THE EFFECT OF ANTIMICROBIAL PEPTIDES ON BACTERIAL BIOFILMS Focusing on prevention of biofilm formation of urinary tract infection isolates by the antimicrobial peptides 1037 and LL-37

Andreas Skovgård Jacobsen Department of Science, Systems and Models, Roskilde University, Denmark June 2013

Master’s thesis

Supervisors: Associate Professor Håvard Jenssen (Roskilde University) Professor Karen Angeliki Krogfelt (Statens Serum Institut)

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Abstract The ongoing development of antibiotic resistant infections is a major obstacle in ensuring the future health and wellbeing. Even today, many people die from infections, which are caused by hospitalacquired multidrug resistant bacteria. This development predicts an imminent inadequacy of applicable antibiotics. Biofilms are bacteria that stick together, forming a community, which is embedded within a self-produced matrix. In urinary tract infections, this way of life is very common. The formation of a biofilm creates resistance and leads to the use of even more antibiotics. Since an overly and extensive use of antibiotics have adverse effects, such as inducible antibiotic resistance, there is a major demand for new antibiotics. Antimicrobial and host-defense peptides have been proposed as a new anti-infective therapeutic strategy. The research of antimicrobial peptides has primarily two motives: understanding the host defense and developing novel antibiotics to fight infections in an increasingly antibiotic resistant world. This master’s thesis focuses on the only human cathelicidin, LL-37, which has a major role in human host defence. LL-37, as well as the novel peptide 1037, possesses strong antibiofilm effects at sub-MIC concentrations. The results show that both LL-37 and 1037 kills clinical strains isolated from the urinary tract. Both tobramycin and tetracycline induce P. aeruginosa biofilm formation close to MIC. LL-37 and 1037 are both active against P. aeruginosa biofilms at 2 µg/ml, inhibiting the biofilm formation 21.9 % and 28.2 %, respectively. LL-37 and 1037 only inhibited K. pneumoniae strain C3091 attachment after two hours, but for LL37, the decrease of attachment was indicated to be due to less bacterial growth. Interestingly, 1037 did not inhibit K. pneumoniae growth at 4x MIC. The effects on biofilms of two E. coli urinary tract isolates were inconsistent. LL-37 was effective against E. coli CFT073 at 2 µg/ml inhibiting the biofilm formation 29.3 %, and 1037 was effective against E. coli 536 at 2 µg/ml inhibiting the biofilm formation 26.9 %. The experiments indicate that LL-37 and 1037 possess antibiofilm activity towards urinary tract isolates.

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Danish summary (Dansk sammendrag) Den igangværende udvikling af antibiotikaresistente infektioner er en stor forhindring for at garantere fremtidens helbred og velvære. Selv i dag dør mange mennesker af hospitalsinfektioner forårsaget af multiresistente bakterier. Denne udvikling varsler om en kommende mangel af brugbare antibiotika. Biofilme er sammenklæbede bakterier som har dannet et samfund, indkapslet i en selvproduceret matrix. I urinvejsinfektioner er denne levemåde almindelig. Dannelsen af en biofilm skaber resistens og fører til endnu mere brug af antibiotika. Da et overdrevent og omfattende forbrug af antibiotika har bivirkninger, såsom induceret antibiotikaresistens, er der en stor efterspørgsel af nye antibiotika. Antimikrobielle og værtsforsvars peptider er foreslået som en ny antiinfektions terapeutisk strategi. Forskningen af antimikrobielle peptider har primært to motiver: forstå værtsforsvaret og udvikle nye antibiotika til at bekæmpe infektioner i en stigende antibiotisk resistent verden. Dette speciale fokuserer på det eneste menneske-cathelicidin, LL-37, hvilket har en større rolle i menneskets immunforsvar. LL-37, samt det nye peptid, 1037, har vist at have antibiofilm effekter ved sub-MIC koncentrationer. Resultaterne viser at både LL-37 og 1037 dræber kliniske stammer isoleret fra urinvejsinfektioner. Både tobramycin og tetracycline inducerer P. aeruginosa biofilmdannelsen tæt på MIC. LL-37 og 1037 er begge aktive mod P. aeruginosa biofilme ved 2 µg/ml, hvor de inhiberer biofilmdannelsen henholdsvis 21.9 % og 28.2 %. LL-37 og 1037 inhiberede kun K. pneumoniae stamme C3091 biofilme efter to timer, men for LL37, blev mindskelsen af biofilm indikeret til at skyldes mindre bakterievækst. Interessant nok inhiberede LL-37 ikke K. pneumoniae vækst ved 4x MIC Effekterne på biofilme af to E. coli isolater fra urinvejsinfektioner var inkonsekvente. LL-37 var effektiv mod E. coli CFT073 ved 2 µg/ml og inhiberede biofilmdannelsen 29.3 %, og 1037 var effektiv mod E. coli 536 ved 2 µg/ml hvor biofilmdannelsen blev inhiberet med 26.9 %. Disse eksperimenter indikerer, at LL-37 og 1037 har en antibiofilm effekt mod isolater fra urinvejsinfektioner.

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Preface This thesis was written as a 60 ECTS point interdisciplinary master’s thesis in medical and molecular biology. All experiments were conducted at Roskilde University in the laboratory of Håvard Jenssen, Anders Løbner-Olesen and Ole Skovgård. My supervisors for this project were Associate Professor Håvard Jenssen, Roskilde University, and Professor Karen Angeliki Krogfelt, Statens Serum Institut.

Since high school I have been interested in antibiotic resistance. I was introduced to antimicrobial peptides at Roskilde University, where I did a project on plectasin. I was also caught by the fact that we are not using antimicrobial peptides therapeutically. Further investigation has showed me how important antimicrobial peptides can be for human health, and I see the great opportunity in optimizing the peptides and prepare them for therapeutic use.

I would like to express my appreciation and sincere gratitude to my supervisors Associate Professor Håvard Jenssen and Professor Karen Angeliki Krogfelt. Håvard Jenssen introduced me to the world of antimicrobial peptides and I am very grateful for this. I would also like to thank Karen Angeliki Krogfelt for being an inspiration and introducing the world of microbes and giving my project a clinical aspect.

I also want to thank the laboratory technicians Christa Persson and Kirsten Olesen from Roskilde University, who advised and assisted me in the laboratory. The project would not have been the same without the presence of the many wonderful people of Håvard Jenssen, Anders Løbner-Olesen and Ole Skovgårds laboratory, Troels Godballe, Biljana Mojsoska, Godefroid Charbon, Jakob Frimodt-Møller, Louise Bjørn and Andreas Thymann.

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List of Abbreviations ACN

Acetonitrile

AHL

Acyl homoserine lactones

AMP

Antimicrobial peptide

BM2

Basal medium 2

BSA

Bovine serum albumin

CFU

Colony-forming unit

DMEM

Dulbecco's Modified Eagle Medium

EPS

Extracellular polymeric substance

ESI

Electrospray ionization

hCAP

Human cationic antimicrobial protein

HPLC

High-pressure liquid chromatography

LB

Lysogeny broth

LPS

Lipopolysaccharide

MH

Mueller Hinton

MIC

Minimum inhibitory concentration

MRSA

Methicillin-resistant Staphylococcus aureus

MS

Mass spectrometry

OD

Optical density

RNA

Ribonucleic acid

TFA

Trifluoroacetic acid

μg

Microgram

μM

Micromolar

UTI

Urinary tract infection

UV

Ultraviolet

VRSA

Vancomycin-resistant Staphylococcus aureus

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Table of content Content Abstract ................................................................................................................................................ 3 Danish summary (Dansk sammendrag) ............................................................................................... 4 Preface .................................................................................................................................................. 5 List of Abbreviations ........................................................................................................................... 6 Table of content ................................................................................................................................... 8 1.

Motivation and aim .................................................................................................................... 10

2.

Introduction ................................................................................................................................ 12

3.

2.1

Antibiotics ........................................................................................................................... 12

2.2

Bacterial antibiotic resistance .............................................................................................. 14

2.3

Bacterial biofilms ................................................................................................................ 17

2.4

P. aeruginosa biofilms ........................................................................................................ 18

2.5

Quorum sensing controls biofilm formation ....................................................................... 20

2.6

Biofilm inhibitors as potential drugs ................................................................................... 21

2.7

Urinary Tract infections ...................................................................................................... 22

2.8

Antimicrobial peptides ........................................................................................................ 23

2.9

Human cathelicidin LL-37 .................................................................................................. 26

2.10

Antibiofilm activity of LL-37 .......................................................................................... 30

2.11

The potential of LL-37 and 1037 as UTI inhibitors ........................................................ 31

2.12

Structure activity relationship analysis ............................................................................ 32

Materials and methods ............................................................................................................... 34 3.1

Bacterial strains ................................................................................................................... 34

3.2

Bacterial growth conditions................................................................................................. 34

3.3

Peptides and antibiotics ....................................................................................................... 34

3.4

Purification of LL-37 with HPLC ....................................................................................... 35

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4.

3.5

Identification of LL-37 with HPLC/MS.............................................................................. 35

3.6

MIC determination .............................................................................................................. 36

3.7

Biofilm experiments ............................................................................................................ 36

3.8

Time-kill kinetics ................................................................................................................ 37

3.9

Statistical analysis ............................................................................................................... 38

Results ........................................................................................................................................ 39 4.1

HPLC purification of LL-37................................................................................................ 39

4.2

MS identification of peptides .............................................................................................. 40

4.3

MIC determination of antibiotics, LL-37 and LL-37 12-residue fragments ....................... 41

4.4

MIC determination of 1037 and synthetic analogues .......................................................... 43

4.5

Effect of LL-37, 1037 and antibiotics on P. aeruginosa biofilms ...................................... 45

4.6

Effect of LL-37 and 1037 on K. pneumoniae biofilms ....................................................... 48

4.7

Effect of LL-37 and 1037 on E. coli biofilms ..................................................................... 52

5.

Discussion .................................................................................................................................. 53

6.

Conclusion ................................................................................................................................. 65

7.

Future perspectives .................................................................................................................... 66

8.

References .................................................................................................................................. 67

Appendix I.......................................................................................................................................... 80 Effect of LL-37, 1037 and antibiotics on P. aeruginosa biofilms ................................................. 80 Effect of LL-37 and 1037 on K. pneumoniae biofilms .................................................................. 83 Effect of LL-37 and 1037 on E. coli biofilms ................................................................................ 87

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1. Motivation and aim Urinary tract infections (UTI) are very common, especially in young women. Urinary catheters are commonly used in hospital and health care facilities and their use make UTI the most common infection acquired in both hospitals and health care facilities. With long term urinary catheterization, the presence of bacteria in the urine, bacteriuria, is developed in all patients and 25 % for patients who have catheters placed from 2 to 10 days (Saint, 2000). These infections can in severe cases lead to death after developing pyelonephritis, urinary stones or perinephric abscesses, yet the contribution of catheter-associated UTI to mortality is unclear (Warren, 2001). Due to the time correlation and infection rate, it has been suggested to limit the use of urinary catheters (Jain et al., 1995). Additional hospital days associated with these infections have great economic and physical costs and thus it is important that we understand how these types of infections develop, how we prevent and cure them. This is thought to be achieved by coating the urinary catheters with antimicrobials. For example, silver coating of catheters has been commercialized for many years (Johnson et al., 1990, Karchmer et al., 2000). A wide variety of infection organisms are isolated from UTIs, but Escherichia coli, stands out as the far most frequent infecting organism. Klebsiella pneumoniae are also a common infecting organism (Nicolle, 2005). A catheter serves as an eminent surface for bacterial attachment, which then connects the bacteria with the lumen of the urethra. Both E. coli and K. pneumoniae can attach to the catheter and form a subsequent biofilm. A biofilm often complicates the clearance of infections. This is experienced with the lung disease cystic fibrosis which causes many (often fatal) Pseudomonas aeruginosa infections. These types of infections are eradicated by intensive antibiotic therapies, but these antibiotics affect the bacteria itself and are not directed towards biofilms. Biofilm also prevents the antibiotics and immune defense from acting on the bacteria. Not only can it be difficult to cure biofilm infections with antibiotics, but the often excessive amount of antibiotics used for treatment will eventually increase the chances of developing bacterial antibiotic resistance, and there are often severe side effects associated with the use of antibiotics.

Antibiotic resistant strains are often isolated from UTI. One of these strains is E. coli sequence type ST131, which was first reported in 2008 (Johnson et al., 2012, Nicolas-Chanoine et al., 2008). Since the discovery of this resistant strain, it has spread and continued to adapt new types of antibiotic resistance and is increasing among older adults and residents of nursing homes (Banerjee

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et al., 2013). This example of resistance development is dangerous and threatens how we will be able to treat urinary tract infections in the future, and it calls for new strategies focusing on preventing and treating UTIs. There are two major approaches to solving this problem: to improve or discover new antibiotics and to develop urinary catheters that cannot serve as a link to infection.

Antimicrobial peptides (AMP) are an evolutionarily conserved component of the immune defense in most complex organisms. Many AMPs have been discovered during the last two decades, but in spite of the lack of discovery of new antibiotics (Figure 1, p.12) no newly discovered AMP has passed clinical trials. But peptide drugs are becoming more and more popular in the pharmaceutical industry and currently, there are 60-70 approved peptide drugs on the market and it is assumed that 100-200 peptide drugs are in clinical trials (Sun, 2013). AMPs have been tried immobilized to biomaterial surfaces to lower side effects and decomposition and they have been clinically trialed as peptide antibiotics to kill multi drug resistant strains (Costa et al., 2011, Mygind et al., 2005). Considering their effects against microbes and with the experience with peptides in medication, peptides are highly qualified as new antibiotics.

This master’s thesis will focus on two AMPs: a highly conserved human AMP, LL-37, and the synthetic AMP, 1037, which are both antimicrobial and antibiofilm against P. aeruginosa. P. aeruginosa is in this study used for evaluation of the antimicrobial potency of the peptides and serves as a model biofilm producing organism for further evaluation of antibiofilm effect of LL-37 and 1037. This antibiofilm effect will finally be examined on a number of UTI pathogens. The aims for this master’s thesis are as listed: 1. Determining the antimicrobial potential of 8 LL-37 derivatives and for 1037, and studying the effect of single residue substitution on antimicrobial activity. 2. Confirming and expanding the knowledge of the antimicrobial and antibiofilm activity of LL-37 and 1037 on P. aeruginosa. 3. Studying the effects on biofilm growth of LL-37 and 1037 on clinical E. coli and K. pneumoniae UTI strains

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2. Introduction The following chapters will explain the historical aspects of antibiotics and how the use of antibiotics consequently has induced antibiotic resistance. Following, bacterial biofilms, quorumsensing and urinary tract infections chapters will argue why we need more and different working antibiotics. Then there will be an introduction to antimicrobial peptides and the two antimicrobial peptides, LL-37 and 1037. The introduction will finally provide an introduction and overview of the conducted experiments.

2.1 Antibiotics Since the 1940s, bacterial infections have become a less considerable problem and many infections which today easily can be treated, would back then often result in death. The decreased number of deaths from bacterial infections is most importantly due to the discovery of antibiotics. With the discovery of the first commercially available antibacterial drugs, Salvarsan (1910) by Paul Ehrlich, the sulfonamide Prontosil (1935) by Gerhard Domagk, and later with the introduction of Penicillin as a therapeutic agent in 1942 by Alexander Fleming, the mortality rate of bacterial infections rapidly decreased and an era had begun. Those three drugs was the beginning of a drug discovery era. Salvarsan and Prontosil was both later replaced by more non-toxic drugs, while penicillin antibiotics still are highly used today. With discovery of penicillin, the foundation of drug discovery the next 20 years had been set. A great number of antibiotics, which are still in use today in either their original or modified form, were commercially introduced shortly afterwards (Figure 1).

Figure 1: Timeline of antibiotic deployment (top) and antibiotic resistance (bottom) (Clatworthy et al., 2007).

