Download Insect-Derived Cecropins Display Activity against

Insect-Derived Cecropins Display Activity against Acinetobacter
baumannii in a Whole-Animal High-Throughput Caenorhabditis
elegans Model
Division of Infectious Diseases, Rhode Island Hospital, Alpert Medical School of Brown University, Providence, Rhode Island, USAa; Massachusetts General Hospital,
Harvard Medical School, Boston, Massachusetts, USAb; Institute of Phytopathology and Applied Zoology, Justus-Liebig University, Giessen, Germanyc
The rise of multidrug-resistant Acinetobacter baumannii and a concomitant decrease in antibiotic treatment options warrants a
search for new classes of antibacterial agents. We have found that A. baumannii is pathogenic and lethal to the model host organism Caenorhabditis elegans and have exploited this phenomenon to develop an automated, high-throughput, high-content
screening assay in liquid culture that can be used to identify novel antibiotics effective against A. baumannii. The screening assay involves coincubating C. elegans with A. baumannii in 384-well plates containing potential antibacterial compounds. At the
end of the incubation period, worms are stained with a dye that stains only dead animals, and images are acquired using automated microscopy and then analyzed using an automated image analysis program. This robust assay yields a Z= factor consistently greater than 0.7. In a pilot experiment to test the efficacy of the assay, we screened a small custom library of synthetic antimicrobial peptides (AMPs) that were synthesized using publicly available sequence data and/or transcriptomic data from
immune-challenged insects. We identified cecropin A and 14 other cecropin or cecropin-like peptides that were able to enhance
C. elegans survival in the presence of A. baumannii. Interestingly, one particular hit, BR003-cecropin A, a cationic peptide synthesized by the mosquito Aedes aegypti, showed antibiotic activity against a panel of Gram-negative bacteria and exhibited a low
MIC (5 ␮g/ml) against A. baumannii. BR003-cecropin A causes membrane permeability in A. baumannii, which could be the
underlying mechanism of its lethality.
A
cinetobacter baumannii is a Gram-negative, opportunistic
bacterium that has recently emerged as a dangerous nosocomial pathogen (1–4). An increasing number of A. baumannii infections in patients have been detected among U.S. military service
members injured in Iraq and Afghanistan (5). The genetic adaptability of A. baumannii allows it to gain resistance to a wide spectrum of commercial antibiotics, and the intrinsic presence of various efflux pumps in A. baumannii also contributes to an
insensitivity to many antibiotics (6–8), resulting in very few viable
treatment options for Acinetobacter infections (9–11). In addition,
most of the clinical strains of A. baumannii also harbor a large
antimicrobial resistance island (RI) of 86 kb that contains several
beta-lactamase genes, conferring resistance to beta-lactam antibiotics (12, 13). The scarcity of antibiotics that can be used against A.
baumannii infections drives the need for new kinds of antimicrobial agents (14).
Empirical drug screening methods traditionally involve in vitro
assays to measure the MICs for various pathogens. This is followed
by in vivo testing of the drugs to measure their toxicity to eukaryotic cells (15). The disadvantage of these traditional assays is that a
significant number of hit compounds show nonspecific toxicity to
eukaryotic cells and are not promising as therapeutics (16). In this
paper, we describe a whole-animal A. baumannii infection model
compatible with large-scale compound screening using the model
organism Caenorhabditis elegans. The nematode C. elegans has
garnered interest among researchers as a model to study innate
immunity as well as microbial pathogenesis due to its genetic tractability, transparency, small size, and conserved defense response
pathways (17–20). In addition, the bacteriovorous C. elegans can
be readily infected with a number of human pathogens and treated
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with small molecules to evaluate curative and cytotoxic effects
(21–24).
