions (22). Cytokine levels were expressed as percent change relative to LPS stimulated control using triplicate samples for each treatment condition.

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In vivo efficacy of peptides in a Caenorhabditis elegans infection model Infection and treatment of C. elegans was done as described previously with some modifications (39, 40). C. elegans AU37 [sek-1(km4); glp-4(bn2) I] was used to test the efficacy of peptides in vivo. This strain has a mutation in the sek-1 of the p38 mitogen-activated protein kinase pathway (p38 MAPK pathway) that makes this particular strain more susceptible to bacterial infection (39, 40). Briefly, synchronized adult worms were transferred to modified NGM (0.35% peptone) agar plates seeded with a lawn of P. aeruginosa PAO1, colistin-resistant P. aeruginosa PAO1, or A. baumannii ATCC BAA-1605, for infection. After infection, worms were collected and washed with PBS three times. Worms (~30 per sample) were transferred either to microcentrifuge tubes or 96-well plates. D-RR4 and colistin (at 8 × MIC) were added, in triplicate, to all wells with one exception; worms treated with colistin (at 1 × MIC, equal to 128 µM) in colistin- resistant P. aeruginosa 1109. Untreated worms served as a negative control. To assess the survival of infected C. elegans, worms were assessed for viability (live worms are sinusoidal with movement, whereas dead worms are rigid rods). In order to assess the bacterial load in worms, worms were washed three times with PBS and subsequently lysed by addition of 200 mg of 1.0-mm silicon carbide particles (Biospec Products, Bartlesville, OK) to each tube and vortexing for one minute. Bacteria were plated onto TSA plates containing 100 µg/ml ampicillin to permit the selective growth of P. aeruginosa and A. baumannii over Escherichia coli. Plates were incubated for 16 hours at 37°C and bacterial colonies were counted.

Statistical analyses

Statistical analyses were performed using GraphPad Prism 6.0 software (GraphPad Software, La Jolla, CA, USA). Comparison between two groups was analyzed using a two-tailed unpaired Student’s t-test. Comparison between three or more groups was analyzed using one-way ANOVA, with post hoc Tukey’s multiple comparisons test. P- values of ˂ 0.05 were considered significant.

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6.4 Results and discussion

Peptide design and antimicrobial activity Herein, we aimed to explore the antibacterial activity of RR against important Gram- negative pathogens and to design modified derivatives of RR focusing on key structural elements that are essential for potent antimicrobial activity and reduced toxicity to eukaryotic cells. To address the latter aim, the following design features were considered, (i) peptides were designed to maintain the amphipathic helices by converging the hydrophobic residues on one side and the hydrophilic residues on the other side of the helical axis as shown in the helical wheel diagram (figure 6.1); (ii) the hydrophobic face of certain peptides was disrupted via inclusion of a single hydrophilic residue (lysine or arginine) in order to decrease toxicity and improve the selectivity toward bacteria (41, 42); (iii) the length of amino acids was kept short (12-14 residues) in order to reduce cost of production and minimize host toxicity; (iv) amino acids known to play a crucial role in the antimicrobial activity of RR were maintained in all modified derivatives. For example, tryptophan and arginine residues can improve antimicrobial activity under physiological conditions (e.g., salts, plasma, and serum) without negatively impacting host toxicity (24, 43, 44); (v) the cationic net charge of the designed analogues was increased by adding one or more polar amino acids, particularly lysine residues, to enhance antibacterial activity (25, 45). The peptides were synthesized as described above, and mass spectrometry confirmed the expected molecular weight (Table 6.1).

The antibacterial activity of RR and its modified derivatives was investigated against a collection of important multidrug-resistant strains of P. aeruginosa and A. baumannii. In the current study, RR displayed weak antibacterial activity against both P. aeruginosa and

A. baumannii with MIC50 values equal to 64 µM and MIC90 values equal to 128 µM (Table 6.2). All modified derivatives, except RR1, demonstrated improved antibacterial activity over RR, as discussed below.

Modification of peptide RR, by replacing hydrophobic residues (leucine) with cationic residues (lysine) to generate RR1, increased the cationic charge from +5 to +7 and decreased the hydrophobicity from 0.45 to 0.24. As a result, RR1 was found to be inactive against all tested bacterial strains (MIC > 256 µM). This may be due to the inability of 155

RR1 to form an alpha helix, as was observed in the presence of membrane mimicking environments (figure 6. 2) due to disruption of amphipathicity when in a helical conformation (figure 6.1). These findings highlight that hydrophobicity is more critical for the antibacterial activity of helical AMPs than increasing the cationic charges. Indeed, the peptide with the highest helical content that maintained amphipathicity was found to have the most potent antibacterial activity. RR4, which also had an increased cationic charge, hydrophobicity and amphipathicity as compared to RR, exhibited very potent activity against both P. aeruginosa and A. baumannii strains with MIC50 and MIC90 values equal to 4 and 8 µM. This represents a 16-fold improvement in the MIC over RR. Replacing a lysine residue of RR4 with histidine and two leucine residues with isoleucine resulted in RR2, which demonstrated more potent antibacterial activity compared to RR

(MIC50 value was equal to 4 µM and its MIC90 value was equal to 8 µM). The placement of two additional cationic amino acids within RR4, one within the hydrophobic face of the peptide, resulted in RR3. This modification increased the net charge (+8) of this peptide and reduced its hydrophobicity to 0.14. RR3 was found to be more potent than RR but less potent than RR4 and RR2, again emphasizing the importance of hydrophobicity in antibacterial activity of helical AMPs.

Susceptibility of peptides to digestion by proteases serves as a major impediment to the clinical application of peptides (15). In order to overcome this limitation, we designed a D-enantiomer of the most active peptide, RR4. D-RR4 was amidated at the C-terminus in order to increase its cationic net charge. The MIC of D-RR4 ranged from 2 to 8 µM with

MIC50 and MIC90 values equal to 2 and 4 µM, respectively. The antimicrobial activity of D-RR4 was one-fold more potent than RR4 against most P. aeruginosa strains. However, D-RR4 was similar to RR4 against most A. baumannii strains and one-fold less potent against two strains (NR-13382 and NR-17777) (Table 6.2). The reason for this minor discrepancy in antimicrobial activity between RR4 and D-RR4 against different bacterial isolates is not clear. Previously it was reported that membrane-targeting antimicrobial peptides work without the need for receptors and therefore D isomers have similar antimicrobial activity to L isomers (46). However, there are several studies that have reported that D isomers demonstrated different antimicrobial responses than their L isomer counterparts against different bacterial isolates. This may be due to the fact that 156 the backbone of D and L isomers of helical AMPs, in three-dimensional space, have opposite rotations and therefore their side chains would appear in different topology (47). For example, Papo et al reported that the antimicrobial peptide diastereomer (Amphipathic-1D) demonstrated improved activity (4-7 fold) over its L isomer (Amphipathic-1L) against all bacterial isolates (including P. aeruginosa). However, Amphipathic-1D demonstrated less activity (13 fold) than the L isomer against A. baumannii (48). Conversely, in another study, a D isoform peptide (IK8-all D) demonstrated improved activity (4 fold) than the L isomer (IK8-all L) against A. baumannii. However, IK8-all D demonstrated less activity (two- fold) than the L isomer against P. aeruginosa (25).

As highlighted above, D-RR4 demonstrated the most potent antibacterial activity with a

MIC50 value of 2 µM (3.73 µg/mL) against A. baumannii and a MIC50 value of 4 µM (7.5 µg/mL) against P. aeruginosa. This is superior to the MIC values obtained for the natural derived human cathelicidin, LL-37, whose MIC50 value was found to be 4 and 32 µM (18 and 144 µg/mL) against A. baumannii and P. aeruginosa, respectively. Bovine indolicidin demonstrated moderate antibacterial activity with MIC50 values of 32 and 64 µM (61 and 122 µg/mL) against A. baumannii and P. aeruginosa, respectively.

Circular dichroism spectroscopy (CD)

The secondary structure of designed peptides in aqueous and membrane mimicking solvents (24, 25), such as TFE and SDS, was investigated using CD spectroscopy. In all, the peptides demonstrated a mostly random coil conformation in the aqueous solvent as demonstrated by a broad minimum peak at 200 nm. The starting peptide, RR, was found to have an increase in helical content in the presence of the membrane-mimicking solvents (TFE and SDS) to 20% and 12%, respectively, as determined by the mean residue ellipticity at 222 nm (figure 6.2). 1b). All of the modified peptides, with the exception of the non-amphipathic RR1, changed their conformation to some extent in the presence of the membrane-mimicking solvents (figure 6.2). Peptide RR2, which contained no leucine residues in the hydrophobic face as compared to the original RR peptide, displayed very modest changes in the CD spectra in the presence of the membrane-mimicking solvents. The pronounced maximum at 190 nm and strong minima at 208 and 222 nm displayed by 157

RR3 and RR4, however, are highly indicative of alpha helicity. RR4 was found to be somewhat more helical than RR with the addition of SDS and TFE, with helical contents of about 30%. In this study, an all D-amino acid version of the RR4 peptide (D-RR4) was prepared with an amidated C-terminus. As expected, the CD spectra of D-RR4 was the mirror image of the RR4 peptide, with mean residue ellipticities that were opposite in sign, but somewhat higher than RR4 with the addition of SDS and TFE (about 40% helical content) due to the addition of the C-terminal amide (figure 6.2).

Antimicrobial activity against colistin-resistant P. aeruginosa

After confirming the potent antibacterial activity of the designed peptides against relevant colistin-sensitive P. aeruginosa isolates, we moved to examine if the peptides’ activity could be retained against a panel of clinical isolates of colistin-resistant P. aeruginosa isolated from cystic fibrosis patients (13). Although these isolates showed high resistance to colistin and several natural peptides, our designed peptides retained their potent antibacterial activity (Table 6.3). The most potent peptide was D-RR4 with an MIC50 value of 4 µM. This represents a 32-fold enhancement in the antimicrobial activity of this peptide compared to RR (MIC50 =128 µM). RR2 demonstrated improved activity over RR with a MIC50 value equal to 32 µM. RR3 and RR4 were found to be more potent than RR2 with MIC50 values equal to 16 and 8 µM, respectively. However, the antimicrobial activity of indolicidin was abolished against all tested isolates. LL-37 showed weak to moderate activity against some strains (MIC50 of 64 µM), while no activity was observed against five colistin-resistant strains (Table 6.3). Colistin is an antibiotic of last resort for multidrug-resistant P. aeruginosa infections (49). The emergence of resistant isolates to colistin, particularly via plasmid-mediated resistance in Enterobacteriaceae (50), highlights the urgent need to discover new antimicrobial agents to address this issue. The discovery that RR4 and D-RR4 have potent activity against colistin-resistant P. aeruginosa isolates indicates a potential advantage of these peptides over several antimicrobial peptides that are ineffective against colistin-resistant P. aeruginosa (13).

Resistance to proteolytic digestion To confirm the stability of D-RR4 to proteolytic digestion, we evaluated its antimicrobial activity after treatment with mammalian (trypsin) and bacterial (proteinase K) proteases 158 for 4 hours. As presented in Table 6.4, the antibacterial activity of the L isoform of RR4 was completely abolished in the presence of both trypsin and proteinase K. However, the MIC of the D isoform of RR4 (D-RR4) was not sensitive to proteolytic degradation when tested against P. aeruginosa. We confirmed the stability of D-RR4 to proteases utilizing HPLC. As demonstrated in figure 6.3, incubation of the L isoform of RR4 with proteases for 4 hours led to the degradation of the intact peptide, resulting in multiple lower molecular weight peaks, hence explaining the loss of antimicrobial activity observed earlier (Table 6.4). However, D-RR4 remained intact after protease treatment, even after 24 hours of incubation with proteases (figure 6.3).

Antimicrobial activity of designed peptides in the presence of salts, serum and acidic pH.

A significant drawback of many antimicrobial peptides is their antibacterial activity is impaired in the presence of biological fluids (serum, plasma, high concentration of salts and acidic pH); this severely limits their clinical utility (16, 17). With this point in mind, we assessed the antibacterial activity of our peptides against P. aeruginosa PAO1 in several biologically relevant conditions. Overall, all designed peptides demonstrated low salt sensitivity (Table 6.4). All peptides showed a two-fold increase in the presence of 150 mM NaCl. In the epithelial cell secretions of a CF patients, the NaCl concentration is approximately 120 mM (18); at this concentration all RR derivatives, particularly RR2, RR4 and D-RR4, maintained good antibacterial activity. Recently, several studies reported that abnormally reduced pH in the airway surface liquid of CF patients (pH 6.8) inhibits the antimicrobial activity of host defense peptides and may impair the host’s defense against pathogens (19, 20). Therefore, we assessed the effect of acidity on the antibacterial activity of our designed peptides by determining the MIC at pH 5.5 and pH 6.5. There was no significant difference in the MICs of most peptides at acidic pH (a one-fold decrease in MIC was observed at pH 5.5, except for RR3 where a four-fold decrease was observed). A one-fold decrease in MIC was observed for RR and RR3 at pH 6.5 while no change in MIC for RR2 was observed. Interestingly, RR4 and D-RR4 demonstrated a one-fold improvement in activity at pH 6.5 (Table 6.4). 159

In addition to salts and acidity, serum and plasma can also impair the antibacterial activity of conventional antibiotics and peptides either via proteolytic digestion or binding to protein or lipid fractions (51). We thus moved to examine whether our peptides would exhibit diminished activity. As presented in Table 6.4, the antimicrobial activity of most peptides was diminished or completely abolished in the presence of fetal bovine serum, human serum, and human plasma. Interestingly, D-RR4, retained its potent anti- Pseudomonas activity under the same test conditions. We suspect D-RR4’s decreased susceptibility to the presence of serum is due to the resistance of the D isoform to enzymatic degradation, as discussed above. Given the designed peptides’, particularly D- RR4, antibacterial activity is not significantly diminished in the presence of high salts, acidic pH, and serum, this indicates a selective advantage for our peptides over many natural and synthetic antimicrobial peptides, particularly in cystic fibrosis conditions (16, 17). The stability of our peptide constructs in physiologically relevant test conditions, may be attributed to the high percentage of tryptophan and arginine residues in the peptides which can improve their antibacterial activity under physiologically relevant conditions (e.g., salts, plasma, and serum) (24, 43, 44).

