AAC Accepted Manuscript Posted Online 8 February 2016 Antimicrob. Agents Chemother. doi:10.1128/AAC.02972-15 Copyright © 2016, American Society for Microbiology. All Rights Reserved.
1
Novel engineered peptides of a phage lysin as effective antimicrobials against multidrug
2
resistant Acinetobacter baumannii
3
Mya Thandar1§, Rolf Lood1,2§, Benjamin Y. Winer1,3§, Douglas R. Deutsch1, Chad W.
4
Euler1,4,5, Vincent A. Fischetti1#
5
1
6
York Avenue, New York, NY 10065, USA
7
2
8
Lund University, Sweden
9
3
Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, 1230
Present address: Department of Clinical Sciences Lund, Division of Infection Medicine,
Present address: 119 Lewis Thomas Laboratory, Washington Road, Princeton, New Jersey,
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NJ, 08544-1014, USA
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4
12
Hunter College, CUNY, New York, NY, 10065, United States of America.
13
5
14
College, New York, NY, 10065, United States of America.
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Short title: Antibacterial peptides active against Acinetobacter baumannii
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# Corresponding author: Vincent A. Fischetti, [email protected]
17
§
Present address: Department of Medical Laboratory Sciences, Belfer Research Building,
Present address: Department of Microbiology and Immunology, Weill Cornell Medical
These authors contributed equally.
18 19
1
20
Abstract
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Acinetobacter baumannii is a Gram-negative bacterial pathogen responsible for a range of
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nosocomial infections. The recent rise and spread of multidrug resistant A. baumannii clones
23
has fueled a search for alternative therapies, including bacteriophage endolysins with potent
24
antibacterial activities. A common feature of these lysins is the presence of a highly
25
positively charged C-terminal domain with a likely role in promoting outer membrane
26
penetration. In the current study, we show that the C-terminal amino acid 108-138 of phage
27
lysin PlyF307, named P307, alone was sufficient to kill A. baumannii (>3-logs). Furthermore,
28
P307 could be engineered for improved activity, the most active derivative being P307SQ-8C
29
(>5-log kill). Both P307 and P307SQ-8C showed high in vitro activity against A. baumannii in
30
biofilms. Moreover, P307SQ-8C exhibited MICs comparable to levofloxacin and ceftazidime
31
and acted synergistically with polymyxin B. While the peptides were shown to kill by
32
disrupting the bacterial cytoplasmic membrane, they did not lyse human red blood cells or B
33
cells; however, serum was found to be inhibitory to lytic activity. In a murine model of A.
34
baumannii skin infection, P307SQ-8C reduced the bacterial burden by ~2-logs in 2 h. This
35
study demonstrates the prospect of using peptide derivatives from bacteriophage lysins to
36
treat topical infections and remove biofilms caused by Gram-negative pathogens.
37
Introduction
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Acinetobacter baumannii is an increasingly significant nosocomial pathogen worldwide (1).
39
Arising from both intrinsic and acquired antibiotic resistance, multi- and pan-drug resistant
40
clones of A. baumannii can readily be isolated from hospital environments (2). A. baumannii
41
has been shown to develop resistance to several classes of antibiotics, including
42
aminoglycosides, cephalosporins, carbapenems, tigecycline and colistin (3). The reasons for
43
high resistance include a high degree of genetic plasticity combined with an intrinsic
2
44
resistance to certain antibiotics due to the presence of β-lactamases, low permeability of the
45
outer membrane, and highly efficient efflux pump systems (4). Furthermore, A. baumannii is
46
prone to develop biofilms on solid surfaces, including medical devices (5). Thus, A.
47
baumannii is not only problematic as an infectious agent, but it is also increasingly difficult
48
to remove from hospital environments – a phenomenon similar to the Gram-positive
49
nosocomial pathogen Staphylococcus aureus.
50
One of the last resort antibiotics for A. baumannii is the antimicrobial peptide polymyxin B
51
(6). The bactericidal effect of polymyxin B is mediated through its positively charged Dab
52
(α,γ-diaminobutyric acid) residues interacting with LPS and destabilizing the outer
53
membrane (7). Many antimicrobial peptides kill in a similar way: clustered cationic residues
54
permeabilize the bacterial membrane to cause lysis and death (8). Due to this mechanism of
55
action, most of the membrane-acting antimicrobial peptides commonly have cytotoxic effects
56
on eukaryotic cells (9). Indeed, polymyxin B has severe side effects: cytotoxicity,
57
nephrotoxicity and neurotoxicity (10). Since careful administration is required to avoid its
58
toxicity, the dose range of polymyxin B is limited and resistant strains of A. baumannii have
59
been documented (11).
60
Recently, there has been a growing interest in the use of bacterial viruses (i.e., bacteriophage
61
therapy) to treat infections by Gram-negative bacteria, including A. baumannii (12-14).
62
Several phages that infect A. baumannii have been identified and characterized. However,
63
their restricted spectrum (killing only ∼60% of A. baumannii isolates) limits the effectiveness
64
of such phages as therapeutic agents (12, 13). Using an alternative bacteriophage-based
65
approach, our group and others have taken advantage of the lytic enzymes (lysins) encoded
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and produced by bacteriophages during lytic proliferation (15-18). Bacteriophage lysins are
67
classified as peptidoglycan hydrolases, being able to cleave a variety of bonds in the bacterial
3
68
peptidoglycan. Cleavage of the cell wall by lysins destabilizes the peptidoglycan and
69
weakens the structural framework, resulting in hypotonic lysis. While purified lysins are
70
effective at killing Gram-positive bacteria (19), the outer membrane of Gram-negative
71
bacteria largely limits lysins from accessing and cleaving the subjacent peptidoglycan.
72
Different strategies have been employed to increase the efficiency of lysins against Gram-
73
negative bacteria, including the use of the chelating agent EDTA (16, 17), and the genetic
74
engineering of lysins to add either highly charged/hydrophobic N-/C-terminal extensions (20)
75
or other membrane translocating domains (21, 22). However, there has been little focus on
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the intrinsic features of certain active lysins against Gram-negative bacteria, and how they
77
function to allow the lysins to cross the outer membrane and reach the subjacent
78
peptidoglycan substrate.
79
Here, we have identified a highly cationic C-terminal domain within an A. baumannii phage
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lysin as a peptide with potent antibacterial activity. We have modified the peptide to further
81
improve its activity, and have proven the high efficiency of such peptides to kill A.
