Content uploaded by Brian P Conlon
Author content
All content in this area was uploaded by Brian P Conlon on Sep 05, 2014
Content may be subject to copyright.
ARTICLE
doi:10.1038/nature12790
Activ ated ClpP kills persisters and
eradicates a chronic biofilm infection
B. P. Conlon
1
, E. S. Nakayasu
2
{, L. E. Fleck
1
, M. D. LaFleur
3
, V. M. Isabella
1
, K. Coleman
3
, S. N. Leonard
4
, R. D. Smith
2
, J. N. Adkins
2
& K. Lewis
1
Chronic infections are difficult to treat with antibiotics but are caused primarily by drug-sensitive pathogens. Dormant
persister cells that are tolerant to killing by antibiotics are responsible for this apparent paradox. Persisters are
phenotypic variants of normal cells and pathways leading to dormancy are redundant, making it challenging to
develop anti-persister compounds. Biofilms shield persisters from the immune system, suggesting that an antibiotic
for treating a chronic infection should be able to eradicate the infection on its own. We reasoned that a compound capable
of corrupting a target in dormant cells will kill persisters. The acyldepsipeptide antibiotic (ADEP4) has been shown to
activate the ClpP protease, resulting in death of growing cells. Here we show that ADEP4-activated ClpP becomes a fairly
nonspecific protease and kills persisters by degrading over 400 proteins, forcing cells to self-digest. Null mutants of clpP
arise with high probability, but combining ADEP4 with rifampicin produced complete eradication of Staphylococcus
aureus biofilms in vitro and in a mouse model of a chronic infection. Our findings indicate a general principle for killing
dormant cells—activation and corruption of a target, rather than conventional inhibition. Eradication of a biofilm in an
animal model by activating a protease suggests a realistic path towards developing therapies to treat chronic infections.
The current antibiotic crisis stems from two distinct phenomena, drug
resistance and drug tolerance. Resistance mechanisms such as drug
efflux or modification prevent antibiotics from binding to their targets
1
,
allowing pathogens to grow. Antibiotic tolerance is the property of
persister cells, phenotypic variants of regular bacteria
2
. Antibiotics kill
by corrupting their targets, but these are inactive in dormant persisters,
leading to tolerance
3,4
. Persisters were discovered by Joseph Bigger in
1944, when he found that a small sub-population of Staphylococcus
aureus survives treatment with penicillin
5
. We identified persisters as
the main component responsible for drug tolerance of biofilms
6
.A
multitude of chronic diseases is associated with biofilms: endocarditis,
osteomyelitis, infections of catheters and indwelling devices, gingivitis
and deep-seated infections of soft tissues
7,8
.InEscherichia coli, which
has served as a model organism for studying persisters, pathways lead-
ing to dormancy are highly redundant and largely depend on the action
of toxin/antitoxin modules
3,9
. Protein synthesis inhibition by the HipA
toxin
10,11
, a kinase
12
that phosphorylates glutamyl-transfer RNA syn-
thetase GltX
11
, and by at least 10 different messenger RNA endonucleases
such as RelE, MazF and YafQ
3,9,13,14
leads to dormancy. Damage of DNA
inducestheSOS responseand expression of the TisB toxin
15
, which is an
endogenous antimicrobial peptide
16
and causes persister formation by
opening an ion channel
17
. This decreases the proton motive force and
ATP levels, leading to target shutdown and a dormant, drug-tolerant
state. The multiplicity of dormancy pathways precludes development
of drugs that could prevent persister formation
18
.
We reasoned that a compound capable of corrupting a target in dorm-
ant, energy-deprived cells will kill persisters. Acyldepsipeptide (ADEP)
activates the ClpP protease, and it was reported to kill growing cells
19
.
Normally, ClpP recognizes and eliminates misfolded proteins with
the aid of ATP-dependent ClpX, C or A subunits
20
. ADEP binds to
ClpP and keeps the catalytic chamber open, allowing entry to peptides
and proteins
21,22
. In the presence of ADEP, proteolysis by ClpP no longer
depends on ATP
23
. Several related ADEP compounds are produced by
Streptomyces hawaiensis
24
, and a more potent derivative, ADEP4 (Fig. 1),
showed good activity against a variety of Gram-positive bacteria
19
. ADEP4
was efficacious in a lethal systemic murine infection of Enterococcus
faecalis and S. aureus and in lethal sepsis caused by Streptococcus pneu-
moniae in the rat
19
. Nascent polypeptides emerging from the ribo-
some, rather than mature folded proteins, were proposed to be primary
targets of ADEP4/ClpP
23
. This would indicate that ADEP4 targets
growing cells with active protein synthesis. A particular mature pro-
tein, FtsZ, has been reported to be a major target of ADEP4/ClpP
25
.
FtsZ forms the cell division ring, suggesting activity of ADEP4 against
growing cells as well.
Here we sought to examine the ability of ADEP4 to activate protein
degradation in non-growing cells and find that in its presence, ClpP
becomes a fairly nonspecific protease. Null clpP mutants are resistant
to ADEP4 (ref. 19), but we find that they are highly susceptible to
killing by a variety of antibiotics. Combining ADEP4 with rifampicin
leads to eradication of persisters in growing, stationary and biofilm
populations of S. aureus in vitro, and clears a deep-seated murine
biofilm infection that is untreatable with conventional antibiotics.
1
Antimicrobial Discovery Center, Department of Biology, Northeastern University, Boston, Massachusetts 02115, USA.
2
Biological Sciences Division, Pacific Northwest National Laboratory, Richland,
Washington 99352, USA.
3
Arietis Corporation, Boston, Massachusetts 02118, USA.
4
Bouve
´
College of Health Sciences, School of Pharmacy, Northeastern University, Boston, Massachusetts 02115, USA.
{Present address: Bindley Bioscience Center, Purdue University, West Lafayette, Indiana 47907, USA.
ADEP4ADEP1 (factor A)
N
O
NH
O
N
O
N
O
O
O
N
H
O
HN
O
F F
N
O
NH
O
N
O
N
O
O
O
N
H
O
HN
O
Figure 1
|
Structures of acyldepsipeptide factor A and its synthetic
derivative ADEP4.
21 NOVEMBER 2013 | VOL 503 | NATURE | 365
Macmillan Publishers Limited. All rights reserved
©2013
ADEP4 causes extensive protein degradation
Previous studies showing that ADEP targets nascent peptides and FtsZ
in particular were performed with short exposure times and with rapidly
growing cells, and we considered the possibility that longer incubation
with ADEP may result in nonspecific degradation of proteins in non-
growing cells. A stationary phase population of S. aureus was chosen to
test this, as cells are not dividing and synthesis of nascent polypeptides
is strongly downregulated
26
. Stationary cells of methicillin-resistant
S. aureus (MRSA) were exposed to ADEP4 for 24 h and the resulting
proteome was compared with that of an untreated control (Fig. 2).