Time-kill assay against logarithmic and stationary phase bacteria

After confirming that the AMPs possessed excellent antimicrobial activity against multidrug-resistant clinical isolates of P. aeruginosa and A. baumannii, we next assessed the killing kinetics of RR and the most promising analogue, D-RR4 against logarithmic phase P. aeruginosa PAO1 and A. baumannii ATCC BAA-1605. D-RR4 demonstrated fast and concentration-dependent killing of both bacteria (figure 6.4). At 5 × MIC, D-RR4 reduced P. aeruginosa and A. baumannii starting inoculum by 2.1 and 2.58 log10, respectively, after one hour and completely eliminated both bacteria within two hours. Treatment with 10 × MIC of RR and D-RR4 completely eliminated both bacteria within one hour (figure 6.4). Similar to RR and D-RR4, colistin demonstrated rapid bactericidal activity. At 5 × and 10 × MIC, colistin completely eliminated P. aeruginosa PAO1 within one hour. However, colistin required three and two hours to eradicate A. baumannii ATCC BAA-1605 culture both at 5 × and 10 × MIC, respectively. Tobramycin and gentamicin, at 10 × MIC, demonstrated rapid bactericidal activity, eliminating P. aeruginosa PAO1 160 within three and two hours, respectively. Tobramycin, at 10 × MIC, eliminated A. baumannii ATCC BAA-1605 after three hours. AMPs with fast bactericidal activity have several advantages including limiting spread of infection, improving treatment prognosis, limiting the emergence of resistance, and reducing duration of treatment (52).

Next, we examined the impact of RR and D-RR4 and select antibiotics against stationary- phase P. aeruginosa and A. baumannii. This population of cells is known to contain a high percentage of persisters that are tolerant to many antimicrobial agents which can contribute to recurring infections (5, 53). Treatment of stationary-phase P. aeruginosa

PAO1 with 10 × MIC D-RR4 resulted in rapid bactericidal activity (3-log10 reduction) of a high starting inoculum (3 × 109 CFU/ml) after three hours (figure 6.4). However, RR had no effect. This most likely is due to its weak antimicrobial activity against a high inoculum of P. aeruginosa. Conventional antibiotics had minimal impact on stationary- phase P. aeruginosa PAO1. Tobramycin reduced the number of stationary-phase P. aeruginosa PAO1 by only 1.87 log10, after 24 hours of exposure, while colistin and gentamicin had no effect. Complete eradication of stationary-phase A. baumannii ATCC BAA-1605 was achieved after treatment with D-RR4 for three hours (figure 6.4). RR reduced the number of stationary-phase A. baumannii ATCC BAA-1605 by three-log10 after three hours; however, regrowth of bacteria occurred after this, most likely due to degradation of the RR peptide. Tobramycin failed to demonstrate bactericidal activity (reduced the number of stationary-phase A. baumannii ATCC BAA-1605 by only 2.39 log10). Colistin did not have any effect, which is in agreement with previous findings (54). The superior activity of D-RR4, when compared to conventional antibiotics, against stationary phase bacteria can be explained by its unique antimicrobial mechanism of action. Many antibiotics require bacteria to be growing and metabolically active in order to inhibit their molecular targets, including protein synthesis (tobramycin and gentamicin) (55). In contrast, as elaborated further below, the positive charge present in D-RR4 serves as a point of attraction with negatively-charged bacterial cell membranes and consequently leads to targeted disruption of the bacterial membrane and leakage of intracellular contents (24, 25). This unique mechanism of action of AMPs does not require cells to be metabolically active and is not impaired by the dormant and quiescent state of 161 bacteria (55). The results obtained provide valuable insight into using D-RR4 as a potential future therapeutic option for treatment of persistent bacterial infections.

Mechanism of action of designed peptides

In order to gain insight into the mode of bacterial killing of D-RR4, we performed a series of experiments to explore its antibacterial mode of action. First, damage to the bacterial outer membrane by D-RR4 was monitored by measuring the fluorescent intensity of P. aeruginosa PAO1 mixed with 1-N-phenylnaphthylamine (NPN) and incubated with varying peptide concentrations. As demonstrated in figure 6.5A, similar to the known membrane-targeting antimicrobial peptide LL-37, there was a significant and dose- dependent increase of fluorescence intensity of P. aeruginosa cultures treated with D-RR4. These findings indicate that D-RR4 likely targets the bacterial outer membrane leading to partitioning of NPN into the outer membrane and increased fluorescence. Damage of the bacterial inner (cytoplasmic) membrane by peptides was monitored by measuring the fluorescence intensity of P. aeruginosa PAO1 mixed with propidium iodide and treated with D-RR4. As demonstrated in figure 6.5B, the fluorescence intensity didn’t change in untreated bacteria confirming cytoplasmic membrane integrity. However, when D-RR4 and LL-37 were added to bacterial cultures, a rapid and significant increase in fluorescence due to access of propidium iodide inside bacteria was evident. At 10 × MIC, D-RR4 was faster and more potent at producing membrane perturbation in comparison to D-RR4 tested at 5 × MIC. To confirm our results of membrane permeabilization, we assayed the ATP level of bacterial cultures after exposure to D-RR4 compared to untreated bacteria. Physiologically, ATP is maintained inside bacterial cells with intact cell membranes. Leakage of ATP into the extracellular environment is indicative of membrane damage. As demonstrated in figure 6.5C, similar to LL-37, there was a significant increase in luminescence intensity of P. aeruginosa PAO1 treated with D-RR4 compared to the untreated control.

To confirm the membrane-damaging effect of D-RR4 on bacteria, transmission electron microscopy was conducted on thin sections of P. aeruginosa PAO1 treated with two different concentrations of D-RR4 (figure 6.6). TEM micrographs of untreated bacteria revealed cell membranes that were intact and a distinct cell wall (figure 6.6 A and B). The 162 cytoplasmic region displayed a homogenous electron density. However, after exposure to D-RR4, severity of membrane damage was observed in a concentration-dependent manner. Bacteria treated with a low concentration of D-RR4 (1 × MIC) exhibited disintegrated membranes with blebs and an increase in the periplasmic space (figure 6.6C and D). Mesosomes, invaginations and condensed materials with similar electron density to that of the cell wall murein layer were also observed under membranes (figure 6.6C and D). Bacteria treated with a higher concentration of D-RR4 (10 × MIC) exhibited a more severe damaging effect on membranes as complete loss of membrane integrity and the cell wall were clearly observed (figure 6.6E, F and G). Leakage of cytoplasmic contents, which result in heterogeneous cytoplasmic densities, were also seen (figure 6.6E and G). Ghost cells due to complete loss of cytoplasmic contents were also evident at higher peptide concentrations (figure 6.6H). Collectively, these data confirm the membrane-destructive effect of D-RR4 against Gram-negative bacteria.

Multi-step resistance selection

To examine if the bacterial membrane-damaging effect of peptides can overcome traditional mechanisms of antibiotic resistance, we serially passaged P. aeruginosa PAO1 with sub-inhibitory concentrations of D-RR4 and three different classes of antibiotics, tobramycin (protein synthesis inhibitor), rifampicin (RNA synthesis inhibitor) and ciprofloxacin (DNA synthesis inhibitor) for ten days. The MIC was determined daily using the broth microdilution assay. As presented in figure 6.7, P. aeruginosa PAO1 was not able to develop resistance to D-RR4 for ten passages. However, treatment of P. aeruginosa PAO1 with rifampicin induced a rapid shift in the MIC within the second passage and resulted in a 32-fold increase in rifampicin’s MIC by the sixth passage. The MIC of ciprofloxacin and tobramycin increased to 8 and 16-fold, respectively by the ninth passage (figure 6.7). The inability of bacteria to develop peptide resistance under laboratory conditions is attributed to the rapid action of peptides and their unique mechanism of action. Peptides disrupt bacterial membranes without the need for a specific receptor, so for bacteria to acquire resistance, significant changes to the membrane lipid composition would need to occur (56). These alterations are extremely difficult for bacteria to produce compared to point mutations in specific (15). The results of this 163 experiment, which was limited in terms of the number of passages, indicates that in vitro resistance emerged rapidly to several commercial antibiotics compared to D-RR4. Further investigation is required to validate these data in suitable animal models.

Combination studies with conventional antibiotics

We next investigated if peptide D-RR4 possesses a synergistic relationship with antibiotics, utilizing the checkerboard assay. As presented in Table 6.5, D-RR4 displayed potent synergistic activity when combined with colistin or polymyxin B against all tested bacterial isolates with FIC indices ranging from 0.38 to 0.5. Colistin is considered to have high nephrotoxicity rates of ∼45 to 55% under the currently recommended dosing regimen (57, 58). Nephrotoxicity is linked to both the total dose of colistin administered and duration of therapy. Decreasing the therapeutic dose of colistin required to treat bacterial infection (by combining it with our peptide) is beneficial as it will potentially reduce adverse side effects in patients receiving colistin treatment.

Combining D-RR4 with aminoglycoside antibiotics (tobramycin, gentamicin and amikacin) resulted in no beneficial effect observed against P. aeruginosa strains. However, a synergistic relationship was evident against A. baumannii ATCC BAA-1605 with tobramycin and amikacin (FIC index, 0.38) but not with gentamicin (FIC index, 1.25). Remarkably, D-RR4 improved the activity of meropenem and cefotaxime, even in the case of meropenem-resistant and cefotaxime-resistant isolates of A. baumannii and P. aeruginosa which resulted in an 8 to 128-fold reduction in their MIC values (FIC indices 0.26-0.38). Thus, this peptide holds promise for potentially prolonging the clinical use of these antibiotics against both antibiotic-sensitive and resistant strains.

Toxicity of designed peptides

A significant limitation for translating AMPs into clinical applications is their toxicity to host cells (59). Herein, we evaluated the cytotoxic effects of our peptide constructs against murine macrophages (J774.1A) and human keratinocytes (HaCaT cells) via the MTS assay. The half maximal effective concentration (EC50) of RR, RR1, RR2, RR3, RR4 and D-RR4 was 128/256, >256/>256, 128/64, 128/128, 64/128 and 64/64 µM, respectively, in the case of J774.1A / HaCaT cells (Table 6.6). Pexiganan had an EC50 equal to 64 µM 164

when tested against J774.1A cells. Melittin, however, had an EC50 equal to 2/4 µM, respectively, when tested against J774.1A / HaCaT cells.

Table 6.6 also presents the therapeutic index (TI) of the peptide constructs. All modified derivatives displayed a higher safety window than the parent peptide, RR. The therapeutic index of RR is 2/4 respectively, in the case of J774.1A / HaCaT cells. RR2, RR3, RR4 and D-RR4 have TI values equal to 32/16, 16/16, 16/32 and 32/32, respectively, in the case of J774.1A / HaCaT cells. These data demonstrate a 2-16 fold improvement in the safety window of the modified derivatives compared to RR. Pexiganan demonstrated a similar therapeutic index to D-RR4 (TI = 32). On the other hand, melittin exhibited a very low therapeutic index of 0.25/0.5 in the case of J774.1A / HaCaT cells, respectively. This indicates the superior safety profile of our peptide constructs over this naturally-produced AMP.

Next, we assessed the hemolytic activity of the peptides on human red blood cells (RBCs).

As demonstrated in Table 6.6, The HC50 (the peptide concentration that causes 50% hemolysis of RBCs) for all peptides is equal to or more than 256 µM. Peptide D-RR4 demonstrated the highest selective index (TI = 128) compared to RR (TI = 8). This translates to a 16-fold improvement in selectivity towards prokaryotic versus eukaryotic cell membranes. Previous reports indicate that pexiganan has a HC50 value in the range of

120-208 µM (60, 61). In contrast, melittin possessed a very low HC50 (4 µM) equal to ½ × MIC of melittin against P. aeruginosa (TI = 0.5). The low toxicity of designed peptides against mammalian cells in relation to their potent antimicrobial activity suggests the peptide constructs exhibit selectivity towards the negatively-charged bacterial membranes compared to the zwitterionic mammalian membranes.

