Accepted Manuscript

Title: The human antimicrobial peptide LL-37 and itsfragments possess both antimicrobial and antibiofilm activitiesagainst multidrug-resistant Acinetobacter baumannii

Author: Xiaorong Feng Karthik Sambanthamoorthy ThomasPalys Chrysanthi Paranavitana<ce:footnoteid="fn1"><ce:note-para id="npar0005">Contributed equallyto this work.</ce:note-para></ce:footnote>

PII: S0196-9781(13)00320-3DOI: http://dx.doi.org/doi:10.1016/j.peptides.2013.09.007Reference: PEP 69070

To appear in: Peptides

Received date: 2-8-2013Revised date: 12-9-2013Accepted date: 12-9-2013

Please cite this article as: Feng X, Sambanthamoorthy K, Palys T, Paranavitana C,The human antimicrobial peptide LL-37 and its fragments possess both antimicrobialand antibiofilm activities against multidrug-resistant Acinetobacter baumannii, Peptides(2013), http://dx.doi.org/10.1016/j.peptides.2013.09.007

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Highlights

Antimicrobial peptide LL-37 and its fragments showed antibacterial activity

against several drug-resistant Acinetobacter baumannii strains

This peptide and two of its fragments also prevented biofilm formation by A.

baumannii

They may be considered as potential therapeutics for A.baumannii infections

Highlights (for review)

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The human antimicrobial peptide LL-37 and its fragments possess 1

both antimicrobial and antibiofilm activities against multidrug-2

resistant Acinetobacter baumannii 3

Journal Name: PEPTIDES 4

Xiaorong Feng Ϯ, Karthik Sambanthamoorthy Ϯ, Thomas Palys, and 5

Chrysanthi Paranavitana * 6

7

Department of Wound Infections, Bacterial Diseases Branch, Walter Reed 8

Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, 9

Maryland, USA, 20910. 10

11

Running Title: Activities of LL-37 and its fragments against A. baumannii 12

*Corresponding Author: Telephone 301-319-9172 13

E-mail: Chrysanthi.M.Paranavitana2.ctr@us.army.mil 14

Ϯ Contributed equally to this work 15

16

ABSTRACT 17

Acinetobacter baumannii infections are difficult to treat due to multidrug resistance. 18

Biofilm formation by A. baumannii is an additional factor in its ability to resist 19

antimicrobial therapy. The antibacterial and antibiofilm activities of the human 20

antimicrobial peptide LL-37 and its fragments KS-30, KR-20 and KR-12 against clinical 21

isolates of multidrug-resistant (MDR) A. baumannii were evaluated. The minimal 22

inhibitory concentration (MIC) of LL-37 against MDR A. baumannii isolates ranged from 23

16 to 32 µg/mL. The MIC of KS-30 fragment varied from 8.0 to16 µg/mL and the KR-20 24

fragment MIC ranged from 16 to 64 µg/mL. LL-37 and KS-30 fragment exhibited 100% 25

bactericidal activity against five A. baumannii strains, including four MDR clinical 26

isolates, within 30 min at concentrations of 0.25-1 µg/mL. By 0.5 hours, the fragments 27

KR-20 and KR-12 eliminated all tested strains at 8 and 64 µg/mL respectively. LL-37 28

and its fragments displayed anti-adherence activities between 32-128 µg/mL. A 29

*Manuscript

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minimum biofilm eradication concentration (MBEC) biofilm assay demonstrated that LL-30

37 inhibited and dispersed A. baumannii biofilms at 32 µg/mL respectively. Truncated 31

fragments of LL-37 inhibited biofilms at concentrations of 64-128 µg/mL. KS-30, the 32

truncated variant of LL-37, effectively dispersed biofilms at 64 µg/mL. At 24 hours, no 33

detectable toxicity was observed at the efficacious doses when cytotoxicity assays were 34

performed. Thus, LL-37, KS-30 and KR-20 exhibit significant antimicrobial activity 35

against MDR A. baumannii. The prevention of biofilm formation in vitro by LL-37, KS-30 36

and KR-20 adds significance to their efficacy. These peptides can be potential 37

therapeutics against MDR A. baumannii infections. 38

39

Keywords: cathelicidin; biofilm; Acinetobacter baumannii; anti-infective 40

41

42

1. Introduction 43

44

Acinetobacter baumannii are Gram-negative, non-fermentative, non-spore forming, 45