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baumannii both in vitro and in vivo in a skin infection model.
83
Materials and Methods
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Bacterial strains and growth conditions
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Acinetobacter baumannii strains in this study include clinical isolates from Hospital for
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Special Surgery in New York (#1775-#1799) (18), Ohio State University (S1, S3 and S5
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provided by Dr. Vijay Pancholi), and ATCC® 17978™ from American Type Culture
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Collection. Bacteria were cultured in Trypticase Soy Broth or Brain Heart Infusion (TSB or
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BHI; Thermo Fisher Scientific, Waltham, MA, USA) at 37°C with aeration (200 rpm).
90
Stationary phase bacteria were cultured overnight while log phase bacteria were grown for 3
4
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h in fresh media from 100x dilutions of overnight cultures. Strains for determining the
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specificity of the antimicrobial peptides were cultured under the same conditions, at the
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temperatures indicated: Bacillus anthracis ΔSterne (30°C), Escherichia coli DH5α (37°C),
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Klebsiella pneumoniae ATCC® 700603TM and 10031TM (37°C), Pseudomonas aeruginosa
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PAO1 (30°C), and Staphylococcus aureus RN4220 (30°C).
96
Biofilms were set up as previously described (18). Briefly, an overnight culture of A.
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baumannii strain #1791 was diluted 1000x (~105 cfu/mL) in TSB containing 0.2% glucose
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and incubated at 37°C for 72 h in ~2.5 cm segments of PVC catheter tubing (CareFusion)
99
with the ends clamped shut to prevent evaporation. The catheter biofilms were washed with
100
autoclaved Milli-Q water to remove planktonic cells before peptide treatment. Crystal violet
101
(0.1%) was used to detect the presence of biofilm.
102
Peptide synthesis and preparation
103
Peptides were synthesized at The Rockefeller University Proteomics Resource Center. All
104
peptides were created using a Protein Technologies Symphony™ peptide synthesizer (PTI
105
Tucson, Arizona, USA) on pre-coupled Wang (p-alkoxy-benzyl alcohol) resin (Bachem,
106
Torrance, CA, USA). Reaction vessels were loaded at 25 μM and peptides were elongated
107
using Fmoc protected amino acids (Anaspec, San Jose, CA, USA) (23). Deprotection of the
108
amine was accomplished with 20% piperidine (Sigma-Aldrich) in NMP (N-
109
methylpyrrolidinone). Repetitive coupling reactions were conducted using 0.6 M HATU/Cl-
110
HOBT (azabenzotriazol tetramethyluronium hexafluorophosphate/6-chloro-1-
111
hydroxybenzotriazole)(P3 Biosystems, Shelbyville, KY, USA) and 0.4 M NMM (N-
112
methylmorpholine) using NMP (EMD) as the primary solvent (24). Resin cleavage and side-
113
chain deprotection were achieved by transferring to a 100 mL round bottom flask and reacted
114
with 4.0 mL concentrated, sequencing grade, trifluoracetic acid (Fisher) with 5
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triisopropylsilane (Fluka), degassed water, and 3,6-dioxa-1,8-octanedithiol (DODT, Sigma-
116
Aldrich) in a ratio of 95:2:2:1 over a 6 h time frame. This was followed by column filtration
117
to a 50 mL round bottom flask and TFA volume reduced to 2 mL using a rotary evaporator.
118
A standard ether precipitation was performed on the individual peptides by transferring to a
119
50 mL falcon tube containing 40 mL cold tert-butyl methyl ether (TBME, Sigma-Aldrich).
120
Samples were placed in an ice bath for 2 h to aid precipitation followed by pellet formation
121
using centrifugation (3300 rpm, 5 min). Excess ether was removed by vacuum aspiration and
122
the peptide pellets were allowed to dry overnight in a fume hood. Dried peptide pellets were
123
resolved in 20% acetonitrile and 10 mL HPLC grade water, subsampled for LC/MS and
124
lyophilized. All crude products were subsequently analyzed by reverse-phase Aquity™
125
UPLC (Waters Chromatography, Milford, MA, USA) using a Waters BEH C18 column.
126
Individual peptide integrity was verified by tandem electrospray mass spectrometry using a
127
ThermoFinnigan LTQ™ (Thermo Fisher, Waltham, MA, USA) spectrometer system.
128
Preparative chromatography was accomplished on a Vydac C18 RP preparative column on a
129
Waters 600 Prep HPLC. Individual fractions were collected in 30 s intervals, characterized
130
using LC/MS and fractions containing desired product were lyophilized. These were stored at
131
-20°C until being re-suspended in autoclaved Milli-Q water for various assays. The stock
132
solutions were then stored at 4°C.
133
Bactericidal assays
134
Unless otherwise indicated, stationary phase bacteria (overnight cultures) were used for the
135
assays. Bacteria were washed in assay buffer, re-suspended to ∼106 cfu/mL, mixed with each
136
antimicrobial peptide and incubated for 2 h at room temperature (22-25°C). After treatment,
137
the reactions were serially diluted and plated for enumeration. Influencing factors on the
138
activities of the peptides (P307 and P307SQ-8C) were examined: buffer (50 mM sodium
6
139
phosphate or Tris-HCl, pH 6.8-8.8; NaCl (0-200 mM); concentration (0-125 μg/mL P307);
140
time (1-120 min); specificity (the bacterial strains mentioned above); and growth phase (log,
141
stationary and biofilm). The buffer system for the experiments with the latter five factors was
142
50 mM Tris-HCl, pH 7.5. The biofilm-associated bacteria were re-suspended by thoroughly
143
scraping the catheter tubing and vortexing for 1 min.
144
To examine the mechanism(s) of action of the peptides, the following bactericidal assays
145
were also conducted. The contribution of disulfide bond formation to the increased activity of
146
P307SQ-8C was investigated by comparing with P307SQ-8A in the presence of the reducing
147
agent TCEP (0.1 and 1 mM) (Tris (2-carboxyethyl) phosphine hydrochloride
148
solution)(Sigma). To examine the importance of ionic interaction, the least sensitive Gram-
149
negative bacterial species at pH 7.5 were treated with P307 and P307SQ-8C at pH 8.8.
150
Experiments were conducted at least in triplicate and representative data are shown as mean
151
± standard deviation. The black horizontal lines mark the limit of detection.
