Modulation of antibiotic sensitivity and biofilm formation in Pseudomonas aeruginosa by interspecies signal analogues

Article abstract of DOI:10.1038/s41467-019-10271-4

Pseudomonas aeruginosa, a significant opportunistic pathogen, can participate in inter-species communication through signaling by cis-2-unsaturated fatty acids of the diffusible signal factor (DSF) family. Sensing these signals leads to altered biofilm formation and increased tolerance to various antibiotics, and requires the histidine kinase PA1396. Here, we show that the membrane-associated sensory input domain of PA1396 has five transmembrane helices, two of which are required for DSF sensing. DSF binding is associated with enhanced auto-phosphorylation of PA1396 incorporated into liposomes. Further, we examined the ability of synthetic DSF analogues to modulate or inhibit PA1396 activity. Several of these analogues block the ability of DSF to trigger auto-phosphorylation and gene expression, whereas others act as inverse agonists reducing biofilm formation and antibiotic tolerance, both in vitro and in murine infection models. These analogues may thus represent lead compounds to develop novel adjuvants improving the efficacy of existing antibiotics.

Full text of DOI:10.1038/s41467-019-10271-4

A R T I C L E
https://doi.org/10.1038/s41467-019-10271-4
OPEN
M o d u l a t i o n o f a n t i b i o t i c s e n s i ti v it y a n d b i o fil m
f o r ma t i o n i n Pseudomonas aeruginosa b y
i nt e rs p e c i e s s i g n a l a n a l o g u e s
S h i - q i A n , J u l i e M u r t a g h , K a t e B . T w om ey , M a n o j K . G u p ta , T i m o t h y P . O ’S u l li v a n , R e b e c c a I n g r a m ,
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1 National Biofilms Innovation Centers, Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK. 2 The Wellcome-Wolfson Institute for
Experimental Medicine, Queen’s University of Belfast, Belfast BT9 7BL, UK. 3 School of Microbiology, Biosciences Institute, University College Cork, Cork T12,
Ireland. 4 School of Chemistry, University College Cork, Cork T12, Ireland. 5 Department of Chemistry, Central University of Haryana, Mahendergarh 123029
Haryana, India. 6 School of Pharmacy, University College Cork, Cork T12, Ireland. 7 State Key Laboratory for Conservation and Utilization of Subtropical Agro-
bioresources, College of Life Science and Technology, Guangxi University, 100 Daxue Road, Nanning 530004, China. Correspondence and requests for
materials should be addressed to S.-q.A. (email: s-q.an@soton.ac.uk) or to M.A.V. (email: m.valvano@qub.ac.uk) or to J.-l.T. (email: jltang@gxu.edu.cn)
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N A T U R E C O M MU N IC A T IO N S | h t t p s : / / d o i . o r g / 1 0 .1 0 3 8 / s 4 14 6 7 - 0 1 9 -1 0 2 71 -4
ntibiotic resistance, coupled with limited development of transmembrane helices (TMHs), and with the N- and the C-
new antibiotic agents, poses a significant global threat to terminus located in the periplasm and cytoplasm, respectively
public health, underscoring the need to find alternative (Fig. 1a). In contrast, TOPPRED predicted a relatively large
A
strategies to fight infection1–4. These strategies include targeting cytosolic loop and a different location in the protein for some of
the signaling pathways that regulate the synthesis of microbial the TMHs. To experimentally assess the PA1396 topology, we
virulence factors and approaches to improve the efficacy of constructed translational fusions expressing hybrid proteins of
existing antibiotics. Bacterial cell–cell communication (quorum the PA1396 input domain with either alkaline phosphatase
sensing), biofilm formation, and cyclic di-GMP signaling were (PhoA) or β-galactosidase (LacZ) reporters as topological probes
proposed as potential targets for interference by small mole- for periplasmic and cytoplasmic locations, respectively. The
cules1–4. Anti-virulence factors are attractive since they do not hybrid proteins were designed such that at least one fusion was
influence bacterial growth, which may reduce the selective pres- placed in each predicted loop facing either the periplasm or the
sure for developing resistance.
cytoplasm (Fig. 1b).