To test the efficacy of the C. elegans-A. baumannii infection
assay, we carried out a pilot screen of 68 insect-derived antimicrobial peptides (AMPs). AMPs are ubiquitously present in many
cells and tissues of invertebrates, plants, and animals (25, 26). The
physical properties of AMPs, including the presence of two or
more positively charged amino acids and a large proportion of
hydrophobic residues that fold into specific secondary structures
with a certain amphipathicity, allow them to intercalate into and
form pores in bacterial membranes, as well as to translocate inside
bacterial cells (27). Moreover, AMPs also target the anionic phospholipid head groups in bacterial membranes by electrostatic interactions (26, 28, 29). These properties of AMPs that allow them
to disrupt membrane architecture make it difficult for target or-
Received 31 August 2014 Returned for modification 3 October 2014
Accepted 2 January 2015
Accepted manuscript posted online 12 January 2015
Citation Jayamani E, Rajamuthiah R, Larkins-Ford J, Fuchs BB, Conery AL, Vilcinskas
A, Ausubel FM, Mylonakis E. 2015. Insect-derived cecropins display activity against
Acinetobacter baumannii in a whole-animal high-throughput Caenorhabditis
elegans model. Antimicrob Agents Chemother 59:1728 –1737.
doi:10.1128/AAC.04198-14.
Address correspondence to Eleftherios Mylonakis, [email protected].
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/AAC.04198-14.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.04198-14
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Elamparithi Jayamani,a,b Rajmohan Rajamuthiah,a,b Jonah Larkins-Ford,b Beth Burgwyn Fuchs,a,b Annie L. Conery,b
Andreas Vilcinskas,c Frederick M. Ausubel,b Eleftherios Mylonakisa,b
Antimicrobial Peptides against A. baumannii by HTS
TABLE 1 Bacterial strains used in this study
Source
Reference(s)
A. baumannii ATCC 19606
A. baumannii ATCC 17978
A. baumannii G5139
A. baumannii G5140
A. baumannii GC7945
A. baumannii GC7951
A. baylyi PS8004
Staphylococcus aureus MW2
P. aeruginosa PA14
E. coli OP50
Reference strain
Reference strain
Drain fluid
Drain fluid
adeB knockout
adeB knockout
Reference strain
Clinical strain MW2
Clinical strain
Lab
64, 76
64, 76
57
57
57
57
64
46
37
46
ganisms to develop resistance and make them novel candidates for
new-drug development (25, 28, 30). In addition to potent antimicrobial activity, AMPs are also known to have immunomodulatory properties, which add to their potential as therapeutic agents
(31, 32).
In this pilot C. elegans-A. baumannii screen of 68 insect-derived AMPs, we identified 15 cecropin or cecropin-like peptides
that prolonged the survival of worms infected with A. baumannii.
The hit peptide BR003-cecropin A isolated from Aedes aegypti
showed higher activity against A. baumannii than did the other
cecropins and caused bacterial membrane perturbation. As a
proof of concept, this small pilot screen of AMPs demonstrated
that the automated, high-throughput C. elegans-A. baumannii
screening assay can be used to screen small-molecule libraries to
identify novel antimicrobials, which could lead to the identification of novel therapeutics for A. baumannii infections.
Sytox orange, a dye that specifically stains dead worms, in M9 was
added to each well using a Thermo Scientific multidrop Combi dispenser (Thermo Scientific, Waltham, MA) to obtain a final Sytox concentration of 0.7 ␮M. The worms were then incubated overnight at
25°C. Stained plates were imaged with a Molecular Devices ImageXpress Micro (Molecular Devices, Sunnyvale, MA) automated microscope with bright-field and tetramethyl rhodamine isothiocyanate
(TRITC) channels (Fig. 2 and 3) (16, 37). AMPs (2 mg/ml) dissolved in
DMSO were screened at a final concentration of 2 ␮g/ml and 1%
DMSO in triplicate. In this screen, 2.5 ␮g/ml polymyxin B (Fluka) in
1% DMSO and 1% DMSO were used as positive and negative controls,
respectively.