152
Minimum inhibitory concentrations (MICs) and synergy
153
The broth microdilution method was used (25) to determine the MICs of levofloxacin,
154
ceftazidime, polymyxin B, P307 and P307SQ-8C for A. baumannii strains #1791, #1798, S5
155
and ATCC17978. For the antibiotics, 1.5–2 fold serial dilutions (three lower and three
156
higher) of the MICs determined by Etest (18) were included. For the peptides, two-fold serial
157
dilutions (2000–31.25 μg/mL) were tested. Overnight cultures were re-suspended to ~105
158
cfu/mL in Mueller Hinton broth (pH 7.9). Antibiotics or peptides were added to a final
159
volume of 100 μL for each dilution. Bacteria were allowed to grow at 37°C for 24 h at 220
160
rpm. The absorbance at 595 nm was then read in a SpectraMax Plus Reader (Molecular
161
Devices). The MICs were determined as the lowest concentrations of antimicrobial agents
7
162
that visibly inhibited bacterial growth. In addition, Alamar®Blue was used to confirm the
163
data obtained from OD595. Each experiment was repeated at least twice in duplicate.
164
To investigate whether synergy exists between each peptide and antibiotic, checkerboard
165
assays were conducted by mixing fractional inhibitory concentrations (FICs) of antibiotics
166
(levofloxacin, ceftazidime and polymyxin B) with those of peptides P307 and P307SQ-8C as
167
previously described (26). Isobolograms were constructed to determine the fractional
168
inhibitory concentration indices (FICIs). Each experiment was repeated at least three times.
169
DNA binding assay
170
The affinity of peptide P307 for DNA was investigated by electrophoretic mobility shift
171
assay (27). P307 was mixed with 50 ng of random DNA (1 kb amplicon of Bdellovibrio
172
genomic DNA) at different peptide to DNA ratios (0:1-15:1) in a binding buffer (5 mM Tris-
173
HCl, pH 8.0, 10 mM EDTA, 5% glycerol, 20 mM KCl, 50 μg/mL BSA). As a positive
174
control, a peptide from Bdellovibrio bacteriovorus that was previously found in our
175
laboratory to have DNA-binding capacity (amino acid sequence –
176
MASKTKKTEYIRERKKATSGKKRKAANRTKGTTKSAKTLFKD) was used
177
(unpublished results). After incubation for 1 h at 22-25°C, the peptide and DNA mixture was
178
analyzed by agarose gel electrophoresis (27).
179
Transmission electron microscopy (TEM)
180
An overnight culture of A. baumannii strain #1791 was collected, washed in 50 mM Tris-HCl,
181
pH 7.5 and re-suspended in the same buffer to ~108 cfu/mL. The bacteria were treated with
182
300 μg/mL P307SQ-8C or buffer (control) for 5 min or 2 h. The samples were fixed with 2.5%
183
glyceraldehyde in 0.1 M sodium cacodylate, and then TEM images were taken at
184
magnification ×2600 or ×5000. Representative figures are shown. 8
185
SYTOX Green uptake assay
186
The permeability of bacterial membrane upon peptide treatment was measured by SYTOX®
187
Green uptake (28). Briefly, overnight cultures of bacteria were washed and re-suspended to
188
∼107 cfu/mL in 50 mM Tris-HCl pH 7.5. Benzonase® nuclease (25 U/mL)(Novagen) and
189
SYTOX® Green (1 μM)(Invitrogen) was added to the bacterial cells, and incubated for 15
190
min at 22-25°C in the dark. Peptides (50 μg/mL) were added, and polymyxin B (2
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μg/mL)(Sigma) was used as a control (29). Relative fluorescence units (RFU) were measured
192
in a SpectraMax Plus reader (Molecular Devices) at 22-25°C (ex: 485 nm, em: 520 nm) for 2
193
h. Experiments were carried out twice in duplicate and representative data are shown as mean
194
± standard deviation.
195
Cytotoxicity assays
196
The hemolytic assays were conducted as previously described (28). Briefly, human blood
197
was gathered in an EDTA-tube, and red blood cells (RBC) were collected through low speed
198
centrifugation. Cells were washed and re-suspended to a 10% RBC solution in PBS, and
199
mixed with 345 μg/mL P307SQ-8C. PBS and 1% Triton X-100 were used as negative and
200
positive controls, respectively. After 1 h incubation at 37°C, the supernatant was collected,
201
and the absorbance at 405 nm was recorded through SpectraMax Plus Reader (Molecular
202
Devices) to measure the release of hemoglobin. The reactions were carried out twice in
203
triplicate and representative data are shown as mean ± standard deviation.
204
A human B lymphoblastoid cell line (LCL) obtained from The Rockefeller University was
205
grown in RPMI media supplemented with L-glutamine and 10% fetal bovine serum. Cells
206
were harvested by low speed centrifugation, washed and re-suspended in PBS to ~5x106 live
207
cells/mL, as determined by trypan blue exclusion tests. LCL cells (~5x105) were incubated
9
208
with 172.5, 345 and 517.5 μg/mL P307SQ-8C at 37°C in a humidified 5% CO2 atmosphere for
209
one hour. The cells were then stained according to manufacturer’s instructions (CellTiter 96
210
Non-radioactive cell proliferation assay, Promega) where live B cells reduced the dye to
211
insoluble formazan for an additional 4 h. Solubilization/Stop solution was added and
212
incubated at 37°C overnight. Next, the absorbance at 570 nm was measured in SpectraMax
213
Plus Reader (Molecular Devices) to quantitate B cell survival. Triton X-100 was used as a
214
positive control. The reactions were carried out in triplicate and data are shown as mean ±
215
standard deviation.
216
Endotoxin release
217
To detect the amount of endotoxin release, strain #1791 (~107 cfu/mL) was incubated at 22-
218
25°C for 2 h with different concentrations of P307 or P307SQ-8C. The samples were briefly
219
centrifuged (2000 × g) for 1 min to remove live cells. All reagents were prepared in
220
endotoxin-free autoclaved Milli-Q water. Endpoint Chromogenic LAL Assay (Lonza) was
221
conducted according to manufacturer’s instructions. Polymyxin B and 100% ethanol were
222
used for comparison and positive control, respectively. The experiment was conducted twice
223
in duplicate and representative data are shown as mean ± standard deviation.