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Antibiotics are classified according to their mode of action, chemical structure and spectrum of activity. Per definition these chemotherapeutic agents should be either bactericidal (killing) or bacteriostatic (inhibiting growth) towards pathogens while having limited side effects when administered to the host. The target(s) of antibiotics should ideally be essential for the viability of the infecting pathogen and should not be similar to structures found in host cells. Often, these essential structures are highly conserved but still contain an evolutionary distance to human counterparts, consequently some antibiotics act against a wide range of pathogenic bacteria and are therefore called broad-spectrum antibiotics. The disruption of these essential mechanisms leads to cell cycle arrest or even better, cell death. Due to the major impact of penicillins they are often used as a model antibiotic. Penicillins are all of the β-lactams antibiotic class which is named due to the presence of a structural β-lactam ring. The target of penicillins is the cell wall of gram-positive and gram-negative bacteria. This cell wall is of critical importance for the bacteria and keeps the integrity and shape. The fundamental feature of the cell wall is the cross-linked polymer, peptidoglycan (Scheffers and Pinho, 2005). Penicillins disrupt the final synthesizing step of peptidoglycan by irreversibly binding to DD-transpeptidases (also named Penicillin binding proteins) which lowers the enzyme activity and disrupts the formation of the cell wall which leads to cell death (Lange et al., 2007). Since the bacterial cell wall differ from the plasma membrane of human eukaryotic cells, the targeted enzymes are conveniently not present in eukaryotic cells wand builds the whole foundation of a suitable antibiotic. Other targets in antibiotic treatment are other targets within the bacterial cell wall, in protein synthesis, DNA replication, transcription and other essential pathways that make the bacteria viable (BrotzOesterhelt and Brunner, 2008, Morar and Wright, 2010), for examples see Table 1. Whereas antibiotics that target cell wall and DNA replication is commonly bactericidal, antibiotics which target the translation are commonly bacteriostatic (except Aminoglycosides) (Table 1).

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Table 1: Simplified diagram of selected classes of antibiotic, their target, mechanism of action and effect (Brotz-Oesterhelt and Brunner, 2008, Morar and Wright, 2010).

Antibiotic class

Examples

Target

Mechanism of action

Effect

β-lactams

Penicillins Cephalosporins

Peptidoglycan biosynthesis

Inhibits the formation of peptidoglycan crosslinking though binding to DD-transpeptidases

Bactericidal

Glycopeptides

Vancomycin Teicoplanin

Peptidoglycan biosynthesis

Inhibits the formation of peptidoglycan by binding to D-Ala-D-Ala.

Bactericidal

Aminoglycosides

Tobramycin Kanamycin

Translation

Protein synthesis and proofreading are disturbed by binding to 16S rRNA near A-site of 30S ribosomal subunit.

Bactericidal

Tetracyclines

Tetracycline Minocycline

Translation

Binds to 16S RNA at the A-site which inhibits the binding of aminoacyl-tRNA which blocks protein synthesis.

Bacteriostatic

Macrolides

Erythromycin Azithromycin

Translation

Binds to the domain V of 23S rRNA of 50S subunit which blocks peptide chain elongation.

Bacteriostatic

Quinolones

Ciprofoxacin

DNA replication

Inhibits and topoisomerases II (DNA gyrase) and IV.

Bactericidal

Polypeptides

Colistin

Outer membrane

Destabilizes the outer membrane

Bactericidal

Bacitracin

2.2 Bacterial antibiotic resistance After the discovery and commercial introduction of antibiotics it was soon observed that usually treatable infections were not affected by the administration of antibiotics and had adapted mechanisms of resistance. The cause for this was lack of experience from other antibiotics and though Alexander Fleming predicted that too low doses would lead to development of penicillin resistance, and though bacterial resistance towards penicillin was actually discovered before it was made available on the market, the usage of penicillin was not restricted by any means (Wright, 2012). After a period in the mid-20th century, it was finally recognized that there should be some antibiotic control, and penicillin and other antibiotics have to some extent been restricted since. Antibiotic resistance is a result of the evolutionary pressure that bacteria undergo. Antibiotic resistance has existed even before the widespread of antibiotics which there are several reasons for (Wright, 2007).

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The environment, in which the microorganisms exist, is not a monoculture but consist of a complex mixture of organisms, where some live in symbiosis, but where many are combatting each other with many toxic compounds. Many of the surrounding compounds would be lethal to the bacteria if consumed, and to overcome the toxicity, the organisms must develop strategic countermeasures, a sort of an environmental selective pressure. Since many antibiotics are uncovered from microorganisms, the microorganisms found in the same environment could potentially have developed specific mechanisms to make the antibiotics less harmful, for example by mutating the antibiotic target or developing antibiotic paralyzing pathways. Resistance occurs for all antibiotics after they are clinical deployed and there is a limit to the number of natural antibiotic substances which fulfill all pharmacokinetic demands. Because of this, much of the antibiotic work done after the 1960s was focused on chemically modifying existing antibiotics to make them more susceptible to resistant pathogens and to improve pharmacokinetics.

Staphylococcus aureus is a major cause of hospital and community-acquired infections and due to drug resistance they are becoming an even bigger health care problem. The antibiotic resistance of this species is well documented, and is in this chapter used to describe how bacteria acquire resistance. The main cause of the antibiotic resistance problem with S. aureus lies within the extensive use of penicillin. When introduced on the market, penicillin was life-changing and extremely efficient in combating bacterial infections, so it became widespread and as a result of bad administering strategies, strains of S. aureus was observed to gain resistance towards penicillin. Today some S. aureus infections are resistant to most antibiotics in the clinic due to their number of adopted resistance genes. When penicillin allergies were reported, antibiotics such as macrolides, lincosamides and streptogramins, which target the bacterial 50S ribosomal subunit, were tried as an alternative treatment. These treatments led to the fast adaption of resistance since strains already carried two form of resistance genes to these antibiotic classes. These two resistance mechanisms are implemented by the methylation of 23S rRNA by ermA, ermB or ermC and by the active efflux mediated by msrA (Schito, 2006). Quinolones, which target topoisomerase II and IV, were only in use for a short time before spontaneous chromosomal mutations in the GrlA subunit of topoisomerase IV and GyrA subunit of

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topoisomerase II occurred (Figure 1). This resulted in reduced quinolone-protein affinity (Lowy, 2003). β-lactams, such as penicillins, bind very effectively to DD-transpeptidases which play an essential role in the formation of peptidoglycan cross-linking. Penicillin resistant S. aureus have either acquired a mutated DD-transpeptidase or a β-lactamase which hydrolyses the β-lactam ring of the drug, thus inactivating it (Sauvage et al., 2008). As for many other antibiotics that faced resistance, natural penicillin was chemical modified to gain an improved antibiotic, methicillin, which was less susceptible to resistance. But as experienced with all other notable antibiotics, there was soon to become methicillin-resistant S. aureus infections, MRSA (Figure 1, p. 12). The resistance gene in MRSA, mecA, is carried by the mobile SCCmec element and encodes a methicillin-resistant penicillin-binding protein which most likely has been exogenously acquired from a distantly related Staphylococcus species (Hiramatsu et al., 2001, Tsubakishita et al., 2010) MRSAs are increasingly observed in infections among persons without established risk factors and they are the most common identifiable cause of skin and soft-tissue infections in several metropolitan areas across the United States (Moran et al., 2006) where they are associated with a higher mortality than methicillin-susceptible S. aureus, MSSA, infections (Cosgrove et al., 2003). Unfortunately, MRSA infections are no longer only hospital and healthcare-associated but an increasing number of infections are seen to be community acquired (Klevens et al., 2007, Naimi et al., 2003). When MSSAs develop into a MRSA, they are only susceptible to intravenously administered glycopeptides antibiotics such as vancomycin. The problem with antibiotic resistance in S. aureus seems to become even more severe since some MRSAs have developed decreased susceptibility towards vancomycin, so called vancomycin-resistant S. aureus (VRSA) strains (Hiramatsu et al., 1997, Sieradzki and Tomasz, 1997). Today, the search for expansion of new antibiotics is focusing on optimizing preexisting antibiotics, discovering new drug targets and investigate new potential groups of antibiotics (Donadio et al., 2010). One qualified target that has been extensively studied is biofilms and will be described in the section below.

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2.3 Bacterial biofilms Bacteria can live in two disparate ways: as single, free-floating cells (planktonic) or in sessile aggregates, so-called biofilms where the bacteria live in organized communities. The production of a biofilm originates with the initial adherence of the bacteria to a surface. Here the bacteria are embedded within a self-produced matrix of extracellular polymeric substance (EPS), which mainly contain polysaccharides, nucleic acids, lipids and proteins (Costerton et al., 1978). Biofilms consist mostly of EPS, 90 %, whereas the cells account for only 10 %. The formation of a biofilm gives the bacteria several advantages: it immobilizes the cells while maintaining a comfortable architecture allowing the cells to communicate, it creates a reservoir of nutrients from lysed cells including DNA, which makes horizontal gene transfer more likely to occur. Of most clinical importance, it also protects the cells from the surroundings such as host immune defence, many antibiotics, ultraviolet radiation and oxidizing or charged biocides (Flemming and Wingender, 2010). The biofilm mode of life is a central infection mechanism and is recognized as the causing or exacerbating feature in many medical infections including dental caries, nosocomial infections, pneumonia, cystic fibrosis, urinary tract infections, and infections related to catheters and medical implants (Costerton et al., 1999, Vuong and Otto, 2002). According to the US National Institutes of Health, biofilms are medically important and they estimate biofilms to account for 80 % of human bacterial infections.

The typical biofilm development is divided into 5 stages: (I) reversible attachment, (II) irreversible attachment, (III) maturation-1, (IV) maturation-2, and (V) dispersion (Figure 2) (Stoodley et al., 2002). Most research has been conducted in P. aeruginosa which is often used as a model biofilm producing organism and is also done so below.

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Figure 2: Biofilm development shown as a five-stage process. (I) initial attachment of cells to a surface (II) EPS production which leads to irreversible attachment (III) early development of biofilm architecture (IV) maturation of biofilm architecture (V) dispersal of cells (Stoodley et al., 2002).

2.4 P. aeruginosa biofilms P. aeruginosa is a gram-negative bacterium which lives in the soil and water, but is also an opportunistic human pathogen causing pneumonia in cystic fibrosis patients, urinary tract infections by contaminating urinary catheters and skin and soft tissue infections after breach of the skin. To form a biofilm, P. aeruginosa has the ability to get to and move across a surface while overcoming the hydrodynamic boundary layer and repulsive forces as the bacteria approach the surface. A single flagellum drives swimming motility in P. aeruginosa and is thought to enable the bacteria to reach the surface where it can attach and form a biofilm (O'Toole and Kolter, 1998, Sauer et al., 2002). Type IV pili contribute to the generation of motile forces, so called twitching motility, which makes the bacteria move across the surface of which the bacteria attaches itself to (Mattick, 2002). In contrast too much twitching movement will make it difficult for the bacteria to settle and form a biofilm (Picioreanu et al., 2007, Singh et al., 2002). These motion effectors are also involved in the initial attachment phase (I) of P. aeruginosa to biotic and abiotic surfaces as they also function as adhesins (Giltner et al., 2006, Jin et al., 2011, O'Toole and Kolter, 1998) . However, the precise nature of that role and its impact on biofilm development varies with 18

environmental conditions (Klausen et al., 2003). Another factor that is related in attachment to surfaces is the CupA fimbriae (Vallet et al., 2001). Other factors are often linked to the attachment but there is a lack of knowledge of how these factors plays a role in the attachment. The irreversible attachment (II) is followed by microcolony formation, but is still not fully described. Though several c-di-GMP associated proteins, such as SadB, SadC and BifA have shown to be involved in transition from reversible to irreversible attachment and microcolony formation (Caiazza and O'Toole, 2004, Merritt et al., 2007, Kuchma et al., 2007, Caiazza et al., 2007). These proteins are thought to be part of a complex system that regulates swarming motility, which regulates the reversible attachment and thus continuation of the process of biofilm formation (Murray et al., 2010), but also regulates EPS production and modulation of flagellar reversal rates via the chemotaxis cluster IV (Petrova and Sauer, 2012). After the formation of reversible attachment and microcolonies, a large amount of EPS is produced (III) and (IV). P. aeruginosa produces three exopolysaccharides, alginate, Psl, Pel, which all are differently expressed depending on strain (Ryder et al., 2007). For example, mucoid strains isolated from Cystic fibrosis patients produce alginate in large amounts but in strains such as PAO1 and PA14 alginate are not a major constituent of EPS (Wozniak et al., 2003). It is known that Psl is a great constitutor to biofilm formation in PAO1 (Jackson et al., 2004) and is also required for the attachment to both biotic and abiotic surfaces in many other strains (Ma et al., 2006, Colvin et al., 2012, Ma et al., 2009). Pel is required for biofilm formation in PA14 (Friedman and Kolter, 2004) where it also is crucial for the cell-cell contact. This does not account for PAO1, where Psl seems to be the primary structural polysaccharide for biofilm maturity (Colvin et al., 2011). The production of these polysaccharides is highly regulated but the precise role of the different components of the biofilm matrix remains to be determined. Overall, it is thought that Pel and Psl are involved in the initial stages of biofilm formation and that alginate serves as the stress response polysaccharide associated with chronic stages of infection (Schurr, 2013, Ghafoor et al., 2011). The relationship between Pel and Psl is not determined and there is significant strain-to-strain variability in the contribution of Pel and Psl to the mature biofilm structure (Colvin et al., 2012). Dispersal (V) enables the bacteria in a biofilm to spread and colonize new surfaces if it is nutrient advantageous (Sauer et al., 2004). It involves a phenotypic change of the bacteria so that bacteria in the dispersion stage are more similar to planktonic bacteria than to maturation-2 stage bacteria (Sauer et al., 2002). One theory propose that programmed cell death, autolysis and reduced

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synthesis of Psl matrix are used to create a center in the biofilm matrix with planktonic-like-bacteria for dispersal (Ma et al., 2009). Clumping dispersal and surface dispersal have also been suggested as possible dispersal mechanisms (Hall-Stoodley and Stoodley, 2005) but the precise mechanism is unknown in P. aeruginosa. In Actinobacillus actinomycetemcomitans, Dispersin B degrades polyN-acetylglucosamine, a biofilm matrix polysaccharide, and mutant colonies fail to release cells and disperse (Kaplan et al., 2003). It is likely that there is a similar enzyme present in P. aeruginosa but it remains to be discovered.

2.5 Quorum sensing controls biofilm formation The conversion of a planktonic cell into the biofilm mode of growth is a drastic event and involves a coordinated phenotypic change of the bacteria in every stage of biofilm formation (Sauer et al., 2002). The reason for this phenotypic change is still an evolving matter of research but is for example known to be influenced by nutritional changes (Hancock et al., 2011, Shrout et al., 2006, Petrova and Sauer, 2012). In the last decade, the knowledge of this feature of bacterial pathogenesis has been improved. We now consider most bacteria to communicate and react coordinated by making use of autoinducers, a system known as quorum sensing (Kjelleberg and Molin, 2002). In many bacteria quorum sensing plays a role in biofilm establishment, growth and maintenance (Irie and Parsek, 2008). P. aeruginosa are one of the best described bacteria which quorum sensing system is linked to biofilm formation. Two systems have been described in P. aeruginosa: las and rhl. Quorum-sensing systems respond to a class of autoinducers named acyl homoserine lactones (AHLs) (Ng and Bassler, 2009). The AHL autoinducer in las quorum-sensing system is synthesized by LasI and is regulated by LasR, a transcriptional activator protein (Pearson et al., 1994). The AHL autoinducer in rhl quorum sensing system is synthesized by RhlI and is regulated by RhlR (Fuqua et al., 2001). rhl quorum sensing system is furthermore controlled by las quorum sensing system (Latifi et al., 1996, Pesci et al., 1997, Medina et al., 2003). Compared to the P. aeruginosa wild type and frequently used laboratory strain, PAO1, lasI mutants form thin biofilms whereas rhlI is unaffected (Davies et al., 1998). Since then, a number of reports have shown that rhl quorum sensing system affects biofilm formation and it can might be explained by different experimental setups (de Kievit, 2009). In PAO1, both las and rhl are also important for eDNA release to the matrix (Allesen-Holm et al., 2006). Inactivation of las compromise late stages of biofilm formation but not earlier stages. This effect, however, might be influenced by environmental conditions (Sauer et al., 2002, Shrout et al.,

20

2006). Both systems are also required for Type IV pilus-dependent twitching motility (Glessner et al., 1999) and in PA14 they regulate pel expression (Sakuragi and Kolter, 2007).