Efficacy of peptides on established biofilms

Biofilms are aggregated bacterial communities covered by a polysaccharide matrix that protects bacteria from host immune defenses and hinders antibiotics from targeting deep- seated bacteria encased within the biofilm (62). Biofilm development has been linked to serious biofilm-related infections including pneumonia in cystic fibrosis patients and colonization of medical devices (63). Biofilms of A. baumannii permit this pathogen to survive on abiotic equipment in hospital settings. This facilitates contact with susceptible 165 patients and has led to outbreaks of ventilator-associated pneumonia (7). Biofilms pose a serious medical challenge, that is difficult to control and there is a critical need to find novel approaches to address this problem. We examined the capability of the most promising peptide analogues, RR4 and D-RR4, and control antibiotics to disrupt established biofilms of P. aeruginosa PAO1 and A. baumannii ATCC BAA-1605. As shown in figure 6.8, RR4 and D-RR4 disrupted mature (24 hour) biofilms more potently than several antibiotics of choice. RR4 and D-RR4 demonstrated a concentration- dependent biofilm-disrupting activity. RR4, at 4, 8, 16 and 32 × MIC, disrupted more than 31, 39, 41 and 61% of biofilm mass, respectively (p < 0.05). D-RR4 disrupted more than 30%, 40%, 60% and 75% of biofilm mass, respectively (p < 0.05), at the same concentrations. Colistin reduced more than 10%, 25%, 28%, and 75% of biofilm mass, respectively (p < 0.05), at the same concentrations. Gentamicin and tobramycin required a concentration equal to 32 × MIC to disrupt 50% of biofilm mass (p < 0.05). Remarkably, RR4 and D-RR4 were capable of reducing 20-35% of P. aeruginosa biofilm mass at very low concentrations equivalent to 0.5 - 1 × MIC, respectively. In contrast, low concentrations of gentamicin and tobramycin enhanced biofilm production (100 - 240% increase compared to untreated samples), which is in agreement with previous findings (64). When examining RR4 and D-RR4 against A. baumannii biofilm, a concentration- dependent activity was observed (figure 6.8). At 8, 16 and 32 × MIC, RR4 disrupted more than 45%, 57% and 66% of biofilm mass, respectively (p < 0.05). D-RR4 reduced 40%, 58% and 76% of biofilm mass, respectively (p < 0.05) at the same tested concentrations. Colistin reduced the biofilm mass by 10%, 36% and 51%, respectively (p < 0.05), at the same tested concentrations. Tobramycin, at 32 × MIC, disrupted only 20% of the biofilm mass (p < 0.05) while gentamicin was not effective as this strain is resistant to gentamicin (figure 5). Collectively, the data demonstrates that peptides RR4 and D-RR4 are superior to conventional antibiotics in penetrating and disrupting adherent P. aeruginosa and A. baumannii biofilms. Recently, multiple AMPs have been reported that demonstrated potent antibiofilm activities compared to conventional antibiotics. For example, de la Fuente-Nunez et al. reported a potent broad-spectrum antibiofilm peptide (peptide 1018), despite its weak 166 antimicrobial activity against planktonic bacteria (65). The antibiofilm mechanism of peptide 1018 was through binding and degradation of the stress-related second messenger, guanosine pentaphosphate (pppGpp) (65). The same group designed a series of more potent antibiofilm peptides that work by the same mechanism. The most potent antibiofilm peptides in the series were two D enantiomeric peptides (DJK-5 and DJK-6). They were proteolysis-resistant and protected invertebrates from lethal P. aeruginosa infections (47). Another group of researchers reported a de novo engineered cationic antimicrobial peptide (WLBU2) that reduced P. aeruginosa biofilm growth on CF airway epithelial cells and protected mice from lethal P. aeruginosa infections (66, 67). Intracellular antibacterial activity of peptides in murine macrophages

P. aeruginosa and A. baumannii were considered to be primarily extracellular pathogens for many years. However, recent studies have revealed that these pathogens can gain entry and persist inside both phagocytic and non-phagocytic eukaryotic cells such as alveolar macrophages and lung epithelial cells (35-37). The intracellular persistence of pathogens constitutes a significant challenge as they are able to avoid host immune defenses. Additionally, intracellular pathogens evade the action of many antibiotics that exhibit low intracellular penetration ability such as aminoglycosides and colistin (35). Eradication of these challenging bacterial pathogens is critical to ensure successful treatment of persistent intracellular bacterial infections. Here, we investigated if our designed peptides have the ability to kill intracellular P. aeruginosa and A. baumannii residing in murine macrophages. As demonstrated in figure 6.9, the D isoform of RR4, but not the L form, showed significant reduction of intracellular P. aeruginosa and A. baumannii at nontoxic concentrations. D-RR4 showed the maximum efficacy as it killed 96.00% ± 1.15 and 100.00% ± 0.00 of intracellular P. aeruginosa PAO1 and A. baumannii ATCC 19606, respectively, at 8 × MIC. In contrast, the L isoform of RR4 produced a 14.60 % ± 5.50 and 25.00% ± 8.90 reduction of intracellular P. aeruginosa PAO1 and A. baumannii ATCC 19606, at 8 × MIC. The enhanced efficacy of D-RR4 compared to RR4 may be related to its resistance to proteolysis by serum and other cellular enzymes, as described earlier. The potent intracellular activity of D-RR4 may be useful in treating certain chronic diseases such as cystic fibrosis where P. aeruginosa persists inside eukaryotic cells as pods leading to failures to antibiotic therapy (36). 167

Lipopolysaccharide (LPS) studies A) Binding of peptides to LPS (LAL assay) A key virulence factor unique to Gram-negative bacterial pathogens is the presence of lipopolysaccharide (LPS). LPS is a principal structure of the outer membrane of Gram- negative bacteria. During bacterial death, cell replication or exposure to certain antibiotics, LPS is released from bacteria leading to stimulation of proinflammatory cytokine release such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) from mononuclear phagocytes. Excessive LPS exposure leads to overproduction of these cytokines which can result in septic shock and death. Annually there are 18 million cases of septic shock globally with a mortality rate of ~30% (68, 69). Therefore, identifying agents that are capable of neutralizing LPS is critical. We investigated if our peptides could bind to and neutralize the effect of LPS. As presented in figure 6.10A, RR and its analog, D-RR4 were able to bind to LPS in vitro as evident by inhibition of the LPS-induced activation of the LAL enzyme (Limulus amoebocyte lysate). RR produced a 21.3%, 43.2%, 59.6% and 82.4% inhibition of the LAL enzyme at 5, 10, 15 and 20 µM, respectively. D-RR4 demonstrated a more significant inhibition than RR, producing a 42.0%, 67.3%, and 85.6% reduction at 5, 10 and 15 µM, respectively. Complete inhibition was achieved at 20 µM. We suspect the high cationic charge of D-RR4, in relation to RR, is responsible for the enhanced binding affinity of this peptide to LPS. Colistin, a known LPS binding agent, showed significant inhibition generating a 51.2% and 83.0% reduction at 5 and 10 µM. Complete inhibition of LPS-induced activation of the LAL enzyme was achieved at both 15 and 20 µM (figure 6.10A). B) Anti-inflammatory effect of peptides on LPS stimulated macrophages (neutralizing the effect of LPS on macrophages) Next, we examined if RR and D-RR4 could reduce the endotoxin-induced proinflammatory cytokine response in murine macrophages by measuring cytokine levels using ELISA. As depicted in figure 6.10B, RR and its analog, D-RR4 were able to inhibit LPS-induced proinflammatory cytokines in macrophages in a manner similar to colistin. The inhibition of cytokine production was found to be concentration-dependent. RR, at 10 µM, decreased TNF-α and IL-6 levels by 24.4% and 75.4%, respectively. At 20 µM, 168

RR inhibited 84.0% and 96.5 % of TNF-α and IL-6 production, respectively. At 10 µM, D-RR4 was superior to RR as it decreased TNF-α and IL-6 production by 64.7% and 96.4% of TNF-α and IL-6 production, respectively. At 20 µM, D-RR4 inhibited 85.6% and 96.5% of TNF-α and IL-6 production, respectively. Colistin, as expected, produced a >95% inhibition of both cytokines at the tested concentration (10 and 20 µM) (figure 6.10B). The capability of the designed peptides to reduce endotoxin-mediated proinflammatory cytokine production provides a potential avenue for their development as antibacterial agents to treat sepsis and also as an adjunctive with antibiotics to overcome sepsis. In vivo efficacy of peptides in a C. elegans infection model. To test the efficacy of the synthetic peptides to protect against infections in vivo, we utilized a C. elegans model of bacterial infection. This model has been used extensively for early-stage evaluation of promising antimicrobials (47, 70, 71). Initially, the lethal dose of D-RR4 against uninfected nematodes was determined. D-RR4 and colistin were not toxic to worms at 8 × MIC (figure 6.11A). D-RR4 and pexiganan at 32 × MIC killed approximately 50% of worms after 4 days (figure 6.11A). However, melittin completely killed C. elegans after 3 days which is in agreement with its known toxicity to mammalian cells. Therefore, a nontoxic concentration of D-RR4 (8 × MIC) was selected for worm efficacy studies.

We next evaluated the ability of D-RR4 to reduce the bacterial burden inside the gut of infected worms. As demonstrated in figure 6.11B, D-RR4 produced a significant reduction of P. aeruginosa PAO1 at 8 × MIC (85%). Similarly, colistin (8 × MIC) produced an 89% reduction in bacterial load. Potent synergism was evident when D-RR4 was combined with colistin as complete clearance of bacteria from worms was achieved. Next, we evaluated their activity against a colistin-resistant P. aeruginosa 1109 strain isolated from a CF patient. Colistin was unable to significantly decrease bacterial burden, in contrast to D-RR4, which produced a 75% reduction. Interestingly, the combination of D-RR4 and colistin showed enhanced killing as the combination reduced 98% of colistin- resistant P. aeruginosa in infected worms (figure 6.11B). When the same test agents were evaluated against A. baumannii, a similar pattern was observed. D-RR4 and colistin, 169 independently decreased bacterial burden by 95% and 96%. When the two agents were tested in combination, a complete eradication of bacterial load was observed (figure 6.11B). Bolstered by these results, we next examined whether D-RR4 could protect infected worms from the cidal activity of bacteria. Interestingly, P. aeruginosa PAO1 killed 80% of untreated worms after 24 hours and 100% mortality was observed after 36 hours (figure 6.11C). D-RR4 and colistin significantly increased the rate of survival for Pseudomonas- infected C. elegans in contrast to untreated worms, giving nearly complete protection for 24 and 36 hours. A 65% to 72% protection was observed up to 48 hours. A combination of D-RR4 and colistin showed complete protection of worms even up to 48 hours (figure 6.11C). The survival experiment with C. elegans was next utilized to examine the impact of colistin-resistant P. aeruginosa 1109. As expected, this strain demonstrated higher virulence than PAO1 as it completely killed C. elegans after 24 hours. While colistin failed to protect worms from killing, D-RR4 significantly increased the survival rate of C. elegans giving nearly complete protection after 24 hours and 85% and 47% protection after 36 and 48 hours, respectively. Interestingly, a combination of D-RR4 and colistin showed enhanced protection as they showed complete protection of worms after 24 and 36 hours and 75% protection after 48 hours (figure 6.11C). A. baumannii killed worms more slowly than P. aeruginosa, killing 75% and 95% of worms after 48 and 72 hours, respectively. A notable protective effect was provided by D-RR4 and colistin as they showed complete protection of worms after 36 hours and 80- 85% protection after 72 hours. Consistent with in vitro data, the combination of D-RR4 and colistin showed complete protection up to 72 hours (figure 6.11C).

6.5 Conclusion

The worldwide increase of antibiotic resistance and limited supply of new antibiotics necessitates the discovery and development of novel alternatives to address this burgeoning challenge. In the present study, we designed modified derivatives of a short synthetic peptide, RR, to enhance its potency and spectrum of activity against Gram- 170 negative pathogens. RR4 and its D-enantiomer, D-RR4, were the most potent analogues of RR, highlighting the significance of hydrophobicity and amphipathicity in the antimicrobial activity of AMPs against Gram-negative bacteria. These modifications successfully improved the antibacterial activity of D-RR4 against both multidrug-resistant P. aeruginosa and A. baumannii and also stabilized D-RR4 to the effect of high salts, acidic pH, human serum, and both bacterial and mammalian proteases. This indicates a selective advantage of our peptides over many natural and synthetic antimicrobial peptides. D-RR4 was found to exert its antibacterial activity via disruption of both the outer and inner membranes of the cell envelope. However, no adverse toxic effect was observed to mammalian cells at concentrations 16 times higher than the MIC, indicating D-RR4 possesses an enhanced safety profile compared to RR. Moreover, D-RR4 displayed a lower propensity to select for resistant bacteria compared to conventional antibiotics. In addition to its potent antibacterial activity, D-RR4 demonstrated significant disruption of established bacterial biofilms more than conventional antibiotics, even at subinhibitory concentrations. Furthermore, D-RR4 was capable of binding strongly to LPS and subsequently reduced the endotoxin-mediated proinflammatory cytokine production in murine macrophage cells. This discovery highlights a potential avenue for development of D-RR4 to treat sepsis. D-RR4 also significantly reduced the burden of intracellular P. aeruginosa and A. baumannii highlighting the potential application of D-RR4 for treatment of certain chronic diseases, such as cystic fibrosis, where P. aeruginosa persists inside eukaryotic cells. Finally, D-RR4’s potent antibacterial activity, alone and in combination with colistin, was confirmed in vivo as it significantly reduced the bacterial burden inside the gut of infected C. elegans and increased the rate of survival for nematodes infected with either P. aeruginosa or A. baumannii. Collectively, the unique antibacterial, antibiofilm, and anti-inflammatory properties of D-RR4 warrant further investigation to assess its safety and efficacy in suitable animal models of infection.

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Table 6.1. Amino acid sequence and physicochemical properties of peptides used in this study

<µH>

eight

Mean

Actual

w weight

Length

Charge

molecular molecular

sequences

Calculated

Amino acid

amino acids

Hydrophobic Hydrophobic

moment hydrophobicity

RR WLRRIKAWLRR 11 1553.92 1553.91 +5 54 % 0.453 0.831

RR1 WKRRIKIWKKIR 12 1711.17 1711.16 +7 41 % 0.242 0.659

RR2 WIRRIKKWIRRVHK 14 1975.46 1975.45 +7 42 % 0.303 0.901

RR3 WLRRIKAWLRRKRK 14 1966.45 1966.44 +8 42 % 0.142 0.652

RR4 WLRRIKAWLRRIKA 14 1866.33 1866.32 +6 57 % 0.436 0.759

D- a wlrrikawlrrika-NH2 14 1866.33 1866.32 +7 57 % 0.436 0.759 RR4

a Small underlined residues represent D-amino acids

177

Table 6.2. Minimum inhibitory concentration (MIC) (µM) of peptides against clinical and drug-resistant isolates of P. aeruginosa and A. baumannii

in

37

-

n

RR4

RR

-

RR1 RR2 RR3 RR4

strain

LL

D

Colistin

designatio Indolicid PAO1 64 >256 4 8 4 2 16 64 0.5 ATCC 9721 64 >256 8 16 8 4 32 >256 0.5 ATCC BAA- 128 >256 16 16 8 8 32 64 0.5 1744

P. ATCC 27853 64 256 8 8 8 4 16 64 0.25 aeruginosa ATCC 35032 64 >256 16 16 8 4 128 >256 0.5 ATCC 25619 64 >256 8 4 8 4 16 >256 0.25 ATCC 9027 64 >256 4 4 4 2 16 32 0.25 ATCC 15442 128 >256 16 16 8 4 32 64 0.25 ATCC 10145 128 >256 8 16 8 4 >256 128 0.5