224
Bactericidal activity in plasma and its components
225
Human blood was collected in a heparin-tube and centrifuged. The supernatant was filtered
226
and stored at 4°C overnight. The filtrate was centrifuged and filtered again to remove any
227
debris. The resulting solution was used as 100% plasma. The components of plasma
228
examined for interference were monovalent cation (Na+), divalent cations (Ca2+ and Mg2+)
229
and albumin. Chloride salts of the cations and albumin from human serum (lyophilized
230
powder, ≥ 97%)(Sigma) were prepared in Milli-Q water and sterile filtered. Bactericidal
10
231
assays, as described above, were conducted in 100% plasma, and in buffered solutions (50
232
mM Tris-HCl, pH 7.5) containing 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, and 50 mg/mL
233
human serum albumin (HSA), using 100 µg/mL P307 or P307SQ-8C. The experiments were
234
conducted at least in triplicate and data are shown as mean ± standard deviation.
235
Mouse in vivo skin model
236
The Rockefeller University institutional animal care and use committee approved all in vivo
237
protocols. Skin infection was induced with A. baumannii as previously described (30).
238
Briefly, the backs of 30 female CD-1 mice (6 to 8 weeks of age; Charles River Laboratories)
239
were shaved with an electric razor. NairTM depilatory cream was applied to the shaved areas
240
to remove any remaining hair. The areas were then disinfected with alcohol wipes, and tape
241
stripped with autoclave tape 20 times in succession, using a fresh piece of tape each time.
242
The tape stripped areas were disinfected again with alcohol wipes. An area of ~ 1 cm2 was
243
then marked and infected with 10 μL of ~108 cfu/mL A. baumannii strain #1791. The bacteria
244
were allowed to colonize for 16-18 h, after which the infected area was either left untreated
245
or treated with 200 μg of P307SQ-8C or 2 μg of polymyxin B in autoclaved Milli-Q water for 2
246
h. To harvest the remaining bacteria on the skin, the mice were sacrificed and the infected
247
skin was processed in 500 μL PBS for 1-2 min in a Stomacher® 80 Biomaster using a
248
microbag (Seward Ltd., UK). The solution was serially diluted and plated on LB agar
249
containing 4 μg/mL levofloxacin and 12 μg/mL ampicillin for selection. The resulting
250
cfu/mL from each animal was plotted as an individual point and the horizontal bars represent
251
the mean values. Data were analyzed using Ordinary one-way ANOVA in GraphPad Prism
252
6.0. The dotted horizontal line marks the limit of detection.
253
Results
11
254
Identification and modification of putative antibacterial peptides from phage lysin PlyF307
255
Previously published works on phage lysins against Gram-negative bacteria (15, 18)
256
suggested that the C-terminal portions of lysins might destabilize the bacterial outer
257
membrane to facilitate access to the lysin’s substrate, i.e., the peptidoglycan. Specifically, the
258
highly cationic C-terminal region of A. baumannii phage lysin LysAB2 was speculated to be
259
involved in permeabilizing the outer membrane (15). We were able to identify a similar
260
highly positively charged sequence in one of our A. baumannii phage lysins, PlyF307 (18).
261
This peptide, termed P307 (Table 1), extends from amino acids 108-138 in PlyF307, and
262
partly resembles the LysAB2 peptide in terms of length and charge, but sharing only 41.2%
263
similarity and 17.6% identity (MacVector ClustalW alignment).
264
We modified P307 with the eight amino acids constituting the full C-terminal part of native
265
PlyF307, forming peptide P307AE-8 (Table 1). In addition, we fused eight amino acids
266
(SQSRESQC) to the C-terminus of P307 and yielded P307SQ-8C (Table 1). This modification
267
was based on the observation of an earlier study showing that the lack of these eight amino
268
acids reduces the activity spectrum of the antimicrobial peptide from Hepatitis B virus core
269
protein (28). We also generated two additional modifications of P307SQ-8C: scrambling the
270
sequence of the last eight amino acids to CSQRQSES (P307CS-8); and changing the last
271
amino acid from C to A (P307SQ-8A) (Table 1). The sequence was scrambled to evaluate the
272
importance of a specific sequence and the cysteine change was introduced to examine the
273
importance of intermolecular disulfide bond formation for activity. A predicted structure of
274
PlyF307, generated through I-TASSER (31-33), suggested that P307 and its derivatives
275
formed a “hairpin-like” di-alpha helical structure connected with a flexible linker region (Fig.
276
1).
277
Comparison of the peptides 12
278
We evaluated the bactericidal activities of the peptides P307, P307AE-8 and P307SQ-8C.
279
P307SQ-8C was the most active, reducing ~106 cfu/mL of bacteria to below the limit of
280
detection (5-
323
log decrease). Biofilm-associated A. baumannii was more resistant (Fig. 4B). Biofilms were
324
treated with 250 μg/mL P307 or P307SQ-8C for 2 or 24 h. After 2 h, ~3- and 4-log decrease in
14
325
cfu/mL was observed with P307 and P307SQ-8C, respectively. After 24 h, an additional ~1.3-
326
log decrease was observed with P307 while there was no further decrease with P307SQ-8C.
327
Minimal inhibitory concentration and synergy
328
In order to compare the efficiency of the peptides with clinically used antibiotics, we
329
performed minimal inhibitory concentration (MIC) assays on four A. baumannii strains:
330
#1791, #1798, S5 and ATCC17978. The strains displayed varying degrees of sensitivity to
331
levofloxacin, ceftazidime and P307 while the MICs of polymyxin B and P307SQ-8C were more
332
consistent (Table 2). P307SQ-8C had a lower MIC than P307, which was in accordance with in
333
vitro killing activity (Fig. 2 and 3).
334
We also examined the synergistic effects between each peptide and levofloxacin, ceftazidime
335
or polymyxin B by the checkerboard method (isobologram). No synergy was observed
336
between either peptide and levofloxacin or ceftazidime (data not shown). However,
337
polymyxin B acted synergistically with both P307 and P307SQ-8C (FICIs = 0.125, 1024 >1873
0.25 0.19
1000 294
62.5 14.5
6 16.6
64 117
0.25 0.19
2000 588
125 29
6 11
0.25 0.19
750 221
125 29
S5 ATCC17978 616
4.3
0.075 0.21
Ceftazidime
Polymyxin B
P307
P307SQ-8C
µg/mL µM µg/mL µM
a
Numbers in bold indicate resistance to the antibiotics.
