Targeting methicillin-resistant Staphylococcus aureus with short salt-resistant synthetic peptides

Article abstract of DOI:10.1128/AAC.02578-14

The seriousness of microbial resistance combined with the lack of new antimicrobials has increased interest in the development of antimicrobial peptides (AMPs) as novel therapeutics. In this study, we evaluated the antimicrobial activities of two short synthetic peptides, namely, RRIKA and RR. These peptides exhibited potent antimicrobial activity against Staphylococcus aureus, and their antimicrobial effects were significantly enhanced by addition of three amino acids in the C terminus, which consequently increased the amphipathicity, hydrophobicity, and net charge. Moreover, RRIKA and RR demonstrated a significant and rapid bactericidal effect against clinical and drug-resistant Staphylococcus isolates, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate S. aureus (VISA), vancomycin-resistant S. aureus (VRSA), linezolid-resistant S. aureus, and methicillin-resistant Staphylococcus epidermidis. In contrast to many natural AMPs, RRIKA and RR retained their activity in the presence of physiological concentrations of NaCl and MgCl₂. Both RRIKA and RR enhanced the killing of lysostaphin more than 1,000-fold and eradicated MRSA and VRSA isolates within 20 min. Furthermore, the peptides presented were superior in reducing adherent biofilms of S. aureus and S. epidermidis compared to results with conventional antibiotics. Our findings indicate that the staphylocidal effects of our peptides were through permeabilization of the bacterial membrane, leading to leakage of cytoplasmic contents and cell death. Furthermore, peptides were not toxic to HeLa cells at 4- to 8-fold their antimicrobial concentrations. The potent and salt-insensitive antimicrobial activities of these peptides present an attractive therapeutic candidate for treatment of multidrug-resistant S. aureus infections. Copyright

Full text of DOI:10.1128/AAC.02578-14

AAC Accepts, published online ahead of print on 5 May 2014
Antimicrob. Agents Chemother. doi:10.1128/AAC.02578-14
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Targeting methicillin-resistant Staphylococcus aureus with short salt-resistant synthetic
peptides
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Mohamed F. Mohamed1, Maha I. Hamed 1, Alyssa Panitch 2, Mohamed N. Seleem1 #
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Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University,
West Lafayette, USA1
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Weldon School of Biomedical Engineering, Purdue University, West Lafayette, USA2
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Running title: Targeting methicillin resistant-Staphylococcus aureus
Key words: antimicrobial peptides, MRSA, VRSA, biofilm, lysostaphin
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# Corresponding Author:
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Mohamed N. Seleem
Department of Comparative Pathobiology
Purdue University College of Veterinary Medicine
625 Harrison Street, Lynn 1298, West Lafayette, IN 47907-2027
Phone: 765-494-0763 Fax: 765-496-2627
Email: mseleem@purdue.edu
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Abstract:
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The seriousness of microbial resistance combined with the lack of new antimicrobials have
increased the interest in the development of antimicrobial peptides (AMPs) as novel therapeutics.
In this study, we evaluated the antimicrobial activity of two short synthetic peptides, namely
RRIKA and RR. These peptides exhibited potent antimicrobial activity against Staphylococcus
aureus and their antimicrobial effects were significantly enhanced by addition of three amino
acids in the C terminus, which consequently increased the amphipathicity, hydrophobicity and
net charge. Moreover, RRIKA and RR demonstrated a significant and rapid bactericidal effect
against clinical and drug-resistant Staphylococcus isolates including methicillin-resistant
Staphylococcus aureus (MRSA), vancomycin-intermediate S. aureus (VISA), vancomycin-
resistant S. aureus (VRSA), linezolid-resistant S. aureus and methicillin-resistant S. epidermidis.
In contrast to many natural AMPs; RRIKA and RR retained their activity in the presence of
physiological concentrations of NaCl and MgCl2. Both RRIKA and RR enhanced the killing of
lysostaphin over 1000-fold and eradicated MRSA and VRSA isolates within 20 minutes.
Furthermore, the peptides presented were superior in reducing adherent biofilms of S. aureus and
S. epidermidis when compared to conventional antibiotics. Our findings indicate that the
staphylocidal effects of our peptides were through permeabilization of the bacterial membrane,
leading to leakage of cytoplasmic contents and cell death. Furthermore, peptides were not toxic
to HeLa cells at 4 to 8 fold their antimicrobial concentrations. The potent and salt-insensitive
antimicrobial activities of these peptides present an attractive therapeutic candidate for treatment
of multidrug-resistant S. aureus infections.
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INTRODUCTION
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Methicillin-resistant Staphylococcus aureus (MRSA) infections have become a major public
health concern in both hospital and community settings (1, 2). Since the late 1990s, community-
associated MRSA (CA-MRSA) emerged as a principal cause of skin and soft-tissue epidemics
throughout the world (3). It has been estimated that mortality rates from MRSA infections in US
hospitals are higher than infections caused by HIV/AIDS and tuberculosis combined (4).
Furthermore, there is a considerable medical challenge for treating MRSA infections due to the
remarkable ability of S. aureus to develop resistance to multiple antibiotics, thus limiting the
number of viable therapeutic options (2, 5). Therefore, there is an urgent need to develop novel
antimicrobials with unique mechanisms of action to combat MRSA.