This study addresses the modulation of Pseudomonas aerugi-
Plasmids carrying hybrid constructs were mobilized into E. coli
nosa by inter-species signaling. P. aeruginosa, a widespread and tested for PhoA and LacZ activity in solid medium
opportunistic human pathogen, uses two N-acyl homoserine containing the substrates for either PhoA (X-Phos) or LacZ (X-
lactones (3-oxo-dodecanoyl- and butanoyl-HSL) and the quino- Gal), which permitted estimation of the enzyme activity of
lone signal PQS (2-heptyl-3-hydroxy-4(1H)-quinolone) as PA1396-PhoA and PA1396-LacZ fusion proteins, respectively. E.
quorum-sensing molecules that regulate the synthesis of virulence coli expressing PhoA fusions to PA1396 at amino acid residues
factors5–8. There are various reports of small molecule inhibition E4, N6, G66, A70, A137, Q139, and Q141 exhibited clear PhoA+
of these pathways with effects on virulence factor synthesis9–11. In activity (Supplementary Table 1), whereas PhoA fusions to
addition to intra-species signaling, P. aeruginosa can participate PA1396 at amino acid residues V37, E38, G107, G109 and L205
in inter-species signaling mediated by molecules of the diffusible gave rise to PhoA− activity (Supplementary Table 1). Conversely,
signal factor (DSF) family, which are cis-2-unsaturated fatty acids. bacteria expressing LacZ fusions to PA1396 at amino acid
In particular, P. aeruginosa senses cis-11-methyl-2-dodecenoic residues V37, E38, G107, G109, L205, T240, L300 and L381
acid (DSF) and cis-2-dodecenoic acid (BDSF), which are pro- exhibited LacZ+ activity (Supplementary Table 1). In contrast,
duced by other bacteria such as Burkholderia species and Steno- LacZ fusions to PA1396 at amino acid residues E4, N6, G66, A70,
trophomonas maltophilia, but not by P. aeruginosa12,13. Sensing A137, Q139 and Q141 resulted in LacZ− activity (Supplementary
occurs through the PA1396 histidine kinase and results in altered Table 1). A summary of the quantitative assays are presented in
biofilm formation and increased tolerance to several antibiotics Supplementary Table 1, fit with a model of a five-TMHs protein
including polymyxin B13,14. Deletion of PA1396 in the model and the topology shown in Fig. 1b.
strain PAO1 and several clinical isolates also leads to increased
To test whether the predicted TMHs are important for DSF
tolerance to polymyxins B and E, suggesting DSF negatively sensing, we constructed PA1396 derivatives with progressive
modulates PA1396 activity13,14. Detection of DSF family mole- truncations at the N-terminus. Each truncated PA1396 gene was
cules in polybacterial infections, such as those associated with cloned into pBBR1MCS and the constructs introduced into a
cystic fibrosis (CF) where P. aeruginosa is present together with PA1396 deletion mutant (herein ΔPA1396). Bacteria expressing
other bacterial species, suggests interspecies signaling occurs each of these transconjugants were examined for their ability to
in vivo and may therefore lead to reduced efficacy of antibiotic respond to exogenous DSF by measuring pmrAB gene expression
therapy13,14
.
using a pmr-gfp fusion, and for polymyxin B resistance (see
Here, we examine in more detail the role of PA1396 in sensing Methods). Removal of the first three TMHs did not affect the
DSF family signals and the potential of structural analogs of these ability of PA1396 to respond to DSF when compared to wild type
signals to modulate PA1396 action. In particular, we focus on the (Fig. 1c). In contrast, any further truncation led to a loss of DSF
effects of DSF analogs on the PA1396-regulated functions of responsiveness (Fig. 1c). Western blot analysis of the truncated
biofilm formation and antibiotic tolerance, both in vitro and in proteins with antisera against the His6 tag showed they were all
murine infection models. We demonstrate that two of the five expressed to the same level as the wild type (Supplementary
transmembrane helices of the input domain of PA1396 are Fig. 1). These results suggested TMHs IV and V have a role in
required for DSF sensing, and DSF binding is associated with DSF sensing by PA1396.
enhanced PA1396 auto-phosphorylation. Furthermore, we found
To identify specific residues necessary for DSF binding and
that certain analogs of DSF block its ability to trigger auto- signal transduction, we first focused on those conserved in
phosphorylation, which coincide with specific alterations in the PA1396 homologs from different bacterial species. Conserved or
expression of a subset of P. aeruginosa genes, reduction in biofilm semi-conserved residues in TMHs IV and V (Supplementary
formation and antibiotic tolerance using both in vitro and in vivo Fig. 2) were examined for functionality by constructing alanine
infection models. Altogether, our findings reveal that DSF- replacements (or serine in the case of alanine residues). Residues
mediated interspecies interactions may be influenced pharmaco- Y116, L117, T121, L123, L128, T135, P136, W138, A140, P142,
logically to improve the treatment of chronic P. aeruginosa M144, L148, M149, V154, I155, P156, and Y158 were replaced
infections.