Proteomic analysis of untreated stationary cells led to the detection
of 1,712 proteins (65% of the predicted open reading frames). Treatment
0
5
10
15
20
25
30
35
40
Ribosome
Purine metabolism
Glycolysis / Gluconeogenesis
Pyruvate metabolism
Phosphotransferase system (PTS)
Galactose metabolism
Pentose phosphate pathway
Propanoate metabolism
Aminoacyl-tRNA biosynthesis
Pyrimidine metabolism
Citrate cycle (TCA cycle)
Cysteine and methionine metabolism
Amino sugar and nucleotide sugar metabolism
One carbon pool by folate
Fructose and mannose metabolism
Oxidative phosphorylation
Glycine, serine and threonine metabolism
Arginine and proline metabolism
Glyoxylate and dicarboxylate metabolism
Fatty acid biosynthesis
Nitrogen metabolism
Alanine, aspartate and glutamate metabolism
Butanoate metabolism
Selenoamino acid metabolism
Bacterial secretion system
Starch and sucrose metabolism
Glycerolipid metabolism
Valine, leucine and isoleucine degradation
Protein export
DNA replication
Valine, leucine and isoleucine biosynthesis
Pathwa
y
(
KEGG
)
Fold enrichment
0
0.0100
0.0200
0.0300
0.0400
0.0500
0.0600
0.0700
0.0800
0.0900
P-value (•)
P > 0.05
P ≤ 0.05
P > 0.05
P ≤ 0.05
–10
–5
0
5
10
–10 –5 0 5 10
Replicate B (log
2
treat/untreat)
Replicate A (log
2
treat/untreat)
Pyk
SerS
Frr
RplI
RpsU
Efp
FtsZ
Tuf
FbaA
Cold shock
protein
GrpE
GreA
RpsA
–10
–5
0
5
10
–10 –5 0 5 10
Replicate B (log
2
treat/untreat)
Replicate A (log
2
treat/untreat)
Tuf
FbaA
Pyk
FtsZ
SerS
Proteins Partially tryptic peptides
ab
c
Figure 2
|
Quantitative proteomic analysis of
S. aureus
cells treated with
ADEP4 reveals extensive protein degradation. S. aureus cells were treated
with ADEP4 in biological duplicates and submitted for global quantitative
proteomic analysis. a, b, The dispersion graphs show the relative abundances
(treated/untreated) of total proteins (a) and partially tryptic peptides
(b) in different biological replicates (n 5 2). The significant changes in
abundances (P # 0.05 and .twofold) are represented in red circles.
c, Function–enrichment analysis of proteins degraded by ADEP4. Functions
overrepresented among proteins degraded by ADEP4 were annotated using
Database for Annotation, Visualization and Integrated Discovery (DAVID)
and the overrepresented pathways compared to the genome background are
shown as columns, whereas their P-values are represented by the black dots.
Bayesian moderated t-test was used to provide P-values that were further
corrected by the data set size.
RESEARCH ARTICLE
366 | NATURE | VOL 503 | 21 NOVEMBER 2013
Macmillan Publishers Limited. All rights reserved
©2013
with ADEP4 resulted in decreased abundance of 243 proteins (P # 0.05
and twofold decrease) (Fig. 2a) (Supplementary Table 1). However,
this is probably an underestimate. The proteome reports changes in the
relative abundance of peptides produced by exogenous trypsin cleav-
age. A protein only cleaved once by ADEP4/ClpP, for example, would
still generate several tryptically derived peptides, and not appear to
show an overall decrease in protein abundance.
To address this, we examined partially tryptic peptides to uncover
additional ADEP4/ClpP targets (Fig. 2b). Partially tryptic peptides
exist following trypsin treatment at certain abundance in cells due
to natural degradation. However, the levels of these peptides changed
markedly due to degradation induced by addition of ADEP4 (red
spots). An increase of partially tryptic peptides indicates ADEP4-
dependent degradation of a protein. This analysis revealed 174 addi-
tional ADEP4/ClpP targets (peptides of increased abundance; Fig. 2b;
Supplementary Table 2), bringing their total numberto 417. A decrease
on the other hand indicates that a particular degradation product,
present at the time of ADEP4 addition, can be further degraded by
ADEP4/ClpP, but these are of less relevance to the study.
Essential ribosomal proteins were among the most strongly dimin-
ished by ADEP4/ClpP, with proteins S21, L9, S1 and ribosomal recyc-
ling factor all showing between 17- and 64-fold reduction in the
ADEP4 treated sample. Elongation factor Tu, pyruvate kinase and
fructose bi-phosphate aldolase were among the proteins with the
largest increase in non-trypsin cleavage sites (Fig. 2b). FtsZ was also
one of the many strongly degraded proteins perhaps because of its
disordered carboxy terminus
27
. Other than the ribosome, degraded
proteins belonged to various functional types, including purine meta-
bolism, glycolysis and aminoacyl-tRNA biosynthesis, among others
(Fig. 2c).
ADEP4 kills persister cells
The proteomic data indicates that ADEP4 forces the cell to self-digest,
and may be effective in killing dormant cells. ADEP4 uncouples ClpP
from the requirement to use ATP, which would help kill persisters
with low energy levels
15
. In a control experiment, ciprofloxacin was
added to an exponentially growing culture of S. aureus, which produced
a typical biphasic killing pattern with surviving persisters (Fig. 3a).
Addition of rifampicin to surviving persisters had no effect on their
viability, in agreement with previous observations on the multidrug
tolerant nature of these cells
3,28
. By contrast, addition of ADEP4 led to
eradication of persisters to the limit of detection (Fig. 3a). Next, we
examined the ability of ADEP4 to kill stationary cells of S. aureus.
Stationary phase S. aureus cells behave as persisters and are extremely
difficult to kill with antibiotics
28,29
, even over a 5-day period (Fig. 3b).
Furthermore, combinations of vancomycin, rifampicin and ciproflox-
acin had limited activity against this population (Extended Data Fig. 1).