Image analysis was carried out using CellProfiler (http://www.cellprofiler
.org/), a free, open-source image analysis software, using a series of image
processing and analysis modules (16, 38). Survival was calculated by CellProfiler based on the ratio of Sytox-stained worm area to total worm area
for each well of the assay plates.
MATERIALS AND METHODS
Bacterial strains, nematode strains, and culture conditions. All bacterial
strains used in this study, shown in Table 1, were routinely cultured in
Luria-Bertani broth (LB) or on LB agar at 37°C. The nematode strain glp-4
(bn2);sek-1 (km4) was used because the glp-4 mutation renders the nematodes incapable of producing progeny at 25°C (33) and because sek-1
mutant animals are relatively immunocompromised, thereby decreasing
the duration of the assay (34–36).
Infection assay. The assay was carried out in 384-well plates (Corning
no. 3712) in a final volume of 70 ␮l. Assay media consisted of 70% M9
buffer, 19% sheath solution (Union Biometrica no. 300-5101-000), 10%
tryptic soy broth (TSB; Becton Dickson Co., NJ, USA), 10 ␮M FeCl3,and
1% dimethyl sulfoxide (DMSO) or test compounds dissolved in 1%
DMSO with a final density of bacteria at an optical density at 600 nm
(OD600) of 0.03 (⬃5 ⫻ 106 CFU/ml). The assay workflow is outlined in
Fig. 1. In brief, preparation of the bacterial culture started with an overnight culture of A. baumannii in LB broth that was spread on LB agar and
incubated at 37°C for 8 h prior to infection assays. The bacteria were then
scraped from the LB agar plates, pelleted, resuspended in M9 buffer containing 10% TSB (Becton Dickinson Co., NJ, USA) and 10 ␮M FeCl3, and
aliquoted into 384-well plates. Fifteen worms were then dispensed into
each well of the assay plates with a complex object parametric analyzer and
sorter (COPAS BioSort; Union Biometrica, Holliston, MA).
Assay plates were sealed with Breathe-Easy membranes (Diversified Biotech, Dedham, MA) and incubated for 120 h with 80 to 85%
humidity at 25°C. Subsequently, plates were washed 6 times with M9
medium using a BioTek ELx405 microplate washer (BioTek, Winooski, VT) to remove bacteria. All 15 worms were consistently recovered after each step in the washing process. After the last wash, ⬃25 ␮l
of assay medium with worms was left in each well. Before aspiration of
the spent liquid, the plate was allowed to rest for 5 min, which is
sufficient time to allow the worms to settle. Finally, 60 ␮l of 0.9 ␮M
March 2015 Volume 59 Number 3
FIG 1 C. elegans-A. baumannii liquid killing assay workflow. Schematic representation of C. elegans-A. baumannii liquid infection assay for highthroughput screening.
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Bacteria
Jayamani et al.
formed by coincubation of nematodes and bacteria in 10% TSB, 90% M9 with
10 ␮M FeCl3 and either 1% DMSO (negative control) or 2 ␮g/ml polymyxin B
and 1% DMSO (positive control) and E. coli OP50 (positive control). The
assay plate was incubated at 25°C for 5 days, and residual bacteria and debris
were removed by washing followed by staining with Sytox orange. The images
shown are TRITC fluorescent images of 6 wells in a column of a 384-well plate.
(B) Optimization of the starting bacterial titer for the liquid killing assay.
Z=-factor calculation. The quality of the HTS was evaluated by calculating the Z=-factor value from positive and negative-control data (39). Z=
factor is calculated as 1 ⫺ [(3␴p ⫹ 3␴n)/(|␮p ⫺ ␮n|)] where ␴p and ␴n are
the standard deviations of the positive and negative controls, respectively,
and ␮p and ␮n are the means of the positive and negative controls, respectively. The assay was validated with a Z= factor of ⬎0.5, which shows that
the assay is consistent and robust enough for large-scale screens. The
negative control was 1% DMSO, and the positive control was polymyxin
B at a final concentration of 2.5 ␮g/ml and 1% DMSO.