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Antimicrobial peptides (AMPs) are key components of the host innate defense against infections
in most creatures (6, 7). The potential therapeutic applications of AMPs are significant (8). They
have rapid and broad spectrum antibacterial activity. In addition, it is extremely difficult for
bacteria to evolve resistance against AMPs that disrupt the microbial membrane, as it would
necessitate fundamental alterations in the lipid composition of the bacterial membranes. Though
AMPs have many important antibacterial properties, several studies have exposed their potential
limitations as therapeutic agents.
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A significant number of natural AMPs are large in size with high host cytotoxicity and moderate
antimicrobial activity. Moreover, their production cost is high (8, 9). In addition, many natural
AMPs lose their antimicrobial activity in physiological salt concentrations (10, 11). These
characteristics have substantially hindered their pharmaceutical development as new therapeutic
agents. Thus, successful development of novel AMPs as future therapeutics requires
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identification of short AMPs demonstrating strong antimicrobial activity, salt tolerance and
minimal toxicity to host tissues.
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Previously, our group has developed short synthetic peptide templates for therapeutic
applications against hyperplasia and inflammation (12-14). However their antimicrobial activity
was never explored. In the present study, we screened four synthetic peptides and identified two
peptides, namely RRIKA and RR, with potent antimicrobial activity against a panel of clinical
and drug resistant Staphylococci strains. In addition, we investigated the synergistic activity of
these peptides in combination with clinically relevant antimicrobials and examined the peptides’
ability to disrupt staphylococcal biofilms. Moreover, we performed a series of experiments to
explore the antibacterial mechanism of action of RRIKA and RR and examined the toxicity of
these peptides toward mammalian cells.
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MATERIALS AND METHODS
Bacterial strains and media
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All Staphylococcus strains tested in this study are presented in table (1). Mueller-Hinton broth
(MHB) and Mueller-Hinton agar (MHA) were purchased from Sigma-Aldrich, while trypticase
soy broth (TSB) and trypticase soy agar (TSA) were purchased from Becton-Dickinson,
Cockeysville, Md.
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Reagents, peptides and antibiotics
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Nisin (Sigma, N5764), melittin from honey bee venom (Sigma, M2272), magainin I (Sigma,
M7152), vancomycin hydrochloride (Gold Biotechnology), linezolid (Selleck Chemicals),
ampicillin sodium salt (IBI Scientific), recombinant lysostaphin (3000 U/ mg)
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from Staphylococcus simulans (Sigma, L9043) and calcein AM (Molecular Probes, Life
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Technologies) were all purchased from commercial vendors.
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Peptide synthesis
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Peptides RRIKA, RR, KAF, and FAK were synthesized on Knorr-amide resin (Synbiosci Corp.)
using standard FMOC chemistry. Two different chemistries were used to couple each amino
acid. The first coupling reagents were N-hydroxybenzotriazole (HoBt)/N, N’-
diisopropylcarbodiimide (DIC) and the second coupling reagents were 2-(1Hbenzotriazole-1-yl)-
1, 1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and lutidine. For FITC labeled
YARA, an aminohexanoic acid spacer was added to the N-terminus to serve as a spacer for the
addition of FITC isomer 1 (Molecular Probes). The FITC isomer was solubilized in 12:7:5
pyridine/DMF/DCM and incubated with the deprotected peptide overnight. A ninhydrin test was
used to check complete coupling of FITC to the peptide. Following synthesis, the peptide was
cleaved from the resin with a trifluoroacetic acid-based cocktail, precipitated in ether, and
recovered by centrifugation. The recovered peptide was dried in vacuo, resuspended in MilliQ
purified water, and purified using an FPLC (ÄKTA Explorer, GE Healthcare) equipped with a
22/250 C18 prep-scale column (Grace Davidson). An acetonitrile gradient with a constant
concentration of 0.1% trifluoroacetic acid was used to achieve purification. Desired molecular
weight was confirmed by time-of-flight MALDI mass spectrometry using a 4800 Plus MALDI
TOF/TOF™ Analyzer (Applied Biosystems).
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Antibacterial assays
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Minimum inhibitory concentrations (MIC) of peptides and antibiotics were evaluated with the
broth microdilution technique in MHB with an initial inoculum of 5×105 cells non-treated
Polystyrene microtiter plates (CytoOne, CC7672-7596) in accordance with the Clinical and
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Laboratory Standards Institute (CLSI) (15). The MIC was interpreted as the lowest concentration
of peptide or antibiotic that completely inhibited the visible growth of bacteria after 16 hr
incubation of the plates at 37°C. Each agent was tested in triplicate in at least two independent
experiments. The highest MIC value was reported.
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Antimicrobial activity in the presence of salts
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To investigate the activity of peptides in the presence of high salt concentrations, the MIC was
again identified as described above except, fixed concentrations of NaCl or MgCl2 were added to
each well of the microtiter plate. For further analysis of the effect of salinity on killing ability of
peptides; logarithmic phase of MRSA USA300 and VRSA VRS13 at 5×105 colony forming
units/ml (CFU/ml) were incubated with 2 × MIC of RRIKA and RR, in MHB, with different
concentrations of NaCl (0, 50, 100, 150 mM) or MgCl2 (0, 1, 2, 3 mM) for 4 hr. Bacteria were
serially diluted and plated, in triplicate, on TSA plates. CFUs were counted after incubation of
plates for 24 hr at 37°C.