strictly aerobic, oxidase-negative coccobacillary organisms. Historically considered a 46

commensal bacterium with a relatively low pathogenicity, A. baumannii is now 47

recognized as a nosocomial pathogen associated with a variety of infections in critically 48

ill, hospitalized patients and those with suppressed immune systems, burns, or major 49

trauma. Recently, A. baumannii has emerged as a pathogen that can develop 50

resistance to many commercially available antibiotics by diverse mechanisms [8]. In 51

military personnel, reports of multidrug-resistant (MDR) A. baumanni infections 52

associated with bacteremia, extremity war wounds, and osteomyelitis have been 53

reported in over 30% of admitted deployed soldiers [8]. Due to the widespread drug 54

resistance of these bacteria, it has been difficult to treat A. baumannii infections [25, 35]. 55

In addition, the ability to form biofilms on abiotic surfaces is a characteristic feature of 56

clinical A. baumannii strains, especially those isolated from bloodstream infections and 57

catheter-associated urinary tract infections [29]. It has been demonstrated that several 58

clinical MDR A. baumannii isolates form biofilms and adhere to epithelial cells, which 59

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may contribute to their survival in the hospital environment [21]. There is a clear need 60

to develop new antimicrobial agents against emerging MDR A. baumannii. 61

62

To counter the impacts of emerging multidrug resistance, new antibiotic alternatives 63

such as antimicrobial peptides (AMPs) are being addressed. AMPs are a large family of 64

naturally occurring peptides found in almost all living organisms as innate immune 65

defenses [3, 41, 42]. Eukaryotic AMPs are cationic peptides with a net positive charge 66

and a molecular weight in the range of 1-5kDa. Due to their amphipathic nature with 67

both hydrophilic and hydrophobic moieties, they form pores in the cytoplasmic 68

membrane, leading to loss of cell contents. Human AMPs consist of defensins, 69

cathelicidins and histatins [3, 41, 42]. The only cathelicidin from humans is LL-37 [2, 3, 70

41, 42]. Cathelicidin hCAP-18 (human cationic antibacterial peptide, 18kD) is produced 71

by epithelia in a prepropeptide form. The full-length hCAP-18 is cleaved by skin-derived 72

kallikreins and/or by proteinase 3 from white blood cells to release a 37-amino acid 73

residue, LL-37, a cationic peptide containing a carboxy terminus [39]. LL-37 has been 74

shown to exhibit a broad spectrum of antibacterial, antifungal and antiviral activity [11, 75

16, 39, 40]. LL-37 forms an alpha-helical structure when associated with the bacterial 76

cell membrane [16]. In addition to its broad range of antimicrobial activity against 77

bacteria, fungi, and viruses, LL-37 has been associated with keratinocyte migration 78

during wound healing and plays a role in wound closure by promoting 79

neovascularization and re-epithelialization of healing skin [1, 4, 14]. 80

81

The mature LL-37 can be further processed to release peptide fragments that possess 82

enhanced antimicrobial activity and can act synergistically with each other. Previous 83

studies show that LL-37 and its fragments are active against other Gram-negative 84

bacteria. LL-37 is also active against Gram-positive bacteria and certain fungi [6, 28, 85

36]. This study investigated the activity of LL-37 and its fragments (KS-30, KR-20 and 86

KR-12) against MDR clinical isolates of A. baumannii. In addition, LL-37 and its 87

fragments inhibited biofilm development. 88

89

2. Materials and methods 90

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91

2.1. Strains and growth conditions 92

Clinical isolates of A.baumannii were obtained from the Multidrug-resistant 93

organism Repository and Surveillance Network (MRSN) at the Walter 94

Reed Army institute of Research (WRAIR). The ATCC 19606T strain was 95

obtained from the American Type Culture Collection ATCC (Manassas, 96

VA, USA). 97

98

2.2. Peptides and fragments 99

LL-37 was purchased from The American Peptide Company (Louisville, KY, USA). The 100

fragments KS-30 (LL-8-37), KR-20 (LL-18-37), and KR-12 (LL-18-29) were purchased 101

from AnaSpec (Fremont, CA, USA). The sequences of these peptides are as follows: 102

LL-37: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES 103

KS-30: KSKEKIGKEFKRIVQRIKDFLRNLVPRTES 104

KR-20: KRIVQRIKDFLRNLVPRTES 105

KR-12: KRIVQRIKDFLR 106

107

2.3. Minimum inhibitory concentration (MIC) assay 108

Minimum inhibitory concentrations (MIC) of LL-37 and its fragments (KS-30, KR-20, and 109