Synthesis of insect-derived antimicrobial peptides. A total of 68 antimicrobial peptides (AMPs) from insects were synthesized based on
publicly available sequence data or sequences generated by transcriptomic analyses of immune-challenged insects, such as the medicinal
maggot Lucilia sericata, the red flour beetle Tribolium castaneum, the
invasive harlequin ladybird Harmonia axyridis, and the greater wax moth
Galleria mellonella (40–43). G. mellonella has been established as a powerful model host for human pathogens and a source for novel anti-infec-
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RESULTS
High-throughput C. elegans-A. baumannii liquid killing assay.
We developed a C. elegans-A. baumannii liquid killing assay by
testing several different media, bacterial concentrations, and incubation conditions. The nonpathogenic strain Escherichia coli
OP50 (a widely used food source for laboratory-reared C. elegans)
was used as a negative control. A medium containing 10% TSB
and 90% M9 showed a good separation between the negative and
positive controls, with 70% survival of worms incubated with E.
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FIG 2 C. elegans-A. baumannii liquid killing assay. (A) The assay was per-
tives (44, 45). The 68 peptides (see the supplemental material) were produced by solid-phase synthesis and purified by reversed-phase
chromatogaphy by GenScript (Piscataway, NJ, USA). The integrity of the
peptides was confirmed by using liquid chromatography-mass spectrometry (LC/MS).
Z score and hit identification. The hits were identified based on Z
scores calculated from the CellProfiler survival ratio data. A Z score is
defined as (x ⫺ ␮)/␴, where x is the raw sample ratio score, ␮ is the mean
for the population, and ␴ is the standard deviation for the population.
Test samples with Z scores of ⬎1.5 were considered hits.
Hit validation assay. The hit compounds that prolonged worm survival in the A. baumannii-C. elegans infection assay were tested at two
different concentrations (5 ␮g/ml and 10 ␮g/ml) in triplicate (data not
shown) (37, 46).
MIC assay. The MIC assay was performed in 384-well plates in triplicate by the broth microdilution method adapted from an established protocol (47). The test compound range was set from 0.16 to 20 ␮g/ml by
performing 2-fold serial dilutions. The assay volume was 70 ␮l containing
M9 buffer, 10% TSB, and A. baumannii ATCC 19606 at an initial density
corresponding to an OD600 of 0.03 (5 ⫻ 106 CFU/ml). DMSO (1%) and
polymyxin B (2.5 ␮g/ml in 1% DMSO) were used as negative and positive
controls, respectively. The bacteria were incubated overnight at 37°C, and
the bacterial growth was measured to determine antimicrobial activity of
the test compounds.
Disc diffusion assay. Mid-log-phase bacteria (50 ␮l) at an OD600 of
⬃0.06 (1 ⫻ 107 CFU/ml) were plated onto LB agar plates and dried for 10
min to create a bacterial lawn. The discs were prepared by adding 10 ␮l of
1 mg/ml stock solutions of cecropin A or polymyxin B in DMSO or
DMSO as controls on 6-mm Whatman filter paper disks (Fisher Scientific). The discs were placed on the bacterial lawn and incubated overnight
at 37°C, and the diameter of the zone of inhibition for each disc was
measured. Each experiment was repeated three times (48).
Membrane permeability assay. A. baumannii ATCC 19606 cultures
were harvested at exponential growth phase, washed twice, and then resuspended in Hanks medium (catalog no. 14025-092; Life Technologies)
supplemented with 10 mM D-glucose at a bacterial concentration corresponding to an OD600 of 0.06 (1 ⫻ 107 CFU/ml) and a final concentration
of 1 ␮M Sytox green. One hundred-microliter aliquots of this suspension
were transferred to 96-well plates. Sytox green fluorescence (excitation
wavelength [␭ex], 485 nm; emission wavelength [␭em], 520 nm) was measured in a fluorescence plate reader (BioTek microplate readers) (14, 49,
50). BRR003-cecropin A and polymyxin B were added at the corresponding 2⫻ MICs, and the fluorescence was monitored for 60 min at 10-min
intervals. DMSO at 1% and 2.5 ␮g/ml polymyxin B were used as negative
and positive controls, respectively. The experiment was repeated three
times.