ADEP4 showed excellent killing,decreasing the cell count of a stationary
culture by 4 log
10
in two days (Fig. 3c), but the population rebounded
after day 3. Null mutants of clpP are resistant to ADEP4 (ref. 19) and
arise with high frequency because ClpP is not essential in S. aureus.No
cross-resistance to marketed antibiotics was identified for ADEP4
(ref. 19). Sequencing of 9 isolates of this culture showed mutations
in clpP, and all of them displayed the temperature-sensitive phenotype
characteristic of null clpP mutants
30
(Extended Data Fig. 2). To sup-
press resistant mutants, ADEP4 was paired with either rifampicin,
linezolid or ciprofloxacin. ADEP4 with rifampicin eradicated a sta-
tionary population of S. aureus to the limit of detection (Fig. 3d). This
shows that ADEP4, unlike conventional antibiotics, has a remarkable
ability to kill drug-tolerant persister cells. The rich Mueller-Hinton
broth (MHB) in which these experiments were performed probably
does not reflect conditions in vivo where pathogens experience nutri-
ent limitation. We therefore tested susceptibility to killing of station-
ary cells in a chemically defined medium
31
. Killing in the minimal
medium by ADEP4 with rifampicin was even more effective than in
MHB, eradicating the population in 24 h (Fig. 3e). Complete steriliza-
tion in these experiments was unexpected—the frequency of clpP
mutants is 10
26
, and in a population of 10
9
cells, there should have
abc
def
6
7
8
9
10
02448
WT
Rifampicin
WT
Linezolid
ΔclpP
Rifampicin
ΔclpP
Linezolid
1
2
3
4
5
6
7
8
0 2 4 6 24 48
Log (c.f.u. ml
–1
)Log (c.f.u. ml
–1
)
Log (c.f.u. ml
–1
)
Log (c.f.u. ml
–1
)
Log (c.f.u. ml
–1
)
Log (c.f.u. ml
–1
)
Time (h)
Time (h)
Ciprooxacin
Ciprooxacin
then rifampicin
Ciprooxacin
then ADEP4
2
3
4
5
6
7
8
9
10
0123
ADEP4
Control
Second
antibiotic
added
1
2
3
4
5
6
7
8
9
10
012345
Time (days)
Time (days)
Time (days)
Control
Rifampicin
Linezolid
Vancomycin
Ciprooxacin
2
3
4
5
6
7
8
9
10
0123
Control
ADEP4
Rifampicin
ADEP4
Linezolid
ADEP4
Ciprooxacin
2
3
4
5
6
7
8
9
Pre-treatment
Control
Vancomycin
Rifampicin
ADEP4
ADEP4 + rifampicin
*
Figure 3
|
ADEP4 kills persisters. a, ADEP4 kills persisters surviving
ciprofloxacin treatment. b, Conventional antibiotics are inactive against
stationary phase S. aureus. c, ADEP4 activity against stationary S. aureus.
d, e, ADEP4 in combination with rifampicin, linezolid or ciprofloxacin
eradicates stationary phase S. aureus to the detection limit in 72 h in MHB
(d) and in 24 h in chemically defined medium (e). The x axis is the limit of
detection. The asterisk represents eradication to the limit of detection.
f, ADEP4 resistant mutants are less tolerant to rifampicin and linezolid than the
parent wild-type strain. Data are representative of 3 independent experiments.
Error bars represent s.d.
ARTICLE RESEARCH
21 NOVEMBER 2013 | VOL 503 | NATURE | 367
Macmillan Publishers Limited. All rights reserved
©2013
been 10
3
survivors. To investigate this, a clpP mutant was examined for
its susceptibility to linezolid and rifampicin (Fig. 3f). The DclpP strain
had the same minimum inhibitory concentration (MIC) as the wild
type, but stationary phase counts were reduced 10- to 100-fold more
than the wild type by linezolid or rifampicin in stationary state. A
mutation in clpP apparently diminishes the fitness of cells and makes
them vulnerable to certain antibiotics. In agreement with this, a clpP
mutant was reported to be avirulent in a murine skin abscess model of
infection
30
. We then tested the eradicating potential of the ADEP4
and rifampicin combination against a variety of S. aureus strains.
These included the laboratory strain SA113, as well as clinical isolates
USA300, UAMS-1 and strain 37. USA300 is a community acquired
MRSA and is the most common cause of staphylococcal skin and soft
tissue infections in the United States
32
. UAMS-1 is a highly virulent
clinical isolate associated with chronic osteomyelitis
33
. Strain 37 was
isolated from a patient undergoing vancomycin therapy who suc-
cumbed to infection
34
. No colonies were detected in any of these
strains after 72 h of incubating stationary cultures with ADEP4 and
rifampicin (Fig. 4).
ADEP4 with rifampicin eradicates biofilm
Biofilms produced by the osteomyelitis-associated strain UAMS-1
displayed a similar tolerance to antibiotics as stationary phase cultures
(Fig. 5). ADEP4 showed considerable killing following 24 h of treat-
ment, but the population rebounded after 72 h. Again, a combination
of ADEP4 with rifampicin resulted in eradication of living cells in the
biofilm to the limit of detection (Fig. 5). The replacement of antibio-
tics with fresh medium did not result in re-growth after 3 days of
ADEP4 and rifampicin treatment, confirming the complete eradica-
tion of living cells. An elimination of a biofilm is unprecedented for
such low, clinically achievable concentrations of compounds.
ADEP4 with rifampicin eradicates infection
Eradication of stationary and biofilm populations was an encouraging
sign that ADEP4 could be a very useful antibiotic against untreatable
chronic infections. To test this, we used a deep-seated mouse thigh
infection model. In a standard thigh model, a mouse is infected with a
low dose of pathogen and antibiotic therapy begins within a few hours
of infection. Under these conditions, conventional antibiotics are very
effective. In the deep-seated model, the mouse is made neutropenic by
treatment with cyclophosphamide, a large dose of pathogen is deliv-
ered and the infection is allowed to develop for 24 h before therapy,
leading to a severe, recalcitrant, deep-seated infection. This model
emulates a difficult to treat human deep-seated chronic infection in
immunocompromised patients. We performed histopathology of the
infected thigh and detected massive aggregates of S. aureus cells with
Gram staining (Fig. 6a). Electron microscopy of cross-sections of the
infected tissue revealed S. aureus growing in biofilms adhered to
muscle cells (Fig. 6a). Administration of vancomycin, rifampicin
or a combination of both decreased the viable counts, but did not
clear the infection (Fig. 6b). Furthermore, no notable difference was
observed between mice treated for 24 h or 48 h with vancomycin in
this model, indicating the presence of a persister subpopulation sur-
viving the antibiotic treatment (Fig. 6b). Remarkably, an ADEP4 and
rifampicin combination led to sterilization of the infected tissue to the
limit of detection within 24 h (Fig. 6c). Based on this efficacious dose
and the mouse pharmacokinetics data
19
, we performed a hollow-fibre
experiment and found that the combination of ADEP4 and rifampicin
also resulted in complete eradication of the pathogen to the limit of
detection (Extended Data Fig. 3).