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Time-kill assay
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An overnight culture of MRSA USA300 was diluted in fresh MHB and incubated until optical
density (OD600) ≈1, was reached, then washed twice with phosphate buffered saline (PBS)
diluted to ~5×105 CFU/ml in MHB. Peptides, vancomycin and linezolid were added at
concentrations equivalent to 5 × MIC. Peptide diluent (sterile water) served as a negative control.
Bacterial cell viability was monitored up to 24 hr. Samples were removed at specific intervals,
serially diluted in PBS, and plated, in triplicate, on TSA plates. CFUs were counted after 24 hr
incubation of plates at 37°C. To study the effect of serum and salinity on the killing kinetics of
the peptides, a time-kill assay was performed as described above with the exception that bacteria
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and peptides at 5 × MIC were incubated in MHB alone or with 10% fetal bovine serum (FBS) or
incubated in PBS (containing 137 mM NaCl).
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Synergy with antimicrobials
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The fractional inhibitory concentration (FIC) index was utilized to determine the relationship
between antimicrobial agents. Peptide MICs against test microorganisms were determined in
triplicate samples. Two-fold serial dilutions of antimicrobials (lysostaphin, vancomycin,
linezolid and nisin) were tested in the presence of a fixed concentration of peptide, equals to ¼ ×
peptide MIC. It is worthy to note that none of the peptides killed the test microorganisms at their
quarter MICs. The FIC index was calculated as follows: FIC = 0.25 + MIC (antibiotic in
combination)/MIC (antibiotic alone); 0.25 is equal to MIC (peptide in combination)/MIC
(peptide alone). An FIC index of ≤ 0.5 is considered to demonstrate synergy. Additive effect was
defined as an FIC index of 1. Antagonism was defined as an FIC index of > 4(16).
To further investigate the synergism of peptides with lysostaphin, the time-kill method of
determining synergy was utilized as described elsewhere (17). MRSA USA300 and VRSA
VRS13 were incubated with ½ × MIC of RRIKA or ½ × MIC of RR alone; or in combination
with ¼ × MIC of lysostaphin (MIC of lysostaphin against MRSA USA300 and VRSA VRS13
were 0.04 and 0.08 µM, respectively). Samples were obtained at different time points, then
diluted and plated on TSA plates. The plates were incubated for 24 hr at 37°C before CFU were
determined. Synergy was defined as a 100-fold or 2-log10 decrease in colony count by the
combination of two agents together as compared to each agent tested alone. Additivity or
indifference was defined as a 10-fold change in CFU and antagonism was defined as a 100-fold
increase in CFU by the combined peptide treatment in comparison to the single treatment.
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Bacterial membrane disruption activity (Bacteriolysis)
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Cell lysis, as indicated by a decrease in OD595, was determined as described before (18). Briefly,
MHB was inoculated with an overnight culture of MRSA USA300 and incubated aerobically at
37°C until an OD595≈0.6 was reached and then diluted to OD595≈0.2 in MHB (equivalent to ≈108
CFU/mL). 200 µL of diluted bacteria was added to all wells of a 96-well plate. RRIKA and RR
were added in concentrations equivalent to 4 × MIC. Nisin at 4 × MIC was used as a positive
control and untreated bacteria served as a negative control. MHB with the same concentrations
of drugs served as blanks. Turbidity was monitored at defined intervals up to 10 hr in a
Molecular Devices Vmax microplate reader at 595 nm absorption. The assay was carried out in
triplicate samples for each treatment regimen.
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Calcein leakage assay
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Membrane permeabilization of S. aureus by peptides was monitored and quantified by the
leakage of the preloaded fluorescent dye, calcein, as described before (19). MRSA USA300 was
grown in MHB to logarithmic phase at 37°C. Cells were then harvested by centrifugation,
washed twice with PBS, and then adjusted spectrophotometrically to an OD600 of 1.0 (≈109
CFU/ml) in PBS containing 10% (vol/vol) MHB. Then MRSA cells were incubated with 3 µM
calcein AM for 1 hr at 37°C. Calcein-loaded cells were harvested by centrifugation (3,000 × g,
10 min), suspended in PBS, and diluted to achieve a final inoculum of 107 CFU/ml. Aliquots of
100 µL were then added into a sterile black-wall 96-well plate. RRIKA and RR were added in
concentrations equivalent to 5 × and 10 × MIC. Bacteria treated with peptide diluent (sterile
water) and nisin (10 µg/mL) served as negative and positive controls, respectively. Calcein
leakage was measured for 120 min using a fluorescence plate reader (FLx800 model BioTek®
Instruments, Inc. Winooski, Vermont). Membrane permeabilization (%) was calculated as the
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absolute percent calcein leakage by peptides with respect to calcein-loaded with no-peptide
treated cells. Experiments were done in triplicate and repeated independently twice.