and the resulting variants assayed for their ability to confer
response to DSF. Bacteria expressing many of the variants did not
exhibit significant changes in DSF responsiveness. We identified
several variants exhibiting decreased sensitivity to DSF, such that
Results
Examining the sensor input domain of PA1396 for DSF higher concentrations were required to activate the pmr-gfp
recognition. Although DSF perception by PA1396 requires its reporter, although significant activation occurred at concentra-
membrane-associated sensor input domain13,14, the underlying tions in excess of 50 µM. These variants had alanine replacements
molecular mechanism is unknown. As a first step to better at T121, L123, L128, P142, L148, M149, V154, and I155. Except
understand DSF sensing by PA1396, topological models of the for P142, all the variants with reduced DSF sensitivity were
protein were generated with various prediction programs. Ana- altered in positions within the last two TMHs of PA1396.
lyses with TMHMM, MEMSAT, SOSUI, and HMMTOP sug- Variants with combinations of alanine substitutions (such as
gested
a topological model for PA1396 comprising five T121A/L123A/L128A) had an even further reduced sensitivity
2
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N A T UR E C O M M UN I CA T I O NS | h t t p s : //d o i . o r g / 10 . 1 0 38 / s 4 1 4 6 7 - 01 9 - 1 0 2 7 1 - 4
A R T I C L E
a
b
4E
13
70A
139Q
66G
62
6N
137A
136
I
I
II
II
II
III
III
IV
IV
IV
V
V
V
141Q
143
TMHMM
MEMSAT
SOSUI
Periplasm
82
I
III
V
I
II
III IV
I
II
III IV
V
TOPPRED
HMMTOP
I
II
III
IV
V
35
40
38E
104
107G
114
162
Cytoplasm
540
37V
50
100
150
200
250
300
350
400
450
500
109G
205L
240T
300L
381L
Cytosol
Transmembrane helix
Periplasmic
c
d
70,000
60,000
50,000
40,000
30,000
20,000
10,000
0
WT
T121A
L123A
L128A
Response to DSF
HisKA
HATPaase_c
REC
I
II III IV V
pmrAB gene
expression (RFU)
Polymyxin B
resistance
T121A/L123A/L128A
0
100
200
300
400
500
25 ꢀg/ml
25 ꢀg/ml
25 ꢀg/ml
25 ꢀg/ml
25 ꢀg/ml
25 ꢀg/ml
2 ꢀg/ml
3.0E + 4 ( 1.1E+3)
3.2E + 4 ( 1.1E+3)
3.1E + 4 ( 1.1E+3)
2.9E + 4 ( 1.1E+3)
3.2E + 4 ( 1.1E+3)
3.1E + 4 ( 1.2E+3)
1.1E + 3 ( 5.0E+2)
9.0E + 2 ( 5.0E+2)
0
35
40
82
104
114
136
143
0
0.01 0.1
1
5
10
50
100 150 200
2 ꢀg/ml
Dose of synthetic DSF (ꢀM)
Fig. 1 F u n c t i o n a l c h a r a c t e r iz at i o n o f D SF s e n s o r P A 1 3 9 6 . a G r a p h i c a l r e p r e s e n t a t i o n o f t h e t o p o l o g ic a l p r ed i c t i on s o f t h e p o s i t i o n o f t r a n s m e mb r a ne h e l ic e s
( T M H s) o f P A 1 3 96 m a d e b y v a r i o u s m e m b r an e t o p o l o gy p r e d i c t i o n p r o gr a m s . T h e l o c a t i o n o f s o l u b l e s e g m e n t s ( c y t o s o li c o r p e r i p l a s mi c ) a n d t h e
p o si t io n s o f p r e d i c t ed T M Hs a r e i n d i c a t ed. T MH s a r e d e s i g n a t e d b y R o m a n n u m e r a l s . b A m o d e l o f m e m b r a n e t o p o l o g y o f P A 1 3 9 6 d e r i v e d f r om r e po r t e r
f u si o n d a t a . P h e n o ty pe s o f phoA o r lacZα r e p o r t e r f u s i o n s t o g et h e r w i t h t h e a m i n o a c i d p o s i t i o n a t w h i ch t h e r e p o r t e r s w e r e f u s e d a r e i n d i c a t e d b y G r e e n
( P h oA a c t i v i t y) a n d P i n k ( L a c Z a c t i vi t y) . N u m b e r s n e x t t o e a c h p r ed i c t e d T M H i n d i c a t e t h e p o s i t i o n o f a m i n o a c i d r e s i d u e s a t t h e p r ed i c t e d b ou nd ar ie s
b e t w e en s o l u b l e a n d m e mb r a n e- em b ed d e d r e gi o n s . c D o m a i n s o f P A 1 3 9 6 a s p r ed i c t e d b y S M A R T . D o m a i n a b b r e vi a t i o n s a r e : H i s K A : H i s K in as e A