KR-12) were determined according to Clinical and Laboratory Standards Institute (CLSI) 110

guidelines as described previously [19, 26]. Briefly, an upper concentration of 512 111

µg/mL was chosen for the peptides with two-fold dilutions in Mueller Hinton Broth (MHB) 112

in a 96-well polypropylene plate. An actively growing bacterial culture obtained from an 113

overnight culture of the appropriate bacterial strain in MHB was washed twice and 114

resuspended in phosphate buffered saline (PBS) with a corresponding absorbance at 115

600nm, resulting in a concentration of 2x105 colony forming units (CFU)/mL. An aliquot 116

of 50 µL was added to each well. The plate was incubated for 24 hours at 37°C. The 117

absorbance was measured in a Spectramax Plus micro plate reader (Molecular 118

Devices, Sunnyvale, CA, USA). 119

120

2.4. Minimum bactericidal concentration (MBC) assay 121

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The antibacterial assay was performed following the protocol described previously [26]. 122

Briefly, overnight cultures of each bacterial strain were harvested, washed with PBS, 123

and suspended in sodium phosphate buffer. The bacterial suspension (5x105 cells/mL) 124

was inoculated into 100 µL of sodium phosphate buffer with or without LL-37, KS-30, 125

KR-20 or KR-12 at different concentrations and incubated aerobically for 0.5, 1, 2, and 126

24 hours at 37 °C. Immediately following incubation, the bacterial mixture was plated 127

onto Luria Broth (LB) agar plates with appropriate dilutions. To measure the total 128

bacterial CFUs, the reaction mixture without peptides was similarly diluted and plated 129

onto LB plates. After counting the colonies grown on the LB plates, MBC was defined as 130

the concentration that killed 99.9% of total bacteria. The number of CFU was 131

determined as the total number of colonies identified on each plate. The antibacterial 132

effect was calculated as the ratio of surviving cells (percentage survival) to the total 133

number of bacteria incubated in control sodium phosphate solution after exposure to 134

antimicrobial peptides. 135

136

2.5. Cytotoxicity assay 137

The HEK-293 cell line (human keratinocyte cell line) (ATCC, Manassas, VA, USA) was 138

used in this study. The cytotoxicity of LL-37 and its fragments (KS-30, KR-20, and KR-139

12) on eukaryotic cells was evaluated by the release of lactate dehydrogenase (LDH) 140

and total cell number assay. The LDH cytotoxicity assay was performed according to 141

the manufacturer's guidelines (CytoTox 96 Non-Radioactive Cytotoxicity Assay, 142

Promega, Madison, WI, USA). After the addition of LL-37 and its fragments for 24 143

hours, the cell culture medium was collected for LDH measurement. An aliquot of 50 µL 144

cell medium was used for LDH activity analysis and the absorption was measured using 145

a UV–visible spectrophotometer. At the end of the LDH assay, the remaining cells were 146

processed according to the manufacturer’s instructions (CytoTox 96 Non-Radioactive 147

Cytotoxicity Assay, Promega, USA) for the total cell number assay. The number of total 148

cells is directly proportional to absorbance values at 490 nm of supernates from lysed 149

cells, which represent LDH activity. All assays were repeated three times, each in 150

triplicate. 151

152

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2.6. Adherence of A. baumannii to abiotic surfaces 153

An initial adherence assay was used to measure the impact of AMPs on the surface 154

binding capacity of A. baumannii. The assay was performed by modifying a microtiter 155

biofilm assay described previously [31]. Briefly, overnight cultures of A. baumannii test 156

strains were diluted to an absorbance value of 0.05 at 595 nm in fresh brain heart 157

infusion broth, and 200 µL was added to each well (polystyrene pre-coated with human 158

plasma) in triplicate. Following 1 hour incubation at 37 °C, the microtiter wells were 159

washed three times with PBS. Adherent cells were then fixed with 200 µL of 100% 160

ethanol for 10 min. The ethanol was removed and the wells were air dried for 2min. 161

Adherent cells were stained for 2 min with 200 µL of 0.41% crystal violet (w/v in 12% 162

ethanol), then washed three times with PBS. The wells were allowed to dry and then 163

eluted with ethanol. Absorbance readings were made at 595 nm using a SpectraMax 164