Discussion
The rise in biofilm infections is a recent phenomenon, mainly a side-
effect of medical intervention
35
. Biofilms form readily on indwelling
devices such as catheters, prostheses and heart valves. Biofilms have
a complex architecture and developmental program
36,37
and form a
protective environment for persisters, shielding them from the immune
system. In patients undergoing cancer chemotherapy or organ trans-
plantation or in the elderly, the immune system is compromised, enab-
ling deep-seated infections in soft tissues to take hold. Even disseminating
infectionsof S. aureusaredifficult to eradicate in immunocompromised
patients. The dormant state of persisters and the multiplicity of the
pathways leading to their formation make treatment of chronic infec-
tions unusually challenging. Our results demonstrate that persister
cells in a biofilm can be killed with a protease-activating antibiotic.
This study shows that persisters are not invulnerable, and helps settle
an important uncertainty surrounding chronic diseases—it has been
unclear whether conventional antibiotics fail owing to their ineffective
killing or simply because they do not reach all pathogens at the site of
infection. We had previously described high-persister (hip) mutants
that are selected in the course of antibiotic treatment in patients with
Candida albicans biofilms
38
or with Pseudomonas aeruginosa in the
lungs of patients with cystic fibrosis
39
. Selection for increased produc-
tion of persister cells suggests that antibiotics effectively reach the
pathogens. Sterilization of a deep-seated biofilm infection with ADEP4,
but not with conventional antibiotics, shows directly that the problem
indeed lies in pathogen tolerance. Pathogens surviving antibiotic treat-
ment in a chronic infection are detrimental not only to a given patient,
but to society as well. A large, lingering population of pathogens is
2
3
4
5
6
7
8
9
10
0123
Log (c.f.u. ml
–1
)
Time (days)
UAMS-1
Strain 37
SA113
USA300
Figure 4
|
ADEP4 with rifampicin eradicates a variety of
S. aureus
strains.
S. aureus was grown in MHB for 16 h and challenged with 103 MIC of ADEP4
and rifampicin. Colony counts were performed every 24 h. The x axis is the
limit of detection. Data are representative of 3 independent experiments. Error
bars represent s.d.
1
2
3
4
5
6
7
Control
Oxacillin
Rifampicin
Linezolid
Vancomycin
Gentamicin
Ciprooxacin
ADEP4
ADEP4 + rifampicin
Log (c.f.u. ml
–1
)
Day 1
Day 3
*
Figure 5
|
ADEP4 kills a
S. aureus
biofilm and in combination with
rifampicin eradicates the population. The x axis is the limit of detection. An
asterisk represents eradication to the limit of detection. Data are representative
of 3 independent experiments. Error bars represent s.d.
RESEARCH ARTICLE
368 | NATURE | VOL 503 | 21 NOVEMBER 2013
Macmillan Publishers Limited. All rights reserved
©2013
fertile ground for the development of resistance
40–42
. The ability to
efficiently eradicate an infection will help reduce the spread of resistance.
ADEP4 is remarkable in its ability to kill dormant cells. Persisters formed
by competitors would present an obvious problem for Actinomycetes,
and it is perhaps not surprising that they evolved compounds capable
of killing both growing and dormant cells. ADEP4 points to a general
principle of killing, activation and corruption of a target (Extended
Data Fig. 4). Apart from ADEP4, other activators of ClpP
43
may be
developed into therapeutics, and additional bacterial proteases such as
Lon could be used as targets for killing specialized survivor cells. This
general principle of killing may be applied to other organisms as well
and prove effective in developing therapeutics to treat fungal infections
and cancer.
METHODS SUMMARY
In all experiments, bacterial cells were cultured in 20 ml of Mueller-Hinton (MH),
brain heart infusion (BHI) broth or chemically defined media
31
at 37 uC and were
aerated at 225 r.p.m in 250 ml flasks. Antibiotics were applied at the following
concentrations, corresponding to 103 MIC: vancomycin 10 mgml
21
, ADEP4
5 mgml
21
, rifampicin 0.4 mgml
21
, linezolid 10 mgml
21
and ciprofloxacin3 mgml
21
.
MICs were the same for each strain examined. The strains used in this study were
S. aureus: ATCC 33591, UAMS-1, USA300, SA113 and strain 37 (ref. 34). Biofilm
survival assays were performed in 96-well polystyrene plates in BHI broth in a
static 37 uC incubator. Biofilm was allowed to develop for 24 h. Wells were gently
washed with PBS and fresh medium containing antibiotics was added to each well.
Biofilms were incubated for either 24 or 72 h. Biofilms were then washed and
sonicated in PBS. Serial dilutions were performed and 10 ml aliquots were spotted
on BHI agar. iTRAQ proteomics was performed on stationary phase ATCC 33591
treated with 103 MIC of ADEP4 for 24 h and compared to an untreated control.
Mouse experiments were performed with female 6-week-old Swiss-Webster (Taconic)
mice that were first rendered neutropenic by cyclophosphamide treatment (150 mg
per kg and 100 mg per kg 96 h and 24 h before infection, respectively). Infection
was allowed to develop for 24 h before commencement of antibiotic therapy. All
antibiotics were delivered intraperitoneally. Animal experiments were carried out
at Northeastern University and conformed to institutional animal care and use
policies.
Online Content Any additional Methods, Extended Data display items and Source
Data are available in the online version of the paper; references unique to these
sections appear only in the online paper.
Received 2 August; accepted 18 October 2013.
Published online 13 November 2013.
1. Alekshun, M. N. & Levy, S. B. Molecular mechanisms of antibacterial multidrug
resistance. Cell 128, 1037–1050 (2007).
2. Lewis, K. Persister cells. Annu. Rev. Microbiol. 64, 357–372 (2010).
3. Keren, I., Shah, D., Spoering, A., Kaldalu, N. & Lewis, K. Specialized persister cells
and the mechanism of multidrug tolerance in Escherichia coli. J. Bacteriol. 186,
8172–8180 (2004).
4. Keren, I., Wu, Y., Inocencio, J., Mulcahy, L. R. & Lewis, K. Killing by bactericidal
antibiotics does not depend on reactive oxygen species. Science 339, 1213–1216
(2013).
5. Bigger, J. W. Treatment of staphylococcal infections with penicillin. Lancet 244,
497–500 (1944).
6. Spoering, A. L. & Lewis, K. Biofilms and planktonic cells of Pseudomonas aeruginosa
have similar resistance to killing by antimicrobials. J. Bacteriol. 183, 6746–6751
(2001).
7. Costerton, J. W., Stewart, P. S. & Greenberg, E. P. Bacterial biofilms: A common
cause of persistent infections. Science 284, 1318–1322 (1999).