2.6 Biofilm inhibitors as potential drugs Some of the advantages of biofilms are that they aid to overcome immune system and create resistance towards antibiotics. When the bacteria are organized in a biofilm they are up to 1,000 times less susceptible to antimicrobial agents compared to the planktonic state (Smith, 2005, Olson et al., 2002). For example the hypochlorite resistance increased 600-fold when S. aureus was grown as a biofilm on an abiotic surface (Luppens et al., 2002). A lower metabolic/slow growing rate (persisters), difficult penetration of biofilm matrix, up regulation of efflux pumps and stress response regulons are often mentioned as the reason (Davies, 2003, Lewis, 2010, Costerton et al., 1999, Stewart, 2002, Stewart and Costerton, 2001). The clinical consequences are that biofilm infections often develop into a chronic infection which is difficult to eradicate (Hoiby et al., 2011, Hoiby et al., 2010). Due to the lack of discovery of new and effective antibiotics, and because biofilms are regarded as a determent for chronic infections and decrease of antibiotic susceptibility, many alternative strategies have been focusing on reducing biofilm formation in infections and preventively incorporate antibiotics into in-dwelling medical devices. There are numerous ways to inhibit biofilm formation but when it comes to an infection where the biofilm has already formed, quorum sensing might turn out to be the most strategic advantageous mechanism to inhibit (de Kievit, 2009). Garlic extracts have for example shown inhibitory effect on quorum sensing and have a synergistic effect with tobramycin (Bjarnsholt et al., 2005b, Rasmussen et al., 2005). Other strategies have focused on decreasing the attachment onto in-dwelling medical devices such as catheters. This is attempted primarily by coating catheters with antibiotics such as minocycline, rifampin and Ampicillin (Darouiche et al., 1999, Raad et al., 1997, Liu et al., 2012) and a wide range of other antimicrobials, for example polymeric materials (Hook et al., 2012), benzalkonium chloride (Jaramillo et al., 2012), sodium fluoride and chlorhexidine (Liu et al., 2012), and many more (Smith, 2005).

21

2.7 Urinary Tract infections Urinary tract infections (UTI) occur through the invasion of microorganisms in the urinary tract, most commonly in women. In women they are the most prevalent hospital acquired type of infection accounting for an estimated 25 % - 40 % of all infections and in elderly women, UTIs are the second most prevalent community acquired infection (Matthews and Lancaster, 2011). It is estimated that 40 % – 60 % of women will get one or more UTI during their lifetime (Salvatore et al., 2011). Recurrences are common in young women and are associated with sexual intercourse, use of spermicidal products, having a first UTI at an early age and having a maternal history of UTIs. In hospitals and long-term care facilities the uropathogen is often introduced to the urinary tract through contaminated urinary catheters which increases the risk of developing a UTI significantly. The infecting pathogens usually arise from ascending infection from the urethra to the bladder. The most prevalent uropathogen is E. coli (80-90 %) and in healthy women they are suggested to primarily originate from the gastrointestinal flora. Other uropathogens such as Klebsiella species, Enterococcus species and P. aeruginosa are less common and are reported to be related to the usage of urinary catheters (Matthews and Lancaster, 2011). The establishment and maintenance of the infection are mediated through the formation of a biofilm by the infecting pathogen (Salo et al., 2009), which in many cases creates hard-to-treat infections (Tenke et al., 2006). To initiate infection and oppose the wash out effect uropathogenic E. coli express several virulence factors (Svanborg and Godaly, 1997), but most important is the Dmannose binding by type 1 fimbriae FimH adhesin which mediates attachment to uroepithelial cells and abiotic surfaces (Finer and Landau, 2004, Klemm and Schembri, 2000). Another virulence factor in E. coli is the expression of proteinaceous cell surface filaments, curli fimbriae. It is thought that curli fimbriae are involved in cell-cell and cell-surface interactions and have been linked to catheter associated infections (Hatt and Rather, 2008). K. pneumoniae form a different form of biofilm than E. coli, since it is a urease producing species. Urease gives the ability to the bacteria to form a crystalline biofilm on urinary catheters. In K. pneumoniae there are two types of fimbriae, type-1 and type-3, both types were equally important for biofilm formation in a catheterized bladder model (Stahlhut et al., 2012) but only type-3 fimbriae have shown to be important for biofilm formation in flow chamber (Schroll et al., 2010).

22

Since catheter associated urinary tract infections are quite common, there is a need for modified or coated catheters to lower the occurrence of UTIs. Many strategies have attempted to solve the issue: integrating antibiotics on urinary catheters is a popular strategy, and it has been attempted many times, such as with sparfoxacin (Kowalczuk et al., 2012), ciprofloxacin, norfloxacin, and ofloxacin (Reid et al., 1994), silver (Liedberg and Lundeberg, 1989) and a fish muscle protein,α-tropomyosin (Vejborg and Klemm, 2008). Some have been clinically introduced and have had a positive effect on reducing catheter associated infections but since infections only are being limited researchers are still looking for better alternatives (Johnson et al., 2006).

2.8 Antimicrobial peptides Antimicrobial peptides (AMPs) or host defense peptides are evolutionarily highly conserved components of the innate immune system and are produced by all complex organisms (Ganz, 2003). Their importance in host defense is indicated in plants and insects which live in non-bacteria free environments without the ability to produce lymphocytes and antibodies. In humans and other mammalians, the significance of peptides in host defense are especially demonstrated by the low risk of infection in the cornea of the eye where they serve as a first line of defense like they do throughout the human body, e.g. in epithelia cells of human colon mucosa (Tollin et al., 2003) and at the skin surface due to sweat glands peptide secretion (Schittek et al., 2001). They are also found in large amounts in granulocytes where they are part of degranulation (Ganz, 2003). AMPs are polypeptides of usually 10-50 amino acids of which the majority of them, due to the positively charged amino acids arginine and lysine, are cationic with an overall charge of +2 to +9. Hydrophobic residues contribute to ≥30% of the peptide which gives the peptides an amphipathic nature with the clustering of cationic and hydrophobic amino acids into distinct domains (Hancock and Lehrer, 1998, Zasloff, 2002). Many antimicrobial peptides have a wide range of activities and they often have broad spectrum antimicrobial activity and some even kills multi drug resistant bacteria at low concentrations. They were originally known for their antifungal, antiviral, antiparasitic and antibacterial properties (Jenssen et al., 2006). Most antimicrobial peptides disrupt the bacterial cell wall by forming pores (Figure 3) and therefore many AMPs show highest activity against gram-positive bacteria. Some AMPs target lipid II or other cell wall biosynthetic processes to disturb peptidoglycan synthesis and translocation (Yount and Yeaman, 2013), other targets are DNA, transcription, translation, replication and essential enzymatic activity Figure 3(Brogden, 2005, Sato and Feix, 2006). There is evidence showing that

23

antimicrobial peptides are not only involved in direct antimicrobial action but also serve as immunomodulatory peptides, functioning as chemokines and/or inducing chemokine production, inhibiting LPS induced pro-inflammatory cytokine production, promoting wound healing, and modulating the responses cells of the adaptive immune response (Bowdish et al., 2005, Oppenheim and Yang, 2005).

Figure 3: Three pore forming models that explain the mechanism of action of membrane-active antimicrobial peptides. (A) Carpet model, (B) toroidal pore model & (C) barrel-stave model (Brogden, 2005).

An ongoing discussion is that the activity of antimicrobial peptides at physiological concentrations is too weak to single-handedly function as mono antibiotics and that the immune stimulating activities are of greater importance. Though many antimicrobial peptides have been tested in clinical trials, no antimicrobial drugs have yet been commercially introduced except cyclic polymyxins. But several non-bacteria combatting immunostimulating peptides and many other peptide drugs, which are not related to the immune system, are already an established part of the pharmaceutical industry (Hancock and Sahl, 2006, Sun, 2013). The advantages of using AMPs as antibiotics are their wide spectrum of activity, target selectivity, high efficacy at low concentrations, anti-LPS activity, often synergistic action with classical antibiotics, and low probability for developing resistance. The reason why AMPs often fail clinical trials is their short half-life, which demands higher concentrations in treatment which leads to side effects, such as lysis of anionic red blood cells. The problem can be overcome by redistributing the AMP only at the site of infection. This has led to the idea, that incorporation of AMP in implants

24

could decrease systemic distribution of AMPs and therefore decrease side effects. There are mainly two ways to achieve this: by leach- or release based systems or covalent attachment. Many covalent immobilization strategies have been suggested (Costa et al., 2011) but it is important that every peptide is to be evaluated individually. The covalent immobilization can increase stability of the peptide while being located at the site of interest for a longer period of time while decreasing toxic side effects. Many things can influence the activity: peptide orientation, secondary structural changes and length, flexibility and type of spacer used to link the AMP and substrate (Hilpert et al., 2009). The covalent linking of AMPs to a substrate also varies a lot, from contact lenses (Willcox et al., 2008), different types of resin beads (Bagheri et al., 2009) to titanium surfaces (Gabriel et al., 2006). Leakage system such as a calcium phosphate-coated assay on titanium surfaces have also been conducted with positive results (Kazemzadeh-Narbat et al., 2010).

The increasing problem with antibiotic resistance demands that we engage these biofilm infections from a new perspective. Antibiofilm drugs have a great potential but will possibly show not to be broad spectrum drugs since the molecular composition of the biofilm seems not to be highly conserved. Since an antibiofilm effect is below the killing concentration, resistance to the peptide as an antibiotic could be a minor risk. In contrast, highly conserved AHLs could be a broad-spectrum target (Bassler, 2002). A new approach to study these AMPs is to study their inhibition of biofilms which have been shown to be accomplished by a numerous number of antimicrobial peptides (Batoni et al., 2011, Jorge et al., 2012) and in many biofilm producing organisms which are of high clinical relevance, such as oral streptococci (da Silva et al., 2013), S. aureus (Hochbaum et al., 2011), E. coli (Hou et al., 2010) and P. aeruginosa (Kapoor et al., 2011). The most studied human AMP, LL-37, is from a family of peptides named cathelicidins. It has shown antibiofilm effect against most of the biofilm producing species mentioned above.

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2.9 Human cathelicidin LL-37 In mammals, there are mainly two major groups of antimicrobial peptides, defensins and cathelicidins. Cathelicidins are a heterogeneous group and vary in length from 12-80 amino acids but the majority is 23-37 amino acid residues long which, in contact with a biological membrane, folds into an amphipathic α-helix. Cathelicidin are classified based on the presence of a highly conserved “cathelin” domain (Pro-region) consisting of approximately 100 amino acids, flanked by an N-terminal signal peptide (Pre-region) and the antimicrobial peptide on the C-terminal end which become active upon cleavage of the prepro-peptide (Zanetti et al., 1995, Gennaro and Zanetti, 2000). Cathelicidins were initially discovered in myeloid stem cells of bone marrow (Bagella et al., 1995, Zanetti et al., 1995) and they are most important of all stored in granules of neutrophils (Cowland et al., 1995). Later, observations of constitutive and inducible expression of cathelicidins has been observed in broad range of mammalian organs but one of the biggest focuses of the research has been on the expression of human cathelicidin in epithelial cells (Zanetti, 2005, Kosciuczuk et al., 2012). Cathelicidin mode of actions includes a broad range of properties such as direct antimicrobial activity, LPS binding, chemoattraction of immune cells, stimulating release of histamine from mast cells, immune stimulating and induction of angiogenesis (Bals and Wilson, 2003, Kosciuczuk et al., 2012).

Some mammals generate various cathelicidins (Zanetti, 2004), but in humans only one member has been identified, hCAP-18. The CAMP gene expresses a prepropeptide consisting of a 30 amino acid signal peptide and hCAP-18 which consists of a 103 amino acid cathelin domain and a 37 amino acid C-terminal peptide, LL-37 (Figure 4) (Larrick et al., 1996, Gudmundsson et al., 1996).

Figure 4: The structure of CAMP product. The product consists of a 30 amino acids signal peptide at the N-terminal (red), a 103 amino acid pro-region (blue) and a 37 amino acid antimicrobial at the C-terminal (green).

26

LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) folds up into an amphiphilic ahelical structure (Wang, 2008) and exists in an equilibrium between monomers and oligomers both in solution and in contact with zwitterionic lipids (Oren et al., 1999). Due to its cationicity (+6) and hydrophobicity, it can interact with negatively charged membranes by a suggested ‘carpet-like’ mode of action (Figure 3), explaining the peptide antibacterial properties (Larrick et al., 1995b): it has later been demonstrated that the peptide also has the potential to interact directly with bacterial LPS (Larrick et al., 1995a, Nagaoka et al., 2001). hCAP-18 is expressed in many types of blood cells (e.g., ɣδ T cells, B cells, monocytes, macrophages (Agerberth et al., 2000) and mast cells (Di Nardo et al., 2003)), but is mainly found in large amounts in granules of polymorphonuclear neutrophils (Cowland et al., 1995) where it is kept inactive until cleavage of the pro-protein by proteinase 3, which yields the active peptide LL-37 (Sorensen et al., 2001). Similarly, hCAP-18 is expressed in a variety of epithelial cells in the colon (Hase et al., 2002) and cells of the gastric mucosa (Hase et al., 2003), urinary tract (Chromek et al., 2006), epididymis (Malm et al., 2000), corneal (Gordon et al., 2005) and lungs (Bals et al., 1998), in many cases resulting in secretion of LL-37 to fluids lining these epithelial layers. Additionally, LL37 has also been found in fluids and tissue from bone marrow, testes (Agerberth et al., 1995), seminal plasma (Andersson et al., 2002), wound and blister fluid (Frohm et al., 1996), sweat (Murakami et al., 2002b), skin (Yamasaki et al., 2006) and salivary glands (Murakami et al., 2002a, Davidopoulou et al., 2012), among others. Cleavage of hCAP-18 by proteinase 3 yielding LL-37 is embraced as the most prevalent fate of hCAP-18. However, at vaginal pH hCAP-18 is cleaved by gastricsin to ALL-38 (Sorensen et al., 2003). This is particularly interesting since hCAP-18 expression is found in seminal plasma but not in vaginal fluid. Hence, the seminal plasma-derived hCAP-18 could become cleaved and activated following sexual intercourse. Furthermore LL-37 have also been found to be cleaved into smaller fragments, such as KR-20, KS-30, RK-31, LL-23, KS-27, LL-29 and KS-22, in sweat and skin cells by kallikrein serine proteases, of which many of these fragments have shown increased antimicrobial activity (Yamasaki et al., 2006, Murakami et al., 2004) and decreased immunostimulatory functions (Braff et al., 2005). The expression of LL-37 is thought to mainly be regulated by vitamin D (Wang et al., 2004, Dixon et al., 2012) but the regulation of LL-37 during immune system activation seems to be controlled by a more complex mechanism. LL-37 has been linked to several biological processes and

27

demonstrates importance in fighting infections during inflammation, cell differentiation and postinjury. Consequently, both elevated expression levels of hCAP-18 and increased release of the active peptide LL-37 have been observed in response to microbial infections and various chronic inflammation diseases. Decrease of expression has also been observed but overall, the expression are most often induced during inflammation (Vandamme et al., 2012, Durr et al., 2006).

The antibacterial activities of LL-37 are often reported to be weak (Durr et al., 2006, Turner et al., 1998) which are often explained by the in vivo presence of other host defence peptides which all act in synergy. The antibacterial activity of LL-37 (and other antimicrobial peptides) is also weakened with increasing NaCl concentrations matching physiological conditions (Bals et al., 1998, Turner et al., 1998). Other antimicrobial actions of LL-37 are anti-fungal and anti-virus activity (den Hertog et al., 2005, Lopez-Garcia et al., 2005, Barlow et al., 2011, Yasin et al., 2000, Gordon et al., 2005, Wong et al., 2011). The anti-fungal activity has not been a major focus of research whereas LL-37’s antiviral effect is often debated and similarity with the antibacterial debate LL-37 might exhibit strongest anti-viral activity in combination with other host defense peptides.

Aside from the antimicrobial effects described above, recent data have suggested conflicting roles for LL-37 in tumor development: LL-37 is shown to suppress colon cancer (Ren et al., 2012), support natural killer cells in their antitumor effect (Buchau et al., 2010) but on the flip side, LL-37 have been suggested to assist breast cancer development (Heilborn et al., 2005). LL-37 have also been connected to wound healing and is thought to assist by creating a barrier from infection, modulating wound healing, assist in apoptosis and stimulating wound closure through reepithelialization (Heilborn et al., 2003, Huang et al., 2006, Koczulla et al., 2003).