M5 microplate spectrophotometer system (Molecular Devices, Sunnyvale, CA). Data 165

shown is the average of three independent experiments. 166

167

2.7. Assessment of biofilm formation 168

Biofilm formation was measured under two static conditions using a quantitative crystal 169

violet assay on polystyrene 96-well and MBEC (Minimum Biofilm Eradication 170

Concentration plates (Biosurface Technologies, Bozeman, MT, USA) as described 171

previously [13, 30]. The MBEC biofilm assay utilizes a 96-well plate cover containing 96 172

polystyrene pegs that fit the wells of a conventional plate. Briefly, overnight cultures of 173

A. baumannii test strains were diluted to an absorbance value of 0.05 at 595nm in fresh 174

BHI. 165 µL was transferred to the wells of a 96-well polystyrene microtiter plate and 175

the MBEC lid was placed on top of the wells. Biofilms were grown on the pegs under 176

shaking conditions for 24 hours. The lid was removed and the pegs were gently washed 177

twice with 200 µL of PBS to remove non-adherent cells. Adherent biofilms on the pegs 178

were fixed with 200 µL of 100% ethanol prior to staining for 2 min with 200 µL of 0.41% 179

(wt vol-1) crystal violet in 12% ethanol (Biochemical Sciences, Swedesboro, NJ). The 180

pegs were washed several times with PBS to remove excess stain. Quantitative 181

assessment of biofilm formation was obtained by the immersion of pegs in a sterile 182

polystyrene microtiter plate which contained 200 µL of 100% ethanol and incubation at 183

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room temperature for 10 min. The absorbance at 595 nm was determined as previously 184

described [32]. Three independent experiments were performed for each of these 185

assays. 186

187

2.8. Biofilm dispersal 188

To determine if the peptides could disperse preformed biofilms, A. baumannii biofilms 189

were developed on MBEC pegs and then exposed to varying concentrations of peptides 190

in fresh media for short time intervals. Briefly, overnight grown cultures of A. baumannii 191

were diluted to an absorbance value of 0.05 at 595nm in fresh BHI, then 165 µL was 192

transferred to the wells of a MBEC microtiter plate and the MBEC lid was placed on top 193

of the wells. Biofilms were grown on the MBEC pegs under shaking conditions for 24 194

hours. The lid was removed and transferred to a new plate with wells filled with 195

antimicrobial peptides to be tested. The pegs were immersed for 1 hour and the lid was 196

gently washed twice with 200 µL of PBS to remove non-adherent cells. Adherent 197

biofilms on the pegs were fixed with 200 µL of 100% ethanol prior to staining for 2 min 198

with 200 µL of 0.41% (wt/vol) crystal violet in 12% ethanol (Biochemical Sciences, NJ 199

USA). The pegs were washed several times with PBS to remove excess stain. 200

Quantitative assessment of biofilm formation was obtained by the immersion of pegs in 201

a sterile polystyrene microtiter plate which contained 200 µL of 100% ethanol. The plate 202

was incubated at room temperature for 10 min and the absorbance at 595 nm was 203

determined using a SpectraMax M5 microplate spectrophotometer system. Results 204

were interpreted by the comparison of peptides on treated biofilms to untreated biofilms 205

of A. baumannii. Experiments were performed in triplicate and three independent 206

experiments were performed for each of these assays. 207

208

3. Results 209

3.1. Determination of the minimal inhibitory concentrations of LL-37 and 210

fragments against A. baumannii 211

The MIC was determined for A. baumannii strains according to CLSI 212

guidelines. The KS-30 fragment had lower MIC values compared to the 213

others, while the shortest fragment KR-12 had the highest value (Table 1). 214

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215

3.2. Kinetics of antimicrobial activity of LL-37 and its fragments KS-30, KR-20, and KR-216

12 against A. baumannii 217

To determine the time kinetics of antimicrobial activity, peptide LL-37 and its fragments 218

KS-30, KR-20, and KR12 were incubated for 0.5, 1, 2, or 24 hours with several A. 219

baumannii strains, and the amount of CFU at respective time points was analyzed. By 220

0.5 hours, both LL-37 and KS-30 demonstrated strong bactericidal activity in a 221

concentration-dependent manner against the two MDR strains and ATCC19606 (Fig. 1 222

A-F). At a concentration of 0.25 µg/mL, LL-37 was able to completely eliminate MDR 223