( p h o s p h o a cc e p t o r ) d o m a i n ; H A TP a s e _c : H is t id i n e k i n a s e - l ik e A T P a s e s ; R E C : C h e Y - h o mo l o go u s r e c e i v e r d o m a i n . B l u e b a r s w i t h R o m a n n u m e r al s i nd ic a t e
d i f f e re n t t r a n s m em b ran e h e l ic e s . T h e l o we r p a r t o f t h e F i gu r e i nd i c a t e s t h e e f fe c t o f p r o gr e s s i ve N - t e r m i na l t r u n ca ti o n o f P A 1 3 9 6 o n t h e a b i l it y o f t h e
s e n s or t o r e sp o n d t o D S F, a s m e a s u r e d b y a c t i va t io n o f pmrAB g e n e e x p r e s si o n a n d r e s i s t a n c e t o p o l y m y x i n B . D N A f r a g m e n t s e x p r e s s i n g d i f f e r e nt H i s 6 -
t a g g e d c o n s tr u ct s i n d i ca te d b y t h e b l a c k l in es w er e c l o n ed i n t o p B BR 1 M C S v e c t o r a n d i n t r o d u c e d i n t o ΔPA1396 m u t a n t s t r a i n . R e s p o n s e t o D S F a nd
a n t i b i o t i c t o l er a n ce a r e r e p o rt e d . d D o s e–r e s p o n s e r e la t i o n s h ip s f o r a c t i v a t i o n o f a pmr-gfp t r a n s c r i pt i o n a l f u s i o n b y D S F i n P. aeruginosa s t r a i n s e x pr e s s i ng
e i t he r w i l d - t y p e P A 1 3 96 o r r e p r e s e n t a t ive v a r i a n t s . V a l u e s a r e t h e a v e r a ge o f t h r e e t e c h n i c a l r e p e a t s ( m e a n s t a n d a r d d ev i a t i o n ) . D i f f e r e n c e s s e e n
b e t w e en m u ta n ts a n d w i l d - t y p e w er e s t a t is t i c a l l y s i g n ific a n t (p-v a l u e < 0 .0 5 , A N O V A )
(Fig. 1d). The observed differences in the responses to DSF by the
The activity of the molecules in this panel was assayed by
reporter strain could not be attributed to differential protein determining the relative fluorescence of the reporter normalized
expression since all replacements were expressed to the same level by bacterial cell density (Fig. 2b). As the minimum concentration
as the parental protein (Supplementary Fig. 1). Altogether, we of DSF (cis-11-methyl-2-dodecenoic acid) for significant induc-
conclude residues in TMH-IV and TMH-V of PA1396 play a tion of pmr-gfp expression was 0.01 µM, the 27 DSF derivatives
critical role in the recognition of the DSF signal.
were examined at this concentration. The results revealed that all
fatty acids with a trans configuration of the double bond at the 2-
position render the molecule inactive (Fig. 2b), consistent with
previous reports indicating this configuration is critical for DSF
activity13–15. However, the presence or the position of the methyl
group at C-11 in DSF was dispensable for activity. Indeed, cis-2-
tridecenoic acid (C9) and cis-2-dodecenoic acid (C2, BDSF)
without the methyl substitution, as well as cis-7-methyl-2-
dodecenoic acid (C6) and cis-8-methyl-2-dodecenoic acid (C5)
where the methyl group was transposed to the C-7 and C-8
positions, were equally effective in the bioassay. In contrast, the
fatty acid chain length played a major role in determining
bioactivity. cis-2-unsaturated fatty acids with chain lengths of
12–14 carbons were active whereas those with longer chains were
not (Fig. 2b). The introduction of a second double bond in the
chain also led to reduced activity, while derivatives with the
carboxylic acid esterified, converted to a hydroxamic acid or
amide derivative or replaced with a 2-amino pyridine moiety were
all inactive. For each of these derivatives, no activity was seen in
the concentration range from 0.01 to 1000 µM. Interestingly, cis-
2-tetradecenoic acid (C11), which is a naturally occurring analog
Physiochemical features of DSF important for recognition by
PA1396. The DSF family comprises cis-2-unsaturated fatty acids
of differing chain length, methyl substitution and saturation12.