8. Kostakioti, M., Hadjifrangiskou, M. & Hultgren, S. J. Bacterial biofilms:
development, dispersal, and therapeutic strategies in the dawn of the
postantibiotic era. Cold Spring Harb. Perspect. Med. 3, a010306 (2013).
9. Maisonneuve, E., Shakespeare, L. J., Jorgensen, M. G. & Gerdes, K. Bacterial
persistence by RNA endonucleases. Proc. Natl Acad. Sci. USA 108, 13206–13211
(2011).
10. Moyed,H. S. & Bertrand, K. P. hipA, a newlyrecognized geneof Escherichia coli K-12
that affects frequency of persistence after inhibition of murein synthesis.
J. Bacteriol. 155, 768–775 (1983).
11. Germain, E., Castro-Roa, D., Zenkin, N. & Gerdes, K. Molecular mechanism of
bacterial persistence by HipA. Mol. Cell 52, 248–254 (2013).
12. Correia, F. F. et al. Kinase activity of overexpressed HipA is required for growth
arrest and multidrug tolerance in Escherichia coli. J. Bacteriol. 188, 8360–8367
(2006).
13. Harrison, J. J. et al. The chromosomal toxin geneyafQ is a determinant of multidrug
tolerance for Escherichia coli growing in a biofilm. Antimicrob. Agents Chemother.
53, 2253–2258 (2009).
14. Maisonneuve, E., Castro-Camargo, M. & Gerdes, K. (p)ppGpp controls bacterial
persistence by stochastic induction of toxin-antitoxin activity. Cell 154,
1140–1150 (2013).
15. Do
¨
rr, T., Vulic
´
, M. & Lewis, K. Ciprofloxacin causes persister formation by inducing
the TisB toxin in Escherichia coli. PLoS Biol. 8, e1000317 (2010).
16. Unoson, C. & Wagner, E. A small SOS-induced toxin is targeted against the inner
membrane in Escherichia coli. Mol. Microbiol. 70, 258–270 (2008).
17. Gurnev, P. A., Ortenberg, R., Dorr, T., Lewis, K. & Bezrukov,S. M. Persister-promoting
bacterial toxin TisB produces anion-selective pores in planar lipid bilayers. FEBS
Lett. 586, 2529–2534 (2012).
18. Lewis, K. Platforms for antibiotic discovery. Nature Rev. Drug Discov. 12, 371–387
(2013).
19. Bro
¨
tz-Oesterhelt, H. et al. Dysregulation of bacterial proteolytic machinery by a
new class of antibiotics. Nature Med. 11, 1082–1087 (2005).
Before
treatment
Untreated
Before
treatment
Untreated
Vancomycin
Vancomycin
48 h
Rifampicin
Vancomycin
+ rifampicin
3
4
5
6
7
8
9
10
Log
10
c.f.u. per g tissueLog
10
c.f.u. per g tissue
ADEP4
ADEP4
+ rifampicin
3
4
5
6
7
8
9
10
*
S. aureus biolm
S. aureus cells within
extracellular matrix
a
b
c
Figure 6
|
ADEP4 in combination with rifampicin eradicates a deep-seated
mouse biofilm infection. a, Histopathology of S. aureus infected thighs reveals
the presence of a biofilm. Gram staining 380 magnification (left); electron
micrograph 38,000 magnification (right). b, Single day (rifampicin 30 mg per
kg once, vancomycin 110 mg per kg twice) treatments with rifampicin and
vancomycin. A second day of vancomycin treatment (vancomycin 48 h) reveals
an antibiotic tolerant subpopulation. c, Single day ADEP4 rifampicin
combination eradicates S. aureus in the deep-seated infection. An asterisk
represents eradication to the limit of detection. Groups of 5 neutropenic Swiss
mice were used for each experiment. Colony-forming units (c.f.u.) from each
mouse are plotted as individual points and error bars represent the deviation in
c.f.u. within an experimental group.
ARTICLE RESEARCH
21 NOVEMBER 2013 | VOL 503 | NATURE | 369
Macmillan Publishers Limited. All rights reserved
©2013
20. Gottesman, S., Roche, E., Zhou, Y. & Sauer, R. T. The ClpXP and ClpAP proteases
degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging
system. Genes Dev. 12, 1338–1347 (1998).
21. Li, D. H. et al. Acyldepsipeptide antibiotics induce the formation of a structured
axial channel in ClpP: A model for the ClpX/ClpA-bound state of ClpP. Chem. Biol.
17, 959–969 (2010).
22. Lee, B. G. et al. Structures of ClpP in complex with acyldepsipeptide antibiotics
reveal its activation mechanism. Nature Struct. Mol. Biol. 17, 471–478 (2010).
23. Kirstein, J. et al. The antibiotic ADEP reprogrammes ClpP, switching it from a
regulated to an uncontrolled protease. EMBO Mol. Med. 1, 37–49 (2009).
24. Michel, K. H. & Kastner, R. E. A54556 antibiotics and process for production
thereof. US patent 4,492 650 (1985).
25. Sass, P. et al. Antibiotic acyldepsipeptides activate ClpP peptidase to degrade the
cell division protein FtsZ. Proc. Natl Acad. Sci. USA 108, 17474–17479 (2011).
26. Michalik, S. et al. Proteolysis during long-term glucose starvation in Staphylococcus
aureus COL. Proteomics 9, 4468–4477 (2009).
27. Buske, P. J. & Levin, P. A. A flexible C-terminal linker is required for proper FtsZ
assembly in vitro and cytokinetic ring formation in vivo. Mol. Microbiol. 89, 249–263
(2013).
28. Keren, I., Kaldalu, N., Spoering, A., Wang, Y. & Lewis, K. Persister cells and tolerance
to antimicrobials. FEMS Microbiol. Lett. 230, 13–18 (2004).
29. Johnson, P. J. & Levin, B. R. Pharmacodynamics, population dynamics, and the
evolution of persistence in Staphylococcus aureus. PLoS Genet. 9, e1003123 (2013).
30. Frees, D., Qazi, S. N., Hill, P. J. & Ingmer, H. Alternative roles of ClpX and ClpP in
Staphylococcus aureus stress tolerance and virulence. Mol. Microbiol. 48,
1565–1578 (2003).
31. Hussain, M., Hastings, J. G. & White, P. J. A chemically defined medium for slime
production by coagulase-negative staphylococci. J. Med. Microbiol. 34, 143–147
(1991).
32. Stryjewski, M. E. & Chambers, H. F. Skin and soft-tissue infections caused by
community-acquired methicillin-resistant Staphylococcus aureus. Clin. Infect. Dis.
46 (Suppl 5), S368–S377 (2008).