ATCC BAA- 32 >256 4 16 2 2 4 32 0.25 1605 ATCC 19606 16 >256 2 16 2 2 2 8 0.125 ATCC BAA- 64 >256 4 8 2 2 8 32 0.25 747 NR-9667 32 >256 4 32 4 4 4 32 0.0625 NR-13374 32 128 4 8 4 4 4 8 0.125 NR-13375 32 >256 2 8 2 2 4 32 0.125 A. NR-13382 32 >256 4 16 4 8 4 32 0.5 baumannii NR-17777 16 >256 4 8 2 4 4 32 0.5 NR-17778 16 >256 4 8 2 2 4 32 0.0625 NR-17780 32 >256 4 8 2 2 4 32 0.25 NR-17783 32 >256 4 8 4 4 4 16 0.0625 NR-17784 64 >256 4 4 2 2 4 64 0.0625 NR-17785 32 >256 4 8 2 2 4 32 0.25 NR-17786 16 >256 4 4 2 2 4 16 0.0625 NR-19298 32 >256 2 4 2 2 2 8 0.25 NR-19299 64 >256 4 32 4 4 8 64 0.25

MIC50 64 >256 4 8 4 2 4 32 0.25

MIC 90 128 >256 8 16 8 4 32 128 0.5 178

Table 6.3. Minimum inhibitory concentration (MIC) (µM) of peptides against clinical isolates of colistin resistant P. aeruginosa isolated from cystic fibrosis patients.

strain RR RR1 RR2 RR3 RR4 D-RR4 LL-37 Indolicidin Colistin

1109 128 >128 16 8 4 2 64 >128 128

1125 128 >128 32 32 8 4 128 >128 32

1571 128 >128 32 8 4 2 16 >128 256

1603 128 >128 16 8 8 4 32 >128 64

16011 >128 >128 32 32 16 4 >128 >128 128

1017 >128 >128 16 64 32 16 >128 >128 >256

1020 128 >128 16 8 4 2 32 >128 256

1133 64 >128 32 32 16 8 32 >128 32

1015 >128 >128 16 8 32 4 >128 >128 >256

1016 >128 >128 32 32 32 16 >128 >128 >256

1131 128 >128 32 16 8 4 16 >128 64

MIC50 128 >128 32 16 8 4 64 >128 128

MIC 90 >128 >128 32 32 32 16 >128 >128 >256

179

Table 6.4. MIC values of designed peptides in the presence of salts, pH 5.5 or pH 6.5, fetal bovine serum (FBS, 2%, 5% or 10%), human serum (2% or 5%), plasma from human (2% or 5%), against P. aeruginosa PAO1

pH

6.5

2% 5%

ontrol

NaCl NaCl NaCl

1:500

pH 5.5

HS 2% HS 5% HS

C

Trypsin

K K 1:500

FBS 2% FBS 5% FBS

100 mM 150 mM

H H plasma H plasma

FBS 10% FBS Proteinase

RR 64 64 128 128 128 64 64 128 256 256 128 256 nd nd

RR2 4 4 8 8 4 8 32 128 128 128 8 16 nd nd

RR3 8 16 16 32 16 16 32 64 32 128 16 16 nd nd

RR4 4 4 8 8 2 4 4 16 16 32 16 32 >64 >64

D-RR4 2 2 4 4 1 2 2 4 2 4 2 2 2 2 nd, not determined

180

Table 6.5. The fractional inhibitory concentration index range of peptide, D-RR4 in combination with antibiotics against bacterial isolates

Fold MIC of reduction in MIC antibiotic in FIC Strain Interpretation antibiotic (µM) combination index concentration with D-RR4 at the FIC

Colistin 0.5 0.125 0.5 Synergism 4

Polymyxin 0.5 0.07 0.39 Synergism 8 B Tobramycin 1 1 1.25 Indifference 0 P. aeruginosa PAO1 Gentamicin 2 2 1.25 Indifference 0

Amikacin 2 2 1.25 Indifference 0

Cefotaxime 32 4 0.38 Synergism 8 Colistin 0.5 0.06 0.38 Synergism 8 Polymyxin 0.5 0.06 0.38 Synergism 8 B P. aeruginosa ATCC Tobramycin 0.5 1 2.25 Indifference 0 BAA 1744 Gentamicin 2 4 2.25 Indifference 0 Amikacin 1 2 2.25 Indifference 0 Meropenem 256 16 0.31 Synergism 16 Cefotaxime 64 4 0.31 Synergism 16 Colistin 0.25 0.06 0.49 Synergism 4 Polymyxin 0.25 0.03 0.38 Synergism 8 B A. baumannii ATCC Tobramycin 2 0.25 0.38 Synergism 8 AB 1605 Gentamicin 256 256 1.25 Indifference 0 Amikacin 4 0.5 0.38 Synergism 8 Meropenem 256 2 0.26 Synergism 128 Cefotaxime 256 4 0.27 Synergism 64

181

Table 6.6. Toxicity and therapeutic index of the designed peptides

a b c d c MIC50 (µM) EC50 (µM) TI HC50 TI Peptide J774.1/ HaCaT J774.1/ HaCaT (µM) RBCs RR 64 128/256 2/4 >256 8 RR1 >256 >256/>256 NA >256 NA RR2 4 128/64 32/16 256 64 RR3 8 128/128 16/16 >256 64 RR4 4 64/128 16/32 256 64 D-RR4 2 64/64 32/32 256 128 Melittin 8 2/4 0.25/0.5 4 0.5 120e- 60-104 Pexiganan 2 64/ND 32/ND 208f a MIC50 (µM) of peptide against P. aeruginosa and A. baumannii isolates (Table 6.2). b EC50 is the peptide concentration that inhibited 50 % of macrophage cell line (J774.1) or human keratinocyte (HaCaT). c d Therapeutic index is the ratio of the EC50 or HC50 value over the MIC50 value. HC50 is the peptide concentration that cause 50 % hemolysis of RBCs. e from reference (60).f from reference (61). ND. not determined. NA. not applicable.

182

Figure 6.1. Helical wheel projections of the designed peptides

Yellow and blue circles represent hydrophobic and hydrophilic residues, respectively. Grey and bright blue circles represent alanine and histidine residues, respectively. The arrows indicate the directions and magnitudes of the hydrophobic moments (µH) determined for each peptide; N, amino terminus; C, carboxy terminus.

183

Figure 6.2. CD analyses of the designed peptides

Peptides at 50 µM concentration were dissolved in deionized water, 30 mM sodium dodecyl sulfate (SDS) or 30% trifluoroethanol (TFE). 184

Figure 6.3. Analytical HPLC spectra demonstrating proteolytic activity of trypsin and proteinase K on RR4 and D-RR4 after 0, 4 and 24 hours of incubation.

185

Figure 6.3 continued

186

A - L o g a rith m ic p h a s e o f P . a e r u g in o s a P A O 1 B - L o g a r ith m ic p h a s e o f A . b a u m a n ii A T C C B A A -1 6 0 5

1 0 1 0 C o n tro l C o n tro l

8 R R 1 0 X 8 R R 1 0 X

)

)

l l

D -R R 4 5 X D -R R 4 5 X

m

m

/

/ U

U 6 D -R R 4 1 0 X 6 D -R R 4 1 0 X

F

F

C

C (

( C o lis tin 5 X C o lis tin 5 X

0

0 4 4 1

1 C o lis tin 1 0 X C o lis tin 1 0 X

g

g o o T o b ra m y c in 1 0 X T o b ra m y c in 1 0 X

2 L

L 2 G e n ta m ic in 1 0 X

0 0 0 1 2 3 4 5 6 0 1 2 3 4 5 6 T im e (h r ) T im e (h r )

C - S ta tio n a r y p h a s e o f P . a e r u g in o s a P A O 1 D - S ta tio n a r y p h a s e o f A . b a u m a n ii A T T C B A A -1 6 0 5

1 2 1 2 C o n tro l C o n tro l 1 0 1 0 R R 1 0 X

R R 1 0 X )

l )

l D -R R 4 1 0 X m

D -R R 4 1 0 X /

m 8

/ 8 U

U C o lis tin 1 0 X

C o lis tin 1 0 X F F

C 6 C

6 ( T o b ra m y c in 1 0 X

( T o b ra m y c in 1 0 X

0

0 1

1 4 G e n ta m ic in 1 0 X 4

g

g

o

o L L 2 2

0 0 0 4 8 1 2 1 6 2 0 2 4 0 4 8 1 2 1 6 2 0 2 4 T im e (h r ) T im e (h r ) Figure 6.4. The killing kinectics of peptides. Time-kill of peptides and antibiotics against logarithmic (A&B) and stationary phase (C&D) cultures of P. aeruginosa PAO1 (A&C) and A. baumannii ATCC BAA-1605 (B&D).

(A&B) Logarithmic phase of bacteria were exposed to D-RR4 and colistin at 5 × and 10 × MIC or RR, tobramycin and gentamicin at 10 × MIC. (C&D) The killing kinectics of stationary phase of bacteria exposed to peptides and antibiotics at 10 × MIC. Untreated samples served as a control. The killing curves were identical (overlapping in the figure A) for RR, D-RR4 and colistin at 10 × MIC and identical for RR, D-RR4 at 10 × MIC in B. The results are given as means ± SD (n = 3); data without error bars indicate that the SD is too small to be seen.

187

A B

)

0

1

g y

o 1 0 8 0 0 0 0

t

i

L

(

s

n

y

t

e i

8 t

s n

i 6 0 0 0 0

n

e

e

t

c

n i

6 n C o n tro l n e g a tive

e e

c D -R R 4 5 X M IC

c 4 0 0 0 0

s n

e D -R R 4 1 0 X M IC e

4 C o n tro l n e g a tive r c

o L L -3 7 1 0 X M IC s

D -R R 4 5 X M IC u

e

l f

r 2 0 0 0 0

o 2 D -R R 4 1 0 X M IC

e

u

v l

L L -3 7 1 0 X M IC i

f

t

a

e

l v

0 e 0

i t

0 1 2 3 4 5 6 R 0 1 2 3 4 5 6 7 8

a

l

e R T im e (m in u te s ) T im e (m in u te s )

C

A T P le a k a g e

s t

i 6 0 0 0 0 C o n tro l n e g a tive

n U

D -R R 4 5 X M IC e

c D -R R 4 1 0 X M IC n

e 4 0 0 0 0

c L L -3 7 1 0 X M IC

s

e

n

i

m u

L 2 0 0 0 0

e

v

i

t

a

l e

R 0

e IC IC IC iv t M M M a g X X X e 5 0 0 1 1 n 4 l R 4 7 ro R R -3 t - R L n D - L o D C Figure 6.5. Permeabilization of bacterial membrane by peptides

A) Permeabilization of the outer membrane of P. aeruginosa PAO1 by antimicrobial peptides as indicated by the enhanced uptake of 1-Nphenylnaphthylamine (NPN). Fluorescence intensity caused by the partitioning of NPN into the outer membrane was monitored after addition of antimicrobial peptides. Data are presented as means ± standard error of the means (n = 3). B) Permeabilization of the inner (cytoplasmic) membrane by antimicrobial peptides is indicated by propidium iodide fluorescence. C) Release of ATP from bacteria treated with peptides was detected using a luminescence assay after mixing with luciferase reagent. Each experiment was performed in duplicate, and the values represent means ± standard deviation.

188

Figure 6.6. TEM micrographs of untreated and peptide-treated P. aeruginosa PAO1

The cells of untreated bacteria are intact, with a well-defined cell membrane (A&B). Bacteria treated with low concentration of D-RR4 peptide (1 × MIC) showed disintegrated membranes with blebs and increases in periplasmic space (black arrow heads), mesosomes and invaginations (dashed lines) and condensed materials under membranes (white arrows) (C&D). Bacteria treated with a higher concentration of D-RR4 peptide (10 × MIC) (E-H) showed complete loss of membranes (black arrows) and leakage of cytoplasmic contents (dashed arrow). Ghost cells due to loss of most cytoplasmic contents were also evident at higher peptide concentrations (H). Lower panel is a higher magnification of square selected area in upper panel. Upper bar, 500 nM; lower bar 100 nM.

189

3 2 R ifa m p ic in 2 8

C T o b ra m y c in I

2 4 M

C ip ro flo x a c in

n i

2 0

e D -R R 4

g 1 6

n a

h 1 2

c

d

l 8 o

F 4

0 0 2 4 6 8 1 0

N u m b e rs o f p a s s a g e s Figure 6.7. Drug-resistance development profile of P. aeruginosa PAO1 exposed to peptides

P. aeruginosa PAO1 exposed to 10 passages in the presence of sub-inhibitory concentrations of D-RR4 peptide and three classes of antibiotics including the protein synthesis inhibitor, tobramycin, the RNA synthesis inhibitor, rifampicin and the DNA synthesis inhibitor, ciprofloxacin.

190

P . a e ru g in o s a P A O 1

3 0 0 C o lis tin 1 0 0 R R 4

) D -R R 4 %

( 8 0

s s

a 6 0

)

m

m

l %

i 4 0

f

(

o

2 0 0 i B

s 2 0 s

a 0

0 .5 1 2 4 8 1 6 3 2 m

C o n c e n tra tio n (X M IC ) m

l C o lis tin i f 1 0 0

o R R 4 i

B D -R R 4 T o b ra m y c in G e n ta m ic in 0 0 .5 1 2 4 8 1 6 3 2

C o n c e n tra tio n (X M IC )

A . b a u m a n ii B A A -1 6 0 5

1 2 0

1 0 0

)

% (

8 0

s

s a

m 6 0

C o lis tin

m

l i

f R R 4

4 0 o

i D -R R 4 B 2 0 T o b ra m y c in G e n ta m ic in 0 0 .5 1 2 4 8 1 6 3 2

C o n c e n tra tio n (X M IC ) Figure 6.8. Efficacy of peptides on established biofilms of P. aeruginosa PAO1 and A. baumannii ATCC BAA-1605

Twenty four hour old biofilms were treated with different concentrations of RR4, D-RR4, colistin, tobramycin and gentamicin and plates were incubated at 37°C for 24 hours. After incubation, wells were washed and biofilms were stained with 0.5% (w/v) crystal violet for 30 minutes. The dye was solubilized with ethanol (95%) and the optical density (OD) of biofilms was measured. Experiments were repeated twice independently and the average values are reported.