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DNA binding assay
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The ability of RRIKA and RR to bind DNA was investigated by the electrophoretic mobility
shift assay as has been previously described (20). In brief, increasing concentrations of RRIKA,
RR and magainin were incubated with 250 ng of plasmid DNA (pUC19) in 30 µL binding buffer
(10 mM Tris-HCl, 1 mM EDTA buffer, pH 8.0) at room temperature for 30 min. After
incubation, the DNA was analyzed by 1% gel electrophoresis. DNA migration was visualized by
ethidium bromide staining.
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Quantification of activity against biofilms
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Biofilm-forming clinical isolates of S. aureus (ATCC 6538) and S. epidermidis (ATCC 35984)
were grown overnight in TSB. After incubation, cultures were diluted 1:100 in TSB
supplemented with 1% glucose. Diluted bacteria were inoculated in either 24- or 96-well flat
bottom cell culture plates (polystyrene) and incubated at 37°C for 24 hr. The culture medium was
subsequently removed and wells were carefully washed with PBS three times to remove
planktonic bacteria before re-filling wells with fresh MHB. Peptides and antibiotics were added
at different concentrations and plates were incubated at 37°C for 24 hr. After the removal of
medium at the end of incubation, wells were rinsed by submerging the entire plate in a tub
containing tap water. Biofilms were stained with 0.1% (w/v) crystal violet for 30 min. After
staining, the dye was removed and the wells washed with water. The plates were dried for at
least 1 hr, prior to addition of ethanol (95%) to solubilize the dye bound to the biofilm. The
optical densities (OD) of biofilms were measured at 595 nm absorbance by using a microplate
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reader (Bio-Tek Instruments Inc.). An inverted microscope (Vista Vision, VWR), with attached
camera and 25X objective was used to photograph the biofilm in 24-well plates.
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Hemolysis assay
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Human red blood cells (RBCs) (Innovative Research “Novi, MI”) were pelleted by
centrifugation at 2000 rpm for 5 min followed by washing three times with PBS. An 8%
suspension (v/v) of RBCs was prepared in PBS and 50 μL of the solution was transferred to a
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96-well plate. Then, 50 μL of different concentration of peptides in PBS were added to give a
final suspension of 4% (v/v) of RBCs. PBS served as a negative control. 0.1 % triton X-100 as
well as 5 µM melittin served as a positive control. The plate was incubated at 37°C for 1 hr.
The plate was subsequently centrifuged at 1000 rpm for 5 min at 4°C. 75 μL aliquots of the
supernatants in each well were carefully transferred to a new sterile 96-well plate. Finally the
hemolytic activity was evaluated by measuring the optical absorbance at OD405 with a microplate
reader (Vmax, Molecular Devices). The hemolysis percentage was calculated based on the 100%
release with 0.1 % triton X-100 or 5 µM melittin. Experiments were done in triplicates.
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Cytotoxicity assay
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The toxicity of RRIKA, RR and melittin against HeLa cells was carried out using the CellTiter
96® AQueous Non-Radioactive Cell Proliferation Assay kit from Promega (21). Briefly, ~ 2 × 104
HeLa cells suspended in 200 µL of DMEM supplemented with 10% FBS, were seeded in 96-
well plates and incubated at 37°C in a 5% CO2 atmosphere for 24 hr. The HeLa cells were further
incubated with different concentration of peptides for 24 hr. At the end of the incubation period,
the culture media was discarded and the cells in each well were washed with PBS. 100 µL of cell
culture media and 20 µL CellTiter 96® AQueous assay reagent were added next (solution reagent
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contains a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-
(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] ). The plate was returned to the incubator for 2
hr to allow color development. The intensity of color was quantified at 490 nm using a 96-well
microplate reader (Vmax, Molecular Devices). Results were expressed as the percentage mean
absorbance by cells upon incubation with peptide with respect to incubation with the control
(untreated wells).
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Statistical analyses
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The mean absorbance of crystal violet in the biofilm reduction was compared between the groups
using the two-tailed Student t test (p 0.05). All statistical analyses were performed with
Microsoft Excel’s statistical program.
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RESULTS:
Antimicrobial activity
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The amino acid sequences and characteristics of the synthetic peptides used in this study are
shown in Table 2. We screened the synthetic peptides against a panel of clinical and drug-
resistant Staphylococcal strains (Table 1). RRIKA inhibited the growth of methicillin-sensitive
S. aureus (MSSA), MRSA, VISA, VRSA, linezolid-resistant S. aureus, methicillin-resistant S.
epidermidis and multidrug-resistant strains from concentrations ranging from 2 to 4 µM. RR
inhibited the same isolates with MICs ranging from 8 to 32 µM. In contrast, KAF and FAK
peptides were inactive against all strains tested up to 64 µM (data not shown).
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Bacterial killing kinetics
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Figure 1 presents the rate of microbial killing by RR, RRIKA, vancomycin and linezolid when
MRSA USA300 was exposed to 5 × MIC of each treatment over a 24 hr incubation period at 37
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ºC. Both RRIKA and RR exhibited a rapid bactericidal effect with 3-log10 reduction (99.9 %
clearance) within 60 and 90 min, respectively. In addition, both peptides completely eliminated
the inoculum (5×105 CFU/mL) within 120 and 180 min, respectively. In comparison,
vancomycin achieved a 3-log10 bacterial reduction only after 24 hr; while linezolid (a protein
synthesis inhibitor) exhibited a bacteriostatic effect producing only a single log bacterial
reduction over the 24 hr incubation. The killing kinetics of RRIKA and RR in PBS (containing
137mM NaCl) did not significantly change when compared to MHB (figure 2). In the presence
of 10% FBS, RRIKA retained its bactericidal activity, though it required longer to kill the
bacteria completely (6h), while RR lost its antibacterial activity at the same condition.