Individual bacteria can produce multiple DSF family signals,
although often a single chemical species predominates. Previous
work showed that the configurational isomer trans-11-methyl-2-
dodecenoic acid was 500-fold less effective than DSF in activation
of pmrAB, whereas the corresponding unsaturated fatty acids
have little or no activity13. To examine the structural features of
the DSF signals required for PA1396 sensing in more detail, we
synthesized a panel of analogs differing from the DSF ‘parent’
molecule in chain length, position and presence of methyl
branching or with a trans configuration of the double bond.
Additional molecules were created by esterification, conversion to
hydroxamic acids or amides, and by incorporation of stable
aromatic substituents to replace the carboxylic acid moieties (see
Fig. 2a). This library of structural derivatives provided a platform
to define key structural features for PA1396 sensing and signal
transduction activity.
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
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(
2
0
1
9
)
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3
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1
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n
a
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c
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a
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s
3

A
R
T
I
C
L
E
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
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I
O
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S
|
h
t
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1
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.
1
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3
8
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4
1
4
6
7
-
0
1
9
-
1
0
2
7
1
-
4
a
b
Acid
12,000
Concentration tested: 0.01 ꢀM
Carbonyl
**
*
Methyl groups
cis-double bond
10,000
*
*
1 DSF
Alkyl chain
8000
6000
4000
2000
0
OH
OH
O
O
O
O
**
OH
OH
**
OH
OH
2 BDSF OH
11
12
13
20
21
O
O
NH OH
3
OH
**
**
** **
**
**
**
**
O
**
**
**
** **
O
O
**
O
O
NH
S
4
22
23
24
25
26
OH
O
Compound ID
O
O
c
OH
70,000
5
14 trans-DSF
NH
O
OH
WT + DSF
WT + C11
O
O
O
O
O
60,000
50,000
40,000
30,000
20,000
10,000
0
OH
OH
T121A/L123A/L128A + DSF
T121A/L123A/L128A + C11
6
15
16
OH
O
7
O
OH
O
O
N
N
O
OH
OH
8
17
NH
NH2
OH
O
O
OH
9
18
27
28
OH
O
O
0
0.001 0.01
0.1
1
5
10
50
100
150
200
Dose of synthetic compound (ꢀM)
10
19
Fig. 2 D i f fu s i b l e s i g n a l f a c t o r ( D S F ) a n a l o g t e s t i n g r e ve a l s f e a t u r e s n e e d e d f o r p e r c e p t i o n b y P A 1 3 9 6 . a T h e s t r u ct u r e o f D S F ( cis- 1 1 - m e t h yl - 2 -d o de c e n oi c
1
a
c
i
d
)
h
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.
B
e
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7
s
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F
,
a
l
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a
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i
d
a
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e
d
b
y
E
S
I
-
M
S
a
n
d
H
N
M
R
s pe c t r a a s d e s c r i b ed i n t h e M et h o d s . b T h e b i o l o g ica l a c t i vi t y o f D S F a n d i t s d e r i v a t i v es i n a c t i v a t i o n o f t h e pmr-gfp f u s i o n . S a m p l e s w e r e t e s te d a t a
c o nc e n tra t i o n o f 0 . 0 1 µM ( t h e m i n i m u m c o n c e n t ra t i o n o f cis- 11 -m e t h yl - 2 - d o d e c e n o i c a c i d r e q u i r e d f o r a c t i v a t i o n o f t h e b i o a s s a y) . V a l u e s a r e t h e a v e r age
o f t hr e e t ec h n i ca l r e p e at s ( m e a n s t a n d a r d d e v ia t i o n ) . A s te r i s k s i n d i c a t e v a l u e s t h a t a r e s i gn ific a n t l y d i f f e r e n t f r o m t h e a p p r o p r i a t e w i l d t y p e (p < 0 . 0 5 ,
S tu d e n t ' s t-t es t ) . c D o se–r e s p o n s e o f D SF (cis- 11 -m e t h yl -2 - d o d e c en o i c a c i d ) a n d C 1 1 ( cis- 2 - t et r a d e ce n o i c a c i d ) o n a c t i v a t i o n o f pmr-gfp t r a ns c r i pt i ona l
f u si o n i n w i l d - t y p e P A 1 3 9 6 a n d a s t r a in e x p r e s s in g t h e v a r i a n t T 1 2 1 A / L 1 2 3 A/ L 1 2 8 A w i t h a l t er a t i o n i n c o n s e r v ed a m i n o a c i d s o f T M H- IV . T h e d at a o f G F P