33. Gillaspy, A. F. et al. Role of the accessory gene regulator (agr)inpathogenesisof
staphylococcal osteomyelitis. Infect. Immun. 63, 3373–3380 (1995).
34. Miyazaki, M. et al. Vancomycin bactericidal activity as a predictor of 30-day
mortality in patients with methicillin-resistant Staphylococcus aureus bacteremia.
Antimicrob. Agents Chemother. 55, 1819–1820 (2011).
35. Lewis, K. Antibiotics: recover the lost art of drug discovery. Nature 485, 439–440
(2012).
36. Hung, C. et al. Escherichia coli biofilms have an organized and complex
extracellular matrix structure. mBio 4, e00645–13 (2013).
37. Vlamakis, H., Chai, Y., Beauregard, P., Losick, R. & Kolter, R. Sticking together:
building a biofilm the Bacillus subtilis way. Nature Rev. Microbiol. 11, 157–168
(2013).
38. LaFleur, M. D., Qi, Q. & Lewis, K. Patients with long-term oral carriage harbor
high-persister mutants of Candida albicans. Antimicrob. Agents Chemother. 54,
39–44 (2010).
39. Mulcahy, L. R., Burns, J. L., Lory, S. & Lewis, K. Emergence of Pseudomonas
aeruginosa strains producing high levels of persister cells in patients with cystic
fibrosis. J. Bacteriol. 192, 6191–6199 (2010).
40. Mwangi, M. M. et al. Tracking the in vivo evolution of multidrug resistance in
Staphylococcus aureus by whole-genome sequencing. Proc. Natl Acad. Sci. USA
104, 9451–9456 (2007).
41. Levin, B. R. & Rozen, D. E. Non-inherited antibiotic resistance. Nature Rev. Microbiol.
4, 556–562 (2006).
42. Howden, B. P. et al. Evolution of multidrug resistance during Staphylococcus aureus
infection involves mutation of the essential two component regulator WalKR. PLoS
Pathog. 7, e1002359 (2011).
43. Leung, E. et al. Activators of cylindrical proteases as antimicrobials: identification
and development of small molecule activators of ClpP protease. Chem. Biol. 18,
1167–1178 (2011).
Supplementary Information is available in the online version of the paper.
Acknowledgements We thank B. Wright and C. Blinn of AstraZeneca for assisting with
the establishmentof the mouse infection model, R. E. Lee, M. Pollastri and Z. Maglika for
critical discussions and advice, I. Keren and S. Rowe for reading of the manuscript,
H. Brewer, V. Petyuk and D. Camp II for assistance with proteomics, and Z. Zheng for
assistance with ChemDraw.Thisworkwas supported by NIH award T-RO1 AI085585 to
K.L., by Arietis Corporation to M.D.L and K.C., by the NIH-NIAID IAA Y1-AI-8401 to J.N.A.
and P41 GM103493-11 to R.D.S. Proteomic analysis was performed in the EMSL, a
DOE-BER national scientific user facility at Pacific Northwest National Laboratory
(PNNL). PNNL is a multi-program national laboratory operated by Battelle Memorial
Institute for the DOE under contract DE-AC05-76RLO 1830.
Author Contributions B.P.C., M.D.L., K.C. and K.L. designed the study, analysed results
and wrote the manuscript. B.P.C. performed in vitro antibiotic susceptibility assays,
collected samples for proteomics, and performed biofilm susceptibility studies and
mouse infection models. V.M.I. assisted with in vitro susceptibility assays. E.S.N. and
J.N.A. performed i-TRAQ proteomics and analysed results. L.E.F. participated in mouse
infection model experiments. S.N.L performed hollow-fibre experiments. M.D.L. was
responsible for histopathology. R.D.S. provided the proteomics measurement
capabilities.
Author Information Reprints and permissions information is available at
www.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of the paper. Correspondence
and requests for materials should be addressed to K.L. (k.lewis@neu.edu).
RESEARCH ARTICLE
370 | NATURE | VOL 503 | 21 NOVEMBER 2013
Macmillan Publishers Limited. All rights reserved
©2013
METHODS
Bacterial strains, plasmids, media and growth conditions. Methicillin-resistant
Staphylococcus aureus strain ATCC 33591 was used for proteome analysis, anti-
biotic killing assays and in vivo infections. USA300, SA113, UAMS-1 and strain
37 (ref. 34) were also used in antibiotic killing assays. Biofilm experiments were
carried out with UAMS-1. Stationary phase populations were prepared as follows:
bacteria from frozen stock were grown in 20 ml of Mueller-Hinton broth (MHB)
or in a chemically defined medium
31
in 250 ml conical flasks with aeration at
225 r.p.m. at 37 uC overnight. Exponential phase cultures were prepared as fol-
lows: a stationary overnight culture was diluted 1:1,000 in MHB and incubated at
37 uC with aeration at 225 r.p.m. until A
600 nm
5 0.5 was reached. Biofilms were
grown in brain heart infusion (BHI) broth. Mueller-Hinton agar (MHA) and BHI
agar were used for colony counts.
Antibiotic susceptibility assays. Overnight, stationary phase, biological tripli-
cates were used in all susceptibility assays. Bacteria were incubated in the presence
of antibiotics at 37 uC with aeration at 225 r.p.m. Antibiotic concentrations, cor-
responding to 103 the minimum inhibitory concentration were as follows:
vancomycin 10 mgml
21
, ADEP4 5 mgml
21
, rifampicin 0.4 mgml
21
, linezolid
10 mgml
21
and ciprofloxacin 3 mgml
21
. Live cell numbers at a given time point
were determined as follows: 100 ml of culture was removed from the flask and
centrifuged at 10,000g for 1 min. The resulting pellet was resuspended in PBS.
Serial dilutions were performed and 10 ml of each dilution was spotted onto
Mueller-Hinton agar plates. Plates were allowed to dry and then incubated over-
night at 37 uC.
Biofilm assays. Overnight, stationary phase, biological triplicates of UAMS-1
were diluted 1:20 in BHI broth. Then 100 ml of this culture was added to each
well of a tissue-culture treated polystyrene 96-well plate. Plates were incubated at
37 uC for 24 h. Medium was carefully removed and wells were gently washed twice
with PBS. Then 100 ml of fresh medium containing 103 MIC of antibiotics was
carefully added to each well. Plates were incubated for either 24 or 72 h. Medium
was carefully removed and wells were gently washed twice with PBS. Then 100 ml
of PBS was added to each well and biofilm was solubilized by sonication for
5 minutes in a sonicating water bath (Fischer Scientific FS30). Serial dilutions
of each well were performed and 10 ml of each dilution was spotted onto BHI
plates and incubated overnight at 37 uC.