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Antimicrobial activity in the presence of salts
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The MIC of RRIKA and RR for MRSA USA300 was determined in the presence of NaCl or
MgCl2. There was no increase in the MICs of RRIKA and only a one fold increase in case of RR
was observed in the presence of 100 mM NaCl. Moreover, the antimicrobial activity of RRIKA
and RR was not impeded in the presence of MgCl2 at a concentration equivalent to reported
physiological conditions (2 mM). To study the effect of salinity on the killing ability of the
peptides, CFU were counted after incubation of MRSA USA300 and VRSA VRS13 strains with
RRIKA and RR (2 × MIC) in increasing concentrations of NaCl or MgCl2. As shown in
supplementary figure 1, there was no significant difference in the bactericidal activity of RRIKA
in the presence of NaCl up to 150 mM. Similar patterns were observed for RR with the exception
of one log10 increase in CFU observed at 150 mM NaCl; however this peptide retained its
bactericidal activity at this concentration (producing a 3-log10 reduction). Furthermore,
physiological concentrations of MgCl2 (1, 2 and 3 mM) had no effect on the killing ability of
both RRIKA and RR.
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Synergy with antimicrobial agents
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Both RRIKA and RR acted synergistically with lysostaphin against MSSA, MRSA and VRSA
strains with a FIC index equal to 0.26 (Table 3). When both peptides were combined together,
only one fold decrease in each peptide’s MIC was observed (FIC index = 0.75). However, an
additive effect was observed when each peptide was combined with vancomycin, linezolid or
nisin against tested strains (data not shown). There were no antagonistic effects observed
between the peptides and antimicrobials against all strains tested.
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Time-kill synergy studies revealed RRIKA and RR, at only ½ × MIC enhanced the killing of
lysostaphin (at ¼ × MIC) more than 1000 fold and completely eradicated MRSA and VRSA
isolates within 20 minutes (figure 3).
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Bacterial membrane disruption activity (Bacteriolysis)
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Since evidence of rapid cell lysis of treated cultures is indicative of membrane damaging activity
of antimicrobial peptides (22, 23), we assessed whether the bactericidal activity of our peptides
was associated with lysis of bacterial cells by measuring culture turbidity over time after
treatment. We monitored the turbidity of MRSA cultures treated with 4 × MIC of peptides over a
10 hr period at OD595 via a microplate reader. As shown in figure 4 both RRIKA and RR caused
a rapid decrease in OD595 even with a high inoculum concentration of 108 CFU/ml. Peptide
RRIKA produced greater than 50% and 86% reduction in turbidity after 2.5 and 6 hr,
respectively. RR produced a similar percentage of turbidity as RRIKA albeit at a slower rate
(after 4.5 and 10 hr). Nisin at 4 × MIC, a polycyclic antibacterial peptide which is known as a
membrane perturbing agent (24), lysed cells at a slightly higher rate than RRIKA with more than
70% reduction in turbidity observed after 2.5 h. In contrast, wells treated with 4 × MIC of
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vancomycin (a cell wall biosynthesis inhibitor) had no effect on culture turbidity within the same
time frame.
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Permeabilization of S. aureus membrane (calcein leakage assay)
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We monitored the effect of the peptides on MRSA membrane integrity by using the calcein
leakage assay as described previously (19, 25). Damage of membrane integrity is indicated by
calcein leakage from bacterial cells, leading to a reduction in fluorescence intensity. Figure 5
demonstrates that both peptides perturb the S. aureus cell membrane leading to leakage of
preloaded calcein in both a concentration- and time-dependent manner. RRIKA was faster and
more potent in comparison to RR in membrane perturbation. At 5 × MIC, RRIKA and RR
caused more than 60% and 30% leakage respectively within 60 minutes. When the concentration
of peptide was increased to 10 × MIC, a significant change in membrane damage was observed
for both peptides. There was a more than 75% and 60% reduction in fluorescence intensity
measured for RRIKA and RR respectively in the same time frame. Nisin at 5 × MIC resulted in
more than 70% calcein leakage from cells, which is comparable to RRIKA at 10 × MIC.
Vancomycin, as expected, had no effect on the bacterial cell membrane integrity even at high
concentration (10 × MIC).
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DNA binding properties
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Since there was no obvious evidence that the antibacterial effect of AMPs was restricted to
perforation of membranes, we explored the feasibility of other intracellular targets, such as DNA
by assessing the DNA-binding properties of peptides (26, 27). As shown in figure 6, both
peptides were able to bind the plasmid DNA and delay its electrophoretic migration into agarose
gel, in a dose-dependent manner. At 8 µM concentration, of RRIKA peptide, a fraction of the
plasmid DNA was still able to migrate into the gel as non-complexed DNA; however at 16 µM,
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the majority of DNA remained in the gel’s wells. At higher concentrations, complete retardation
of DNA migration was exhibited, implying that the DNA was aggregated by peptide. Similar
patterns of migration were observed with the RR peptide except that free DNA was still seen at
16 µM concentration. In contrast, magainin, a cationic antimicrobial peptide of amphibian skin,
lacks this ability to bind DNA even at concentrations up to 80 µM.