flu o re s ce n c e s h o w n i s f r o m o n e r e p r e s e nt a t i v e e x p e r im e n t ( n = 3 ) . D i f f e r e n c e s s e e n b e t w e e n w i l d t y p e a n d D S F , a n d m u t a n t a n d D S F w a s s t a t is t i c a ll y
s ig n ific a n t ( p-v a l u e < 0 . 0 5 , A N OV A ) . D if fe r e n c e s s e e n b e t w ee n w i l d t y p e a n d C 1 1 , a n d m u t a n t a n d C 1 1 w a s s t a t i s t ic a l l y s i gn i fic a n t ( p-v a l u e < 0 . 0 5 ,
A N O VA )
first detected in Xylella fastidiosa15, was most effective in from the accessibility of ATP to the kinase site, which allows
activating the bioassay (Fig. 2c). Strains carrying the T121A/ auto-phosphorylation without disruption of the liposomes. Basal
L123A/L128A replacement mutants of PA1396 showed similar auto-phosphorylation of PA1396 in the liposomes was seen in the
reduced responsiveness to both C11 and DSF (Fig. 2c).
absence of any signal molecule (Fig. 3c and Supplementary
Table 2). However, the addition of DSF to PA1396-loaded
liposomes led to a robust increase in the level of auto-
phosphorylation, an effect not seen in the presence of C23
(Fig. 3c and Supplementary Table 2). These findings suggest C23
blocks the recognition of DSF by PA1396, which normally
mediates increased kinase activity. The observation that the
T121A/L123A/L128A mutant protein had a similar level of basal
auto-phosphorylation as the parental PA1396, which could not
increase upon addition of DSF, also supports this conclusion
(Fig. 3c and Supplementary Table 2). Further, the latter result
agrees with the observation that T121A/L123A/L128A has a
substantially reduced response to DSF in vivo as measured by the
pmr-gfp reporter bioassay (Fig. 1d).
We previously found that DSF alters the expression levels of P.
aeruginosa genes implicated in virulence, biofilm formation and
stress tolerance13. In this study, we assessed the effect of C23 on
DSF-regulated functions by quantitative reverse transcription
PCR (qRT–PCR) using a subset of 11 DSF-regulated genes. These
included genes involved in iron uptake (PA4358, PA4359) and
antibiotic resistance (PA4599, PA4774–PA4777). For these
experiments, P. aeruginosa was grown in artificial CF sputum
medium with and without DSF supplementation, and with both
DSF and C23. Cultures were assayed at early log phase (OD600 of
DSF analogs interfere with DSF signaling in P. aeruginosa. The
structural analogs unable to trigger DSF-dependent responses
were assessed for inhibitory activity against DSF signaling. For
these experiments, the molecules were screened at 10μM using P.
aeruginosa grown with 50 μM DSF. Several compounds, espe-
cially C12, C23, and C24 effectively repressed pmr-gfp expression
(Fig. 3a), possibly acting as PA1396 receptor antagonists. None of
the compounds affected planktonic bacterial growth (Supple-
mentary Fig. 3). Dose–response assays for each of the three
compounds revealed that compound 23 ((Z)-11-methyl-N-
(methylsulfonyl)dodec-2-enamide; C23) was the most potent DSF
antagonist showing substantial inhibition at 0.1 μM (Fig. 3b).
To examine if these compounds interfere directly with signal
transduction through PA1396, we assessed the effects of DSF and
the putative antagonists on PA1396 auto-phosphorylation. For
these experiments, MycHis-tagged PA1396 was expressed,
purified under native conditions, and reconstituted in liposomes
where the protein adopts an inside-out orientation in which the
cytoplasmic histidine acceptor, histidine kinase and receiver
domains are surface exposed. The incorporation of PA1396 into
liposomes was confirmed by western blot using anti-Myc
antibody, whereas the inside-out orientation can be surmised
4
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
(
2
0
1
9
)
1
0
:
2
3
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1
0
.
1
0
3
8
/
s
4
1
4
6
7
-
0
1
9
-
1
0
2
7
1
-
4
|
w
w
w
.
n
a
t
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.
c
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n
a
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c
a
t
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s

N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
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I
O
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|
h
t
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1
0
.