26
1
Novel engineered peptides of a phage lysin as effective antimicrobials against multidrug
2
resistant Acinetobacter baumannii
3
Mya Thandar1§, Rolf Lood1,2§, Benjamin Y. Winer1,3§, Douglas R. Deutsch1, Chad W.
4
Euler1,4,5, Vincent A. Fischetti1#
5
1
6
York Avenue, New York, NY 10065, USA
7
2
8
Lund University, Sweden
9
3
Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, 1230
Present address: Department of Clinical Sciences Lund, Division of Infection Medicine,
Present address: 119 Lewis Thomas Laboratory, Washington Road, Princeton, New Jersey,
10
NJ, 08544-1014, USA
11
4
12
Hunter College, CUNY, New York, NY, 10065, United States of America.
13
5
14
College, New York, NY, 10065, United States of America.
15
Short title: Antibacterial peptides active against Acinetobacter baumannii
16
# Corresponding author: Vincent A. Fischetti, [email protected]
17
§
Present address: Department of Medical Laboratory Sciences, Belfer Research Building,
Present address: Department of Microbiology and Immunology, Weill Cornell Medical
These authors contributed equally.
18 19
1
20
Abstract
21
Acinetobacter baumannii is a Gram-negative bacterial pathogen responsible for a range of
22
nosocomial infections. The recent rise and spread of multidrug resistant A. baumannii clones
23
has fueled a search for alternative therapies, including bacteriophage endolysins with potent
24
antibacterial activities. A common feature of these lysins is the presence of a highly
25
positively charged C-terminal domain with a likely role in promoting outer membrane
26
penetration. In the current study, we show that the C-terminal amino acid 108-138 of phage
27
lysin PlyF307, named P307, alone was sufficient to kill A. baumannii (>3-logs). Furthermore,
28
P307 could be engineered for improved activity, the most active derivative being P307SQ-8C
29
(>5-log kill). Both P307 and P307SQ-8C showed high in vitro activity against A. baumannii in
30
biofilms. Moreover, P307SQ-8C exhibited MICs comparable to levofloxacin and ceftazidime
31
and acted synergistically with polymyxin B. While the peptides were shown to kill by
32
disrupting the bacterial cytoplasmic membrane, they did not lyse human red blood cells or B
33
cells; however, serum was found to be inhibitory to lytic activity. In a murine model of A.
34
baumannii skin infection, P307SQ-8C reduced the bacterial burden by ~2-logs in 2 h. This
35
study demonstrates the prospect of using peptide derivatives from bacteriophage lysins to
36
treat topical infections and remove biofilms caused by Gram-negative pathogens.
37
Introduction
38
Acinetobacter baumannii is an increasingly significant nosocomial pathogen worldwide (1).
39
Arising from both intrinsic and acquired antibiotic resistance, multi- and pan-drug resistant
40
clones of A. baumannii can readily be isolated from hospital environments (2). A. baumannii
41
has been shown to develop resistance to several classes of antibiotics, including
42
aminoglycosides, cephalosporins, carbapenems, tigecycline and colistin (3). The reasons for
43
high resistance include a high degree of genetic plasticity combined with an intrinsic
2
44
resistance to certain antibiotics due to the presence of β-lactamases, low permeability of the
45
outer membrane, and highly efficient efflux pump systems (4). Furthermore, A. baumannii is
46
prone to develop biofilms on solid surfaces, including medical devices (5). Thus, A.
47
baumannii is not only problematic as an infectious agent, but it is also increasingly difficult
48
to remove from hospital environments – a phenomenon similar to the Gram-positive
49
nosocomial pathogen Staphylococcus aureus.
50
One of the last resort antibiotics for A. baumannii is the antimicrobial peptide polymyxin B
51
(6). The bactericidal effect of polymyxin B is mediated through its positively charged Dab
52
(α,γ-diaminobutyric acid) residues interacting with LPS and destabilizing the outer
53
membrane (7). Many antimicrobial peptides kill in a similar way: clustered cationic residues
54
permeabilize the bacterial membrane to cause lysis and death (8). Due to this mechanism of
55
action, most of the membrane-acting antimicrobial peptides commonly have cytotoxic effects
56
on eukaryotic cells (9). Indeed, polymyxin B has severe side effects: cytotoxicity,
57
nephrotoxicity and neurotoxicity (10). Since careful administration is required to avoid its
58
toxicity, the dose range of polymyxin B is limited and resistant strains of A. baumannii have
59
been documented (11).
60
Recently, there has been a growing interest in the use of bacterial viruses (i.e., bacteriophage
61
therapy) to treat infections by Gram-negative bacteria, including A. baumannii (12-14).
62
Several phages that infect A. baumannii have been identified and characterized. However,
63
their restricted spectrum (killing only ∼60% of A. baumannii isolates) limits the effectiveness
64
of such phages as therapeutic agents (12, 13). Using an alternative bacteriophage-based
65
approach, our group and others have taken advantage of the lytic enzymes (lysins) encoded
66
and produced by bacteriophages during lytic proliferation (15-18). Bacteriophage lysins are
67
classified as peptidoglycan hydrolases, being able to cleave a variety of bonds in the bacterial
3
68
peptidoglycan. Cleavage of the cell wall by lysins destabilizes the peptidoglycan and
69
weakens the structural framework, resulting in hypotonic lysis. While purified lysins are
70
effective at killing Gram-positive bacteria (19), the outer membrane of Gram-negative
71
bacteria largely limits lysins from accessing and cleaving the subjacent peptidoglycan.
72
Different strategies have been employed to increase the efficiency of lysins against Gram-
73
negative bacteria, including the use of the chelating agent EDTA (16, 17), and the genetic
74
engineering of lysins to add either highly charged/hydrophobic N-/C-terminal extensions (20)
75
or other membrane translocating domains (21, 22). However, there has been little focus on
76
the intrinsic features of certain active lysins against Gram-negative bacteria, and how they
77
function to allow the lysins to cross the outer membrane and reach the subjacent
78
peptidoglycan substrate.
79
Here, we have identified a highly cationic C-terminal domain within an A. baumannii phage
80
lysin as a peptide with potent antibacterial activity. We have modified the peptide to further
81
improve its activity, and have proven the high efficiency of such peptides to kill A.
82
baumannii both in vitro and in vivo in a skin infection model.