191

P . a e r u g in o s a P A O 1 A . b a u m a n n ii A T C C 1 9 6 0 6 e

e 1 0 0 1 0 0

g

g

a

a

t

t

n

n

e

e

c

c

r

r

e

e

p

p

l

l 5 0 5 0

a

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The effect of RR4 and D-RR4, at 8 × MIC, to kill intracellular P. aeruginosa PAO1 and A. baumannii ATCC 19606 inside infected J774A.1 cells after treatment for 24 hours. Statistical analysis was calculated using one-way ANOVA, with post hoc Tukey’s multiple comparisons test. P values of ˂ 0.05 were considered significant. One asterisk (*) indicates significance from the negative control. Results are expressed as means from three biological replicates ± standard deviation.

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L I N 2 0 T 2 0 * * * * * * * * 0 * 0 l l o o r S r S M M t P M M M M M M t P M M M M m m n L   0    0  n L   0   o 0 0 0 0 o 0 0 0 0 0 + 1 1 1 2 2 2 + 1 1 1 2 c c 2 2 l 4 n 4 n l 4 d o R i R i d o R in R 4 n r R t R t r R t i e t R R e t R R R t t R is R is t R is s a n - l - l a n - l R i l o l o - l u D o D o u D o o C C C C C D C im im t t s s n n U U Figure 6.10. Anti-inflammatory effect and LPS binding activity of peptides

A) LPS binding activity of peptides. Peptides at different concentrations were incubated with one endotoxin unit (EU) of LPS at 37°C for 30 minutes. Colistin was used as positive control due to its high binding affinity for LPS. The binding of peptides with LPS is expressed as percent change relative to untreated samples. B) Anti-inflammatory effect of peptides on LPS stimulated macrophage. J774A.1 cells were stimulated with LPS (150 ng/mL final concentration) in the presence of different concentrations of peptides. Cells stimulated with LPS alone and untreated cells served as controls. Cells were incubated for six hours at 37 °C after which the supernatants of the medium from each treatment were collected. Cytokine detection of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in supernatants was analyzed using ELISA. Cytokine levels are expressed as percent change relative to the LPS stimulated control. Statistical analysis was calculated using one-way ANOVA, with post hoc Tukey’s multiple comparisons test. P values of ˂ 0.05 were considered significant. One asterisk (*) indicates significance from the negative control.

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Figure 6.11. Evaluation of toxicity and efficacy of peptides in Caenorhabditis elegans.

A). Evaluation of toxicity of peptides in Caenorhabditis elegans. C. elegans strain glp- 4;sek 1 was grown for four days in the presence of peptides at different concentrations. Worms were monitored daily and the live worms were counted. Results are expressed as a Kaplan-Meier survival-curve. B) Evaluation of antimicrobial activity of peptides on reducing the bacterial count inside infected Caenorhabditis elegans. C. elegans strain glp- 4; sek 1 were infected with P. aeruginosa PAO1, colistin-resistant P. aeruginosa 1109 or A. baumannii ATCC BAA-1605. After infection, worms were treated with D-RR4, colistin, or a combination of both agents. After treatment, worms were lysed and bacteria were counted. One asterisk (*) indicates significance from the negative control. Two asterisks (**) indicate significance from the monotherapy treatment (P < 0.05). Results are expressed as means from three biological replicates ± standard deviation. C) Efficacy of peptides in a Caenorhabditis elegans model of bacterial infection. C. elegans strain glp-4;sek 1 were infected with P. aeruginosa PAO1, colistin resistant P. aeruginosa 1109 or A. baumannii ATCC BAA-1605. After infection, worms were treated with D-RR4, colistin, or a combination of both. Worms were monitored daily and the live worms were counted. Results are expressed as a Kaplan-Meier survival-curve. C. elegans receiving no treatment served as a control.

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Figure 6.11 continued

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Figure 6.11 continued

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CHAPTER 7. TARGETING BIOFILMS AND PERSISTERS OF ESKAPE PATHOGENS WITH P14KANS, A KANAMYCIN PEPTIDE CONJUGATE

(Mohamed, M.F., Brezden A., Mohammed H, Chmielewski J, and Seleem, M.N. Targeting biofilms and persisters of ESKAPE pathogens with P14KanS, a kanamycin peptide conjugate. BBA General Subjects, under review)

7.1 Abstract

The worldwide emergence of antibiotic resistance represents a serious medical threat. The ability of these resistant pathogens to form biofilms that are highly tolerant to antibiotics further aggravates the situation leading to recurring infections. Thus, new therapeutic approaches with novel mechanisms of action are urgently needed. To address this significant problem, we conjugated the antibiotic kanamycin with a novel antimicrobial peptide (P14LRR) to develop a kanamycin peptide conjugate (P14KanS). P14KanS showed enhanced and potent antimicrobial activity against ESKAPE pathogens. P14KanS demonstrated at least 128 fold improvement in the MIC over kanamycin against kanamycin resistant strains. Mechanistic studies confirmed that P14KanS exerts its antibacterial effect by selectively disrupting the bacterial cell membrane. P14KanS was not toxic or hemolytic at concentrations much higher than required for antibacterial activity. In contrast to many antibiotics, P14KanS demonstrated rapid bactericidal activity against stationary phases of Gram-positive and Gram-negative pathogens, Furthermore, P14KanS was superior in disrupting adherent bacterial biofilms and killing intracellular pathogens when compared to conventional antibiotics. Of note, P14KanS demonstrated potential anti-inflammatory activity by suppression of LPS-induced proinflammatory cytokines. Finally, P14KanS protected C. elegans from lethal infections of Gram-positive and Gram-negative pathogens. In conclusions, this study show that equipping kanamycin with selective membrane activity is a promising way for tackling biofilms and bacterial resistance to aminoglycosides. 197

7.2 Introduction

The emergence of antibiotic resistance is a notorious problem worldwide [1]. In the United

States alone, antibiotic-resistant bacteria infect at least two million people, killing 23,000 patients annually [2]. Currently, members of the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii,

Pseudomonas aeruginosa, and Enterobacter species) represent one of the most significant threats to human health as strains exhibit resistance to multiple antibiotic classes[3].

Compounding the situation further, global reports of extensively drug-resistant (XDR) and pandrug-resistant (PDR) isolates of P. aeruginosa and A. baumannii have been increasing [4-6]. The ability of these resistant pathogens to form biofilms that are highly tolerant to antibiotics further aggravates the situation, ultimately leading to persistent and recurring infections [7, 8]. Hence, the development of therapeutic alternatives that have novel mechanisms of action are urgently needed in order to effectively address this problem.

Aminoglycosides are a class of valuable antibiotics that are used in the treatment of several microbial infections [9-11]. They bind to the 16S rRNA component of the bacterial ribosome, leading to mistranslation and bacterial death. However, the worldwide epidemic of resistance to aminoglycosides, to kanamycin in particular, has diminished their widespread clinical use [9-12].

We recently conjugated kanamycin with the antibacterial peptide P14LRR to achieve a dual agent (P14KanS) (Figure 7.1). P14LRR is a small de novo proline rich antimicrobial peptide that forms a cationic amphiphilic polyproline helix and demonstrates broad- spectrum efficacy against pathogenic bacteria [13, 14]. P14LRR is non-hemolytic and 198 highly stable to proteases. These characteristics contributed to the selection of P14LRR for conjugation to kanamycin. However, much remains to be examined with P14KanS including its spectrum of activity against Gram-positive and Gram-negative pathogens, its mechanism of action, its ability to disrupt bacterial biofilms, its efficacy against stationary phase bacteria, and confirming its potent antibacterial activity in a suitable animal model of infection.

In the present study, we investigated the activity of the P14KanS conjugate against important multidrug-resistant strains of the ESKAPE family. Additionally, we explored the conjugate’s mechanism of action against kanamycin-resistant isolates. Moreover, the conjugate’s ability to eradicate preformed biofilms and to kill stationary phases of pathogens was examined. Furthermore, the immune-modulatory activity of the conjugate was explored. Finally, the antibacterial activity of the conjugate was validated in vivo using a Caenorhabditis elegans model of bacterial infection. Taken altogether, our findings indicate that the conjugate is a promising candidate that warrants further investigation as a therapeutic agent for multidrug-resistant bacterial pathogens.

7.3 Materials and methods

Bacterial isolates, drugs and reagents

Clinical isolates used in the study are presented in Table 1. P14LRR peptide and the peptide kanamycin conjugate (P14KanS) (figure 7.1) were synthesized as described previously (13, 14). Melittin was purchased from Sigma-Aldrich (St. Louis, MO). Antibiotics were purchased from commercial vendors. Mueller-Hinton broth (MHB), Dulbecco’s phosphate buffered saline (PBS), fetal bovine serum (FBS), and Dulbecco's Modified Eagle's medium (DMEM) were purchased from Sigma-Aldrich (St. Louis, MO). MTS reagent were purchased from Promega (Madison, WI, USA). Brain heart infusion 199 broth (BHIB), trypticase soy broth (TSB) and trypticase soy agar (TSA) were purchased from Becton-Dickinson (Cockeysville, MD). Enzyme-linked immunosorbent assay (ELISA) development kits for cytokine detection were purchased from R&D Systems, Inc. (Minneapolis, MN).

Determination of minimum inhibitory concentration (MIC) values

The broth microdilution technique was utilized to assess the minimum inhibitory concentrations of compounds according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) (15). Bacterial isolates were diluted to 5×105 colony forming unit (CFU/ml) in MHB (except E. faecium, diluted in BHIB). Compounds were added to 96-well plates at specified concentrations and subsequently serially diluted. The MIC was defined as the lowest concentration of compound that prevented visual bacterial growth.

Mechanism of action studies

A) Outer membrane permeabilization assay

Permeation of the bacterial outer membrane by compounds was assessed using the non- polar fluorescent dye 1-N-phenylnaphthylamine (NPN) as described elsewhere (16). P. aeruginosa PAO1, in logarithmic growth phase was centrifuged for 10 minutes at 4000 × g, washed, and adjusted to OD600 of 0.5. A small aliquot (100 µl) of bacteria was added to black wall 96-well plates. NPN was mixed with bacteria at a final concentration of 10 μM. Compounds were added, in triplicates, at specified concentrations. Plates were monitored using a microplate reader (Molecular Devices, Sunnyvale, CA) at excitation and emission wavelengths of 350 nm and 420 nm, respectively. Data was corrected for background fluorescence and plotted as percentage ± standard deviation. Experiments was done in triplicate and was repeated twice independently.

B) Inner (cytoplasmic) membrane permeabilization assay

Damage of the bacterial inner (cytoplasmic) membrane after exposure to compounds was evaluated using the fluorescent propidium iodide dye, as described elsewhere (17). P. aeruginosa PAO1 in logarithmic growth phase, was centrifuged for 10 min at 4000 × g, washed, and adjusted to OD600 of 0.2. An aliquot (100 µl) of bacterial suspension was added to black 96-well plates. Propidium iodide dye was added at a final concentration of 200

10 μM. Compounds were added, in triplicates, at specific concentrations. Bacteria treated with compounds diluent (distilled water) served as a negative control. Plates were read using a microplate reader at excitation and emission wavelengths of 585 nm and 620 nm, respectively. Data was corrected for background fluorescence and plotted as percentage ± standard deviation. Experiments was done in triplicate and was repeated twice independently.

C) Calcein leakage assay

Membrane permeabilization of S. epidermidis (ATCC 35984) by the peptide kanamycin conjugate was monitored and quantified by the leakage of the preloaded fluorescent dye, calcein, as described before(18, 19).

Time-kill assay against logarithmic and stationary phase bacteria

S. aureus ATCC 6538, S. epidermidis ATCC 35984, P. aeruginosa PAO1 and A. baumannii ATCC BAA-1605 were grown in MHB with aeration at 250 rpm at 37 ºC overnight, then diluted in fresh MHB and incubated aerobically at 37 ºC until the suspension reached a logarithmic phase of growth (OD600 = 0.2). Next, bacteria were diluted to 5×105 CFU/ml in MHB. Compounds were added at desired concentrations (10 µM for S. aureus and S. epidermidis and 20 µM for P. aeruginosa and A. baumannii), in triplicate. Bacteria were incubated aerobically at 37 ºC in a shaking incubator at 250 rpm. Aliquots at specified time points were taken, serially diluted in phosphate-buffered saline (PBS) and plated in triplicate on TSA. CFUs were counted after incubation of plates for 24 hours at 37 ºC. In order to determine the kinetics of killing against stationary phase bacteria S. aureus ATCC 6538, S. epidermidis ATCC 35984, P. aeruginosa PAO1 and A. baumannii ATCC BAA-1605 were grown in MHB with aeration at 250 rpm at 37 ºC overnight. Bacteria were subsequently exposed to compounds at similar concentrations used in logarithmic phase. Aliquots were taken and plated as described above.

Toxicity of compounds against mammalian cell lines

Compounds were assayed for potential in vitro toxicity against human keratinocytes (HaCaT) and HeLa cells as described before (20) with the following modifications. Cells 201 were treated with compounds at various concentrations for 24 hours. Compounds were assayed for hemolytic activity against human red blood cells as described previously (18).

Efficacy of compounds on bacterial biofilms

To examine the compounds’ ability to disrupt bacterial biofilms, the microtiter dish biofilm formation assay was employed (21). Briefly, overnight cultures of S. aureus (ATCC 6538) and S. epidermidis (ATCC 35984) were diluted 1:100 in TSB supplemented with 1% glucose. Overnight cultures of P. aeruginosa PAO1 and A. baumannii ATCC BAA-1605 were diluted 1:100 in M63 minimal medium supplemented with magnesium sulfate, glucose and casamino acids. Bacterial suspensions were incubated in 96-well plates at 37°C for 24 hours. After removing media, wells were rinsed with PBS to remove planktonic bacteria before re-filling wells with fresh media. Compounds were added at specific concentrations and plates were incubated at 37°C for 24 hours. After incubation, wells were washed and biofilms were stained with 0.5% (w/v) crystal violet for 30 min.

The dye was solubilized with ethanol (95%) and the biofilm mass was quantified at OD595. Experiments was done in triplicate and was repeated twice independently.