Fluorescence microscopy. A. baumannii was grown to mid-log phase
at 37°C and treated with 5⫻ the MIC of cecropin A, the positive control (2
␮g/ml polymyxin B), or the negative control (1% DMSO). After 2 h of
incubation at 30°C, the treated cultures were harvested and stained with 1
␮g/ ml FM4-64, 2 ␮g/ ml DAPI, and 0.5 ␮M Sytox green (Molecular
Probes/Invitrogen). A 1-␮l portion of stained cells was transferred to a
1.2% agarose pad containing 20% LB medium for observation by using
confocal laser microscopy (TCS NT; Leica Microsystems) (51, 52).
Antimicrobial Peptides against A. baumannii by HTS
elegans with or without 2 ␮g/ml polymyxin B. Image analysis was carried out using CellProfiler; worms identified in the bright field are outlined in red, and dead
worms identified in the Sytox orange fluorescent image are in green. (B) The Z= factor was calculated from survival percentage determined by CellProfiler analysis
of positive- and negative-control images. The calculated Z= factor for the particular screening assay shown was 0.7. (C) The left half of the 384-well plate shows
live worms incubated with A. baumannii in the presence of polymyxin B as a positive control, and the right half shows dead worms in the presence of DMSO as
a negative control.
coli OP50 and 30% survival with A. baumannii after 5 days (data
not shown).
Although iron acquisition mechanisms vary among Acinetobacter strains (53), recent studies by Eijkelkamp et al., Nwugo et
al., and Dorsey et al. showed that the concentration of iron in the
medium has a significant influence on Acinetobacter virulence factors (54–56). Therefore, we tested the addition of iron to the medium as a means of enhancing killing by A. baumannii ATCC
19606. Addition of 10 ␮M FeCl3 to the assay medium increased
killing efficiency in the liquid assay, decreasing worm survival to
5% (Fig. 2A). In addition, we found that the starting bacterial titer
influenced the killing kinetics of worms. Worms treated with an
initial concentration of A. baumannii ATCC 19606 or E. coli OP50
corresponding to an OD600 of 0.03 in medium supplemented with
10 ␮M FeCl3 exhibited the maximum difference between survival
on E. coli OP50 (80%) and A. baumannii (⬍3%) after 5 days
incubation (Fig. 2B).
Liquid killing assay quality assessment. The reproducibility
and reliability of the liquid killing assay was evaluated by calculating the Z= factor from survival ratios based on CellProfiler analysis
of bright-field and Sytox images using 1% DMSO and 2 ␮g/ml
polymyxin B as negative and positive controls, respectively (Fig.
3A and C). In 3 replicates, the Z= factor ranged from 0.6 to 0.7,
March 2015 Volume 59 Number 3
confirming that the assay is robust and could be used for largescale screening of small-molecule libraries (Fig. 3B).
Screening and identification of compounds effective against
A. baumannii. The C. elegans-A. baumannii liquid killing assay
was used to perform a pilot screen with a small library of insectderived antimicrobial peptides. The library includes 68 synthetic
peptides belonging to different families of insect AMPs biosynthesized by a variety of different insect species.
The AMP library was screened in triplicate at a final AMP concentration of 2 ␮g/ml. We identified hits by calculating Z scores,
i.e., the number of standard deviations by which a sample well
deviated from a negative-control well, derived from the percentage survival data. The average Z score for the polymyxin B positive-control wells was 1.5, and we set that value as a threshold for
“hit” wells. We identified 15 AMPs that prolonged worm survival
upon exposure to A. baumannii that had a Z score of ⱖ1.5. Significantly, all of the hits were either cecropin (11) or cecropin-like
(4) peptides (Table 2; also, see Fig. S1 in the supplemental material). In fact, all cecropins or cecropin-like peptides present in the
peptide library were identified as hits.