However, the inducible expression of hCAP-18 during inflammation demonstrates an immense immunological role for LL-37 and in the latest years this has been one of the hottest LL-37 topics. LL-37 modulates the immune system in several ways of which many are still unclear, though most evidence points towards LL-37 as an alarmin. LL-37 are proposed to bind to formyl peptide receptor like 1, causing Ca2+ mobilization and chemoattraction of immune cells such as monocytes, neutrophils and T lymphocytes (De et al., 2000). LL-37 is also chemotactic for mast cells via a suggested Gi protein–phospholipase C signaling pathway (Niyonsaba et al., 2002), which mobilizes

28

the intracellular Ca2+ and induces histamine release (Niyonsaba et al., 2001). In the presence of serum, LL-37 antibacterial and cytotoxic activity is decreased due to a conformational change (Johansson et al., 1998). Furthermore LL-37 exhibits many other immune stimulatory effects such as cytokine release and modulation of adaptive immunity (Vandamme et al., 2012, Wuerth and Hancock, 2011). For an overview of LL-37s effects see Figure 5.

Figure 5: Multifunctional effects of LL-37. LL-37 has antimicrobial properties and can target the microbes directly at the surface (1), inside the host (2) or by binding to LPS (3). Additionally, LL-37 also functions as an immunostimulating peptide, which counter acts and regulates the release of pro-inflammatory cytokines from macrophages (4). LL-37 also stimulates histamine release from mast cells (5), and recruitment of both monocytes and polymorphonuclear leukocytes from the blood (6). The polymorphonuclear leukocytes will also promote phagocytosis of invading bacteria (7). Increased concentrations of LL-37 will also stimulate dendritic cells (8) and indirectly drive antigen processing and expression of co-stimulatory molecules that ultimately stimulates the T-cell population (9). Additionally, it has been demonstrated that LL-37 has an angiogenic effect (10) and also promote wound repair through stimulation of re-epithelialization, fibroblast growth and adherence (11) (Jacobsen and Jenssen, 2012).

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2.10

Antibiofilm activity of LL-37

Although antibacterial activity of LL-37 has been shown in several studies it is still questionable how secreted LL-37 function in vivo. The activity of antimicrobial peptides in environments with high concentrations of NaCl has been center of debate among scientists where many believe that NaCl has a negative effect on antibacterial effect of peptides (Bals et al., 1998). Therefore it is questionable if LL-37 has a significant antibacterial effect in salt containing environments where LL-37 is secreted into. It is argued that the secretion of LL-37 into the sweat and urine could be a way to get rid of the peptide or that LL-37 is further degraded into more active peptides and finally it is also be argued that it functions as a persistent immune stimulator when secreted. Another answer implicates LL-37s effect on reducing attachment and biofilm formation. The first research that showed LL-37 to exhibit an antibiofilm was done on P.aeruginosa biofilms and is relatively new (Overhage et al., 2008) but yet it is well documented, that LL-37 exhibit an antibiofilm effect towards a wide range of bacterial species (Table 2) (Jacobsen and Jenssen, 2012). The most distinctive characteristic is the antibiofilm activity LL-37 shows at sub-MIC concentrations. For example is LL-37 active against Francisella novicida biofilms as low as at 3.8 ng/ml whereas the MIC is 250 µg/ml (Amer et al., 2010) and in P.aeruginosa the inhibition was as low as 0.5 µg/ml which was 128 times lower than their detected MIC value (Overhage et al., 2008). This sub-MIC antibiofilm activity goes again for a wide range of common pathogenic bacteria (Table 2). LL-37 inhibits the initial attachment of the bacteria to abiotic surfaces and inhibits preformed P.aeruginosa biofilms (Hell et al., 2010, Dean et al., 2011b, Dean et al., 2011a, Overhage et al., 2008). The antibiofilm mechanisms of LL-37 are not thoroughly studied but in P.aeruginosa Las and Rhl quorum sensing system are affected by LL-37 which consequently decreases biofilm relevant gene expression. LL-37 also increases the twitching motility in P.aeruginosa which makes the bacteria unable to settle on a surface, but in contrast, LL-37 is not influencing the swimming and swarming in P.aeruginosa (Overhage et al., 2008, Dean et al., 2011b). In E. coli, the decreased attachment can be explained by LL-37's ability to inhibit the curli structure development by preventing the polymerization of the major curli subunit (CsgA) even in low concentrations, and thereby minimize attachment to epithelial cells (Kai-Larsen et al., 2010). LL-37 is also able to bind to cell wall polysaccharides of the fungus Candida albicans which prevents the adherence of the fungus in an in vivo mouse urinary bladders model (Tsai et al., 2011).

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In addition, LL-37 also binds to EPS polysaccharide alginate of P.aeruginosa and the capsular polysaccharide K40 of K. pneumoniae, which consequently increases the MIC of LL-37 and the survivability of the bacteria (Herasimenka et al., 2005, Foschiatti et al., 2009, Benincasa et al., 2009) Table 2: In vitro effect of LL-37 on biofilm formation. The studies represent the percentage inhibition of LL-37 after ≈24h incubation. †Inhibition indicates the percent inhibition of bacterial biofilm formation, compared with untreated controls.

Bacteria strain Inhibition† (%)

LL-37 concentration

Reference μg/ml

μM

S. epidermidis

43

1

0.22

(Hell et al., 2010)

F. novicida

>80

0.2

0.05

(Amer et al., 2010)

S. aureus

>40

10

2.23

(Dean et al., 2011a)

E. coli

80

11.2

2.5

(Kai-Larsen et al., 2010)

P. aeruginosa

40

0.5

0.11

(Overhage et al., 2008)

P. aeruginosa

≈50

1

0.22

(Dean et al., 2011b)

P. aeruginosa

≈35

4.5

1

(de la Fuente-Nunez et al., 2012a)

P. aeruginosa

57

13.5

3

(Mohanty et al., 2012)

The antibiofilm effects of LL-37 are indicated not to be a unique feature of the cathelicidin peptide family. Whereas Indolicidin, a bovine cathelicidin, showed to inhibit P.aeruginosa biofilm formation, CRAMP, a mice cathelicidin, did not affect P.aeruginosa biofilm formation (Overhage et al., 2008, Dean et al., 2011b).

2.11

The potential of LL-37 and 1037 as UTI inhibitors

The study by Kai-Larsen et al. 2010 shows that LL-37 prevents the assembly of curli fimbriae from a UTI isolated E. coli, which indicates that LL-37 antibiofilm properties plays an active preventive role in establishment of infection by minimizing epithelial cell attachment. Another peptide, 1037, has shown great antibiofilm potential, inhibiting P.aeruginosa biofilms with 78 % at ½x MIC (MIC 304 µg/ml) (de la Fuente-Nunez et al., 2012a). The major aim for these experiments is to show that the biofilm formation of three UTI isolates, one K. pneumoniae and two E. coli isolates can be inhibited by LL-37 and peptide 1037. The experiments are initiated by confirming and determining

31

the MIC and biofilm inhibition on P.aeruginosa by a chosen spectrum of LL-37 derivatives and 1037 analogs. The experimental setup is outlined in Figure 6.

Figure 6: Experimental outline of the conducted experiments.

Based on statements and studies from a previous experiment (Pompilio et al., 2011) tetracycline was used as a negative control and tobramycin was used as a positive control for P.aeruginosa biofilm inhibition. All biofilm experiments are performed as abiotic static surface assay in 96-well microtiter plates. Static biofilm systems make it possible to study early stages of biofilm formation, but more importantly, the peptide amount needed for these assays are smaller than in continuous flow cell biofilm assays, and the continuous effect to the peptide in the bacterial environment can be determined.

2.12

Structure activity relationship analysis

To analyze the contribution of the specific residues to the structure and function of 1037, an alanine-scanning technique was used. Alanine-scanning is a commonly used technique to determine epitopes and identify the contribution of specific amino acids in proteins and peptides. The method allows a wild type amino acid to be substituted with alanine, either by mutagenesis or peptide synthesis, to deduce the roles of the wild type side chain (Weiss et al., 2000). The alanine side chain consists of a non-reactive methyl group and is therefore non-bulky. Alanine is chosen due to its 32

simplicity, and because the simple side chain of glycine would lead to a conformational change, thus in most cases alanine substitutions will mimic the secondary structure, but in cases where the secondary structure is affected and need to be conserved, amino acids with larger side chains, such as leucine and valine, can be applied (Morrison and Weiss, 2001). When synthesized, it is easy to detect the critical positions within the peptide sequence in relation to antimicrobial activity by measuring and comparing MIC values. An increase in MIC would mean that the amino acid in question is of major importance for the antimicrobial function of the peptide. This could both be due to a lack in binding contact with peptide and bacteria or the secondary structure could be disrupted. Most amphipathic cationic antimicrobial peptides are linear and rely on the conservation of their linear α-helical structure to disrupt of the bacterial membrane (Powers and Hancock, 2003) and this makes a substitution of hydrophobic side chains difficult to conduct without losing its secondary structure. Alanine scanning of fallaxin have given rise to peptides with higher antimicrobial activity but common for all of them was a higher hemolytic activity (Nielsen et al., 2007) thus, the technique is primarily thought to result in decrease of antimicrobial activities.

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3. Materials and methods 3.1 Bacterial strains A panel of strains was chosen due to their popularity as laboratory strains (Table 3). P. aeruginosa PAO1 are frequently used for peptide experiments and was mainly chosen to replicate previous results. To my knowledge no one has conducted experiments with LL-37 and K. pneumoniae C3091, E. coli 536 and E. coli CFT073 and these strains were all chosen due to their clinical background.

Table 3: Bacterial strains used for the experiments

Name

Strain

Clinical data

Reference

P. aeruginosa

PAO1

Burn-wound infection (Stover et al., 2000)

K. pneumoniae C3091

UTI

(Struve et al., 2009)

E. coli

536

UTI

(Hochhut et al., 2006)

E. coli

CFT073 UTI

(Welch et al., 2002)

3.2 Bacterial growth conditions All strains were grown overnight in lysogeny broth (LB) at 37 °C in a shaking waterbath, diluted in 25 % glycerol and kept at -80 °C until use. Before individual experiments, the bacteria were plated on a LB agar plate which was kept at 5 °C. The day before the experiments, single colonies was picked from the plate and grown overnight in 5 ml of selected media for each experiment in a shaking water bath at 37 °C. For biofilm experiments the following media was used: LB, BM2 (62 mM potassium phosphate buffer (pH 7), 7 mM (NH4)2SO4, 2mM MgSO4, 10 µM FeSO4, 0.4% (wt/vol) glucose and 0.5% (wt/vol) Casamino Acids), M9 minimal (42mM Na2HPO4, 22 mM KH2PO4, 8.5 mM NaCl, NH4Cl 19 mM, 2 mM MgSO4, 0.1 mM CaCl2 and 0.4 % glucose) and DMEM (Cat.-No.: 31885-023 Gibco®).

3.3 Peptides and antibiotics Peptide LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) was made using Fmoc SPPS and purchased from GenScript as a crude product. This peptide was purified with HPLC and purity and identification was determined with HPLC and MS. The amount of HPLC purified

34

peptide was too low and therefore peptide was given as a gift from R.E.W Hancock Laboratory, University of British Columbia. Seven 12 residue LL-37 fragments and a 12 residue fragment with a valine substitution with Cterminal amidation were purchased from GenScript. Peptide 1037 (KRFRIRVRV) and 9 peptides with a alanine substitution in the individual residues (1037 A1 – A9) and 7 other synthetic analogues of 1037 were synthesized with a C-terminal amidation by Solid-phase synthesis using Fmoc chemistry by Håvard Jenssen, Roskilde University. The peptides were stored in autoclaved MilliQ water and stored at -20 °C. The peptides were prepared as two fold dilutions containing 0.01% acetic acid and 0.2% BSA in 10 times higher concentration than desired and stored in polypropylene 96 well microtiter plates and wrapped in parafilm. Tetracycline was dissolved in ethanol and stored at 5 °C, and tobramycin was dissolved in autoclaved MilliQ and stored at 5 °C, both having a stock concentration of 1 mg/ml. They were prepared in 10x test concentration with 0.01% acetic acid and 0.2% BSA in polystyrene 96 well microtiter plates, wrapped in parafilm and stored at 5 °C for maximum a week.

3.4 Purification of LL-37 with HPLC Purchased LL-37 was purified with high performance liquid chromatography (HPLC). 20 mg LL37 crude extract dissolved in 20 % acetonitrile (ACN) was injected into the sample loop. For the reversed-phase chromatography a 201SP™ C18 250x10mm 10um column was used with buffer A, H2O and 0.1 % TFA and buffer B, acetonitrile. The flow rate was 5 ml/min and all products were collected in fractions of 5 ml which were stored at 5 °C before freeze drying.

3.5 Identification of LL-37 with HPLC/MS Purity of the LL-37 HPLC purified product was analyzed with HPLC/MS with a Luna C18(2) column. A small portion of each fraction of interest was dissolved in 23 % acetonitrile. The two solvents were 1 % HCOOH, 94 % H2O and 5 % acetonitrile and methanol. The mass was determined by ESI-MS with a Finnigan™ LTQ™. Due to a systemic breakdown, 1037 alanine scanning peptides was not purified by HPLC and the purity was not determined with HPLC/MS. The mass was determined directly by ESI-MS spray of a peptide concentration of 0.1 mg/ml.

35

The mass weight was identified by using this formula:

Where p is read as mass per charge at the x-axis (m/z), Mw is the mass weight of the peptide and z is the charge at x protons.

3.6 MIC determination The minimum inhibitory concentration, MIC, was determined by broth microdilution (Wiegand et al., 2008). All strains were grown overnight in MH broth (Cat.-No.: 275730 BD Difco™) in a shaking water bath at 37 °C. The culture was diluted 11 times in MH broth and grown exponentially in a shaking water bath at 37 °C until the optical density (600 nm) ≈ 0.400, which are corresponding to 1 x 108 CFU/ml. The bacteria were then diluted 500 times yielding the desired bacterial test concentration 5 x 105 CFU/ml. For verifying this cell number the bacteria were diluted 500 times in 0.9 % NaCl and 100 µl suspension was plated in duplicates. The plates were prepared with 10 µl peptide or antibiotic solution in two fold dilutions containing 0.01% acetic acid and 0.2% BSA in 10 times higher concentration than desired test concentration. The peptides were prepared in range of 2.56 mg/ml to 0.5 mg/ml and the antibiotics were prepared in the range of 50 mg/ml to 0.05 mg/ml. 90 µl bacteria (5 x 105 CFU/ml) were added to each well with a multichannel pipette. Positive controls were 10 µl autoclaved MilliQ water with 0.01% acetic acid and 0.2% BSA and 90 µl bacteria (5 x 105 CFU/ml). Negative controls were 10 µl autoclaved MilliQ water with 0.01% acetic acid and 0.2% BSA and 90 µl medium used for dilutions. All MIC

experiments were conducted in polypropylene 96-well microtiter plates (Cat.-No.: 650201 Greiner Bio-One) and replicated three times. The MIC was defined as the minimal concentration needed to inhibit all visible growth after 48 hours.

3.7 Biofilm experiments Biofilm formation was analyzed in an abiotic static surface assay in polystyrene 96-well microtiter plates (Cat.-No.: 655101 Greiner Bio-One) and for P. aeruginosa in polypropylene 96-well microtiter plates (Cat.-No.: 650201 Greiner Bio-One) (O'Toole et al., 1999, Merritt et al., 2005). Single colonies was inoculated in 5 ml appropriate medium and grown overnight at 37 °C in a shaking water bath. 96-well microtiter plates were prepared with 10 μl peptide/antibiotic solution in 2 fold dilutions in concentrations 10 times higher than desired final concentration. P. aeruginosa 36

biofilm experiments were conducted in LB medium, K. pneumoniae experiments were conducted in M9 and BM2 media, and E. coli biofilm experiments were conducted in DMEM and LB media. The overnight cultures were diluted 100 times and 90 μl of the suspension was distributed in the wells, with positive controls in three wells and negative control with medium only, which data was deducted from all data. The suspension was diluted 105 and 100 μl was plated on LB agar plates to disconfirm contamination. The microtiter plates were closed tightly with parafilm and incubated at 37 °C for ≈24 hours. After ≈24 hours the planktonic bacteria were gently removed, and the remaining were stained with 125 μl, 1 % crystal violet (0.1 % for P. aeruginosa) and incubated for 10 minutes at room temperature. Excess crystal violet was removed and the wells were washed with 200 μl phosphate buffered saline (PBS) 2 times. For dissolvent of attached crystal violet, 200 μl 95 % ethanol was added to each well and incubated for 10 minutes at room temperature. The content was briefly mixed by pipetting, and 125 μl of the solubilized crystal violet was transferred to a separate well in an optically clear flat-bottom 96-well polystyrene plate. The OD595 was measured by a Bio-Tek Synergy HT Microplate Reader in all experiments except for tetracycline and tobramycin experiments where OD600 due to an induction of biofilm formation. The initial attachment was tested for K. pneumoniae and P. aeruginosa as described above but with an incubation time of 2 hours. The effects of LL-37 and 1037 on preformed K. pneumoniae biofilms were analyzed by letting the biofilm form in 24 hours on polystyrene 96-well microtiter plates. After removal of the planktonic bacteria, fresh medium with the desired peptide concentrations were added and incubated 3 hours at 37 °C followed by staining as described above. The effect of LL-37 and 1037 on preformed P. aeruginosa biofilms was analyzed by letting the biofilm form in 24 hours on polypropylene 96-well microtiter plates. After removal of the planktonic bacteria, fresh medium with the desired peptide concentrations were added and incubated 24 or 2 hours at 37 °C followed by staining as described above. All biofilm percentage means with standard deviations can be found in Appendix I.