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Quantification of activity against biofilms
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In order to evaluate the efficacy of RR and RRIKA against established biofilms, we measured
the biofilm mass post-treatment by crystal violet staining biofilms formed by clinical isolates of
S. aureus and S. epidermidis. As observed in figure 7a, both peptides significantly reduced S.
aureus biofilms as compared to the antibiotics of choice. RRIKA and RR, at only 2 × MIC,
dispersed more than 65% of mature biofilms (p < 0.01), while vancomycin and linezolid, at 16 ×
MIC, was only capable of reducing 40% of biofilm mass (p < 0.01). There was significant
difference between peptide and antibiotics treated biofilms (p < 0.01).
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With regards to S. epidermidis, both RR and RRIKA, at 32 and 64 × MIC, were able to reduce
more than 50% of biofilm mass respectively (p < 0.01) (figure 7b). Vancomycin and linezolid,
on the other hand, reduced only 9.5 and 10.7% of biofilm mass, respectively (p < 0.01) at
concentrations equivalent to 256 × MIC. Also we found statistical significance between peptide
and antibiotics treated biofilms (p < 0.01).
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Hemolysis assay and cell toxicity
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We assessed the release of hemoglobin from human erythrocytes exposed to different
concentrations of peptides. As depicted in figure 8, even at concentrations as high as 300 µΜ,
RRIKA and RR exhibited minimal hemolysis against RBCs (maximum of 10% hemolysis
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observed). In contrast, melittin completely lysed RBCs (100% hemolysis) at a significantly lower
concentration of 5 µM.
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The cytotoxic effect of the peptides on HeLa cells was evaluated by the MTS assay. As depicted
in figure 9, neither RRIKA nor RR was toxic to the mammalian cells tested up to concentrations
of 32 µM and 64 µM, respectively. These values correlate to 8 and 4 × MIC for RRIKA and RR,
respectively. The half maximal effective concentration (EC50) of RRIKA and RR against
macrophage cell lines was 64 and 128 µM respectively. On the other hand, melittin showed high
toxicity even at bacteriostatic concentration with EC50 less than 5 µM.
354
DISCUSSION:
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361
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363
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368
In this study, we evaluated the antimicrobial activity of two short synthetic peptides, and
revealed their potential mechanisms of action. We observed that RRIKA and RR exhibited
potent antibacterial activity against all tested S. aureus isolates. Moreover, our peptides
demonstrated activity against multiple clinical isolates of MRSA, particularly MRSA USA300, a
community-associated strain which is responsible for outbreaks of staphylococcal skin and soft-
tissue infections (SSTI) in the United States (28). Similarly, the potent bactericidal activity was
observed in other clinical MRSA isolates (USA100, USA200 and USA500) that are resistant to
various antibiotic classes including macrolides, aminoglycosides, lincosamides, and
fluoroquinolones. Additionally, we found that these peptides retained their activity against
strains of S. aureus which have resistance to drugs of choice in treating MRSA infections,
namely linezolid (MRSA NRS119) and vancomycin (VRSA strains). It is worthy to note that, the
C terminus of the RR peptide was modified to contain three additional amino acids (isoleucine,
lysine and alanine) which made the RRIKA peptide more amphipathic and hydrophobic and also
increased its positive net charge. These modifications augmented the antibacterial activity of
16

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371
RRIKA which exhibited MICs two to eight folds lower than those of RR. The enhanced efficacy
of RRIKA may reveal a functional structure activity relationship and provide a template for
future peptide synthesis.
372
373
374
375
Time-kill kinetics revealed a major advantage of the synthetic peptides over tested antibiotics.
While RRIKA and RR eliminated MRSA within 1-2 hr after treatment, vancomycin required 24
hr to reduce the bacterial CFU by 3-log10. Linezolid, on the other hand, exhibited only a
bacteriostatic effect.
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
Growth kinetic measurements of MRSA exposed to peptides clearly demonstrated that RRIKA
and RR peptides exhibited kinetic behavior similar to membrane lytic peptides such as the
lantibiotic nisin (24) and amphibian magainin (23) but different from non-lytic peptides such as
fungal plectasin (29) and amphibian buforin II (23). These data suggest that our peptides act by
disrupting the integrity of the bacterial membrane. To validate this hypothesis, we studied the
effect of peptides on MRSA membranes using a calcein leakage assay. Similar to the well-
known membrane damaging peptide, nisin, both RRIKA and RR permeabilized S. aureus cells in
a concentration and time-dependent manner. These observations clearly validate that the
mechanism of bacterial killing by our peptides is mediated by pore formation and disruption of
the bacterial cell membrane, leading to leakage of cytoplasmic contents and ultimately cell lysis.