83
Materials and Methods
84
Bacterial strains and growth conditions
85
Acinetobacter baumannii strains in this study include clinical isolates from Hospital for
86
Special Surgery in New York (#1775-#1799) (18), Ohio State University (S1, S3 and S5
87
provided by Dr. Vijay Pancholi), and ATCC® 17978™ from American Type Culture
88
Collection. Bacteria were cultured in Trypticase Soy Broth or Brain Heart Infusion (TSB or
89
BHI; Thermo Fisher Scientific, Waltham, MA, USA) at 37°C with aeration (200 rpm).
90
Stationary phase bacteria were cultured overnight while log phase bacteria were grown for 3
4
91
h in fresh media from 100x dilutions of overnight cultures. Strains for determining the
92
specificity of the antimicrobial peptides were cultured under the same conditions, at the
93
temperatures indicated: Bacillus anthracis ΔSterne (30°C), Escherichia coli DH5α (37°C),
94
Klebsiella pneumoniae ATCC® 700603TM and 10031TM (37°C), Pseudomonas aeruginosa
95
PAO1 (30°C), and Staphylococcus aureus RN4220 (30°C).
96
Biofilms were set up as previously described (18). Briefly, an overnight culture of A.
97
baumannii strain #1791 was diluted 1000x (~105 cfu/mL) in TSB containing 0.2% glucose
98
and incubated at 37°C for 72 h in ~2.5 cm segments of PVC catheter tubing (CareFusion)
99
with the ends clamped shut to prevent evaporation. The catheter biofilms were washed with
100
autoclaved Milli-Q water to remove planktonic cells before peptide treatment. Crystal violet
101
(0.1%) was used to detect the presence of biofilm.
102
Peptide synthesis and preparation
103
Peptides were synthesized at The Rockefeller University Proteomics Resource Center. All
104
peptides were created using a Protein Technologies Symphony™ peptide synthesizer (PTI
105
Tucson, Arizona, USA) on pre-coupled Wang (p-alkoxy-benzyl alcohol) resin (Bachem,
106
Torrance, CA, USA). Reaction vessels were loaded at 25 μM and peptides were elongated
107
using Fmoc protected amino acids (Anaspec, San Jose, CA, USA) (23). Deprotection of the
108
amine was accomplished with 20% piperidine (Sigma-Aldrich) in NMP (N-
109
methylpyrrolidinone). Repetitive coupling reactions were conducted using 0.6 M HATU/Cl-
110
HOBT (azabenzotriazol tetramethyluronium hexafluorophosphate/6-chloro-1-
111
hydroxybenzotriazole)(P3 Biosystems, Shelbyville, KY, USA) and 0.4 M NMM (N-
112
methylmorpholine) using NMP (EMD) as the primary solvent (24). Resin cleavage and side-
113
chain deprotection were achieved by transferring to a 100 mL round bottom flask and reacted
114
with 4.0 mL concentrated, sequencing grade, trifluoracetic acid (Fisher) with 5
115
triisopropylsilane (Fluka), degassed water, and 3,6-dioxa-1,8-octanedithiol (DODT, Sigma-
116
Aldrich) in a ratio of 95:2:2:1 over a 6 h time frame. This was followed by column filtration
117
to a 50 mL round bottom flask and TFA volume reduced to 2 mL using a rotary evaporator.
118
A standard ether precipitation was performed on the individual peptides by transferring to a
119
50 mL falcon tube containing 40 mL cold tert-butyl methyl ether (TBME, Sigma-Aldrich).
120
Samples were placed in an ice bath for 2 h to aid precipitation followed by pellet formation
121
using centrifugation (3300 rpm, 5 min). Excess ether was removed by vacuum aspiration and
122
the peptide pellets were allowed to dry overnight in a fume hood. Dried peptide pellets were
123
resolved in 20% acetonitrile and 10 mL HPLC grade water, subsampled for LC/MS and
124
lyophilized. All crude products were subsequently analyzed by reverse-phase Aquity™
125
UPLC (Waters Chromatography, Milford, MA, USA) using a Waters BEH C18 column.
126
Individual peptide integrity was verified by tandem electrospray mass spectrometry using a
127
ThermoFinnigan LTQ™ (Thermo Fisher, Waltham, MA, USA) spectrometer system.
128
Preparative chromatography was accomplished on a Vydac C18 RP preparative column on a
129
Waters 600 Prep HPLC. Individual fractions were collected in 30 s intervals, characterized
130
using LC/MS and fractions containing desired product were lyophilized. These were stored at
131
-20°C until being re-suspended in autoclaved Milli-Q water for various assays. The stock
132
solutions were then stored at 4°C.
133
Bactericidal assays
134
Unless otherwise indicated, stationary phase bacteria (overnight cultures) were used for the
135
assays. Bacteria were washed in assay buffer, re-suspended to ∼106 cfu/mL, mixed with each
136
antimicrobial peptide and incubated for 2 h at room temperature (22-25°C). After treatment,
137
the reactions were serially diluted and plated for enumeration. Influencing factors on the
138
activities of the peptides (P307 and P307SQ-8C) were examined: buffer (50 mM sodium
6
139
phosphate or Tris-HCl, pH 6.8-8.8; NaCl (0-200 mM); concentration (0-125 μg/mL P307);
140
time (1-120 min); specificity (the bacterial strains mentioned above); and growth phase (log,
141
stationary and biofilm). The buffer system for the experiments with the latter five factors was
142
50 mM Tris-HCl, pH 7.5. The biofilm-associated bacteria were re-suspended by thoroughly
143
scraping the catheter tubing and vortexing for 1 min.
144
To examine the mechanism(s) of action of the peptides, the following bactericidal assays
145
were also conducted. The contribution of disulfide bond formation to the increased activity of
146
P307SQ-8C was investigated by comparing with P307SQ-8A in the presence of the reducing
147
agent TCEP (0.1 and 1 mM) (Tris (2-carboxyethyl) phosphine hydrochloride
148
solution)(Sigma). To examine the importance of ionic interaction, the least sensitive Gram-
149
negative bacterial species at pH 7.5 were treated with P307 and P307SQ-8C at pH 8.8.
150
Experiments were conducted at least in triplicate and representative data are shown as mean
151
± standard deviation. The black horizontal lines mark the limit of detection.