5075 and ATCC 19606 in 30 min while for MDR 5711 the concentration was 1 µg/mL. 224

By 24 hours, 2 µg/mL was able to maintain 100% bactericidal activity for the three 225

strains tested (Fig. 1 A-C). At 0.5 hours, 1 µg/mL of the KS-30 peptide achieved 100% 226

bactericidal activity for the two MDR strains 5075 and 5711, while 0.25 µg/mL was 227

sufficient to eliminate the ATCC 19606 strain (Fig. 1 D-F). By 24 hours, 2 µg/mL of KS-228

30 was able to maintain 100% bactericidal activity for the three strains (Fig. 1 D-F). Both 229

these peptides showed similar activity against two other MDR A. baumannii strains 230

tested (data not shown). In 0.5 hours, 8 µg/mL of KR-20 peptide destroyed 100% of 231

MDR 5075 while the other two strains were eliminated in 2 hours (Fig. 1 G-I). Two other 232

MDR strains were tested with KR-20 with similar results (data not shown). For the 233

smallest fragment, KR-12, 64 µg/mL achieved 100% killing of all the tested strains in 30 234

min (Fig. 1 J-L). Similar results were obtained with two other MDR strains tested (data 235

not shown). 236

237

3.3 Cytotoxicity assays for LL-37, KS-30, KR-20, and KR-12 in eukaryotic cells 238

Results from the cell-mediated cytotoxicity assay are presented in Fig. 2. Human 239

keratinocyte HEK393 cells were treated with LL-37 and their fragments, KS-30, KR-20, 240

and KR-12, for 24 hours. Cytotoxicity was determined by LDH release according to the 241

manufacturer’s instructions (Fig. 2A) and the total cell number assay (Fig. 2B). LL-37, 242

KS-30, and KR-20 did not cause significant toxicity to cells below the concentration of 243

128 µg/mL. Higher concentrations of LL-37 and KS-30 (256 and 512 µg/mL) showed 244

toxicity levels of >20%. KR-20, at the highest concentration of 512 µg/mL, showed 245

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toxicity of <20%. Interestingly, KR-12 did not cause any toxicity to the cells at 246

concentrations that ranged from 32 to 512 µg/mL (Fig. 2A). 247

248

3.4. Impact of LL-37 on A. baumanni attachment to abiotic surface 249

Biofilm formation is a complex process that generally involves three stages: (1) primary 250

adhesion to surfaces, (2) accumulation of multilayered clusters of cells, and (3) 251

detachment. Experiments were performed to determine which stage of biofilm 252

formation LL-37 disrupts. Using an adherence assay, the ability of LL-37 to inhibit the 253

cell attachment in the presence of host proteins was measured by coating the plates 254

with human plasma. After various concentrations ranging from 1-64 µg/mL were tested, 255

it was found that LL-37 significantly impaired the attachment of A. baumannii strains to 256

abiotic surfaces (Fig. 3). It was important to test initial adherence to surfaces with 257

plasma coating since the binding of host proteins is a major contributor to primary 258

adhesion. 259

260

3.5. Impact of LL-37 on A. baumanni biofilm development 261

Next, it was determined if the MBEC biofilm assay could show whether LL-37 262

possessed anti-biofilm activity against A. baumannii strains. This system consisted of a 263

microtiter plate with 96 corresponding pegs attached to the plate lid. These pegs were 264

immersed in the bacterial culture to provide a surface for biofilm formation. For these 265

experiments, the biofilm-forming A. baumannii strains 5711 and 5075 were chosen. 266

From the results obtained from adherence assays, 32 µg/mL was selected as the 267

relevant concentration to test for biofilm inhibition. It was found that LL-37 significantly 268

reduced biofilm development in both strains when used at that concentration (Fig. 4). 269

270

3.6. Dispersion of preformed A. baumannii biofilms by LL-37 271

In the experiments thus far, the peptides were added concurrently with inoculation of 272

bacteria. To determine if these compounds dispersed preformed biofilms, A. baumannii 273

biofilms were developed on MBEC pegs, then exposed to varying concentrations of 274

peptides in fresh media for short time intervals. After removal of the pegs, the amounts 275

of bacteria that remained on the pegs were quantified by crystal violet staining method 276

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as described in the materials and methods section. Compared to the control, 277

preformed biofilms treated with LL-37 at 64 µg/mL increased biofilm dispersion (Fig. 5). 278