1
0
3
8
/
s
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1
4
6
7
-
0
1
9
-
1
0
2
7
1
-
4
A
R
T
I
C
L
E
a
b
12,000
10,000
8000
6000
4000
2000
0
12,000
Compound concentration: 10 µM
DSF concentration: 50 µM
**
**
**
**
10,000
8000
6000
4000
2000
0
*
**
**
*
**
**
*
**
**
**
*
WT
WT + DSF
WT + DSF + C12
WT + DSF + C23
WT + DSF + C24
**
**
**
**
**
**
0
0.01
0.1
1
5
10
50
100
Dose of synthetic compound (µM)
Compound ID
c
d
10.0
PAO1 + DSF
PA1 + DSF + C23
–DSF
+DSF
+DSF
8.0
6.0
PA1396
4.0
**
+DSF and C23
+DSF
**
**
**
**
**
2.0
PA1396
PA1396
0.0
–DSF
–DSF
–2.0
–4.0
–6.0
–8.0
**
**
*
T121A/L123A/L128A
+DSF and C23
PA1396
T121A/L123A/L128A
REC
Fig. 3 D i f fu s i b l e s i g n a l f a c t o r ( D S F) a n a l o g s i n h i b i t P A 1 3 9 6 a c t i vi t y . a T h e e f fe c t s o f a n a l o g s ( a t 1 0 μM ) o n a c t i v a t i o n o f t h e pmr-gfp t r a n s c r i pt i o n al f us io n b y
D S F ( 5 0 μM ) w a s m e a s u r e d a s G F P flu o r e s c e n ce . D a t a s h o wn a r e m e a n s o f t h r e e r e p l i c a t e s a n d e r r o r b a r s i n d i c a t e t h e s t a n d a r d d ev i a t i o n s . T h e a s t e r i s k s
i n di c a t e t h o se a n a l o g s f o r w h ic h t h e l ev e l o f gfp e x p r e s s io n w a s s i gn ific a n t l y d i f f e r e n t f r o m t h a t o f D S F a l o n e ( p < 0 .0 1 , S t u d e n t 's t-t e s t ) . b D o s e–r e s po ns e
r e l a ti o n s h i p s o f t h e e f f ec t s o f s e l e c t e d a n a l o g s o n D SF a c t i va t i o n o f t h e pmr-gfp t r a n s c r i pt i o n a l f u s i o n i n w i l d - t y p e P. aeruginosa. D S F w a s p r es e nt a t 5 0 μM .
T h e a n a l o g s t es t e d w e re C 1 2 (cis-1 3 -m et h y l t e t r a d e c e n oi c a c i d ) , C 2 3 (cis-1 1 - m e t h y l d o d e c e n o y l m e t h y l s u l fo n a m i d e ) , a n d C 2 4 (cis-1 1 - m e t h y l d od e c e n oy l
c y c lo p r o p an a m i d e ) . V a l u e s a r e t h e a v e r a ge o f t h r e e t e c h n ic a l r e p e a t s ( m e a n s t a n d a r d d e v ia t i o n ) . T h e o b s e r v e d v a l u e s w e r e s i gn ific a n t l y d i f fe r e nt f r o m
t he a p p r o p ria t e w i l d t y p e i n a l l c a s e s (p < 0 . 0 5, A N OV A ). c P A 1 3 9 6 a u t o - p h o sp h o r y l a t i o n i n r e s p o n s e t o D S F a n d a n a l o g s . L e ft p a n e l : S c h e m at i c
r e p re s e nt a ti o n o f t h e o r i en t a t io n o f P A 1 3 9 6 i n t h e l ip o s o m e a s s a y . D o m a i n a b b r e v ia t i o n s a r e : H i s K A : H i s K i n a s e A ( p h o s p h o a cc e p t o r ) d o m a i n ; H A T P a s e _ c :
H i s t i d i n e k i n a se - l i k e A T Pa s e s ; R E C : C h e Y - h o m o lo go u s r e c e i ve r d o m a i n . B l u e b a r s i n d i c a t e d i f f e r e n t t r a n s m e m b r an e h e l i ce s . R i g h t p a n e l : P A 13 9 6 a u t o -
p h os p h o ry l a t e s i n t h e l i p o s o m e ( l a n e s 1 –3 ; w h ic h a r e t r ip li c a t e a s s a y s) , b u t i n c r e a s e d p h o s p h o r y l a t i o n i s s e e n i n r e s p o n s e t o 1 0 μM D S F ( l an e s 4–6 ) ;
P A1 3 96 p h o s p h o ry l a t i o n i n r e s p o n s e t o 1 0 μM D SF ( l a n e s 1 –3 ) i s a b r o g a t ed i n t h e p r es e n ce o f C 2 3 ( l a n e s 4 –6 ) ; P A 1 3 9 6 ( T 1 2 1 A / L 12 3 A / L1 2 8 A ) v a r i an t
p ro t e i n a u t o - p h o s p h o ry l a t es b u t d o e s n o t r e s p o n s e t o t h e p r e s e n c e o f D S F a n d / o r C 2 3 . d D i f f e r e n t i a l e x p r e s si o n o f s e l e c t e d g e n e s i m p l i c a t e d i n v i r u le nc e,