3.8 Time-kill kinetics Time-kill kinetics was performed in polystyrene 96-well microtiter plates with BM2 medium. The same setup as for the biofilm experiments was used. The suspension was removed from each well at each time points, diluted and plated on LB plates. The experiment was performed as in singles with no replication.

37

3.9 Statistical analysis Two tailed Student's t-test was used for the determination of statistical significance with a threshold for statistical significance at p = 0.01. Statistical analyses were accomplished with GraphPad Prism 5.

38

4. Results 4.1 HPLC purification of LL-37 LL-37 was purchased as an unpurified crude extract and was therefore purified with HPLC, which with the chosen HPLC column, using reverse phase, segregates the injected content according to its hydrophobic/hydrophilic nature. LL-37, dissolved and injected into the system, are in a mobile phase but when the reversed-phase chromatography column is reached, the solution is in a stationary phase. In the column, the content is allowed to run through the column based on its hydrophobic nature and based of a gradient of acetonitrile. The purification of LL-37 with HLPC is plotted on Figure 7. A peptide bond is detected by UV absorption at a wavelength of 214 nm which is represented by the red line and aromatic amino acids are detected by UV absorption at a wavelength of 280 nm which is represented by the blue line. The area under the peaks often can be correlated with purity of the solution. In this case the solution would seem to be very impure but taken in mind that LL-37 often forms polymers (Oren et al., 1999), it might not be the case.

Figure 7: RPC-HPLC of LL-37 with a flow rate of 5 ml/ml in a gradient of ACN. Gradient I: 20 % to 70 % ACN over 20 column volumes (5 ml fractions). Step II: Instant jump to 100 % ACN over 2 column volumes (12 ml fractions). Gradient III: 100 % ACN over 2 column volumes (12 ml fractions). Step IV: 100 % - 20 % ACN. Gradient V: 20 % ACN over 5 column volumes (12 ml fractions). Lines are as followed: UV at 280nm (Blue), UV at 214 nm (Red) and gradient of acetonitrile (Green). Red numbers at xaxis represent the fraction collection tube.

39

4.2 MS identification of peptides HPLC/MS was carried out on HPLC fractions 33, 40, 41, 62 and 69. By using the formula described in chapter of methods and the molecular weight of LL-37 (4493.33 g/mol), the theoretical mass per charge (m/z) was calculated (Table 4). Table 4: Theoretical mass per charge (m/z) of LL-37

z

4

5

6

7

8

m/z 1124.33 899.66 749.88 624.90 562.66

Figure 8 represent the trend of most of the analyzed fractions. All analyzed fractions was found to be very pure with only a single peak, as in Figure 8A. All analyzed fractions except peak 41, which was slight larger in size than LL-37, was found to be a more or less pure and contained LL-37. The chosen example, Figure 8B, differ from the theoretical m/z of LL-37 at z = 4 with 0.06 which is considered satisfactory. Fraction 41 had a difference in m/z of 3.45 and was therefore not considered being LL-37. The LL-37 derived peptides (Table 5, p. 42) were purchased purified and was therefore not MS analyzed. Peptide 1037 and the 1037 single residue substitution peptides (Table 6, p. 44) were confirmed by direct ESI-MS and, due to breakdown of machinery, the purity was not examined.

Figure 8: HPLC/MS of HPLC fraction 40. Figure A shows HPLC identified purity of the fraction. Figure B shows the mass spectrum of LL-37 identified with ESI-MS.

40

4.3 MIC determination of antibiotics, LL-37 and LL-37 12-residue fragments P. aeruginosa strain PAO1 is a popular laboratory strain and is frequently used in MIC determination and biofilm experiments. To verify the functionality of the broth microdilution assay and to compare the antimicrobial activity of peptides and antibiotics with other experiment, PAO1 was chosen as a model organism. Antibiotics, such as tobramycin and tetracycline, have been reported to reduce or induce the biofilm formation, respectively, and they were therefore included as controls (Linares et al., 2006, Pompilio et al., 2011). The MIC of LL-37 was 32 µg/ml for P. aeruginosa and K. pneumoniae and 16 µg/ml for both E. coli strains (Table 5). This corresponds to the MIC for P. aeruginosa which has been described elsewhere (de la Fuente-Nunez et al., 2012a) and that LL-37 usually is more sensitive towards E. coli than P. aeruginosa (De Smet and Contreras, 2005). The LL-37 12-mer fragments were in this project only screened for potential antimicrobial activity against P. aeruginosa and K. pneumoniae. It is notable that the MIC of KR-12 is decreased 8-fold compared to FK-12 which is overlapping with the addition of N-terminal phenylalanine and deletion of the cationic C-terminal arginine. The cationicity of antimicrobial peptides is thought to often be correlated with antimicrobial activity. VQ-12 supports that hypothesis since the substitution of the anionic aspartic acid with a hydrophobic valine, VQ-12V6, increases the antimicrobial activity. The relationship between antimicrobial activity and charge of the antimicrobial peptide is illustrated on Figure 9A, and the relationship between antimicrobial activity and peptide hydrophobicity is illustrated on Figure 9B. The trend line of Figure 9A supports the theory (r2 = 0.5741) whereas the hydrophobicity shows no correlation with antimicrobial activity for LL-37(r2 = 0.0161).

Figure 9: Correlation between the MIC of P. aeruginosa of LL-37 related peptides and A, Charge (r2 = 0.5741), and B, Hydrophobicity (r2 = 0.0161). The MIC values >200 was edited to 200.

41

Table 5: MIC determination of LL-37 native fragments, a synthetic analogue of LL-37, tobramycin and tetracycline. a Generally accepted nomenclature. b Number of the first and last residue in native LL-37 of the derivate. c Single letter amino acid code of the peptide. d MIC determination of the tested antimicrobials with least 3 replicates. e Charge of the peptide at pH 7. f Ratio of hydrophobic/total number of residues (%) calculated with innovagen peptide calculator. g Mean relative hydrophobic moment (mH) calculated with HydroMCalc using Eisenberg scale. Strains were P. aeruginosa PAO1, K. pneumoniae C3091, E. coli 536 and CFT073. Underscore is used to make comparison easier. Not determined MIC is marked ND.

Name

a

Residue

b

Sequence

MIC (µg/ml)d

c

PAO1

C3091

536

CFT073

Chargee

%f

mHg

LL-37

1-37

LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES

32

32

16

16

6

35

0.44

LL-12

1-12

LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES

>200

>200

ND

ND

2

33

0.38

SK-12

9-20

LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES

>200

>200

ND

ND

3

25

0.49

FK-12

17-28

LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES

100

100

ND

ND

3

50

0.68

KR-12

18-29

LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES

12.5

>200

ND

ND

4

41

0.75

VQ-12

21-32

LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES

>200

>200

ND

ND

2

58

0.6

VQ-12V6 21-32V26

LLGDFFRKSKEKIGKEFKRIVQRIKVFLRNLVPRTES

50

>200

ND

ND

3

58

0.47

IK-12

24-35

LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES

>200

>200

ND

ND

2

50

0.43

DF-12

26-37

LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES

>200

>200

ND

ND

0

41

0.4

Tobramycin

0.1

0.2

0.8

0.8

Tetracycline

25

6.2

1.6

1.6

42

4.4 MIC determination of 1037 and synthetic analogues The MIC of 1037 against P. aeruginosa was 16 µg/ml which in comparison to the original publication of 1037 (MIC 304 µg/ml), is extremely low (de la Fuente-Nunez et al., 2012a). Since a high genomic diversity of laboratory P. aeruginosa strains is known to occur (Klockgether et al., 2010), the MIC was replicated on the P. aeruginosa strain from the original experiment and the MIC was confirmed to 16 µg/ml. E. coli strains was more susceptible towards 1037 (MIC 8 µg/ml) which is a trend that goes again in these experiments. To determine the amino acids that are most important for the antimicrobial activity of 1037, several 1037 synthetic analogues was synthesized including 9 alanine single-substitution peptides (Table 6 & Figure 10).

Figure 10: Overview of the MIC of 1037 alanine scanning library (A-D). Letters (x-axis) represent the amino acids of 1037 which have been substituted with alanine at each position, starting with K1. The specific antimicrobial activity (µg/ml) of the individual peptides are plotted on the y-axis. Arrows indicate MIC >256. The MIC was determined for P. aeruginosa (A), E. coli 536 (B), K. pneumoniae (C) and E. coli CFT073 (D).

Both K1 and R2 showed only a slight involvement in determining the MIC. This observation led to the synthesizing of 1037 with 1 and 2 deleted N-terminal residues. Of the 7 C-terminal residues, four were hydrophobic and three of four of these, F3, I5 and V7, were shown to be very important or essential for antimicrobial activity. Of the three arginine residues R6 sustained most antimicrobial activity whereas the MIC of R4 and R8 increased 4-8 folds. For further study and to support some of the hypothesis created by the alanine scan, a new list of peptides was synthesized. Deletion of the first residue (1037 1x) showed an actual decrease of MIC 43

whereas deletion of the first two residues (1037 2x) showed a slight increase. It should be considered that these peptides were not synthesized in parallel with 1037 alanine scanning peptides and that these peptides have not but purified. I suspected that the substitution of the hydrophobic residues led to a destabilization of the secondary structure, and the I5 was substituted with V (1037 V5) or L (1037 L5). These substitutions showed no complete disruption of antimicrobial activity but it was also showed that these amino acids were not ideal replacements. The insertions of a P in a peptide will lead to a break in the amino acids structure. P was substituted for R4 and R6 – P6 completely disrupted the antimicrobial activity of the peptide whereas P4 showed only a slight decrease in antimicrobial activity compared to the respective alanine analog. Table 6: MIC determination of 1037 and synthetic analogues. a Generally accepted nomenclature. b Single letter amino acid code of the peptide. c MIC determination of the tested antimicrobials with least 3 replicates. d Charge of the peptide at pH 7.

e

Ratio of hydrophobic/total number of residues (%) calculated with innovagen peptide calculator. f Mean relative

hydrophobic moment (mH) calculated with HydroMCalc using Eisenberg scale. Strains were P. aeruginosa PAO1, K. pneumoniae C3091, E. coli 536 and CFT073.

Name

a

Sequence

MIC (µg/ml)c

b

Charged %e mHf

PAO1

C3091

536

CFT073

1037

K

R

F

R

I

R

V

R

V

16

16

8

8

5

44

0.3

1037 A1

A

R

F

R

I

R

V

R

V

32

16

32

16

4

55

0.12

1037 A2

K

A

F

R

I

R

V

R

V

16

32

16

32

5

55

0.4

1037 A3

K

R

A

R

I

R

V

R

V

>256

>256

128

256

5

44

0.25

1037 A4

K

R

F

A

I

R

V

R

V

128

128

64

64

4

55

0.33

1037 A5

K

R

F

R

A

R

V

R

V

>256

>256

128

256

5

44

0.36

1037 A6

K

R

F

R

I

A

V

R

V

64

32

16

32

4

55

0.52

1037 A7

K

R

F

R

I

R

A

R

V

256

64

64

64

5

44

0.27

1037 A8

K

R

F

R

I

R

V

A

V

128

64

32

64

4

55

0.15

1037 A9

K

R

F

R

I

R

V

R

A

64

32

16

16

5

44

0.31

R

F

R

I

R

V

R

V

16

16

8

8

4

50

0.17

F

R

I

R

V

R

V

64

128

64

64

3

57

0.33

1037 1x 1037 2x 1037 P6

K

R

F

R

I

P

V

R

V

>256

>256

256

256

4

44

0.48

1037 P4

K

R

F

P

I

R

V

R

V

128

256

128

128

4

44

0.31

1037 V5

K

R

F

R

V

R

V

R

V

64

128

16

16

5

44

0.32

1037 L5

K

R

F

R

L

R

V

R

V

64

32

16

8

5

44

0.32

1037 Reverse

V

R

V

R

I

R

F

R

K

32

32

8

16

5

44

0.3

44

4.5 Effect of LL-37, 1037 and antibiotics on P. aeruginosa biofilms P. aeruginosa formed a strong biofilm with the vast majority growing on the sides of the wells at the air/liquid interface. After staining with crystal violet, the average untreated wells were measured to 2.26 at OD595. It is important to note that crystal violet is not selectively binding to the biofilm but binds to all substances in the well. This assay is therefore a measurement of biomass, including both attached cells and EPS. The presences of EPS also influence the number of bacteria which is able to attach, so a high value corresponds to high level of biofilm formation. LL-37 showed to decrease the amount of P. aeruginosa biofilm by 56 % at MIC (32 µg/ml) and the inhibition was statistically significant down to a concentration of 2 µg/ml (1/16x MIC) where the biofilm was reduced by 31.9 % (Figure 11A). 1037 decreased P. aeruginosa biofilm formation with 57.1 % at MIC (16 µg/ml) and a 77.8 % inhibition at 128 µg/ml. Inhibition of biofilm formation was observed at concentrations as low as 2 µg/ml (1/8 of MIC) but was showed not to have an effect at 4 µg/ml (Figure 11B). Tobramycin did not inhibit P. aeruginosa biofilm formation but was increased by 42.4 % at 2x MIC (Figure 11C). Tetracycline showed no reduction in P. aeruginosa biofilm formation but at ½x MIC, tetracycline increased P. aeruginosa biofilm formation with 34. 6 % (Figure 11D).

Figure 11: Effect of LL-37, 1037, Tobramycin and Tetracycline on P. aeruginosa biofilms (A-D). Inhibition of P. aeruginosa biofilm formation was demonstrated by LL-37 (A) and 1037 (B), while tobramycin (C) and Tetracycline (D) showed no inhibition. The data is expressed as percentage of the positive control with mean ± standard derivation and is the result of at least three independent trials. Statistical significance is indicated with asterisks (* p < 0.01, ** p < 0.001, *** p < 0.0001).

45

For further examination of LL-37 and 1037s antibiofilm effect, the incubation time was changed to two hours to determine a potential correlation with inhibition of initial attachment. The attachment of P. aeruginosa was decreased in the presence of LL-37 by 35.8 % at MIC (32 µg/ml) but at subMIC concentrations the majority of the samples were close to 0 % (Figure 12A). 1037 was more potent and inhibited the initial attachment by 29.5 % at MIC (16 µg/ml) and no attachment was detected at 128 µg/ml.

Figure 12: Effect of LL-37 and 1037 on P. aeruginosa initial attachment (A-B) and on preformed biofilms (C-F). Inhibition of LL-37 (A) and 1037 (B) on P. aeruginosa initial attachment (no replication). LL-37 and 1037 were added to P. aeruginosa biofilms that have been growing for 24h and was incubated for 24h (C+E) or 2h (D+F), done in quadruplets (no replication). Statistical significance is indicated with asterisks (*** p < 0.0001).

46

The effect of LL-37 on preformed P. aeruginosa biofilms was indicated to be statistically significant at 16 µg/ml with a biofilm reduction of 55.6 % (Figure 12C). At 4 µg/ml, the amount of biofilm was reduced 17.2 % and was statistically just above the significance level. The effect after 2 hours of incubation showed a slight increase in biofilm produced, thus LL-37 was indicated not to be fast acting (Figure 12D). 1037 was indicated to not affect preformed biofilms, both at 2h and 24h incubation time (Figure 12E-F). Whereas the first two residues of 1037 were shown to be less important for antimicrobial activity, the alanine scanning library of 1037 revealed that, they were very important for the antibiofilm effect (Table 7). This was indicated to be the case with most of the residues. R6 was indicated to have the least importance in the antibiofilm, since 1037 A6 inhibited P. aeruginosa biofilms with 36.2 % at 8 µg/ml. At some concentrations, the biofilm was even shown to increase the amount of biofilm formed (Table 7). a

Table 7: Effect of 1037 Alanine library against P. aeruginosa biofilm formation. c

Generally accepted nomenclature.

b

Single

d

letter amino acid code of the peptide. MIC against P. aeruginosa. Inhibition of biofilm formation in percentage compared to the positive control at ½ MIC and at 8 µg/ml. + indicates an increase in the biofilm formation. For MIC that exceeds the 256 µg/ml detection limit, the MIC was taken as to 256 µg/ml when calculated ½ MIC. The data is the result of a single experiment with no duplicates.