279

3.7. Impact of truncated LL-37 on A. baumanni adhesion, biofilm development and 280

dispersion 281

Further investigations were carried out to determine whether the truncated versions of 282

LL-37 demonstrated anti-adherence, anti-biofilm, and biofilm dispersal properties. A. 283

baumannii strain 5711 was selected for biofilm studies with the truncated peptides due 284

to its highly reproducible biofilm development. Three truncated fragments of LL-37, 285

namely KR-12, KR-20, and KS-30, were analyzed. All three truncated fragments 286

impacted attachment of A. baumannii to an abiotic surface (Fig. 6). In addition, all three 287

truncated peptides inhibited A. baumannii biofilms when grown on a MBEC system 288

(Fig.7). Next, it was determined whether truncated peptides dispersed preformed A. 289

baumannii biofilms. Only KS-30 effectively dispersed A. baumannii upon 1 hour of 290

exposure, and KR-12 and KR-20 did not disperse biofilms even when tested at high 291

concentrations (Fig. 8). 292

293

4. Discussion 294

Multidrug resistant A. baumannii infections are prevalent in hospitalized patients. In 295

addition, A. baumannii forms biofilms that make it difficult to eradicate infections, 296

especially in immunocompromised patients. Sanchez et al. [33] reported that A. 297

baumannii biofilm producers were more often found to be resistant to antibiotics 298

compared to weak biofilm producers [33]. In addition, due to its ability to survive on inert 299

surfaces, A. baumannii contributes to the contamination of hospital equipment and 300

surfaces, resulting in infectious outbreaks in hospitals. MDR A. baumanni infections 301

have also been reported among military personnel from combat regions [15, 23]. 302

303

The current study was done to evaluate the effectiveness of antimicrobial peptide LL-304

37, against drug resistant A. baumannii, in order to discover alternate therapeutics 305

against wound infections. LL-37 possesses a broad range of antimicrobial activity 306

against bacteria, fungi, and viruses [11, 16, 39, 40], and the role of LL-37 in protection 307

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against bacterial infections has also been shown in vivo [10, 20, 28]. The potent 308

bactericidal activity of LL-37 against different bacteria involves the interaction with 309

negatively charged molecules on the surface of bacteria and disruption of bacterial 310

membrane with changes in the membrane lipids and pore formation [2], resulting in 311

direct killing of bacteria. The cathelicidin family of antimicrobial peptides has also been 312

shown to contribute to anti-inflammatory responses associated with LPS toxicity by 313

inhibition of TNFα [24] and blocking LPS binding to LPS binding protein [34]. In addition, 314

LL-37 is associated with immuno modulatory activities such as inducing chemotactic 315

activity of neutrophils, monocytes and T cells via formyl peptide receptor-like 1 (FPRL1) 316

receptor [9]. These functions may ultimately contribute to pathogen clearance and 317

protective effects against bacterial infections. 318

319

320

In addition, Murakami et al. showed that LL-37 was processed into fragments after 321

secretion in human sweat and suggested that its fragments, KS-30 and KR-20, may 322

occur naturally, but are less abundant in sweat [22]. 323

324

In this study, the antibacterial activity of LL-37 and its fragments, KS-30, KR- 20, and 325

KR-12, were tested against clinical strains of multidrug-resistant A. baumannii. LL-37, 326

KS-30, KR-20, and KR-12 were effective against multidrug-resistant A. baumannii in 327

vitro with both antimicrobial and antibiofilm activities. Both LL-37 and KS-30 showed 328

exceptional killing activities towards multidrug-resistant strains of A. baumannii. The 329

killing abilities of the four peptides against A. baumannii were KS-30 > LL-37 > KR-20 > 330

KR-12. Again, work by Murakami et al. [22] showed antimicrobial activity after 331

processing of LL-37 to KS-30, and in another fragment, RK-31, which showed increased 332

activity against Staphylococcus aureus. Compared to LL-37, KS-30 was more effective 333

against Gram-negative A. baumannii. 334

For therapeutic application of peptides, cell cytotoxicity is of critical importance. Even 335

though LL-37 and KS-30 were found to be extremely efficient in killing A. baumannii, 336

they both showed cytotoxicity at higher concentrations. The KR-20 fragment showed 337

less efficient killing compared to these, yet the cytotoxicity levels were much lower. In 338