All the hit compounds were retested at 5 and 10 ␮g/ml in the
liquid killing assay and showed rescue at both concentrations,
suggesting that even at relatively high concentrations, cecropins
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FIG 3 Image data for the C. elegans-A. baumannii assay and Z= factor calculation. (A) Bright-field and Sytox orange-stained images of A. baumannii-infected C.
Jayamani et al.
TABLE 2 AMP hits in HTS assay based on Z scores of triplicates
AMP
Z1, Z2, Z3
Hyalophora cecropia
Aedes aegypti
Lucilia sericata contig 10925
Lucilia sericata contig 26125
Lucilia sericata contig 2999
Lucilia sericata contig 9468
Lucilia sericata contig 16625
Lucilia sericata contig 1380
Lucilia sericata contig 489
Lucilia sericata contig 1845
Lucilia sericata contig 21136
Sarcophaga peregrina
Stomoxys calcitrans
Lucilia sericata contig 25628
Lucilia sericata contig 25628
Cecropin A
Cecropin A
Cecropin
Cecropin
Cecropin
Cecropin
Cecropin
Cecropin
Cecropin
Cecropin
Cecropin
Sarcotoxin IA
Stomoxyn
Stomoxyn start A
Stomoxyn start G
1.8, 1.8, 1.8
1.7, 1.7, 1.6
1.8, 1.8, 1.8
1.7, 1.6, 1.7
1.6, 1.8, 1.8
1.7, 1.8, 1.8
1.8, 1.8, 1.8
1.8, 1.8, 1.8
1.8, 1.8, 1.7
1.7, 1.8, 1.6
1.7, 1.7, 1.6
1.8, 1.8, 1.7
1.8, 1.8, 1.8
1.8, 1.8, 1.7
1.8, 1.8, 1.8
are not toxic to worms (data not shown). To further address the
toxicity of cecropin, we tested and compared the toxic effects of
cecropins and polymyxin B by treating C. elegans with doses of
increasing concentration of the drugs up to 10⫻ the MIC. The
assay was carried out over 10 days with E. coli as the food source. In
order to address the concerns that worms might starve due to the
killing of the E. coli by the peptides, food was supplemented every
day without drastically altering the assay volume. The assay was
performed in triplicate with similar results. There was no drastic
loss in viability for worms treated with cecropin A at a concentration of 10⫻ the MIC for 10 days. However, at a similar concentration, polymyxin B displayed significant toxicity toward worms
(data not shown).
Antibacterial activity of cecropin A and cecropin-like family.
To determine if AMPs identified in the screen were acting directly
on A. baumannii, we tested their ability to inhibit bacterial growth.
We found that one particular hit peptide, BR003-cecropin A isolated from Aedes aegypti, had a very low MIC, ⬍5 ␮g/ml (Table 3),
and had activity against other Gram-negative bacteria as well, including E. coli and Pseudomonas aeruginosa, but not the Grampositive pathogen methicillin-resistant Staphylococcus aureus
(MRSA) MW2 (Table 4).
TABLE 3 MICs of cecropins and cecropin-like family of peptides
Compound
MIC (␮g/ml)
Polymyxin B
BR001
BR002
BR003
BR005
BR029
BR030
BR031
BR032
BR033
BR034
BR035
BR036
BR037
BR043
BR044
2.5
10
10
5
20
20
⬎20
20
20
20
20
10
20
20
⬎20
⬎20
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TABLE 4 In vitro antimicrobial activity of cecropin A
MIC (␮g/ml)
Compound
Vancomycin
Polymyxin B
Gentamicin
BR003-cecropin A
E. coli
(OP50)
MRSA
(MW2)
P. aeruginosa
(PA14)
A. baumannii
1.2
2.25
0.6
3.5
75
0.6
10
4.5
An in vitro disc diffusion assay showed a clear zone of inhibition caused by discs containing BR003-cecropin A and polymyxin
B (Fig. 4B). We further tested the activity of BR003-cecropin A on
a panel of Acinetobacter species, including Acinetobacter baylyi and
clinical strains of Acinetobacter baumannii such as A. baumannii
G5139 and G5140, which overexpress the AdeABC efflux pump,
and A. baumannii GC7945 and GC7951, which lack the AdeABC
efflux pump (57). Interestingly, BR003-cecropin A displayed similar activities against all tested strains (Fig. 4A).