Proteomic analysis. Stationary phase cultures of MRSA cells were treated with
103 MIC of ADEP4 for 24 h at 37 uC. Biological duplicates of untreated control
and ADEP4-treated cells were harvested and lysed in 100 mM NH
4
HCO
3
,1mM
PMSF, 2 mM N-ethylmaleimide (NEM) and 5 mM ETDA, by mechanical dis-
ruption by vigorous vortexing in the presence of 0.1 mm diameter silica/zirconia
beads. A buffer exchange was performed on the cell lysates through an Amicon
10-kDa MWCO filter into 100 mM NH
4
HCO
3
. The lysate was denatured/
reduced in 100 mM NH
4
HCO
3
, 8 M urea, 5 mM DTT for 30 min at 60 uC, and
then diluted to obtain a final concentration of 1 M urea, and digested with trypsin
for 3 h at 37 uC. The resulting peptides were desalted using C18 SPE cartridges
(Discovery C18, 1 ml, 50 mg, Sulpelco), labelled with 4-plex iTRAQ reagent
(Applied Biosystems) following the manufacturer recommendations, and each
of the labelled samples was mixed in equal amounts based on total peptide con-
centrations measured by BCA assay (Thermo Scientific). The peptide mix was
then fractionated into 96 fractions by high pH reverse phase chromatography and
concatenated into 24 fractions as previously described
44
, and analysed by liquid
chromatography-tandem mass spectrometry (LC–MS/MS) analysis in a LTQ
Orbitrap Velos mass spectrometer (Thermo Fisher Scientific). Peptides were
loaded into capillary LC columns (75 mm 3 65 cm, Polymicro) packed with
C18 beads (3 mm particles, Phenomenex) connected to a custom-made 4-column
LC system
45
. The elution was performed in an exponential gradient from 0–100%
B solvent (solvent A: 0.1% formic acid; solvent B: 90% acetonitrile/0.1% formic
acid) with a constant pressure of 10,000 psi and flow rate of ,300 nl min
21
. Full-
MS scans were obtained for m/z 400–2,000 with the six most intense ions selected
for HCD fragmentation using a 2 m/z isolation width and 45% normalized
collision energy.
Raw mass spectrometry data were converted to peak lists (DTA files) using the
DeconMSn
46
(version 2.2.2.2, http://omics.pnl.gov/software/DeconMSn.php) and
searched with MSGF1
47
against the S. aureus COL NC 002951 (2,615 sequences),
bovine trypsin and human keratin sequences (all in correct and reverse orienta-
tions, 5,362 total sequences). Searching parameters included tryptic digestion in at
least one of the peptidetermini (partially tryptic), 10 p.p.m.peptide mass tolerance,
methionine oxidation as variable modification, and cysteine alkylation with NEM
and N terminus and lysine labelling with iTRAQ reagent as fixed modifications.
Peptides were filtered with an MSGF score # 10
29
, resulting # 1% false-discovery
rate at protein level. For the quantitative analysis, the iTRAQ report ion intensities
were extracted with MASIC
48
(MS/MS Automated Selected Ion Chromatogram
Generator, version v2.5.3923, http://omics.pnl.gov/software/MASIC.php). Peptides
yielding multiple spectra had their iTRAQ reporter ions intensities summed to
remove redundancy and to improve signal to noise ratio. For protein quantifica-
tion, the reporter intensities of different fully tryptic peptides belonging to the
same proteins were also summed. Peptides and proteins with missing data were
excluded from the analysis. Because two replicates were analysed, a Bayesian
moderated t-test (available through ‘limma’ BioConductor package) was applied
to determine the differentially abundant proteins.
Mouse thigh infection. Six-week-old female Swiss-Webster mice (Taconic) were
used in groups of five in these experiments. A group size of five mice was chosen
to provide statistically significant results based on the projected outcome of
experiments. Neither randomization nor blinding was deemed necessary for this
animal infection model. Mice were rendered neutropenic by cyclophosphamide
therapy
49
. A stationary culture of S. aureus ATCC 33591 was centrifuged and
resuspended in PBS. Then 100 ml of a 1:100 dilution (2 3 10
6
cells) was injected to
the right thigh of each mouse. Infection was allowed to progress for 24 h and mice
displayed measurable increase in thigh diameter. Mice were then treated with
vancomycin (Hospira), rifampicin (Pfizer), or ADEP4 (custom synthesized by
WuXi AppTec). ADEP4 and rifampicin were solubilized in 100% PEG400.
Vancomycin was solubilized in water. Vancomycin was dosed intraperitoneally
at 110 mg per kg every 12 h. Rifampicin was dosed intraperitoneally at 30 mg per
kg every 24 h. ADEP4 was dosed intraperitoneally at 25 mg per kg followed by a
second 35 mg per kg dose 4 h later. Control mice were sacrificed 24 h after infec-
tion (before treatment) and 48 h after infection (untreated). Thighs were asepti-
cally removed and homogenized in PBS using a Bullet Blender homogenizer.
Homogenates were serially diluted and samples were plated on BHI agar and
incubated at 37 uC overnight. Animal experiments were carried out at Northeastern
University and conformed to institutional animal care and use policies.
Microscopy. Histopathology was performed at the Boston University School of
Medicine Experimental Pathology Laboratory Service Core.
For the Gram stain, infected thigh tissues were aseptically dissected and fixed
overnight at 4 uC in 10% formalin. Samples were dehydrated using a graded
alcohol series from 70–100%, cleared with xylene to remove the dehydrant,
and infiltrated with paraffin. Processed tissue was embedded in paraffin, cut in
5-mm sections, and placed on microscope slides. Slides were baked at 67 uC for
36 min. After cooling, slides were washed twice with xylene for 5 min, twice with
100% alcohol for 5 min, twice with 95% alcohol for 2 min each, with 70% alcohol
for 2 min, and left in distilled water until staining. Slides were stained using a
Gram stain kit from Poly Scientific R&D Corp. Slides were stained with crystal
violet for 1 min and washed thoroughly with distilled water. Next, Gram’s iodine
was applied for 30 s and the slides were washed thoroughly with distilled water.
Slides were discolourized with Gram’s decolourizer until the crystal violet was
washed away. Slides were rinsed with distilled water and counterstained with
Gram’s safranin O counterstain. Slides were washed with distilled water and air
dried before a coverslip was applied. Slides were digitized at 340 using Ventana
iScan Coreo AU slide scanner and viewed using Image Viewer v.3.1.