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the study by Murakami et al. [22], KR-20 also stimulated much lower levels of the 339

proinflammatory cytokine IL-8. Therefore, KR-20 may be the best option among LL-37 340

and its fragments for further testing in an animal model for therapeutic activity against 341

MDR A. baumannii infections. Our results show that LL-37 and all its fragments blocked 342

the formation of a biofilm measured with the MBEC technology. The biofilm inhibition by 343

LL-37 was previously reported by others [18, 27] and by Dean et al. [10] who showed a 344

43% reduction in biofilm formation with LL-37-treated Pseudomonas aeruginosa 345

cultures. Although LL-37 dispersed A. baumannii biofilms, surprisingly of all three 346

fragments tested, only KS-30 was effective at dispersing preformed biofilms of A. 347

baumannii. Interestingly, previous work on Burkholderia pseudomallei [17] showed that 348

the 31 amino acid (LL-31) fragment eradicated biofilms effectively while the 25 amino 349

acid (LL-25) fragment did not, suggesting that more than 25 amino acid residues may 350

be required for biofilm dispersal. In conclusion, our results show that LL-37 and its 351

fragments were effective against A. baumannii and its biofilms. Specifically, KR-20 352

showed lower toxicity and good killing activity, suggesting that it may have therapeutic 353

potential against A. baumannii infections. 354

355

Antimicrobial peptides produced in the oral mucosa, including LL-37, have been 356

considered as alternative therapeutics against other pathogens such as oral 357

microorganisms [7]. Others have reported effective testing of antimicrobial peptides or 358

their fragments in vivo against several pathogens such as P. aeruginosa biofilm [5], 359

methicillin-resistant S. aureus [12], and MDR Klebsiella pneumoniae [37], among 360

others. Several other peptides are in therapeutic development for preclinical or clinical 361

use, as reviewed by Yount and Yeaman [38]. 362

363

In conclusion, the work described here showed that the antimicrobial peptide LL-37 and 364

its fragments KS-30 and KR-20 are very effective against MDR A. baumannii with both 365

antimicrobial and anti biofilm activities. They may be considered for development of 366

therapeutics against A. baumannii infections. 367

368

Acknowledgments 369

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The findings and opinions expressed herein belong to the authors and do not 370

necessarily reflect the official views of the WRAIR, the U.S. Army, or the Department of 371

Defense. This work was supported by a Military Infectious Diseases Research Program 372

(MIDRP) grant awarded to Dr. C.P. The authors would like to thank Dr. Daniel Zurawski 373

and Mr. Mitchell Thompson at WRAIR for providing comments and suggestions when 374

necessary and Ms. Amy Michels for editing the manuscript. 375

376

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Figure 1

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Figure 1. Time-kill assays for LL-37 against A. baumannii 5075 (A), Type

strain ATCC 19606 (B) and 5711 (C). Time kill assays for KS-30 against

A. baumannii 5075 (D) Type strain ATCC 19606 (E) and 5711 (F). Time

kill assays for KR-20 against A. baumannii 5075 (G) Type strain ATCC

19606 (H) and 5711 (I). Time kill assays for KR-12 against A. baumannii

5075 (J) Type strain ATCC 19606 (K) and 5711 (L).

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Figure 2. Cytotoxicity assays for LL-37 peptide and fragments KS-30,

KR-20 and KR-12 in human keratinocyte cell line HEK293 at 24 hrs. LDH

assay was used to measure released LDH from cells treated with

peptides. Percent cytotoxicity was derived from fold change of LDH

Figure 2

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produced by cells treated with LL-37/fragments compared to the negative

control (N-CON). Positive control cells (P-CON) were treated with lysis

buffer; negative control (N-CON) comprised of culture medium (CM) only

(A). Total cell number assay was represented by optical densities at 490

nm of supernates from lysed cells in remaining wells after the LDH assay

(B).

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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Figure 8

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1

1

2

3

Table 1. The Range of minimum inhibitory concentrations (MICs; µg/mL) 4

for LL-37 and its fragments against four clinical MDR isolates of A. 5

baumannii and the type strain ATCC 19606. 6

7

8

LL-37 KS-30 KR-20 KR-12

AB5075 16 8 16 128

AB5711 32 16 32 256

AB#4 16 8 16 128

AB4795 32 16 32 256

AB-ATCC 32 16 64 256

Table 1

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REFERENCE: 69070

Editorial reference: PEP_PEPTIDES-D-13-00377 To be published in: Peptides

Missing figure captions:

Figure 1. Time-kill assays for LL-37 against A. baumannii 5075 (A), Type

strain ATCC 19606 (B) and 5711 (C). Time kill assays for KS-30 against

A. baumannii 5075 (D) Type strain ATCC 19606 (E) and 5711 (F). Time

kill assays for KR-20 against A. baumannii 5075 (G). Type strain ATCC

19606 (H) and 5711 (I). Time kill assays for KR-12 against A. baumannii

5075 (J) Type strain ATCC 19606 (K) and 5711 (L).