Sequence analysis of cecropin A and cecropin like family
from AMPs library. Sequence analysis of the cecropins that were
hits in the screening assay showed the presence of tryptophan at
the 1 or 2 position at the N terminus of many of the cecropins,
although the most effective peptide BR003-cecropin A lacked the
tryptophan (Fig. 5). The absence of tryptophan, as well as an increased number of positively charged amino acids in BR003-cecropin A may contribute to the low MIC of this cecropin against A.
baumannii.
Antimicrobial activity of cecropin A. The bactericidal efficiencies of cecropin A and other cationic peptides have been previously reported to be due to their ability to form pores in bacterial
membranes (50, 58). We tested if treatment of A. baumannii
A9844 with BR003-cecropin A caused increased membrane permeability by using fluorescence microscopy to observe the bacterial cells stained with the membrane dye FM4-64 and with Sytox
green, a dye that penetrates only cells with compromised membranes (51). Bacteria treated with DMSO were short rods and
showed membranes stained with FM4-64 but not Sytox green
(Fig. 6A). Treatment with cecropin A caused the bacteria to be
elongated and show bright Sytox staining (Fig. 6A). Interestingly,
the positive-control polymyxin B showed a bacterial shape and
fluorescence pattern similar to those of cecropin A (Fig. 6A). Maximum accumulation of Sytox green caused by cecropin A treatment was achieved at 10 ␮g/ml, 2-fold above the MIC (data not
shown). To evaluate the kinetics of membrane permeability, fluorometric measurements were taken at 10-min intervals for 60 min.
Membrane permeability induced by cecropin A and polymyxin B
was observed to be time dependent, suggesting that cecropin A
rapidly kills bacteria by disrupting membrane integrity (Fig. 6B).
DISCUSSION
Drug-resistant A. baumannii is a National Institute of Allergy and
Infectious Diseases (NIAID) category C pathogen and is also a
member of ESKAPE (Enterococcus faecium, Staphylococcus aureus,
Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas
aeruginosa, and Enterobacter spp.), a group of bacteria thought to
be the most dangerous multidrug-resistant (MDR) microbes (59–
61). Treatment options are limited due to the variety of resistance
traits harbored by these pathogens, including a spectrum of ␤-lac-
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Source
Antimicrobial Peptides against A. baumannii by HTS
tamases, carbapenemases, aminoglycoside modification enzymes,
and modifications in the penicillin-binding protein and outer
membrane proteins (2, 10). The evolution of resistance to most
conventional antibiotics within 5 years of their introduction has
been a major threat to human health (11, 29), and polymyxin B
and colistin are often treatments of last resort (9, 62, 63).
Previous studies have used C. elegans as a model host to study
the interaction of A. baumannii and Candida albicans by coinfec-
FIG 5 Amino acid sequence analysis of cecropin A and cecropin-like peptides. The color scale highlights the level of conservation of specific amino acid residues.
The distributions of certain hydrophilic and hydrophobic amino acids are conserved in all cecropins and cecropin-like peptides and are characteristic of cationic
peptides.
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FIG 4 Antimicrobial activity of cecropin A against A. baumannii. (A) MIC of BR003-cecropin A compared with that of polymyxin B with a panel of Acinetobacter
strains. (B) Disc diffusion assay to measure zones of inhibition due to bactericidal activity of BR003-cecropin A against A. baumannii. The discs were prepared
by adding 10 ␮l of 1-mg/ml stock solutions of cecropin A or polymyxin B in DMSO by using DMSO alone as a control.