Intracellular antibacterial efficacy of compounds in human keratinocytes

In order to assess the compounds’ ability to kill intracellular staphylococci, human keratinocytes were infected, as described in previous studies (14, 22). Briefly, HaCaT cells was seeded and incubated in DMEM + 10% FBS. Following incubation, cells were infected with S. aureus (multiplicity of infection 100:1) in DMEM + 10% FBS for two hours. After infection, the wells were washed with 200 µl media containing gentamicin (50 µg/ml) and further incubated for 30 minutes with gentamicin to kill any remaining extracellular bacteria. Compounds were diluted in DMEM + 10% FBS to the desired concentrations and wells were treated with 100 µl of DMEM +10 FBS containing compounds for 24 hours. Medium alone was used as a negative control. After incubation, the media was aspirated and washed twice with PBS to remove any residual test agent. Next, 100 µl of PBS containing 0.01% triton X-100 was added to lyse cells. Subsequently, bacteria were diluted and plated on TSA plates. Plates were incubated at 37ºC for 16 hours. After incubation, bacteria were enumerated and analyzed. Experiments were repeated 202 twice independently and the average percent reduction of bacterial growth for each treatment regimen was determined.

Lipopolysaccharides (LPS) studies

A) Binding of compounds to LPS (LAL assay) To determine if the compounds could bind to LPS, a Limulus amoebocyte lysate (LAL) kit (Genscript, USA Inc.) was utilized as described in previous studies (23). Compounds were dissolved in pyrogen-free water and serially diluted in the same solvent. The compounds were then incubated with one endotoxin unit (EU) of LPS at 37 °C for 30 minutes in order to permit the compounds to bind LPS. The reaction mixture (50 μl) was added to 50 μl of LAL reagent, and further incubated for 10 min. Next, 100 μl of LAL chromogenic substrate was added. The mixture was incubated at 37 °C for 6 minutes.

Samples were measured at OD545 using a microplate reader. Colistin was used as a positive control. The binding of compounds with LPS was expressed as percent change relative to the untreated control.

B) Anti-inflammatory effect of compounds on LPS stimulated macrophages (Neutralizing the effect of LPS on macrophage)

To investigate the anti-inflammatory effect of compounds on LPS stimulated macrophages, J774A.1 cells were seeded and incubated as described above. Next, the cells were stimulated with LPS (150 ng/ml final concentration) in the presence of 10 µM of compounds. Cells that were stimulated with LPS alone and untreated cells served as controls. Cells were incubated for six hours at 37°C and supernatants from each treatment were collected and stored at -20°C until use. Cytokine detection of tumor necrosis factor- α (TNF-α) and interleukin-6 (IL-6) in supernatants was done using ELISA as described before following the manufacturer’s instructions (24, 25). Cytokine levels were expressed as percent change relative to LPS stimulated control using triplicate samples for each treatment condition.

Multi-step resistance selection

To evaluate the ability of S. aureus to acquire resistance to the peptide kanamycin conjugate and control antibiotics, a multi-step resistance selection experiment was 203 employed. S. aureus ATCC 6538 was serially passaged in the presence of a sub-inhibitory concentrations of kanamycin, P14LRR, P14KanS and three different classes of antibiotics, ampicillin, rifampicin, and ciprofloxacin for eight serial passages. The MIC was monitored daily using the broth microdilution assay. Results were presented as the change in MIC measurement observed after each passage in comparison to the initial MIC value.

In vivo efficacy of compounds in a C. elegans infection model Infection and treatment of C. elegans was done as described previously with some modifications (26, 27). C. elegans AU37 [sek-1(km4); glp-4(bn2) I] was used to test the efficacy of peptides in vivo. This strain has a mutation in the sek-1 gene of the p38 mitogen-activated protein kinase pathway (p38 MAPK pathway) that makes this particular strain more susceptible to bacterial infection (26, 27). Briefly, synchronized adult worms were transferred to modified NGM (0.35% peptone) agar plates seeded with a lawn of P. aeruginosa PAO1, A. baumannii ATCC BAA-1605, S. aureus ATCC 6538 or S. epidermidis ATCC 35984 for infection. After infection, worms were collected and washed with PBS three times. Worms (~30 per sample) were transferred to 96-well plates. Compounds were added at desired concentrations (10 µM for S. aureus and S. epidermidis and 50 µM for P. aeruginosa and A. baumannii), in triplicate. Untreated worms served as a negative control. To assess the survival of infected C. elegans, worms were assessed for viability (live worms are sinusoidal with movement, whereas dead worms are rigid rods).

Statistical analyses

Statistical analyses were performed using GraphPad Prism 6.0 software (GraphPad Software, La Jolla, CA, USA). Comparison between two groups were analyzed using two- tailed unpaired Student t-tests. Comparison between three or more groups was analyzed using one-way ANOVA, with post hoc Tukey’s multiple comparisons test. P-values of ˂ 0.05 were considered significant.

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7.4 Results and discussion

Antimicrobial activity

The antibacterial activity of kanamycin and the conjugate was investigated against ESKAPE pathogens. Herein, P14LRR displayed moderate to weak antibacterial activity against ESKAPE pathogens with MIC range from 8-64 µM (Table 1). Kanamycin demonstrated activity only against two strains (S. aureus ATCC 6538 and MRSA USA400). However, other bacteria were resistant to kanamycin (MIC, 128 to >256 µM). P14KanS demonstrated a very potent broad spectrum antibacterial activity over kanamycin and the peptide (P14LRR). The MIC of P14KanS was 1 to 2 µM against all strains tested. This represents a 2-4 fold improvement in the MIC over kanamycin against kanamycin sensitive strains and 128 to >256 fold improvement in the MIC over kanamycin against kanamycin resistant strains. Interestingly, P14KanS demonstrated potent activity against several clinical multidrug-resistant isolates including methicillin resistant S. aureus (MRSA USA400), methicillin resistant S. epidermidis (MRSE) and vancomycin resistant E. faecium (VRE) (Table 1). Of particular note, P14KanS demonstrated potent activity against A. baumannii ATCC BAA-1605 which is a multidrug-resistant isolate obtained from the sputum of a Canadian soldier serving in Afghanistan. This strain is resistance to numerous antibiotics including kanamycin, meropenem, imipenem, ceftazidime, ciprofloxacin, piperacillin, ticarcillin, cefepime, aztreonam and gentamicin (Table 1). Moreover, P14KanS demonstrated potent activity against colistin-resistant P. aeruginosa (1109) isolated from cystic fibrosis patients (28). This isolate showed high resistance to colistin (MIC = 128 µM), kanamycin and several antimicrobial peptides (28). Colistin is an antibiotic of last resort for multidrug-resistant P. aeruginosa infections (29). The emergence of resistant isolates to colistin, particularly via plasmid-mediated resistance in Enterobacteriaceae (30), highlights the urgent need to discover new antimicrobial agents to address this issue. The discovery that P14KanS have potent activity against colistin-resistant P. aeruginosa indicates a potential therapeutic advantage of P14KanS over several antimicrobials that are ineffective against colistin- resistant P. aeruginosa (28).

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Mechanism of action of P14KanS

In order to gain insight into the mode of action of P14KanS and its activity against kanamycin resistant strains, we performed a series of experiments on a kanamycin resistant bacteria to explore its antibacterial mode of action. First, damage to the bacterial outer membrane by P14KanS was monitored by measuring the fluorescent intensity of P. aeruginosa PAO1 mixed with 1-N-phenylnaphthylamine (NPN) and incubated with varying concentrations. As demonstrated in figure 7.2a, similar to the known membrane targeting antibiotic, colistin, there was a significant and dose-dependent increase of fluorescence intensity of P. aeruginosa cultures treated with P14KanS but not kanamycin nor the P14LRR peptide. Remarkably, P14KanS demonstrated more potent activity than colistin. These findings indicate that P14KanS likely targets the bacterial outer membrane leading to partitioning of NPN into the outer membrane and increased fluorescence. Damage of the bacterial inner (cytoplasmic) membrane by P14KanS was monitored by measuring the fluorescence intensity of P. aeruginosa PAO1 mixed with propidium iodide and treated with P14KanS. As demonstrated in figure 7.2b, the fluorescence intensity didn’t change in untreated bacteria and with kanamycin and P14LRR treated bacteria, confirming the cytoplasmic membrane integrity. However, when P14KanS and colistin were added to bacterial cultures, a rapid and significant increase in fluorescence due to access of propidium iodide inside the bacteria was evident. The membrane damaging effect was both concentration-and time-dependent. At 10 × MIC, P14KanS demonstrated more potent membrane perturbation than at 5 × MIC.

To confirm our results of membrane permeabilization against a Gram-positive pathogen, we studied the effect of P14KanS on S. epidermidis (kanamycin resistant) membranes using a calcein leakage assay. Calcein AM is a non-fluorescent derivative that is able to penetrate cell membranes; once inside the cells it is hydrolyzed by intracytoplasmic esterases to a fluorescent calcein, which is non-permeable to membranes (19, 31). Damage of membrane integrity is indicated by calcein leakage from bacterial cells, which is in turn proportional to a reduction of fluorescence intensity. In a similar way like the well-known membrane damaging peptide, nisin (figure 7.2c); P14KanS damage the S. epidermidis cell membrane led to fast leakage of preloaded calcein within seconds (figure 7.2c). However, 206

P14LRR demonstrated no leakage implying a non-lytic mechanism of action. Collectively, these data confirm the membrane-destructive effect of P14KanS against Gram-negative and Gram-positive bacteria and clarify why P14KanS demonstrated efficacy against kanamycin resistant isolates.

Time kill assay against logarithmic and stationary phase bacteria

After confirming that P14KanS possessed excellent antimicrobial activity against a broad spectrum of multidrug-resistant clinical isolates, we next assessed its killing kinetics against logarithmic phase of selected Gram-positive (S. aureus ATCC 6538 and S. epidermidis ATCC 35984) and Gram-negative pathogens (P. aeruginosa PAO1 and A. baumannii ATCC BAA-1605). As demonstrated in figure 7.3, P14KanS demonstrated fast killing of Gram-positive bacteria. At 10 µM, P14KanS completely eradicated logarithmic cultures of S. aureus ATCC 6538 and S. epidermidis ATCC 35984 starting inoculum after 0.5 and 2 hours, respectively. However, kanamycin required 6 hours to eradicate S. aureus and was not effective against S. epidermidis. P14LRR was not effective at the same tested concentrations. In contrast, vancomycin demonstrated slower killing kinetics. As shown in figure 7.3, vancomycin caused more than 2.5 log reduction after 6 hours. Linezolid showed a bacteriostatic effect.

Similar to Gram-positive pathogens, P14KanS demonstrated fast killing of Gram-negative bacteria. At 20 µM, P14KanS completely eradicated logarithmic cultures of P. aeruginosa and A. baumannii starting inoculum after one hour. However, kanamycin and P14LRR were not effective. Tobramycin and gentamicin eliminated P. aeruginosa PAO1 within 2- 3 hours. Tobramycin eliminated A. baumannii ATCC BAA-1605 after three hours. Antimicrobials with fast bactericidal activity have several advantages including limiting spread of infection, improving treatment prognosis, limiting the emergence of resistance, and reducing duration of treatment (32).

Next, we examined the impact of P14KanS and select antibiotics against the stationary- phase of bacteria. This population of cells is known to contain a high percentage of persisters that are tolerant to many antimicrobial agents and contributes to recurring infections (7, 33-35). As shown in figure 7.3, P14KanS completely eradicated bacterial cultures of S. aureus after 4 hours and demonstrated rapid bactericidal activity (3-log10 207 reduction) against S. epidermidis. On the other hand, antibiotics were not able to show bactericidal effect on stationary phases, despite their bactericidal activity against logarithmic bacteria. Kanamycin demonstrated only1.3 log10 reduction of S. aureus after 6 hr. Vancomycin and linezolid were not effective against stationary bacteria in same time frame (figure 7.3). Treatment of stationary-phase P. aeruginosa and A. baumannii with 20 µM of P14KanS resulted in rapid bactericidal activity (3-log10 reduction) and complete eradication of a high starting inoculum (109 CFU/ml) after three hours and six hours, respectively. (figure 7.4). Conventional antibiotics had minimal impact on stationary-phase bacteria which is in agreement with previous findings (7, 33-35).

The superior activity of P14KanS when compared to conventional antibiotics, against stationary phase bacteria may be explained by its unique antimicrobial mechanism of action. Many antibiotics require bacteria to be growing and metabolically active in order to inhibit their molecular targets, including protein synthesis (linezolid, tobramycin and gentamicin) and cell wall synthesis (vancomycin) (36). In contrast, as mentioned above, the positive charge present in P14KanS serves as a point of attraction with negatively- charged bacterial cell membranes and consequently leads to targeted disruption of the bacterial membrane and leakage of intracellular contents (37, 38). This unique mechanism of action of P14KanS would not require cells to be metabolically active and would not be impaired by the dormant and quiescent state of bacteria (36). The results obtained above provide valuable insight into using P14KanS as a potential future therapeutic option for persistent and chronic bacterial infections.