152
Minimum inhibitory concentrations (MICs) and synergy
153
The broth microdilution method was used (25) to determine the MICs of levofloxacin,
154
ceftazidime, polymyxin B, P307 and P307SQ-8C for A. baumannii strains #1791, #1798, S5
155
and ATCC17978. For the antibiotics, 1.5–2 fold serial dilutions (three lower and three
156
higher) of the MICs determined by Etest (18) were included. For the peptides, two-fold serial
157
dilutions (2000–31.25 μg/mL) were tested. Overnight cultures were re-suspended to ~105
158
cfu/mL in Mueller Hinton broth (pH 7.9). Antibiotics or peptides were added to a final
159
volume of 100 μL for each dilution. Bacteria were allowed to grow at 37°C for 24 h at 220
160
rpm. The absorbance at 595 nm was then read in a SpectraMax Plus Reader (Molecular
161
Devices). The MICs were determined as the lowest concentrations of antimicrobial agents
7
162
that visibly inhibited bacterial growth. In addition, Alamar®Blue was used to confirm the
163
data obtained from OD595. Each experiment was repeated at least twice in duplicate.
164
To investigate whether synergy exists between each peptide and antibiotic, checkerboard
165
assays were conducted by mixing fractional inhibitory concentrations (FICs) of antibiotics
166
(levofloxacin, ceftazidime and polymyxin B) with those of peptides P307 and P307SQ-8C as
167
previously described (26). Isobolograms were constructed to determine the fractional
168
inhibitory concentration indices (FICIs). Each experiment was repeated at least three times.
169
DNA binding assay
170
The affinity of peptide P307 for DNA was investigated by electrophoretic mobility shift
171
assay (27). P307 was mixed with 50 ng of random DNA (1 kb amplicon of Bdellovibrio
172
genomic DNA) at different peptide to DNA ratios (0:1-15:1) in a binding buffer (5 mM Tris-
173
HCl, pH 8.0, 10 mM EDTA, 5% glycerol, 20 mM KCl, 50 μg/mL BSA). As a positive
174
control, a peptide from Bdellovibrio bacteriovorus that was previously found in our
175
laboratory to have DNA-binding capacity (amino acid sequence –
176
MASKTKKTEYIRERKKATSGKKRKAANRTKGTTKSAKTLFKD) was used
177
(unpublished results). After incubation for 1 h at 22-25°C, the peptide and DNA mixture was
178
analyzed by agarose gel electrophoresis (27).
179
Transmission electron microscopy (TEM)
180
An overnight culture of A. baumannii strain #1791 was collected, washed in 50 mM Tris-HCl,
181
pH 7.5 and re-suspended in the same buffer to ~108 cfu/mL. The bacteria were treated with
182
300 μg/mL P307SQ-8C or buffer (control) for 5 min or 2 h. The samples were fixed with 2.5%
183
glyceraldehyde in 0.1 M sodium cacodylate, and then TEM images were taken at
184
magnification ×2600 or ×5000. Representative figures are shown. 8
185
SYTOX Green uptake assay
186
The permeability of bacterial membrane upon peptide treatment was measured by SYTOX®
187
Green uptake (28). Briefly, overnight cultures of bacteria were washed and re-suspended to
188
∼107 cfu/mL in 50 mM Tris-HCl pH 7.5. Benzonase® nuclease (25 U/mL)(Novagen) and
189
SYTOX® Green (1 μM)(Invitrogen) was added to the bacterial cells, and incubated for 15
190
min at 22-25°C in the dark. Peptides (50 μg/mL) were added, and polymyxin B (2
191
μg/mL)(Sigma) was used as a control (29). Relative fluorescence units (RFU) were measured
192
in a SpectraMax Plus reader (Molecular Devices) at 22-25°C (ex: 485 nm, em: 520 nm) for 2
193
h. Experiments were carried out twice in duplicate and representative data are shown as mean
194
± standard deviation.
195
Cytotoxicity assays
196
The hemolytic assays were conducted as previously described (28). Briefly, human blood
197
was gathered in an EDTA-tube, and red blood cells (RBC) were collected through low speed
198
centrifugation. Cells were washed and re-suspended to a 10% RBC solution in PBS, and
199
mixed with 345 μg/mL P307SQ-8C. PBS and 1% Triton X-100 were used as negative and
200
positive controls, respectively. After 1 h incubation at 37°C, the supernatant was collected,
201
and the absorbance at 405 nm was recorded through SpectraMax Plus Reader (Molecular
202
Devices) to measure the release of hemoglobin. The reactions were carried out twice in
203
triplicate and representative data are shown as mean ± standard deviation.
204
A human B lymphoblastoid cell line (LCL) obtained from The Rockefeller University was
205
grown in RPMI media supplemented with L-glutamine and 10% fetal bovine serum. Cells
206
were harvested by low speed centrifugation, washed and re-suspended in PBS to ~5x106 live
207
cells/mL, as determined by trypan blue exclusion tests. LCL cells (~5x105) were incubated
9
208
with 172.5, 345 and 517.5 μg/mL P307SQ-8C at 37°C in a humidified 5% CO2 atmosphere for
209
one hour. The cells were then stained according to manufacturer’s instructions (CellTiter 96
210
Non-radioactive cell proliferation assay, Promega) where live B cells reduced the dye to
211
insoluble formazan for an additional 4 h. Solubilization/Stop solution was added and
212
incubated at 37°C overnight. Next, the absorbance at 570 nm was measured in SpectraMax
213
Plus Reader (Molecular Devices) to quantitate B cell survival. Triton X-100 was used as a
214
positive control. The reactions were carried out in triplicate and data are shown as mean ±
215
standard deviation.
216
Endotoxin release
217
To detect the amount of endotoxin release, strain #1791 (~107 cfu/mL) was incubated at 22-
218
25°C for 2 h with different concentrations of P307 or P307SQ-8C. The samples were briefly
219
centrifuged (2000 × g) for 1 min to remove live cells. All reagents were prepared in
220
endotoxin-free autoclaved Milli-Q water. Endpoint Chromogenic LAL Assay (Lonza) was
221
conducted according to manufacturer’s instructions. Polymyxin B and 100% ethanol were
222
used for comparison and positive control, respectively. The experiment was conducted twice
223
in duplicate and representative data are shown as mean ± standard deviation.