Jayamani et al.
tion in worms. These studies revealed that A. baumannii inhibits
C. albicans filamentation, a key component of C. albicans virulence, highlighting the importance of pathogenic prokaryotic and
eukaryotic interaction in the host (64). Using an agar-based C.
elegans assay to evaluate an insertion mutant library of A. baumannii, Peleg et al. and Smith et al. identified many genes encoding A.
baumannii virulence factors (64–66). Although these agar-based
assays are useful for studying virulence factors of pathogenic bacteria and to carry out small-scale genetic screens, they are not
appropriate for large-scale small molecule screens. In order to
carry out large-scale chemical screens, we developed a C. elegans-A. baumannii liquid infection assay that could be used to
screen large small-molecule libraries to identify new drugs that
may be useful in treating A. baumannii infections. A key feature of
our C. elegans assay is that it is able to assess antimicrobial activity
of a small molecule while concurrently evaluating its toxicity.
Liquid killing assays for high-throughput screens in C. elegans
have been well established for Enterococcus faecalis, methicillinresistant Staphylococcus aureus (MRSA), and Pseudomonas aeruginosa (16, 37, 46). The C. elegans-E. faecalis infection assay utilizes
worms preinfected with a pathogen followed by treatment. The
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rationale is that E. faecalis persistently colonizes the C. elegans
intestine, which allows screening for compounds that cure an established infection. In contrast to E. faecalis, however, P. aeruginosa, MRSA, and A. baumannii do not form persistent infections
in C. elegans. Moreover, in the case of P. aeruginosa, MRSA, and A.
baumannii, preinfection of the worms with the pathogen cannot
be used due to the high risk of contamination of the equipment
during the dispensing of preinfected worms into 384-well plates
and the difficulties of disinfecting equipment after each use (67).
Therefore, we developed an automated, high-throughput liquid
killing assay of C. elegans mediated by A. baumannii that involves
dispensing worms into plates containing A. baumannii and compound.
Using this assay, we performed a pilot proof-of-principle
screen of a small library of 68 AMPs. Due to the fact that AMPs
exploit fundamental conserved features of the bacterial cell wall,
they are able to act against a broad spectrum of pathogens, including Salmonella enterica, E. coli, K. pneumoniae, P. aeruginosa, and
Listeria monocytogenes (58, 68, 69). Also, because of the mechanism of action of AMPs, the incidence of bacterial resistance is
very low. Moreover, they also exhibit anti-endotoxic effects and
Antimicrobial Agents and Chemotherapy
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FIG 6 Cell permeability assay. (A) A. baumannii cells treated with BR003-cecropin A and polymyxin B stained with the red membrane dye FM4-64 and the
membrane-impermeative dye Sytox green. Treatment with DMSO caused cells to be short rods stained only with FM4-64, whereas treatment with either cecropin
A or polymyxin B caused cells to be elongated rods stained with both FM4-64 and Sytox green. Bar, 2 ␮m. (B) The time course of membrane permeation by
BR003-cecropin A and polymyxin B was measured by following the increase in Sytox green fluorescence (␭ex ⫽ 485 nm; ␭em ⫽ 520 nm). The concentrations of
BR003-cecropin A and polymyxin used corresponded to 2⫻ their MICs.
Antimicrobial Peptides against A. baumannii by HTS
March 2015 Volume 59 Number 3
ACKNOWLEDGMENT
We acknowledge NIH grant R01 AI085581.
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immunomodulatory effects in hosts owing to their ability to bind
lipopolysaccharides (LPS) (14, 25, 30). These qualities make
AMPs suitable candidates for further evaluation of their antimicrobial and therapeutic potential.
From the pilot screen, we identified 15 cecropin and cecropinlike AMPs that prolonged worm survival in the presence of A.
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