Efficacy of P14KanS on established biofilms

Biofilms are aggregated bacterial communities covered by polysaccharide matrix that protects bacteria from host immune defenses and hinders antibiotics from targeting deep- seated bacteria encased within the biofilm (39). Furthermore, biofilms act as an infectious niche with sustained release of bacteria inside the host which leads to disease relapses and therapy failure(40). Biofilm development has been linked to serious biofilm-related infections including pneumonia in cystic fibrosis patients and colonization of medical devices and urinary tract infections (41). Biofilms pose a serious medical challenge, that is difficult to control and there is a critical need to find novel approaches to address this 208 problem. We examined the capability of P14KanS and control antibiotics to disrupt established biofilms of four different bacterial isolates responsible for major biofilm infections; S. aureus, S. epidermidis, P. aeruginosa and A. baumannii. As shown in figure 7.5, P14KanS disrupted the 24 hour old biofilms more potently than several antibiotics of choice. P14KanS was superior to kanamycin and the peptide P14LRR against the kanamycin sensitive isolate (S. aureus). P14KanS demonstrated a concentration- dependent biofilm-disrupting activity. P14KanS, at low concentration (1 µM) disrupted more than 65 % of biofilm mass of S. aureus (p < 0.05); whereas kanamycin and P14LRR were not effective at the same concentrations (figure 7.5A). At higher concentrations, P14KanS disrupted more than 75 and 82% of biofilm mass of S. aureus at 4 and 8 µM, respectively, (p < 0.05). Kanamycin, vancomycin and linezolid, at 8 µM, reduced approximately 50% of biofilm mass (p < 0.05); whereas, P14LRR was not effective (figure 7.5A). We further assessed the efficacy of P14KanS against biofilms of a clinical multi-drug resistant S. epidermidis ATCC 35984, a high slime producing strain that was isolated from septicemic patients with colonized intravascular catheters in Tennessee, US (42) This strain is a multi-drug resistant as it shows resistance to methicillin, erythromycin, kanamycin, gentamicin, clindamycin and trimethoprim. The great thickness of the exopolysaccharide matrix of S. epidermidis biofilms cause it to become extremely resilient to antibiotics penetration (39, 43-45). Indeed, the 24 hr old biofilms of S. epidermidis were less susceptible to vancomycin and linezolid even at 64 µM (64-128 X MIC) showing 25-30% biofilm reduction (figure 7.5B). However, P14KanS disrupted more than 30, 50, 70, 80% of biofilm mass at 8, 16, 32 and 64 µM, respectively, (p < 0.05). P14LRR was not effective except at 64 µM, disrupting approximately 50% of biofilm mass. Kanamycin didn’t disrupt biofilms (figure 7.5B). Remarkably, P14KanS was capable of eradicating P. aeruginosa and A. baumannii biofilms at 32 µM, however, kanamycin was not effective (figure 7.5C). P14LRR (32 µM) was not effective against P. aeruginosa biofilms and demonstrated only 30% reduction of A. baumannii biofilms (figure 7.5C). Collectively, the data demonstrate that P14KanS is superior to conventional antibiotics in penetrating and disrupting adherent biofilms of pathogens. 209

Toxicity of P14KanS against mammalian cells

A significant limitation for translating novel membrane targeting antimicrobials into clinical applications is their toxicity to host cells (46). Herein, we assessed the hemolytic activity of P14KanS on human red blood cells (RBCs). As demonstrated in figure 7.6a, P14KanS and P14LRR displayed no hemolysis even at very high concentrations (256 µM) (Therapeutic index “TI” >256). In contrast, melittin completely lysed RBCs at very low concentrations (8 µM) equal to ½ × MIC of melittin against P. aeruginosa (TI = 0.5). Failure of P14KanS to lyse human RBCs confirms the selectivity of P14KanS toward negatively charged bacterial membrane.

Next, we evaluated the cytotoxic effects of P14KanS against human keratinocytes (HaCaT cells) and HeLa cells via the MTS assay. The half maximal effective concentration (EC50) was 128/256 µM, respectively, in the case of HeLa / HaCaT cells (figure 7.6B). This confirms that P14KanS displayed a higher safety window. The therapeutic index of P14KanS is 128/256 respectively, in the case of HeLa / HaCaT cells. The data indicate the superior safety profile of P14KanS and its selectivity toward bacterial cells.

Intracellular antibacterial activity of P14KanS

S. aureus was considered to be primarily an extracellular pathogen for many years. However, extensive studies have revealed that these pathogens can gain entry and persist inside both phagocytic and non-phagocytic eukaryotic cells, such as alveolar macrophages and keratinocytes (47-49). The intracellular persistence of pathogens constitutes a significant challenge as they are able to avoid host immune defenses. Additionally, intracellular pathogens evade the action of many antibiotics that exhibit low intracellular penetration ability such as aminoglycosides and vancomycin (50). Eradication of these challenging bacterial pathogens is critical to ensure successful treatment of persistent intracellular bacterial infections. Here, we investigated if P14KanS had the ability to kill intracellular S. aureus residing in human keratinocytes. As demonstrated in figure 7.7, P14KanS showed significant reduction of intracellular S. aureus and MRSA USA400 at nontoxic concentrations. P14KanS showed the maximum efficacy as it reduced intracellular S. aureus and MRSA USA400 by 2.52±0.37 and 1.24±0.26 log, respectively at 15 µM. In contrast, peptide P14LRR was not effective. Kanamycin and vancomycin 210 produced approximately 0.4-0.6 log reduction of intracellular S. aureus and MRSA USA400. The potent intracellular activity of P14KanS may be useful in treating certain chronic exacerbating skin diseases such as Darier’s disease (keratosis follicularis) where S aureus persists inside keratinocytes leading to failures to antibiotic therapy and recurrent infections in affected patients (51).

3.7. Anti-inflammatory effect of P14KanS A key virulence factor unique to Gram-negative bacterial pathogens is the presence of lipopolysaccharide (LPS). LPS is a principal structure of the outer membrane of Gram- negative bacteria. During bacterial death, cell replication or exposure to certain antibiotics, LPS is released from bacteria leading to stimulation of proinflammatory cytokine release such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) from mononuclear phagocytes. Excessive LPS exposure leads to overproduction of these cytokines which can result in septic shock and death. Annually there are 18 million cases of septic shock globally with a mortality rate of ~30% (52, 53). Therefore, identifying agents that are capable of neutralizing LPS is critical. We investigated if P14KanS could bind to and neutralize the effect of LPS. As presented in figure 7.8A, P14KanS and the peptide P14LRR were able to bind to LPS in vitro as evident by inhibition of the LPS-induced activation of the LAL enzyme (Limulus amoebocyte lysate). P14KanS, P14LRR and colistin (a known LPS binding agent) produced 60.8 ± 6.5, 71.2 ± 5 and 79.76 ± 4.6 % inhibition of the LAL enzyme at 10 µM, respectively. However, kanamycin demonstrated only minimal inhibition (6.9% ± 3.5) at the same concentration. Next, we examined if P14KanS and the peptide, P14LRR could reduce the endotoxin- induced proinflammatory cytokine response in murine macrophages by measuring cytokine levels using ELISA. As depicted in figure 7.8B, P14KanS and P14LRR were able to inhibit LPS-induced proinflammatory cytokines in macrophages in a manner similar to colistin. P14KanS, P14LRR and colistin, at 10 µM, decreased TNF-α levels by 65.53% ± 2.45, 68.4% ± 5.43, 97.53% ± 0.32, respectively; and IL-6 levels by 74.71% ± 9.10, 81.92% ± 0.78 and 95.04% ± 0.56%, respectively. However, kanamycin demonstrated 11.41% ± 4.5 and 10% ± 3.30 inhibition of TNF-α and IL-6 levels, respectively. The capability of P14KanS to reduce endotoxin-mediated proinflammatory 211 cytokine production provides a potential avenue for their development as antibacterial agents to treat sepsis and also as an adjunctive with antibiotics to overcome sepsis.

Multi-step resistance selection

To examine if the dual mechanism of action of P14KanS can overcome traditional mechanisms of antibiotic resistance, we serially passaged a kanamycin sensitive S. aureus isolate (ATCC 6538) with sub-inhibitory concentrations of P14KanS, kanamycin, P14LRR and three different classes of antibiotics, ampicillin (cell wall synthesis inhibitor), rifampicin (RNA synthesis inhibitor) and ciprofloxacin (DNA synthesis inhibitor) for eight days. The MIC was determined daily using the broth microdilution assay. As presented in figure 7.9, S. aureus did not develop resistance to P14KanS for six passages and only a fourfold change in MIC was observed after eight passages. However, treatment of S. aureus with kanamycin induced a rapid shift in the MIC within the fourth passage and resulted in 64-fold increase in kanamycin’s MIC by passage seven. The MIC of P14LRR increased by 4 and 16 fold by passage 6 and 8, respectively. The MIC of rifampicin and ampicillin increased to 64-fold and ciprofloxacin increased to 16 fold by the sixth passage (figure 7.9). Thus, the conjugation of kanamycin with P14LRR proved to decrease the emergence of resistance. This is attributed to the unique mechanism of action of P14KanS. P14KanS disrupted bacterial membranes, so for bacteria to acquire resistance, significant changes to the membrane lipid composition would need to occur (54). These alterations are extremely difficult for bacteria to produce compared to point mutations in specific genes (36, 55). This result thus provides strong evidence that the selective membrane-disrupting activity of P14KanS can effectively overcome conventional mechanisms of antibiotic resistance.

In vivo efficacy of P14KanS in a C. elegans infection model. To test the efficacy of the synthetic peptides to protect against infections in vivo, we utilized a C. elegans model of bacterial infection. This model has been used extensively for early-stage evaluation of promising antimicrobials (56-58). Moreover, this model demonstrated that bacteria develop a biofilm infections inside the gut of C. elegans (59- 61). 212

Bolstered by in vitro results, we examined whether P14KanS could protect infected worms from the cidal activity of bacteria. Interestingly, P14KanS significantly increased the rate of survival for P. aeruginosa and A. baumannii infected C. elegans in contrast to monotherapy, giving complete protection for 72 hours (figure 7.10). However, kanamycin and P14LRR failed to protect the worms. (P. aeruginosa PAO1 and A. baumannii completely killed untreated worms after 36 and 72 hours, respectively) (figure 7.10). The survival experiment with C. elegans was next utilized to examine the impact of biofilm forming S. aureus. While kanamycin and P14LRR failed to protect worms from killing by a kanamycin sensitive isolate (S. aureus ATCC 6538), P14KanS at 10 µM, significantly increased the survival rate of C. elegans, giving nearly complete protection for 72 hours (figure 7.10). Next, we evaluated the activity of P14KanS to rescue C. elegans from infection by the multi-drug resistant clinical isolate, S. epidermidis ATCC 35984 (NRS 101). Indeed, this strain demonstrated higher virulence than S. aureus as it completely killed C. elegans after 72 hours. A notable protective effect was provided by P14KanS, but not with kanamycin and P14LRR. P14KanS rescued 80% of infected worms after 72 hours. Interestingly, P14KanS was superior to drugs of choice (vancomycin and linezolid) as they protected approximately 70% and 60% of S. aureus and S. epidermidis infected worms, respectively at the same tested concentrations (10 µM) (figure 7.10). Collectively, these data confirm the potent therapeutic efficacy of P14KanS in vivo against notorious biofilm infections of Gram-positive and Gram-negative pathogens.

7.5 Conclusion

The worldwide increase of antibiotic resistance and limited supply of new antibiotics necessitates the discovery and development of novel approaches to address this burgeoning challenge. In the present study, we conjugated the aminoglycoside antibiotic, kanamycin with the antibacterial peptide P14LRR to enhance the antibiotic’s potency and spectrum of activity against resistant bacterial strains. This conjugation successfully improved the antibacterial activity of kanamycin against both kanamycin-sensitive and kanamycin-resistant strains of key ESKAPE pathogens. P14KanS was found to exert its 213 antibacterial activity via disruption of both the outer and inner membranes of the cell envelope. However, no adverse toxic effect was observed to mammalian cells at concentrations 128 times higher than the MIC, indicating P14KanS possesses a selective membrane-active effect to bacterial cells. The rapid, membrane-disruptive nature of P14KanS against bacteria shows low propensity for resistance development in comparison to kanamycin and conventional antibiotics. In addition to its potent antibacterial activity, P14KanS significantly disrupted established bacterial biofilms more so than conventional antibiotics. Furthermore, P14KanS was capable of binding strongly to LPS and subsequently reduced the endotoxin-mediated proinflammatory cytokine production in murine macrophage cells. This discovery highlights a potential avenue for development of P14KanS to treat sepsis. P14KanS also significantly reduced the burden of intracellular MRSA potentially opening an avenue for exploring P14KanS for treatment of certain chronic skin diseases, such as Darier’s disease. Furthermore, P14KanS’s potent antibacterial activity was confirmed in vivo as it significantly increased the rate of survival for nematodes infected with both Gram-positive and Gram-negative pathogens. The conjugation of kanamycin with a cell-penetrating peptide has great potential to facilitate delivery of drugs directly to the site of infection (lungs) via intrapulmonary delivery [62]. Clearly, further investigation of potential clinical applications is necessary in more advanced animal models of infection. The success of this approach will open the door for further testing of other aminoglycosides and different classes of antibiotics conjugated to antimicrobial peptides.

214

7.6 References

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Table 7.1. Minimum inhibitory concentration (MIC) (µM) of compounds against clinical and drug-resistant bacterial isolates.

colistin S. S. resistant A. K. VRE epidermidi P. MRSA aureus pneumoni E. Compoun s aerugino P. baumannii USA400 ATCC ae BAA- faecium d ATCC sa PAO1 6538 aerugino BAA-1605 1706 ATCC 35984 sa 1109 700221 Resistant Biofilm Prototype Biofilm Isolated A Resistant Resistant to forming biofilm producin from multidrug to to methicilli strain, producer, g strain. cystic resistant kanamyci kanamyc fibrosis n and methicill Resistant to Resistant strain; n in and patient. tetracycli in methicillin, to Resistant resistant to teicoplan ne sensitive gentamicin, kanamyc to kanamycin, in kanamycin, in colistin ceftazidim erythromyc and e, in, kanamyc gentamicin clindamyci in, , ticarcillin, Phenotype n and piperacillin trimethopri , m aztreonam, cefepime, ciprofloxac in, imipenem, and meropene m P14LRR 64 16 32 64 64 32 32 8 Kanamyci 4 2 >256 >256 >256 128 256 >256 n P14KanS 1 1 1 2 2 1 2 2 Fold enhancem ent 4 2 >256 >128 >128 128 128 >128 compared to kanamycin

220

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Figure 7.5. Efficacy of P14KanS on established biofilms of A) S. aureus ATCC 6538; B) S. epidermidis ATCC 35984; C) P. aeruginosa PAO1 and A. baumannii ATCC BAA-1605. Twenty-four hour old biofilms were treated with different concentrations of P14KanS, P14LRR, kanamycin and control antibiotics and plates were incubated at 37°C for 24 hours. After incubation, wells were washed and biofilms were stained with 0.5% (w/v) crystal violet for 30 minutes. The dye was solubilized with ethanol (95%) and the optical density (OD) of biofilms was measured. Experiments were repeated twice independently and the average values are reported. Statistical analysis was calculated using one-way ANOVA, with post hoc Tukey’s multiple comparisons test. P values of ˂ 0.05 were considered significant. One asterisk (*) indicates significance from the negative control. Two asterisks (**) indicate significance of P14KanS compared to P14LRR and kanamycin. Results are expressed as means from three biological replicates ± standard deviation.