224
Bactericidal activity in plasma and its components
225
Human blood was collected in a heparin-tube and centrifuged. The supernatant was filtered
226
and stored at 4°C overnight. The filtrate was centrifuged and filtered again to remove any
227
debris. The resulting solution was used as 100% plasma. The components of plasma
228
examined for interference were monovalent cation (Na+), divalent cations (Ca2+ and Mg2+)
229
and albumin. Chloride salts of the cations and albumin from human serum (lyophilized
230
powder, ≥ 97%)(Sigma) were prepared in Milli-Q water and sterile filtered. Bactericidal
10
231
assays, as described above, were conducted in 100% plasma, and in buffered solutions (50
232
mM Tris-HCl, pH 7.5) containing 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, and 50 mg/mL
233
human serum albumin (HSA), using 100 µg/mL P307 or P307SQ-8C. The experiments were
234
conducted at least in triplicate and data are shown as mean ± standard deviation.
235
Mouse in vivo skin model
236
The Rockefeller University institutional animal care and use committee approved all in vivo
237
protocols. Skin infection was induced with A. baumannii as previously described (30).
238
Briefly, the backs of 30 female CD-1 mice (6 to 8 weeks of age; Charles River Laboratories)
239
were shaved with an electric razor. NairTM depilatory cream was applied to the shaved areas
240
to remove any remaining hair. The areas were then disinfected with alcohol wipes, and tape
241
stripped with autoclave tape 20 times in succession, using a fresh piece of tape each time.
242
The tape stripped areas were disinfected again with alcohol wipes. An area of ~ 1 cm2 was
243
then marked and infected with 10 μL of ~108 cfu/mL A. baumannii strain #1791. The bacteria
244
were allowed to colonize for 16-18 h, after which the infected area was either left untreated
245
or treated with 200 μg of P307SQ-8C or 2 μg of polymyxin B in autoclaved Milli-Q water for 2
246
h. To harvest the remaining bacteria on the skin, the mice were sacrificed and the infected
247
skin was processed in 500 μL PBS for 1-2 min in a Stomacher® 80 Biomaster using a
248
microbag (Seward Ltd., UK). The solution was serially diluted and plated on LB agar
249
containing 4 μg/mL levofloxacin and 12 μg/mL ampicillin for selection. The resulting
250
cfu/mL from each animal was plotted as an individual point and the horizontal bars represent
251
the mean values. Data were analyzed using Ordinary one-way ANOVA in GraphPad Prism
252
6.0. The dotted horizontal line marks the limit of detection.
253
Results
11
254
Identification and modification of putative antibacterial peptides from phage lysin PlyF307
255
Previously published works on phage lysins against Gram-negative bacteria (15, 18)
256
suggested that the C-terminal portions of lysins might destabilize the bacterial outer
257
membrane to facilitate access to the lysin’s substrate, i.e., the peptidoglycan. Specifically, the
258
highly cationic C-terminal region of A. baumannii phage lysin LysAB2 was speculated to be
259
involved in permeabilizing the outer membrane (15). We were able to identify a similar
260
highly positively charged sequence in one of our A. baumannii phage lysins, PlyF307 (18).
261
This peptide, termed P307 (Table 1), extends from amino acids 108-138 in PlyF307, and
262
partly resembles the LysAB2 peptide in terms of length and charge, but sharing only 41.2%
263
similarity and 17.6% identity (MacVector ClustalW alignment).
264
We modified P307 with the eight amino acids constituting the full C-terminal part of native
265
PlyF307, forming peptide P307AE-8 (Table 1). In addition, we fused eight amino acids
266
(SQSRESQC) to the C-terminus of P307 and yielded P307SQ-8C (Table 1). This modification
267
was based on the observation of an earlier study showing that the lack of these eight amino
268
acids reduces the activity spectrum of the antimicrobial peptide from Hepatitis B virus core
269
protein (28). We also generated two additional modifications of P307SQ-8C: scrambling the
270
sequence of the last eight amino acids to CSQRQSES (P307CS-8); and changing the last
271
amino acid from C to A (P307SQ-8A) (Table 1). The sequence was scrambled to evaluate the
272
importance of a specific sequence and the cysteine change was introduced to examine the
273
importance of intermolecular disulfide bond formation for activity. A predicted structure of
274
PlyF307, generated through I-TASSER (31-33), suggested that P307 and its derivatives
275
formed a “hairpin-like” di-alpha helical structure connected with a flexible linker region (Fig.
276
1).
277
Comparison of the peptides 12
278
We evaluated the bactericidal activities of the peptides P307, P307AE-8 and P307SQ-8C.
279
P307SQ-8C was the most active, reducing ~106 cfu/mL of bacteria to below the limit of
280
detection (5-
323
log decrease). Biofilm-associated A. baumannii was more resistant (Fig. 4B). Biofilms were
324
treated with 250 μg/mL P307 or P307SQ-8C for 2 or 24 h. After 2 h, ~3- and 4-log decrease in
14
325
cfu/mL was observed with P307 and P307SQ-8C, respectively. After 24 h, an additional ~1.3-
326
log decrease was observed with P307 while there was no further decrease with P307SQ-8C.
327
Minimal inhibitory concentration and synergy
328
In order to compare the efficiency of the peptides with clinically used antibiotics, we
329
performed minimal inhibitory concentration (MIC) assays on four A. baumannii strains:
330
#1791, #1798, S5 and ATCC17978. The strains displayed varying degrees of sensitivity to
331
levofloxacin, ceftazidime and P307 while the MICs of polymyxin B and P307SQ-8C were more
332
consistent (Table 2). P307SQ-8C had a lower MIC than P307, which was in accordance with in
333
vitro killing activity (Fig. 2 and 3).
334
We also examined the synergistic effects between each peptide and levofloxacin, ceftazidime
335
or polymyxin B by the checkerboard method (isobologram). No synergy was observed
336
between either peptide and levofloxacin or ceftazidime (data not shown). However,
337
polymyxin B acted synergistically with both P307 and P307SQ-8C (FICIs = 0.125, 1024 >1873
0.25 0.19
1000 294
62.5 14.5
6 16.6
64 117
0.25 0.19
2000 588
125 29
6 11
0.25 0.19
750 221
125 29
S5 ATCC17978 616
4.3
0.075 0.21
Ceftazidime
Polymyxin B
P307
P307SQ-8C
µg/mL µM µg/mL µM
a
Numbers in bold indicate resistance to the antibiotics.
26