Biofilm inhibitiond Name

a

Sequence

b

MIC (µg/ml)

c

½ MIC

8 µg/ml

1037

K

R

F

R

I

R

V

R

V

16

46.5

46.5

1037 A1

A

R

F

R

I

R

V

R

V

32

6.2

6.6

1037 A2

K

A

F

R

I

R

V

R

V

16

9.5

9.5

1037 A3

K

R

A

R

I

R

V

R

V

>256

52.4

+21.7

1037 A4

K

R

F

A

I

R

V

R

V

128

+8.2

14.7

1037 A5

K

R

F

R

A

R

V

R

V

>256

54.5

6.2

1037 A6

K

R

F

R

I

A

V

R

V

64

25.7

36.2

1037 A7

K

R

F

R

I

R

A

R

V

256

39.2

20.5

1037 A8

K

R

F

R

I

R

V

A

V

128

20

9.3

1037 A9

K

R

F

R

I

R

V

R

A

64

13.7

16.7

47

4.6 Effect of LL-37 and 1037 on K. pneumoniae biofilms The inhibition of K. pneumoniae biofilms by LL-37 was first assessed in five different media (Figure 13A-E). K. pneumoniae C3091 have previously shown to form a biofilm in M9 minimal medium (Struve et al., 2009), but to my knowledge this medium is not used for peptide experiments. BM-2, TB, MH and LB media have all three been used to show an antibiofilm effect of LL-37 and was therefore found qualified to be included for screening (Overhage et al., 2008, Kai-Larsen et al., 2010, Dean et al., 2011b, Pompilio et al., 2011).

Figure 13: The effect of LL-37 on K. pneumoniae biofilm formation in different media (A-E). The experiments were conducted in round-bottomed polypropylene 96-well microtiter plates as singles with at least three replications and incubated ≈24h. Experiments that were considered as major outliers were removed from the graph. The different media were BM2 (A), M9 (B), LB (C), MH (D) and TB (E).

48

Since polystyrene microtiter plates are charged, they can bind some peptides. Therefore the first assessment of LL-37s antibiofilm activity was carried out in polypropylene microtiter plates. K. pneumoniae formed a biofilm on the bottom of the plate. The result was that much of the bound biofilm was removed by pipetting. The results from Figure 13A-E clearly show a great variance within many of the samples. Some of the results were also deleted due to the positive control was too low and was considered interrupted, which in particular was a big problem with M9 minimal medium. With this taken into account, the subsequent experiments were conducted in flat bottomed polystyrene plates. With the object to analyze the time growth of K. pneumoniae biofilm formation, K. pneumoniae was grown in BM2 and M9 media and analyzed after 2, 4, 6 and 24 hours (Figure 14A-B). BM2 medium produced the highest amount of biofilm after 2 and 24 hours (Figure 14B) but the variation after 24 hours was rather big.

Figure 14: The time growth of K. pneumoniae biofilm formation in M9 (A) and BM2 (B) media. The experiment was run as parallel on the same 96-well microtiter polystyrene plate and shows the amount of biofilm produced by K. pneumoniae at different time points in M9 (A) and BM2 (B) media.

Due to this observation and due to the no-inhibition trend (Figure 13), a single experiment with LL37 and 1037 was conducted in polystyrene plates with BM2 medium to verify this trend. For LL-37 (Figure 15A), the trend seems to be no inhibition at all. The highest concentrations, 128 and 64 µg/ml shows a slight increase, while the two of the lowest concentrations, 2 and 4 µg/ml, show a slight decrease. For 1037 (Figure 15B), the trend seems to be a slight decrease of biofilm at the highest concentrations, but nothing that occur significant.

49

To see if the initial of K. pneumoniae biofilm formation is interrupted, the incubation time was adjusted to two and four hours. K. pneumoniae was significant effected by LL-37 down to 2 µg/ml (Figure 15E), but after four hours, the amount of biofilm was completely restored (Figure 15C). The two hour effect of 1037 was more potent (Figure 15F), and the effect was indicated to last for a longer period of time (Figure 15D).

Figure 15: The effect of LL-37 and 1037 on K. pneumoniae biofilms in 24h (A-B), 4h (C-D) and 2h (E-F). The experiments were conducted in flat-bottomed polypropylene 96-well microtiter plates. For 24h (A-B) and 4h (C-D) samples, no replication was carried out. For 2h incubation (E-F), the data is the representative for 3 individual experiments. Statistical significance is indicated with asterisks (* p < 0.01, ** p < 0.001, *** p < 0.0001).

50

Since LL-37 and 1037 seems to inhibit the biofilm formation at short time of incubation, the effect on preformed K. pneumoniae biofilms was examined with a three hour incubation time, but the result showed a slight increase in biofilm, Figure 16.

Figure 16: Effects of LL-37 and 1037 on preformed K. pneumoniae biofilms. The biofilm was grown for 24h and incubated with peptide and fresh medium for 3h. Experiment was done in quadruplets but with no replication.

Too see if this temporary reduction of attached cells and biofilm produced after 2 hours, a time-kill experiment was conducted (Figure 17). LL-37 was very efficient at 2x and 4xMIC but less efficient at 1xMIC (Figure 17A). After 24 hours, the CFU/ml was restored and was comparable with the positive control. 1037 showed no decrease in CFU/ml at all, not even at 4xMIC (Figure 17B). This seems strange, and it the experiment should be reproduced.

Figure 17: Time-kill kinetics of LL-37 (A) and 1037 (B) on K. pneumoniae. The bacteria were grown in the same condition as for the biofilm experiment. After each time point, the bacteria was isolated from the wells and diluted and plated on LB agar plates.

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4.7 Effect of LL-37 and 1037 on E. coli biofilms E. coli biofilms was observed, as for K. pneumoniae, to grow on the bottom of the microtiter plate. The average growth (OD595) for E. coli strain 536 was 0.696 in LB and 0.435 in DMEM and for E. coli strain CFT073 it was 0.937 in LB and 0.321 in DMEM. By observation, it seemed that the effect by the peptides on the biofilm was the same for the two media. The same hurdle as for K. pneumoniae, where the biofilm growth on the bottom of the plate lead to an assay source of error, was observed for E. coli. The biofilm growing on the bottom made the experiment less reliable and some data was, due to a low positive control, removed. The experiment was conducted in two different media, LB and DMEM, which was combined to present the data (Figure 18). LL-37 was shown to have a statistically significant effect on both E. coli strains (Figure 18A-B). The inhibitory concentration was as low as 2 µg/ml where E. coli strain CFT073 was inhibited by 29.3 %. 1037 was shown to be more efficient versus E. coli strain 536 than CFT073 (Figure 18C-D). For E. coli strain 536 the inhibitory concentration was 2 µg/ml where the inhibition was 26.9 %.

Figure 18: Effects of LL-37 (A-B) and 1037 (C-D) on E. coli biofilm formation. The antibiofilm effect of LL-37 on E. coli strains 536 (A) and CFT073 (B) and 1037 on E. coli strains 536 (C) and CFT073 (D). The data are the result of at least 3 replications in either LB or DMEM media with incubation of 21h. Statistical significance is indicated with asterisks (* p < 0.01, ** p < 0.001, *** p < 0.0001).

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5. Discussion With the lack of discovery and development of antibiotics the last decades, Figure 1, scientists are looking for ways to expand the quantity of anti-infective drugs. This is a challenge that is becoming increasingly crucial for keeping up with the development of resistant pathogenic bacteria. AMPs are by many scientists presented as a future solution to this problem but their clinical potential is often rejected or met with skepticism. The first AMPs were discovered in the 1980ies, including a wide variety of AMPs secreted by frog skin (Zasloff, 1987, Bevins and Zasloff, 1990, Ge et al., 1999). The most notable characteristic of frog skin AMPs is that they are found to be secreted in much larger amounts than in mammalians. Since frogs live in constant contact with bacteria, the secretion of AMPs serve as a strong indication of their immunological importance. Since this observation, Dr. Michael Zasloff has put a lot of effort in optimizing and bringing frog magainins into the pharmaceutical industry and has developed Pexiganan, an AMP which is active against various clinical isolates (Ge et al., 1999). Pexiganan was in the late 1990ies in clinical trials but is currently pending and the project is seeking further funding. Meanwhile other (unrelated) peptides, such as a synthetic analog of indolicidin, MBI 226, and a synthetic analog of an AMP (plectasin) isolated from Pseudoplectania nigrella fungus, NZ2114, have in the latest years caught attention (Sader et al., 2004, Xiong et al., 2011). All three peptides have in common that they have been in phase trials, they are efficient against clinical drug resistant isolates, and interestingly they are all derivatives of original isolated AMPs.

In these experiments LL-37 and 8 LL-37 analogs, seven derivatives and one derivative with a single residue substitution, are screened for P. aeruginosa and K. pneumoniae antimicrobial activity. The antimicrobial activity of LL-37 was weak against P. aeruginosa and K. pneumoniae but good against E. coli (Table 5, p. 42). Recently, other studies have found the LL-37 to be 12.5 µg/ml, 31 µg/ml, 64 µg/ml and 112 µg/ml (25 µM) (Bucki et al., 2008, Overhage et al., 2008, de la FuenteNunez et al., 2012a, Nagant et al., 2012). This indicates that the MIC value should be treated with cautious consideration. The experimental setup can vary depending on laboratory, for example the MIC was 31 µg/ml in BM2 (de la Fuente-Nunez et al., 2012a), 12.5 µg/ml and 112 µg/ml in MH (Bucki et al., 2008, Nagant et al., 2012). Taken in mind that the two studies use P. aeruginosa PAO1, it seems unlikely that the MIC can vary from 12.5 µg/ml to 112 µg/ml. It is possible that the high MIC of LL-37 is the result of the abundant use and widespread of P. aeruginosa PAO1 which

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is causing an ongoing microevolution of genotype and phenotype (Klockgether et al., 2010). The experiment which determinates the MIC of LL-37 to 112 µg/ml should be treated with caution since all their tested peptides show decreased antimicrobial activities (Nagant et al., 2012). It is likely that the MIC values for LL-37 shown here are more correct, since they are in range with the previous discussed values. But it is important to notice that the MIC value is not a static measurement. For example did I observe some outliers in my experiments, and one replicate showed an MIC at 64 µg/ml. My results support the conception of AMPs as cooperative components of the innate host defense whose effects are additive and synergistic, resulting in an efficient defense against intruding microorganisms. These additive effects are best illustrated in the saliva of the oral cavity where 45 AMPs, such as defensins, histatins and LL-37 have been identified, whereas some are present in low concentrations (Fabian et al., 2012, Gorr, 2012).

Compared to LL-37, KR-12 (KRIVQRIKDFLR) was the only derivative which showed an increased antimicrobial activity, but only for P. aeruginosa (12.5 µg/ml) and not for K. pneumoniae (>200 µg/ml) (Table 5, p. 42). Compared to FK-12 (FKRIVQRIKDFL), which is overlapping with only one N-terminal residue, the MIC of KR-12 is decreased 8-fold. This argues for residue R29 being important for antimicrobial activity and F17 less important. The improvement of antimicrobial activity should be seen in contrast with KR-12 having a molecular weight more than half as low as LL-37, and when converted into molar concentration, the MICs are almost the same, 8 µM for KR12 and 7.1 µM for LL-37. It also shows the limitations of the micro-broth dilution method where the test concentrations are doubled – theoretically, LL-37 could have an MIC against P. aeruginosa at 17 µg/ml in contrast to the observed 32 µg/ml. VQ-12 (VQRIKDFLRNLV) is overlapping KR-12 C-terminal wise and the antimicrobial activity is completely lost (MIC >200). The three amino acids, KRI18-20, appear to be more important for antimicrobial activity than previously discussed R29. These results are in agreement with novel structural experiments of LL-37 derivative GF-17 ((G)FKRIVQRIKDFLRNLV) and GE-18 ((G)EFKRIVQRIKDFLRNLV) which show that K18, R19, and R29 were important for antimicrobial activity (Wang et al., 2012). The results from Table 5 show that fragments without K18, R19, and R29 lose antimicrobial activity. In the study by Wang et al, 2012, these residues were by alanine scanning not indicated to be the most important residues, but only doubled the MIC. R23 was most important in killing S. aureus whereas R23 and K25 were more important in killing E. coli. K18, R19,

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and R29 were all shown to be on the hydrophilic surface of the peptide, and it is possible that the deletion of these residues could alter the structure of the peptides tested in these experiments. The observed importance of R29 to LL-37s killing mechanism was supported by another study which identified KR-12 (KRIVQRIKDFLR) as the smallest LL-37 fragment with maintained antimicrobial activity (Wang, 2008). KR-10 (KRIVQRIKDF), KR-11 (KRIVQRIKDFL) and RI-10 (RIVQRIKDFL) showed no or little antimicrobial activity, which supports our observations with FK-12 (lacking R29) and VQ-12 (lacking KRI18-20). These results also make sense in contests to naturally found derivatives of LL-37, such as KR-20 (KRIVQRIKDFLRNLVPRTES) which is processed after secretion onto the skin surface and show higher antimicrobial activity than LL-37 (Murakami et al., 2004). These results also indicate that D26 is less important for antimicrobial activity since VQ-12V6 (VQRIKVFLRNLV) has >4 times lower MIC for P. aeruginosa. This is likely to be because VQ-12 (VQRIKDFLRNLV) is less cationic and a small fragment of LL-37 and replacement of anionic D26 increases the cationicity, thus increasing antimicrobial activity. Since the activity of many AMPs is based on their amphipathic nature, correlations between cationicity and hydrophobicity and antimicrobial activity have been suggested (Hilpert et al., 2009). The measured MICs (Table 5) are mostly very high, based on very few peptides and it is therefore difficult to make anything but weak indications based on these results. This taken in mind, the correlation between cationicity and MIC for P. aeruginosa cannot be completely denied (r2 = 0.5741), and in contrast, the correlation between hydrophobicity, the mean relative hydrophobic moment and the tested LL-37 derivatives are non-exciting (r2 = 0.0161) (Eisenberg et al., 1982), see Figure 9A-B and Table 5.