Figure 2. Cytotoxicity assays for LL-37 peptide and fragments KS-30,

KR-20 and KR-12 in human keratinocyte cell line HEK293 at 24 hours.

LDH assay was used to measure released LDH from cells treated with

peptides. Percent cytotoxicity was derived from fold change of LDH

produced by cells treated with LL-37/fragments compared to the negative

control (N-CON). Positive control cells (P-CON) were treated with lysis

buffer; negative control (N-CON) comprised of culture medium (CM) only

(A). Total cell number assay was represented by optical densities at 490

nm of supernates from lysed cells in remaining wells after the LDH assay

(B).

Figure 3. Initial adherence assays. Standardized overnight cultures of the strains AB

5711 and AB 5075 in the presence and absence of LL-37 were incubated for 1 hour at

37°C in plasma-coated microtiter wells. Adherent cells were then fixed with ethanol and

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stained with crystal violet. Next, Ethanol was used to elute the wells and absorbance

was measured. Numbers in parentheses denotes the peptide concentration µg/mL.

Results represent the mean ± SEM from three independent experiments. (*denotes

statistical significance of P < 0.05).

Figure 4. Biofilm inhibition assays. Standardized overnight cultures of the strains AB

5711 and AB 5075 were grown on MBEC pegs in the presence and absence of LL-37

and incubated overnight at 37°C. Adherent cells were then fixed with ethanol, stained

with crystal violet and eluted with ethanol and the absorbance was measured. Number

in parenthesis denotes the peptide concentration µg/mL. Results represent the mean ±

SEM from three independent experiments. (*denotes statistical significance of P < 0.05).

Figure 5. Biofilm dispersion assays. Standardized overnight cultures of the strains AB

5711 and AB 5075 were grown on MBEC pegs and incubated overnight at 37°C to

develop mature biofilms. The pegs were immersed in wells containing LL-37 for short

intervals to allow the biofilm to disperse from the pegs to the wells below and

absorbance was measured. Number in parenthesis denotes the peptide concentration

µg/mL. Results represent the mean ± SEM from three independent experiments.

(*denotes statistical significance of P < 0.05).

Figure 6. Adhesion assay for truncated peptides. Standardized overnight cultures of

the strain AB 5711 were grown in the presence or absence of KR-12, 20 and KS- 30 on

MBEC pegs and incubated overnight at 37°C. Adherent cells were then fixed with

ethanol, stained with crystal violet and eluted with ethanol and absorbance was

measured. Numbers in parentheses denotes the peptide concentrations µg/mL. Results

represent the mean ± SEM from three independent experiments. (*denotes statistical

significance of P < 0.05).

Figure 7. Biofilm inhibition assay on truncated peptides. Standardized overnight

cultures of the wild type strain AB 5711 in the presence and absence of KR-12, 20 and

KS- 30 were grown on MBEC pegs incubated for overnight at 37°C. Adherent cells were

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then fixed with ethanol, stained with crystal violet and eluted with ethanol and

absorbance was measured. Numbers in parentheses denotes the peptide

concentrations µg/mL. Results represent the mean ± SEM from three independent

experiments. (*denotes statistical significance of P < 0.05).

Figure 8. Biofilm dispersion assay on truncated peptides. Standardized overnight

cultures of the wild type strain AB 5711 in the presence and absence of KR-12, 20 and

KS- 30 were grown on MBEC pegs incubated for overnight at 37°C. Adherent cells were

then fixed with ethanol, stained with crystal violet and eluted with ethanol and

absorbance was measured. Numbers in parentheses represent the peptide

concentrations µg/mL. Results represent the mean ± SEM from three independent

experiments. (*denotes statistical significance of P < 0.05).

Table 1. The range of minimum inhibitory concentration (MIC) values for LL-37 and its

fragments against four clinical MDR isolates of A. baumannii and the type strain ATCC

19096.