For electron microscopy, 2 mm cross-sections of infected thigh were fixed
overnight at 4 uC in 2.5% glutaraldehyde/2.0% paraformaldehyde in 0.1 M sodium
cacodylate buffer. Samples were post-fixed 1 h in 1.0% osmium tetroxide in 0.15 M
cacodylate buffer at room temperature, dehydrated through a graded acetone
series, and embedded in epoxy resin. Sections were cut at 70 nm, stained with
uranyl acetate and lead citrate, and examined in a JEOL electron microscope at
80 kV. Images were recorded using a Gatan side mounted 11 megapixel digital
camera.
In vitro
hollow-fibre model. In vitro pharmacokinetic/pharmacodynamic model-
ling experiments were performed over a 96 h period using a hollow-fibre model
(Fibrecell Systems) with a culture of ,10
7
c.f.u. ml
21
as a starting inoculum. Fresh
MHB was continuously supplied by a peristaltic pump (Masterflex; Cole-Parmer)
set to simulate the half-lives of the antibiotics. After inoculation of the bacteria into
the extracapillary space of the hollow-fibre cartridge, antibiotic was infused into
the reservoir chamber via a dosing port. Free drug concentrations of vancomycin
(1 g every 12 h: fC
max
:30mgml
21
, fC
min
: 7.5 mgml
21
, half-life: 6 h; at 50% protein
binding for vancomycin these levels correspond to a total C
max
of 60 mgml
21
and
C
min
of 15 mgml
21
) and rifampicin (300 mg every 8 h: fC
max
:0.8mgml
21
, half-life:
3 h; at 80% protein binding for rifampicin these levels correspond to a total C
max
of
4 mgml
21
) were dosed to simulate human pharmacokinetics while ADEP4 (25 mg
per kg followed by 35 mg per kg 4 h later—repeated every 24 h: C
max
for 25 mg per
kg: 11.7 mgml
21
,C
max
for 35 mg per kg: 16.4 mgml
21
, half-life 1.5 h) was dosed to
simulate mouse pharmacokinetics. Mouse pharmacokinetics were used for ADEP4
because there are no human pharmacokinetic data, nor are there sufficient animal
pharmacokinetic data for an allometric conversion. Antibiotic regimens tested
included ADEP4 alone, vancomycin alone, rifampicin alone, ADEP4 combined
with rifampicin, and vancomycin combined with rifampicin. Model simulations
involving two drugs with different half-lives were performed using a previously
ARTICLE RESEARCH
Macmillan Publishers Limited. All rights reserved
©2013
validated method
50
. All experiments were performed at 37 uC in triplicate, using
biological replicates, to ensure reproducibility.
Samples (1 ml) were removed at 0, 1, 2, 4, 8, 24, 28, 32, 48, 56, 72 and 96 h,
serially diluted, plated on BHI agar, and incubated at 37 uC with a lower limit of
detection of 10
2
c.f.u. ml
21
. Antibiotic concentrations were verified by bioassay
using antibiotic medium 19 and S. aureus ATCC 33591 for ADEP4, antibiotic
medium 5 and B. subtilis for vancomycin, and Mueller Hinton Agar and K. rhizophila
ATCC 9341for rifampicin. Only models usinga single agent had pharmacokinetics
verified while combination models were performed using the verified method
described above. Pharmacokinetic parameters were analysed using WinNonlin
modelling software (Pharsight). Pharmacokinetic values from the models were
all within 10% of targets. Overall activity of regimens over the 96 h period was
compared by calculating the area under the bacterial kill curve for each regimen
using SigmaPlot software (version 11.1,Systat Software). The areas under the curve
were then compared using analysis of variance (ANOVA) with Tukey’s post-hoc
test with IBM SPSS Statistics (Version 19.0, SPSS Inc).
44. Wang, Y. et al. Reversed-phase chromatography with multiple fraction
concatenation strategy for proteome profiling of human MCF10A cells.
Proteomics 11, 2019–2026 (2011).
45. Livesay, E. A. et al. Fully automated four-column capillary LC–MS system for
maximizingthroughput in proteomic analyses. Anal. Chem. 80, 294–302 (2008).
46. Mayampurath, A. M. et al. DeconMSn: a software tool for accurate parent ion
monoisotopic mass determination for tandem mass spectra. Bioinformatics 24,
1021–1023 (2008).
47. Kim, S. et al. The generating function of CID, ETD, and CID/ETD pairs of tandem
mass spectra: applications to database search. Mol. Cell Proteomics 9,
2840–2852 (2010).
48. Monroe, M. E., Shaw, J. L., Daly, D. S., Adkins, J. N. & Smith, R. D. MASIC: a software
program for fast quantitation and flexible visualization of chromatographic profiles
from detected LC-MS(/MS) features. Comput. Biol. Chem. 32, 215–217 (2008).
49. Zuluaga, A. F. et al. Neutropenia induced in outbred miceby a simplified low-dose
cyclophosphamide regimen: characterization and applicability to diverse
experimental models of infectious diseases. BMC Infect. Dis. 6, 55 (2006).
50. Blaser,J. In-vitro model for simultaneous simulation of the serum kinetics of two drugs
with different half-lives. J. Antimicrob. Chemother. 15 (Suppl A), 125–130 (1985).
RESEARCH ARTICLE
Macmillan Publishers Limited. All rights reserved
©2013
Extended Data Figure 1
|
Combinations of conventional antibiotics against
stationary phase
S. aureus
. Data are representative of 3 independent
experiments. Error bars represent s.d.
ARTICLE RESEARCH
Macmillan Publishers Limited. All rights reserved
©2013
Extended Data Figure 2
|
ADEP4 resistant strains are heat sensitive.
Wild-type S. aureus ATCC 33591 and 9 ADEP4 resistant isolates with
mutations in clpP were grown for 20 h in MHB at 44 uC in 96-well polystyrene
plates. Data are representative of 3 independent experiments. Error bars
represent s.d.
RESEARCH ARTICLE
Macmillan Publishers Limited. All rights reserved
©2013
Extended Data Figure 3
|
ADEP4 with rifampicin eradicates
S. aureus
in a
hollow-fibre infection model. Antibiotics were delivered at concentrations
mimicking human dosing, while the concentration of ADEP was varied over
time to match the pharmacokinetics in the mouse model. Data are
representative of 3 independent experiments. Error bars represent s.d.
ARTICLE RESEARCH
Macmillan Publishers Limited. All rights reserved
©2013
Extended Data Figure 4
|
Conventional bactericidal antibiotics target active
processes in bacterial cells (green) resulting in death. In a dormant persister
(blue), the antibiotic binds an inactive target, producing no effect. ADEP4
activates and dysregulates ClpP in growing cells and in dormant persisters,
resulting in eradication of the bacterial population.
RESEARCH ARTICLE
Macmillan Publishers Limited. All rights reserved
©2013