The MIC of the 9-residue peptide, 1037, was determined to 16 µg/ml, which is in striking contrast to the originally determined MIC value at 304 µg/ml. As mentioned above, the MIC at 16 µg/ml was confirmed using the same strains as in the original experiment (de la Fuente-Nunez et al., 2012a). Their study was based on a screening of a peptide library which originated with the consensus peptide, FRIRVRV. 1037 (KRFRIRVRV) was by far the worst peptide, but interestingly, peptide 1029 (KQFRIRVRV) had a MIC of 10 µg/ml, peptide HH10 (KRFRIRVAVRRA) had a MIC of 0.8 µg/ml and HH15 (KRFRIRVRVIRK) had a MIC of 12 µg/ml. 1037 and 1029 differ in sequence only in R2 which would mean that Q2 of 1029 was of crucial importance to antimicrobial activity. In contrast it was shown that HH15 (KRFRIRVRVIRK), which was 1037 with the Nterminal addition of IRK, and the KR sequences is thereby indicated not to negatively affect

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antimicrobial activity. The only plausible reasons for this observation could be that the peptides coincidently have antimicrobial activity by two difference mechanisms, or that this peptide is structural dependent and that 1037 is more affected by the BM2 medium, though it seems very unlikely. This study has already shown that peptides with a single overlapping residue can have completely different antimicrobial activities (Table 5), and it needs to be taken into consideration that the original study determined the MIC of all peptides in BM2 medium. Even this taken into account, it is unlikely that only one peptide of a set of related peptides shows strong media dependence. The alanine scanning of 1037 shows that K1 and R2 residues of 1037 are of minor importance, and even with the removal of K1, 1037 1x (RFRIRVRV), the antimicrobial activity was preserved, and this further questions the previous results, since R2 show not to decrease antimicrobial activity. The only apparent explanation to these crucial differences is that, one of the studies has weighted the peptide wrongfully. Their studies show that 1037 decrease P. aeruginosa biofilm formation with 78 % at 152 µg/ml, and the peptide was identified with the highest biofilm inhibition at their stated ½x MIC. This is exactly the same inhibition observed in this study (Figure 11). Thus it is most likely that the original study of 1037 wrongfully determined the MIC of 1037 and that the biofilm inhibition potential of 1037 at ½x MIC is worse than originally measured. The results from Figure 10 and Table 6 show that the hydrophobic residues F3, I5 and V7 are crucial for antimicrobial activity of 1037 and that the hydrophobic V9 is of less importance. This could be because that 1037 depends on its secondary structure, and α-helical peptides are often helical in the center and not organized in the ends, which in this case probably would mean a single helical turn in the center. The positively charged residues, K1, R2, R4, R6 and R8 are not affected as much as the hydrophobic residues when replaced with alanine. The decrease in antimicrobial activity can be explained by a decreased electrostatic attraction between the positively charged peptides and negatively charged bacterial membrane which contains anionic molecules such as LPS in Gram-negative bacteria, and teichoic acids in Gram-positive bacteria (Powers and Hancock, 2003). But since the cationicity is high for 1037, it is more likely to be explained by loss of structure by making the peptide structure unstable. An insertion of a proline will make a break and completely disrupt peptide structure. The MIC of 1037 A4 and 1037 A6 is increased when R4 and R6 are replaced with proline, with the R6 being

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affected most. This argues for the structure being important for the peptide function and for a structure, which is not highly dependent on hydrophilic residues. The antimicrobial function was almost restored when I5 was replaced with leucine and valine. The hypothetical helical wheel (Figure 19) shows that the hydrophobic residues cluster and that the 1037 peptide forms into an amphipathic nature. Often smaller hydrophobic amino acids are expected to adopt an α-helical conformation with 3.6 amino acids pr. turn, and larger residues such as phenylalanine, isoleucine and valine can adopt both β-sheet and α-helical conformations (Munoz and Serrano, 1994). Even though 1037 consist of large hydrophobic residues that often adopts a βsheet conformation, it is unlikely that 1037 forms a β-sheet since β-sheets form adjacent hydrogen bonds and would essentially need to make a β-turn, which often requires a proline or glycine. However, since a related peptide from the same peptide library, 1018 (VRLIVAVRIWRR), was indicated to form an α-helical conformation (Wieczorek et al., 2010), I suggest that 1037 is also has the ability to form an α-helix.

Figure 19: Helical wheel diagram for 1037. Orange circles mark hydrophobic residues and green circles mark hydrophilic residues. Constructed by using the helical wheel plotting program from http://www-nmr.cabm.rutgers.edu.

The formation of biofilms has a huge clinical impact on the medical capability to treat infections. A biofilm protects the bacteria from polymorphonuclear leukocytes, adds a protective layer that restricts the access of antibiotics and up-regulates resistance genes (Hoiby et al., 2010, Whiteley et al., 2001). More importantly, cells deep within a biofilm are dormant, and since many antibiotics only are able to target metabolic active cells, these cells are called persisters. Consequently, cells living in a biofilm need a higher antibiotic dosage to be eradicated (Hoiby et al., 2011).

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The argument to study the antibiotics tobramycin and tetracycline, were based on results from an independent study (Pompilio et al., 2011), which showed tobramycin to be active against P. aeruginosa and S. aureus biofilms at ½x MIC. In this study, tobramycin and tetracycline were not observed to reduce P. aeruginosa biofilms, in contrast the biofilm formation was induced by 42.4 % at 2x MIC tobramycin and 34 % by tetracycline at ½x MIC (Figure 11C-D). In the biofilm experiments a higher bacteria load and different media were used compared to the MIC experiments, which make it possible to study the antibiotics and peptides in concentrations above MIC. Tetracycline is a bacteriostatic drug and a gradient of pellet size indicated that the samples differed in cell number, but there was not, however, conducted a time-kill kinetics experiment to verify inhibition of cell growth. It is evident that different strains respond differently to the subinhibitory concentrations of tetracycline and tobramycin. In a study, only half of the P. aeruginosa clinical isolates from cystic fibrosis patients showed sub-MIC inducible biofilm formation by tobramycin (Elliott et al., 2010). In an environmental P. aeruginosa strain, RhlI/R quorum sensing system was inhibited by tobramycin (Babic et al., 2010) and with P. aeruginosa PAO1, tobramycin induced biofilm formation by inducing aminoglycoside response regulator gene (arr) gene which effects cell adhesion (Hoffman 2005). Tetracycline activates rhlA and lasB quorum sensing in P. aeruginosa at subinhibitory concentrations (Liang et al., 2008) and at sub-MIC levels, β-Lactam antibiotics induce S. aureus biofilm formation (Kaplan et al., 2012).

For LL-37 P. aeruginosa biofilms were inhibited 58.4 % at ½x MIC (16 µg/ml) and statistically significant down to 2 µg/ml where the biofilm formation was reduced 32.1 % (Figure 11A). These results are comparable with the previous determined antibiofilm activity of LL-37 (Table 2, p. 31). There was no effect of LL-37 on P. aeruginosa initial attachment below MIC (32 µg/ml) (Figure 12A). The effect of LL-37 on pregrown biofilms at 16 µg/ml was 55.6 % inhibition (Figure 12C). Previous studies have shown inhibition of pregrown biofilms down to concentration of 4 µg/ml, but these experiments were performed in flow-cells (Overhage et al., 2008). Pregrown biofilms were shown not to be affected by the presence of LL-37 after 2 hours of incubation (Figure 12D). This indicates that LL-37 does not affect P. aeruginosa biofilms in the short term. In contrast a study shows that LL-37 inhibited the 1 hour attachment and connected it to the LL-37 mediated induction of twitching motility (Overhage et al., 2008).

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1037 have previously been shown to inhibit P. aeruginosa PAO1 and PA14, Burkholderia cenocepacia and Listeria monocytogenes biofilms by inhibiting the flagella-dependent swarming motility and the twitching motility (de la Fuente-Nunez et al., 2012a). At concentrations 10 µg/ml, 15 µg/ml and 25 µg/ml, P. aeruginosa PAO1 was reduced by ≈ 60 % and 78 % at 152 µg/ml. These results were somewhat confirmed in this study. At concentration 8 µg/ml and 16 µg/ml the amount of biofilm was reduced by 46.5 % and 57.1 %, respectively, and the inhibition was even greater at 32 µg/ml and 64 µg/ml (Figure 11B). The initial attachment was indicated not to be inhibited to the same extend and it was indicated that 1037 had no effect on pregrown biofilms at 2h and 24h after (Figure 12BDF). All alanine scanning peptides showed less antibiofilm potency at 8 µg/ml. The cationic residues were shown to be of less importance and the hydrophobic residues were of greater importance in antimicrobial activity (Figure 10A-D). In contrast there was no similar trend for the antibiofilm activity (Table 7).

Many antimicrobial peptides and proteins are secreted from urinary tract epithelial cells producing an antibacterial shield of the human urinary tract (Zasloff, 2007, Spencer et al., 2013). LL-37 is constituently expressed in urinary tract epithelial cells and can be detected in urine with median concentration at 0.3 ng/ml but the level is increased 8 times in children with pyelonephritis or cystitis (Chromek et al., 2006). Nevertheless, it is thought that the concentration of LL-37 and other AMPs is significantly higher in the fluid layer which is in direct contact with the luminal surface. The rapid increase of LL-37 mRNA expression after E. coli exposure suggests that this peptide is highly regulated by bacteria (Chromek et al., 2006). The same study also showed that CRAMP-deficient mice have more bacterial bladder attachment.

The study of K. pneumoniae biofilms were shown to be highly affected by the assay itself. In polypropylene plates, the biofilm was very unstable and caused high standard deviations (Figure 13A-E). This consequently changed the setup of the assay, and a polystyrene microtiter plate had to be employed instead. These plates are not optimal for studying cationic AMPs, since polystyrene and tissue-culture treated plates are negatively charged and can be a deciding determinant on antimicrobial assays (Wiegand et al., 2008). Another affected deciding determinant is the medium used for the experiments. Previously K. pneumoniae biofilms were observed to grow well in M9 medium (Struve et al., 2009) but this medium is not popular for peptide effect determination. Since

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BM2 have been used to identify LL-37 and 1037 inhibition of P. aeruginosa biofilms (de la FuenteNunez et al., 2012a), and since biofilms were observed to grow faster (Figure 14A-B), this medium was chosen for further experiments. In polystyrene microtiter plates, the 24 hour inhibition of 1037 and LL-37 on K. pneumoniae biofilms was initially denied (Figure 15A-B). LL-37 was indicated to moderately inhibit the biofilm at 2 µg/ml and 4 µg/ml but remarkably, the biofilm formation was increased with the concentration of LL-37. 1037 showed low or no inhibition of the biofilm formation. In relation to the polypropylene experiment, there might be an effect of LL-37 on K. pneumoniae biofilms (Figure 13B), but this trend was not confirmed in polystyrene plates. Since K. pneumoniae C3091 is an UTI isolate, and taken in mind that LL-37 is expressed in the urinary tract, it is possible that the cells have developed resistance towards AMPs. Consequently, these experiments focused on the 2 and 4 hour effect on the initial attachment and biofilm formation (Figure 15C-F). Both LL37 and 1037 strongly inhibited the initial attachment, both as low as 2 µg/ml (Figure 15E-F). This effect was somewhat neutralized for LL-37 after 4 hours at low concentrations (Figure 15C), but it was maintained for 1037 (Figure 15D). This indicated that these peptides could be efficient against K. pneumoniae, but only at the initial stages of infection. In contrast, there was no effect when the biofilm were pregrown and incubated for 3 hours with LL-37 or 1037 (Figure 16). It was hypothesized that these AMPs would have an effect on the cell growth, even close to “MIC”. Since the medium and bacterial load were different, the inhibition on cell growth were assessed over time at 1x, 2x and 4x MIC (Figure 17A-B). LL-37 was shown to initially inhibit the cell growth, but after 24 hours the number of cells was restored (Figure 17A). 1037 showed surprisingly no inhibition of planktonic growth at all, even at 4x MIC (Figure 17B). This is truly hard to believe, but it should be taken into account, that this result was not replicated and needs to be replicated for further analysis. It might be due to the peptide being strongly influenced by the salt content of the medium, which could explain the MIC of 304 µg/ml from the previous mentioned experiment (de la Fuente-Nunez et al., 2012a), but it seems unlikely.

LL-37 slightly inhibited E. coli biofilms (Figure 18A-B). This effect was dose dependent for strain 536 but seemed random for strain CFT073 biofilms. 1037 was more effective than LL-37 (Figure 18C-D), but only against 536 biofilms. Another study has shown an 80 % inhibition at 11.2 µg/ml on an E. coli isolate from a child with pyelonephritis (Kai-Larsen et al., 2010). This inhibition was in fact not found to be the case with E.

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coli 536 and CFT073. LL-37 could potentially be almost good against E. coli biofilm, but in these experiments the standard deviations were too big, and the biofilm inhibition trend was often not comparable between the experiments.

The biofilm results expose the inadequacy of this type of experiment. Evidently, the results from this type of experiment are reflected by the type of abiotic material used for bacterial attachment and peptide disablement, the medium, bacterial load and an essential thing such as pipetting. The pipetting for these experiments makes up for the biggest source of error, since it only took little force to the well, before the biofilm fell off. My biggest mistake for these experiments was that I initially washed the biofilms too much which consequently removed most of the biofilm from the wells. As a result, the protocol for these biofilm experiments was changed so the washing procedure became as simple as possible. The succeeding experiments (the results presented) still showed a big variation. Most important was the positive control, since bad pipetting of the positive control would affect the experiment as a whole. Therefore most experiments were performed with three positive controls, but mistakenly no replica of the test wells. This is something I would improve if the experiments were to be reproduced. The focus should only be on sub-MIC concentrations, maybe even as low as ¼ MIC, since the peptide should not affect the reproduction of the cells. On the other hand, another study has shown big variations in an in vivo attachment assay indicating that the standard deviations in these experiments are common (Tsai et al., 2011).

These results do not strongly support an antibiofilm mode of action by LL-37 and 1037 against the tested strains. All three strains are clinical isolates and it is possible that they have developed resistance towards LL-37 and other AMPs since they are required to tolerate exposure antimicrobial peptides at the site of infection. The MIC of E. coli strains correlates with more invasive infection of the upper urinary tract, pyelonephritis, compared to infection of the lower urinary tract, cystitis (Chromek et al., 2006). Resistance of the tested strains towards LL-37 and 1037 seems unlikely since the MICs are relatively low (Table 5 & Table 6). Contrarily, if LL-37 was proven to efficiently fight the bacteria in vitro, it could be the result of the immune system suffering from local stagnation as a result of local temperature decrease or decreased expression of LL-37, which in fact has been linked to some types of infections (Vandamme et al., 2012).

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To sum up, LL-37 significantly inhibits E. coli CFT073 biofilms down to a concentration of 2 µg/ml and E. coli 536 at 32 µg/ml. 1037 inhibits E. coli 536 biofilms at 2 µg/ml but does not affect E. coli CFT073 at any concentration. K. pneumoniae C3091 was inhibited at 2 µg/ml by LL-37 and 1037 after 2 hours but this effect could for LL-37 be explained by a reduced cell number. No timekill kinetics experiments were conducted on E. coli but the effect of LL-37 and 1037 on cell replication at 2 µg/ml after 24 hours would most likely be low and it cannot explain the reduced biofilm production. As for P. aeruginosa, it could be strategically clever to target quorum sensing in E. coli and K. pneumoniae but those species have been observed to secrete low or no AHLs when forming a biofilm on indwelling urethral catheters (Stickler et al., 1998) Another effective strategy would be to block the adhesion to the epithelial surface or the indwelling catheter (Klemm et al., 2010), by blocking the mannose binding pocket of FimH to prevent adhesion of type 1 fimbriae (Chen et al., 2009, Wang et al., 2010) or by blocking the type 1 fimbriae. The curli fimbriae major subunit (CsgA), have previously been shown to be targeted by LL-37 but it is likely that LL-37 and 1037 have several targets (Kai-Larsen et al., 2010). The essential question to answer is how big the therapeutically potential of AMPs, such as LL-37 and 1037 are. This question encompasses important factors such as resistance, resistance development, pharmacokinetics and pharmacodynamics.

The biofilm results presented here also show that the AMPs differ in level of activity when tested on different bacterial species and strains. The elementary component of the bacterial cytoplasmic membrane is the amphipathic phospholipid bilayer which provides a target for the AMP. The net charge of the bacterial membrane is based upon its composition and architecture which differ among bacteria species. A well-known resistance strategy is to alter the bilayer chemically, for example altering Lipid A of LPS, which makes the membrane less anionic and therefore the peptide less likely to interact with the membrane (Yeaman and Yount, 2003, Gutsmann et al., 2005). Other resistance strategies have had direct effect on peptides from this study. For example is the Slayer of Caulobacter crescentus providing a barrier from LL-37 and 1037 which increases the MIC (de la Fuente-Nunez et al., 2012b). In K. pneumoniae, LL-37 is specifically bound to the capsular K40 polymer and EPS polysaccharide alginate of P.aeruginosa (Herasimenka et al., 2005, Foschiatti et al., 2009, Benincasa et al., 2009). This is an example of the fact that peptides, such as LL-37, needs selectivity for their specific target. Since strong biofilm forming bacteria will secrete a

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lot of polysaccharides, it is not very convenient to target that part of the biofilm. The peptide concentration is stagnant or decreasing and cannot prevent the biofilm to form at increasing polysaccharide secretion. The interaction with polysaccharides could explain LL-37’s weak activity towards multidrug resistant clinical strains that produce a strong biofilm (Pompilio et al., 2011). But the most crucial factor is whether the peptide is stable when administered, considering that the peptide stability is challenges both by the host and pathogen responses. For example, OmpT, an outer membrane protease of E. coli , cleaves LL-37 between R7-K8 and K18-R19, which according to my results in Table 5 would inactivate the peptide (Thomassin et al., 2012). The expression levels of OmpT were furthermore shown to be high in some E. coli strains and low in others. Also human host proteases have been mentioned to cleave LL-37, for example in the skin, but it is not known whether this is a type of inactivation and if this type of bacteria-peptide dynamic taking place anywhere else in the human body. In the liver, many AMPs would also be neutralized by proteases, especially peptides containing L-amino acids. Another downside is that many AMPs have been reported to be hemolytic. This problem arises from the negative charge of the red blood cells of which positively charged amino acids are likely to interact with and consequently lyse. Therefore many scientific experiments determine the hemolytic activity of AMPs to evaluate the therapeutic potential. However, this assay is misleading since peptides can bind to many components of the blood. For example, LL-37 have shown not to be cytotoxic to red blood cells when tested in serum due to the binding of lipoproteins, but this consequently also disabled the antibacterial activity of LL-37 (Johansson et al., 1998, Larrick et al., 1995a). It is also convenient that lysed LL-37 yielding KR-20 show no hemolytic activity, indicating that KR-12 would be less toxic (Murakami et al., 2004).