However, recent evidence suggests that lysis of bacterial cells by pore formation is not the only
mechanism of microbial killing of some AMPs and there are other possible intracellular targets,
such as aggregation of DNA (26). To further explore this possibility, we examined the bacterial
DNA-binding ability of the peptides by electrophoretic gel retardation. Our data indicated that
both RRIKA and RR bind with bacterial DNA and alter its electrophoretic mobility in agarose
gels. Although a detailed study on the interaction between peptides and nucleic acids in vivo is
17

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396
needed, the current results lead us to suggest that RRIKA and RR might inhibit the bacterial
functions by binding to bacterial DNA after perturbing the cell membranes, resulting in an
augmented effect by two different mechanisms. While RRIKA and RR are not toxic to
mammalian cells at the levels used here, possible crossing of the peptides to the nuclear
membrane and binding to eukaryotic DNA must be examined further.
397
398
399
400
401
402
403
404
405
406
407
408
Major limitations with the use of AMPs for systemic applications are their possible inactivation
by serum or physiological concentrations of salts. Serum inactivate peptides either through
cleavage by proteases or through binding with proteins or lipids (30) We observed that RRIKA
but not RR retained its bactericidal activity, although at a slower rate, in the presence of 10%
FBS. Furthermore, both RRIKA and RR retain their antibacterial activity when tested in
increasing concentration of NaCl and MgCl2; in contrast to many well studied AMPs (such as
LL-37, human β-defensin-1, gramicidins, bactenecins, and magainins) which showed
substantially reduced antibacterial activities under the same conditions (11). Previously, Turner
et al. reported that LL-37 and HNP-1 demonstrated a 12-fold and 100-fold increase in the MIC
of MRSA, respectively when 100 mM NaCl was added to the test medium (10). The ability to
resist the effects of salt and serum provide a selective advantage for our peptides for potential
therapeutics in physiological solutions.
409
410
411
412
413
414
After identifying that our peptides were capable of inhibiting bacterial growth alone, we wanted
to explore their ability to be used in combination with other antimicrobials such as lysostaphin.
Lysostaphin is a zinc metalloenzyme that specifically cleaves the abundant pentaglycine cross-
bridges of the staphylococcal cell wall. Several studies have reported the potential applications
of lysostaphin in the treatment of staphylococcal infections, however, S. aureus has developed
resistance against lysostaphin via different mechanisms (31). When we tested both RR and
18

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420
421
422
RRIKA in combination with lysostaphin, we observed a synergistic relationship demonstrated by
complete eradication of MRSA and VRSA within 20 minutes (at very low concentrations). The
observed synergistic effect may be due the cleaving of the cell wall peptidoglycan by
lysostaphin, which allows more access of peptides to the bacterial membrane. The synergistic
relationship observed between our peptides and lysostaphin is important as it has the advantage
of reducing the emergence of bacterial resistance to both agents while also minimizing drug-
associated toxicity (by lowering the therapeutic dose needed for each antimicrobial agent to
effectively treat MRSA infection).
423
424
425
426
427
428
429
430
431
One of the difficult challenges facing current antibiotics is bacterial biofilms, which are vital in
the pathogenesis of staphylococcal infections. Biofilms hinder the penetration of antimicrobials
to access bacteria, leading to failure of treatments (32). Our data showed that both RR and
RRIKA are capable of disrupting S. aureus and S. epidermidis biofilms more efficiently than
drugs of choice (vancomycin and linezolid). Most bacteria living in biofilms are either slow-
growing or non-growing dormant cells that are difficult to treat with antibiotics that normally
inhibit macromolecular synthesis in growing cells (33). However our peptides target mainly the
microbial cell membrane, a characteristic that is not only in dividing organisms, but also in
quiescent cells or stationary phase bacteria (34).
432
433
434
435
436
Cell toxicity is one of the major limitations in the development of antibacterials, particularly if
the target of action is the cell membrane (7). We observed minimal hemolysis (less than 10%)
with our peptides even with high concentrations (300 µΜ). On the other hand, 5 µM of melittin
completely lysed human RBCs (100% hemolysis). Furthermore, RRIKA and RR were not toxic
to HeLa cells at 8 and 4 × MIC, respectively.
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454
455
456
In conclusion, our findings reveal the potent bactericidal action of peptides RRIKA and RR
against MRSA. The mechanism of action of the peptides is mainly due to bacterial lysis as a
consequence of bacterial membrane disruption and possibly by binding of the peptides to
bacterial DNA, which interferes with necessary cellular functions vital for microbial survival.
Such effects are extremely challenging for pathogens to overcome by developing resistance;
unlike current antibiotics, which usually inhibit metabolic pathways that can lead to bacterial
resistance (8). To date, issues such as poor pharmacokinetics have limited the potential systemic
applications of therapeutic AMPs (7, 8, 30). Therefore, AMPs which have advanced into
preclinical or clinical trials, are indicated for topical treatment of bacterial skin infections (7, 8,
30) such as Pexiganan (Access Pharmaceuticals, Inc.) for curing diabetic foot ulcers (35). S.
aureus is the most frequently isolated microorganism in diabetic foot infections, and those
caused by MRSA are associated with the worse outcomes and more frequent amputations (36-
41). Additionally, bacterial biofilms appear to play an important role in increasing the difficulty
of treating these ulcers (40). Furthermore, it has been proven recently that the clinical severity of
S. aureus skin infection is driven by the inflammatory response to the bacteria, rather than
bacterial burden (42-44). Taken together, the characteristics of the presented peptides with
combined bactericidal, anti-biofilm and anti-inflammatory effect may offer an effective way for
treating Staphylococcal skin infection. Therefore, these results support the potential for further
study and development of RRIKA and RR as topical therapeutics particularly in an era of
emerging drug resistance.