225

Figure 7.5 continued

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C o n c e n tra tio n ( M ) C o n c e n tra tio n ( M ) Figure 7.6. Toxicity of P14KanS against mammalian cells. (A) The release of hemoglobin in the supernatant of human erythrocytes after treatment with increasing amounts of P14KanS, P14LRR, and melittin was measured at 415 nm. Triton X-100 served as positive control. (B) Cytotoxicity assay showing the percent mean absorbance at 490 nm after incubating human keratinocyte (HaCaT) or HeLa cells with compounds at different concentrations. Sterile water (compounds diluent) served as negative control. Cell viability was measured by MTS assay. Results are expressed as means from three measurements ± standard deviation.

Figure 7.7. Intracellular antibacterial activity of P14KanS in human keratinocytes (HaCaT). The effect of kanamycin, P14LRR, P14KanS and vancomycin, at 15 µM, to kill intracellular S. aureus ATCC 6538 and MRSA USA400 inside infected HaCaT cells after treatment for 24 hours. Statistical analysis was calculated using one-way ANOVA, with post hoc Tukey’s multiple comparisons test. P values of ˂ 0.05 were considered significant. One asterisk (*) indicates significance from the negative control. Two asterisks (**) indicate significance of P14KanS compared to P14LRR and kanamycin. Results are expressed as means from three biological replicates ± standard deviation. 227

A L P S b in d in g

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l S n R S n l S n R S n o i ti o i ti r P c R n r P c R n t L y L a is t L y L a is n l n l + m 4 K o + m 4 K o o 1 4 o 1 4 c l a P 1 C c l a P 1 C o n o n d r a P d r a P e t e t t n K t n K a o a o l l u C u C im im t t s s n n U U Figure 7.8. Anti-inflammatory effect and LPS binding activity of P14KanS. A) LPS binding activity of compounds. Compounds at 10 µM were incubated with one endotoxin unit (EU) of LPS at 37°C for 30 minutes. Colistin was used as positive control due to its high binding affinity for LPS. The binding of compounds with LPS is expressed as percent change relative to untreated samples. B) Anti-inflammatory effect of compounds on LPS stimulated macrophage. J774A.1 cells were stimulated with LPS (150 ng/mL final concentration) in the presence of 10 µM of compounds. Cells stimulated with LPS alone and untreated cells served as controls. Cells were incubated for six hours at 37 °C after which the supernatants of the medium from each treatment were collected. Cytokine detection of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in supernatants was analyzed using ELISA. Cytokine levels are expressed as percent change relative to the LPS stimulated control. 228

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c

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Figure 7.9. Drug-resistance development profile of S. aureus. S. aureus ATCC 6538 exposed to eight passages in the presence of sub-inhibitory concentrations of kanamycin, P14LRR, P14KanS and three classes of antibiotics including the cell wall synthesis inhibitor, ampicillin, the RNA synthesis inhibitor, rifampicin and the DNA synthesis inhibitor, ciprofloxacin.

229

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W W 0 1 2 3 0 1 2 3 D a y s D a y s Figure 7.10. Efficacy of P14KanS in a Caenorhabditis elegans model of bacterial infection. C. elegans strains glp-4;sek 1 were infected with P. aeruginosa PAO1, A. baumannii ATCC BAA-1605, S. aureus ATCC 6538 and S. epidermidis ATCC 35984. After infection, worms were treated with P14KanS, P14LRR, kanamycin and control antibiotics. Worms were monitored daily and the live worms were counted. Results are expressed as a Kaplan-Meier survival-curve. C. elegans receiving no treatment served as a control.

230

VITA

Mohamed F. K. Mohamed

Education:

Purdue University Ph.D. (Microbiology) 2012-2016

Beni-Suef University M.V.Sc. (Microbiology) 2009-2011

Beni-Suef University B.V.Sc. (1st class, honor) 2002-2007

Honors and awards:

First poster award (Conference of Research Workers in Animal Diseases, Chicago, IL) 2016 Second poster award (IB ASM Annual Meeting, IN) 2016 PhD scholarship (Egyptian Cultural and Educational Bureau in Washington DC) 2012-2016 Master scholarship (Beni-Suef University) 2008-2011 Teaching assistantship (Beni-Suef University) 2008-2012 Ideal student award (The Education Day, Beni-Suef University) 2005

Professional activities:

PhD student, College of Veterinary Medicine, Purdue University 2012-2016 Teaching assistant of microbiology, College of Veterinary Medicine, 2008 -2012 Beni-Suef University (Teaching diagnostic bacteriology, mycology and immunology to undergraduates)

Professional associations:

 American Society for Microbiology  ASM-Indiana branch  Phi Zeta Omicron Chapter 231

Presentations:

Oral presentations:

Mohamed, M.F., Maha I. Hamed, Allyssa Panitch, Mohamed N. Seleem (2014). New Drug for Bad Bug: Combatting Methicillin-Resistant Staphylococcus aureus (MRSA) with De Novo Peptides. Comparative pathobiology seminars, October 3, Purdue University, West Lafayette, IN, USA.

Mohamed, M.F., G. Kenitra Hammac, Lynn Guptill and Mohamed N. Seleem (2015). Novel approaches to combat antibiotic resistance: evaluation of short antimicrobial peptides against clinical isolates of multi-drug resistant Staphylococcus pseudintermedius from infected dogs. Comparative pathobiology seminars, November 16, Purdue University, West Lafayette, IN, USA.

Mohamed, M.F., Anna Brezden, Jean Chmielewski and Mohamed N. Seleem (2015). Targeting Intracellular Pathogenic Bacteria with a Kanamycin Antibiotic Peptide Conjugate. Comparative pathobiology seminars, October 29, Purdue University, West Lafayette, IN, USA.

Mohamed, M.F., Anna Brezden, Jean Chmielewski and Mohamed N. Seleem (2016). Targeting Intracellular Pathogenic Bacteria with a Kanamycin Antibiotic Peptide Conjugate.2016 IB ASM Annual Meeting, IN, USA.

Poster presentations:

Mohamed, M.F., J. Brugnano, Y. Yeo, A. Panitch, M. Seleem (2012). When going gets tough, peptide gets going. Next Generation Scholars’ Research Fair, Purdue, November 30, West Lafayette, IN, USA.

Mohamed, M.F., J. Brugnano, Y. Yeo, A. Panitch, M. Seleem (2013). In vitro antibacterial activity of novel synthetic peptides against multiple drug resistant bacteria. Sigma Xi, the scientific research society Purdue, February 13, West Lafayette, IN, USA.

Mohamed, M.F., J. Brugnano, Y. Yeo, A. Panitch, M. Seleem (2013). Targeting multi- drug resistant bacteria with two novel peptides. The 26th Annual Phi Zeta Research Day, Purdue College of Veterinary Medicine. April 15, West Lafayette, IN, USA.

Mohamed, M.F., Maha I. Hamed, Allyssa Panitch, Mohamed N. Seleem (2014). Targeting methicillin-resistant Staphylococcus aureus with short de novo peptides. Sigma Xi, the scientific research society Purdue, February 12, West Lafayette, IN, USA.

Mohamed, M.F., Maha I. Hamed, Allyssa Panitch, Mohamed N. Seleem (2014). New Drug for Bad Bug: Combatting Methicillin-Resistant Staphylococcus aureus (MRSA) with De Novo Peptides. Purdue Interdisciplinary Graduate Program Spring Reception. April 2, West Lafayette, IN, USA. 232

Mohamed, M.F., Maha I. Hamed, Allyssa Panitch, Mohamed N. Seleem (2014). Targeting methicillin-resistant Staphylococcus aureus with short synthetic peptides: coupling membrane disruption with DNA binding. 26th Annual Phi Zeta Research Day, Purdue College of Veterinary Medicine. April 14, West Lafayette, IN, USA.

Mohamed, M.F., G. Kenitra Hammac, Lynn Guptill and Mohamed N. Seleem (2015). Novel approaches to combat antibiotic resistance: evaluation of short antimicrobial peptides against clinical isolates of multi-drug resistant Staphylococcus pseudintermedius from infected dogs. Sigma Xi, the scientific research society Purdue, February 18, West Lafayette, IN, USA.

Mohamed, M.F., Maha I. Hamed, G. Kenitra Hammac, Lynn Guptill, Allyssa Panitch and Mohamed N. Seleem (2015). Efficacy of novel antimicrobial peptides against methicillin-resistant Staphylococci. Health and Disease: Science, Culture and Policy research poster session. March 5, West Lafayette, IN, USA

Anna Brezden, Mohamed, M.F., Mohamed N. Seleem, and Jean Chmielewski (2015). Targeting Intracellular Pathogenic Bacteria with a Kanamycin Antibiotic Peptide Conjugate. 8th Annual Negishi-Brown Lectures, Purdue University, West Lafayette, IN, USA.

Anna Brezden, Mohamed, M.F., Mohamed N. Seleem, and Jean Chmielewski (2015). Targeting Intracellular Pathogenic Bacteria with a Kanamycin Antibiotic Peptide Conjugate. 24th American Peptide Symposium, Orlando, FL, USA. (Poster Finalist)

Anna Brezden, Mohamed, M.F., Mohamed N. Seleem, and Jean Chmielewski (2015). Targeting Intracellular Pathogenic Bacteria with a Kanamycin Antibiotic Peptide Conjugate. Gordon Research Conference of Antimicrobial Peptide, Italy.

Mohamed, M.F. and Mohamed N. Seleem (2015). Efficacy of short novel antimicrobial and anti-inflammatory peptides in a mouse model of methicillin-resistant Staphylococcus aureus (MRSA) skin infection. Purdue Interdisciplinary Graduate Program Spring Reception. April 1, West Lafayette, IN, USA

Mohamed, M.F., G. Kenitra Hammac, Lynn Guptill and Mohamed N. Seleem (2015). Targeting methicillin-resistant Staphylococcus pseudintermedius (MRSP) with novel antimicrobial peptides. Conference of Research Workers in Animal Diseases, Annual Meeting, December 6, Chicago, IL

Mohamed, M.F., Anna Brezden, Jean Chmielewski and Mohamed N. Seleem (2015). Targeting Intracellular Pathogenic Bacteria with a Kanamycin Antibiotic Peptide Conjugate. Conference of Research Workers in Animal Diseases, Annual Meeting, December 6, Chicago, IL

233

Mohamed, M.F., Anna Brezden, Jean Chmielewski and Mohamed N. Seleem (2016). Targeting Intracellular Pathogenic Bacteria with a Kanamycin Antibiotic Peptide Conjugate. Sigma Xi, the scientific research society Purdue, March 2, West Lafayette, IN, USA.

Mohamed, M.F., Anna Brezden, Jean Chmielewski and Mohamed N. Seleem (2016). Targeting Intracellular Pathogenic Bacteria with a Kanamycin Antibiotic Peptide Conjugate.29th Annual Phi Zeta Research Day, Purdue University, College of Veterinary Medicine. April 11, West Lafayette, IN, USA.

Nepal M., Mohamed, M.F., Seleem, M.N, and Chmielewski J. A library approach to develop novel antibacterial agents that target intracellular bacteria. 9th Annual Negishi- Brown Lectures, Purdue University, April 15, West Lafayette, IN, USA. (H.C. Brown Graduate Student Research Award Winner)

Mohamed, M.F., Anna Brezden, Jean Chmielewski and Mohamed N. Seleem (2016). Targeting Intracellular Pathogenic Bacteria with a Kanamycin Antibiotic Peptide Conjugate.2016 IB ASM Annual Meeting, IN, USA. (Second poster award)

234

PUBLICATIONS

Mohamed, M.F., Hamed, M.I., Panitch, A., Seleem, M.N (2014) Targeting methicillin- resistant Staphylococcus aureus with short salt-resistant synthetic peptides. Antimicrobial agents and chemotherapy doi:10.1128/AAC.02578-14

Mohamed, M.F., Seleem, M.N (2014) Efficacy of short novel antimicrobial and anti- inflammatory peptides in a mouse model of methicillin-resistant Staphylococcus aureus (MRSA) skin infection. Drug Design, Development and Therapy 2014:8; 1979–1983.

Mohamed, M.F., Hammac G.K., Guptill, L. and Seleem, M.N (2014) Antibacterial activity of novel cationic peptides against clinical isolates of multi-drug resistant Staphylococcus pseudintermedius from infected dogs. PLoS ONE 9(12): e116259. doi: 10.1371/journal.pone.0116259

Mohamed, M.F., Ahmed Abdelkhalek and Seleem, M.N (2016) Evaluation of short synthetic antimicrobial peptides for treatment of drug-resistant and intracellular Staphylococcus aureus. Scientific reports, Scientific Reports 6, Article number: 29707 (2016) doi: 10.1038/srep29707

Brezden A., Mohamed, M.F., Nepal M., Harwood J.S., Kuriakose J., Seleem, M.N, and Chmielewski J. (2016) Dual Targeting of Intracellular Pathogenic Bacteria with a Cleavable Conjugate of Kanamycin and an Antibacterial, Cell Penetrating Peptide. Journal of the American Chemical Society, DOI: 10.1021/jacs.6b04831

Mohamed, M.F., Brezden A. Mohammed H, Chmielewski J, and Seleem, M.N. A short D-enantiomeric antimicrobial peptide with potent immunomodulatory and antibiofilm activity against multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii (Antimicrobial agents and chemotherapy, accepted with revision)

Mohamed, M.F., Brezden A., Chmielewski J, and Seleem, M.N. Targeting biofilms and persisters of ESKAPE pathogens with a kanamycin antibiotic peptide conjugate. (BBA General Subjects, under review)

Nepal M., Mohamed, M.F., Seleem, M.N, and Chmielewski J. A library approach to develop novel antibacterial agents that target intracellular bacteria (In preparation to Journal of the American Chemical Society)

Pei,Y., Mohamed, M.F., Seleem, M.N, and Yeo, Y. Particle Engineering for intracellular delivery of vancomycin to methicillin-resistant S. aureus (MRSA)-infected macrophages (In preparation)