On the contrary AMPs have many advantages that antibiotics often lack. They can be used as single antibiotics or in combination with other antibiotics. Evidently it would be effective to combine treatments with antibiofilm and conventional drugs. Studies on P. aeruginosa have shown promising results for the future. Inhibiting of las quorum sensing would be the most efficient way to decrease the biofilm formation since las quorum sensing controls the rhl system (Latifi et al., 1996, Pesci et al., 1997, Medina et al., 2003). P. aeruginosa tolerance to tobramycin, hydrogen peroxide and polymorphonuclear leukocytes is quorum sensing dependent (Bjarnsholt et al., 2005a), and when quorum sensing is inhibited in P. aeruginosa, tobramycin are more efficient (Christensen et al., 2012, Brackman et al., 2011).

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AMPs could ideally also function as solely administered peptide antibiotics and especially against biofilm infections because they are often not dependent on the cells being metabolic active, which in contrast many conventional antibiotics are (Batoni et al., 2011). LL-37 has for example shown to effectively kill MRSA at low concentrations (Larrick et al., 1995b, De Smet and Contreras, 2005, Turner et al., 1998) and other AMPs have shown to have an synergistic effect with antibiotics on MRSA (Mataraci and Dosler, 2012) . The biggest challenge blocking AMPs for therapeutically use is the administration challenges mentioned above. Basically, three ways to overcome these challenges have been suggested. Coating of indwelling catheters with peptides, topical administration of the AMP or chemically modifying the AMP to increase the half-life while maintaining antibacterial activity. Peptides such as Pexiganan and MBI 226, which was thought to prevent catheter infections, (Sader et al., 2004, Ge et al., 1999) were/are both drug candidates for topical deployment, but they have both failed FDA approval due to various problems with the experimental setup. Topical administration in the urinary tract would be rather inconvenient and impossible in many complicated infections but prevention of attachment would be more evident.

Since most UTI

strains form a biofilm (Subramanian et al., 2012), it would be most strategic to target the adherence to the indwelling catheter and the following biofilm formation. This also solves the problem with per oral and intravenous absorption and turnover. An approach to optimize peptide antibiotics so it can be administered orally or intravenously is to alter the amino acids chemically so that proteases or gastric acid in the stomach cannot neutralize the peptide. This could be altered by using D-amino acids to target the biofilm formation. This has in fact already been shown in a study that used D-amino acids in LL-37 to maintain the antibacterial potential while being stable to trypsin (Dean et al., 2011b).

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6. Conclusion The results show that both peptide LL-37 and 1037 kill clinical strains isolated from UTI. The low antimicrobial activity of KR-12 for P. aeruginosa was not observed for K. pneumoniae. The activity study of 1037 is not completely consistent the theoretical expectation of an optimal αhelix, but since the peptide is too small to form a β-strands, I suggest that 1037 form in a helical matter. Both tobramycin and tetracycline showed to induce P. aeruginosa biofilm formation close to MIC. LL-37 and 1037 were both active against P. aeruginosa biofilms at 2 µg/ml inhibiting the biofilm formation 21.9 % and 28.2 %, respectively. 1037 showed no effect on against pregrown P. aeruginosa biofilms, and for LL-37, the effect was shown not to occur in the first two hours of incubation. LL-37 and 1037 showed only to inhibit K. pneumoniae biofilms after two hours of incubation and for LL-37, the decrease of biofilm was indicated to be due to less bacterial growth. Interestingly, 1037 showed not to inhibit K. pneumoniae growth at 4x MIC. The effects on E. coli strains were inconsistent. LL-37 was effective against E. coli CFT073 at 2 µg/ml inhibiting the biofilm formation 29.3 %, and 1037 was effective against E. coli 536 at 2 µg/ml inhibiting the biofilm formation 26.9 %. The experiments indicate that LL-37 and 1037 are active against several strains isolated from urinary tract.

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7. Future perspectives There are some challenges in developing AMPs as future antibiotics that need to be addressed. The poor bioavailability when a peptide drug is administered orally limits the commercial applications. In a chemically optimized peptide, the advancement away from the originally structure might increase with more chemically adjustments and as discussed above, the structure is important for maintaining the antimicrobial effect of AMPs. In 2012, Pergamum, a biopharmaceutical company, began phase I/II trials of LL-37 for the treatment of patients with hard-to-heal venous leg ulcers. This brings hope to the possibility that LL-37 at some point can be exploited for its antimicrobial effects. Further analysis of LL-37 could show that the KR-12 fragment of LL-37 could be more effective than LL-37 while lowering the production cost.

Regarding the prevention of bacterial attachment in the urinary tract, screening more strains could reveal if the isolates have developed a tolerance threshold to overcome the effect of LL-37. It could in particular be interesting to measure the content of LL-37 fragments in the urine of infected individuals. Coating of urinary tract catheters could ideally prevent a number of UTIs but it would be too optimistic to expect all catheter associated infections to be prevented. The cost of coating indwelling catheters with peptides would also be high and complicated. If proven efficient, the usage could be focused on individuals in the risk group. Further studies could be applying circular dichroism to determine whether 1037 forms in an αhelical matter or as a random coil. The potential of 1037 as an antibiotic could be even better than LL-37, but future studies should determine whether 1037 is able to inhibit multi drug resistant strains.

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Appendix I Below, the plotted data from the biofilm experiments is listed.

Effect of LL-37, 1037 and antibiotics on P. aeruginosa biofilms Table 8: Effect of LL-37 on P. aeruginosa biofilms. The mean is given as the percentage of untreated samples with standard deviation (SD) and number of replicates (N).

Concentration Mean (%) (µg/ml)

SD

N

0

100.00

16.65

15

0.5

110.60

13.26

2

1

78.14

23.53

5

2

67.84

24.37

5

4

53.73

23.00

5

8

52.69

9.01

5

16

41.64

25.26

4

32

44.08

7.00

4

64

36.39

27.91

4

128

51.94

31.33

3

Table 9: Effect of 1037 on P. aeruginosa biofilms. The mean is given as the percentage of untreated samples with standard deviation (SD) and number of replicates (N).

Concentration Mean (%) (µg/ml)

SD

N

0

100.00

9.21

9

2

71.83

22.43

3

4

93.56

28.02

3

8

53.48

19.67

3

16

42.86

33.80

3

32

31.20

20.27

3

64

28.06

35.84

2

128

22.15

27.48

2

80

Table 10: Effect of tobramycin on P. aeruginosa biofilms. The mean is given as the percentage of untreated samples with standard deviation (SD) and number of replicates (N).

Concentration Mean (%) (µg/ml)

SD

N

0

100.00

21.43

15

0.01

97.42

30.52

5

0.025

110.20

24.22

5

0.05

91.96

35.95

5

0.1

92.42

29.21

5

0.2

142.40

40.31

5

Table 11 Effect of tetracycline on P. aeruginosa biofilms. The mean is given as the percentage of untreated samples with standard deviation (SD) and number of replicates (N).

Concentration Mean (%) (µg/ml)

SD

N

0

100.00

14.16

15

0.1

118.20

25.45

3

0.2

107.10

17.44

3

0.4

88.13

16.56

5

0.8

93.29

30.98

5

1.6

104.20

46.96

5

3.1

97.09

41.61

5

6.2

107.00

44.22

5

12.5

134.60

36.57

4

Table 12: Effect of LL-37 on P. aeruginosa initial attachment. The inhibition shown as % of one untreated sample.

Concentration (µg/ml)

1

2

4

8

16

32

64

128

% of control

143.84

99.29

118.72

117.77

105.45

64.22

29.14

9.24

Table 13: Effect of 1037 on P. aeruginosa initial attachment. The inhibition shown as % of one untreated sample.

Concentration (µg/ml)

2

4

8

16

32

64

128

% of control

108.53

91.79

78.58

70.53

70.85

52.82

0

81

Table 14: The 24h effect of LL-37 on pregrown P. aeruginosa biofilms. The mean is given as the percentage of untreated samples with standard deviation (SD) and number of replicates (N).

Concentration Mean (%) (µg/ml)

SD

N

0

100

10.24

8

4

82.77

15.32

4

16

44.36

12.54

4

Table 15: The 2h effect of LL-37 on pregrown P. aeruginosa biofilms. The mean is given as the percentage of untreated samples with standard deviation (SD) and number of replicates (N).

Concentration Mean (%) (µg/ml)

SD

N

0

100

32.77

8

4

114.8

14.86

4

16

120.8

28.98

4

Table 16: The 24h effect of 1037 on pregrown P. aeruginosa biofilms. The mean is given as the percentage of untreated samples with standard deviation (SD) and number of replicates (N).

Concentration Mean (%) (µg/ml)

SD

N

0

100

12.91

8

2

116.7

9.97

4

8

91.74

3.861

4

Table 17: The 2h effect of 1037 on pregrown P. aeruginosa biofilms. The mean is given as the percentage of untreated samples with standard deviation (SD) and number of replicates (N).

Concentration Mean (%) (µg/ml)

SD

N

0

100

29.70

8

2

96.31

30.13

4

8

83.90

34.14

4

82

Effect of LL-37 and 1037 on K. pneumoniae biofilms Table 18: The effect of LL-37 in BM2 medium on K. pneumoniae biofilms in polypropylene microtiter plates. The mean is given as the percentage of untreated samples with standard deviation (SD) and number of replicates (N).

Concentration (µg/ml)

4

8

16

32

64

128

Mean (%)

155.8

113.8

139.3

171.1

161.9

206.1

SD

110.5

67.96

79.21

113.0

103.8

208.0

N

5

5

5

5

5

5

Table 19: The effect of LL-37 in M9 minimal medium on K. pneumoniae biofilms in polypropylene microtiter plates. The mean is given as the percentage of untreated samples with standard deviation (SD) and number of replicates (N).

Concentration (µg/ml)

4

8

16

32

64

128

Mean (%)

53.32

57.43

51.88

86.18

89.07

57.44

SD

6.723

20.34

27.66

36.25

53.15

23.90

N

4

4

4

4

4

4

Table 20: The effect of LL-37 in LB medium on K. pneumoniae biofilms in polypropylene microtiter plates. The mean is given as the percentage of untreated samples with standard deviation (SD) and number of replicates (N).

Concentration (µg/ml)

4

8

16

32

64

128

Mean (%)

92.46

123.0

97.64

171.4

158.5

186.4

SD

32.37

81.68

39.89

149.2

118.5

98.26

N

4

4

4

4

4

4

Table 21: The effect of LL-37 in MH medium on K. pneumoniae biofilms in polypropylene microtiter plates. The mean is given as the percentage of untreated samples with standard deviation (SD) and number of replicates (N).

Concentration (µg/ml)

4

8

16

32

Mean (%)

81.83

89.64

74.10

98.36

SD

11.56

89.60

27.29

60.23

N

4

4

4

4

83

Table 22: The effect of LL-37 in TB medium on K. pneumoniae biofilms in polypropylene microtiter plates. The mean is given as the percentage of untreated samples with standard deviation (SD) and number of replicates (N).

Concentration (µg/ml)

4

8

16

32

64

128

Mean (%)

107.2

102.9

109.2

120.9

101.7

90.94

SD

13.98

25.15

51.34

56.19

42.57

39.91

N

5

5

5

5

5

5

Table 23: The time growth of K. pneumoniae biofilm formation in M9. The mean is given as the OD595 with standard deviation (SD) and number of replicates (N).

Time (h)

2

4

6

24

Mean

0.113

0.293

0.293

0.225

SD

0.018

0.085

0.033

0.072

N

7

7

7

7

Table 24: The time growth of K. pneumoniae biofilm formation in BM2. The mean is given as the OD595 with standard deviation (SD) and number of replicates (N).

Time (h)

2

4

6

24

Mean

0.173

0.228

0.182

0.507

SD

0.040

0.042

0.037

0.176

N

7

7

7

7

Table 25: The 24h effect of LL-37 on K. pneumoniae biofilms. The inhibition shown as % of one untreated sample.

Concentration (µg/ml)

1

2

4

8

16

32

64

128

% of control

107.04

71.36

71.95

101.98

89.79

92.77

114.77

121.31

Table 26: The 24h effect of 1037 on K. pneumoniae biofilms. The inhibition shown as % of one untreated sample.

Concentration (µg/ml)

2

4

8

16

32

64

128

% of control

85.23

126.83

114.32

89.97

88.61

88.95

75.76

Table 27: The 4h effect of LL-37 on K. pneumoniae biofilms. The inhibition shown as % of one untreated sample.

Concentration (µg/ml) % of control

1 90.90

2 110.33

84

4

8

98.30

98.76

16 126.52

32 79.10

64 29.37

128 23.13

Table 28: The 4h effect of 1037 on K. pneumoniae biofilms. The inhibition shown as % of one untreated sample.

Concentration (µg/ml)

2

4

8

16

32

64

128

% of control

59.67

55.28

78.64

61.98

67.30

41.17

19.89

Table 29: Effect of LL-37 on K. pneumoniae initial attachment. The mean is given as the percentage of untreated samples with standard deviation (SD) and number of replicates (N).

Concentration Mean (%) (µg/ml)

SD

N

0

100.00

10.83

12

1

73.41

23.68

2

2

72.20

17.80

4

4

71.33

12.19

4

8

62.90

17.20

4

16

34.97

23.61

4

32

14.66

19.94

4

64

7.29

14.41

4

128

0.17

5.94

4

Table 30: Effect of 1037 on K. pneumoniae initial attachment. The mean is given as the percentage of untreated samples with standard deviation (SD) and number of replicates (N).

Concentration Mean (%) (µg/ml)

SD

N

0

100.00

16.80

12

2

54.43

13.12

4

4

49.08

19.33

4

8

39.60

18.57

4

16

46.96

24.51

4

32

28.55

14.48

4

64

19.85

15.26

4

128

13.04

11.59

4

Table 31: The 3h effect of LL-37 on pregrown K. pneumoniae biofilms. The mean is given as the percentage of untreated samples with standard deviation (SD) and number of replicates (N).

Concentration Mean (%) (µg/ml)

SD

N

4

116.2

12.15

4

16

115.0

6.375

4

85

Table 32: The 3h effect of 1037 on pregrown K. pneumoniae biofilms. The mean is given as the percentage of untreated samples with standard deviation (SD) and number of replicates (N).

Concentration Mean (%) (µg/ml)

SD

N

8

103.3

12.45

4

32

125.4

6.642

4

86

Effect of LL-37 and 1037 on E. coli biofilms Table 33: Effect of LL-37 on E. coli biofilms. The mean is given as the percentage of untreated samples with standard deviation (SD) and number of replicates (N).

Concentration (µg/ml)

E. coli 536

E. coli CFT073

Mean

SD

N

Mean

SD

N

0

100.00

14.80

15

100.00

16.22

14

1

113.30

16.73

4

94.68

42.87

4

2

102.50

10.18

4

70.66

10.55

4

4

92.33

6.89

4

63.04

18.45

4

8

94.15

6.35

4

74.99

21.23

4

16

76.34

20.84

4

83.28

34.45

4

32

67.22

7.93

4

52.94

25.61

3

64

75.40

11.17

4

66.83

13.01

4

128

68.40

9.20

2

-

-

-

Table 34: Effect of 1037 on E. coli biofilms. The mean is given as the percentage of untreated samples with standard deviation (SD) and number of replicates (N).

Concentration (µg/ml)

E. coli 536

E. coli CFT073

Mean

SD

N

Mean

SD

N

0

100.00

9.35

16

100.00

17.10

15

2

73.06

16.00

3

101.40

28.15

4

4

79.00

19.66

4

85.48

15.95

4

8

79.79

16.10

4

111.70

29.07

4

16

70.58

13.93

4

80.53

13.61

4

87