457
ACKNOWLEDGEMENT
458
459
Mohamed F. Mohamed and Maha I. Hamed are supported by a scholarship from the Egyptian
Cultural and Educational Bureau (ECEB) in Washington DC, USA. We would like to thank the
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Network of Antimicrobial Resistance in Staphylococcus aureus (NARSA) program supported
under NIAID/NIH Contract # HHSN272200700055C for providing Staphylococcus aureus
strains used in this study. The authors wish to acknowledge Mr Haroon Mohammed for editing
the manuscript.
464
465
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497
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23

10
8
C o n tro l
R R IK A
6
R R
4
V a n c o m y c in
L inezolid
2
0
0
4
8
12
16
20
24
T im e (h r)
Figure 1: Bacterial killing kinetics of RRIKA, RR, vancomycin and linezolid at 5X
MIC against MRSA USA300 in MHB (Mueller Hinton broth). Samples treated with
peptide diluent (sterile water) were used as a control. The results are given as means ±
SD (n = 3; data without error bars indicate that the SD is too small to be seen)

(A ) R R IK A
(B ) R R
10
8
10
8
C o n tro l
6
6
C o n tro l
R R IK A in M H B
R R IK A in P B S
R R in M H B
R R in P B S
R R + 1 0 % F B S
4
4
R R IK A + 1 0 % F B S
2
2
0
0
0
2
4
6
8
10
12
14
0
2
4
6
8
10
12
14
T im e (h r)
T im e (h r)
Figure 2: Effect of culture conditions on killing kinetics of RRIKA (A), RR (B) at 5X MIC
against MRSA USA300. Abbreviations, MHB “Mueller Hinton broth”, PBS “phosphate
buffered saline” or MHB+10% FBS “fetal bovine serum”. Untreated samples were used as
a control was. The killing curves were identical (overlapping in the figure) for RRIKA in
MHB and in PBS. Each experiment was done in triplicate, and the values represent means ±
SD (data without error bars indicate that the SD is too small to be seen).

(A ) M R S A "U S A 3 0 0"
(B ) V R S A "V R S 1 3 "
10
8
10
8
C o n tro l
C o n tro l
R R IK A 0.5 X M IC
R R IK A 0.5 X M IC
R R 0 .5 X M IC
R R 0 .5 X M IC
6
6
L y s o s ta p h in 0 .2 5 X M IC
L y s o s ta p h in 0 .2 5 X M IC
R R IK A 0.5 X M IC
+
4
4
R R IK A 0.5 X M IC
+
L y s o s ta p h in 0 .2 5 X M IC
L y s o s ta p h in 0 .2 5 X M IC
R R 0 .5 X M IC
+
2
2
R R 0 .5 X M IC
+
L y s o s ta p h in 0 .2 5 X M IC
L y s o s ta p h in 0 .2 5 X M IC
0
0
0
100
200
300
400
0
100
200
300
400
T im e (m in u tes)
T im e (m in u tes)
Figure 3: Synergistic killing of RRIKA and RR with lysostaphin by time kill assay. MRSA
USA300 (A) and VRSA VRS13 (B) were incubated with 0.5X MIC of RRIKA or RR alone or
in combination with 0.25X MIC of lysostaphin. Samples were obtained at different time
points, then plated and counted. Control was untreated samples. The killing curves were
identical (overlapping in the figure) for RRIKA and RR in combination with lysostaphin. The
results are given as means ± SD (n = 3; data without error bars indicate that the SD is too small
to be seen)

0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
C ontrol
R R IK A
RR
N isin
V a n co m ycin
0
2
4
6
8
10
T im e (h r)
Figure 4: Killing kinetics of MRSA USA300 exposed to 4X MIC of RRIKA, RR, nisin,
vancomycin and sterile water (control) as observed by measuring OD595 by microplate
reader over time. Results are representative of two separate experiments, each was done in
triplicate. Error bars represent standard deviation values (data without error bars indicate that
the SD is too small to be seen)

80
60
40
20
0
C ontrol
N isin 5  M IC
R R IK A 10  M IC
R R IK A 5  M IC
R R 10  M IC
R R 5  M IC
V a n co m ycin 1 0  M IC
10
20
30
40
50
60
T im e (m in )
Figure 5: Permeabilization of the cytoplasmic membrane of MRSA USA300 as a function of
peptide concentration, indicated by percent of calcein leakage for 60 min exposure. The
results are given as means ± SD (n = 3; data without error bars indicate that the SD is too
small to be seen).

Figure 6: Interaction of peptides with plasmid DNA.
Binding was assayed by measuring inhibition of
migration by plasmid DNA. Different concentrations of
peptides were incubated with 250 ng for 1 hr at room
temperature prior to electrophoresis on a 1.0% agarose
gel. The numbers above the lanes represent the
concentration (in µM) of magainin, RRIKA or RR. Lane
C, control consisting of plasmid DNA only.

Fig. 7a