a n t i b i o t i c r es i s ta n c e , o r b iofil m f o r m a t i o n i n t h e p r e s e n c e D SF , C 2 3 , a n d D S F i n c o m b i n a t i o n w i t h C 2 3 . T h e q R T –P C R d a t a w e r e n o r m a l i z ed t o proC a r e
p re s e n t ed a s t h e f o l d c h a n ge w it h r e s p e c t t o t h e w il d t y p e f o r e a ch g e n e . D a t a ( m e a n s s t a n d a r d d e v ia t i o n ) a r e f r o m o n e r e p r e s e nt a t i v e e x p e r i m e n t ( n =
3 ) . A p-v a l u e o f < 0 . 0 5 w a s c o n s i d e r e d s t a t is t ic a l l y s i g n i fic a n t a n d i s d e s i gn a te d i n t h e fig u r e s w i t h a n a s t e r i s k . D o u b l e a s t e r i s k s i n d i c a t e p-v a l u e s o f < 0 . 0 1
0.6). For several of the genes tested, the addition of C23 in the also caused a reduction of biofilm development (Fig. 4a). In
growth medium reduced the level of expression seen with DSF contrast, ΔPA1396 formed more biofilm than the parental strain,
alone and in some cases (e.g. PA2966, PA5505) the effects on but biofilm formation was not significantly altered by either DSF
expression were reversed (Fig. 3d). These experiments were or C23 (Supplementary Fig. 5a). In a second set of assays, the
repeated with the ΔPA1396 mutant strain, demonstrating that the effects of DSF and C23 on biofilm formation on glass surfaces
addition of DSF or C23 had no effect on the level of gene were examined. For this, bacteria were grown either in glass tubes
expression (Supplementary Fig. 4). Altogether, the results from or in µ-chambers16 and biofilm development quantified by crystal
in vitro auto-phosphorylation experiments and transcriptional violet staining. Biofilm biomass in glass tubes was quantified by
profiling of a subset of PA1396-regulated genes indicate C23 dye elution whereas biofilms in µ-chambers were visualized by
interferes with the PA1396 ability to sense DSF.
confocal microscopy and biomass quantified by COMSTAT
software (see Methods). DSF and C23 had comparable effects on
biofilm biomass in glass tubes as seen in the co-culture experi-
ments with epithelial cells: DSF addition led to an increased
biofilm biomass, whereas C23 reversed this effect (Fig. 4b). Fur-
thermore, C23 alone was able to inhibit biofilm formation, but
not to the same extent as in the presence of DSF. These effects
were not seen in experiments using ΔPA1396 mutant strain
(Supplementary Fig. 5b). In µ-chambers, the wild-type strain
formed a flat mat biofilm (Supplementary Fig. 6). Addition of
DSF led to the development of a denser flat biofilm whereas
Interference with DSF sensing decreases biofilm formation and
alters virulence of P. aeruginosa in vivo. DSF stimulates the
development of Pseudomonas aeruginosa biofilms in vitro and
promotes persistent infection in vivo12–14. We examined if C23
interferes with biofilm formation using a co-culture system in
which P. aeruginosa biofilms were allowed to form on human
lung epithelial cells upon addition of 1 µM DSF14. C23 at con-
centrations as low as 0.5 µM led to reduced biofilm formation in
the presence of DSF (Fig. 4a). Intriguingly, addition of C23 alone
N
A
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E
C
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M
M
U
N
I
C
A
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(2
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9
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1
0
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N A T U R E C O M MU N IC A T IO N S | h t t p s : / / d o i . o r g / 1 0 .1 0 3 8 / s 4 14 6 7 - 0 1 9 -1 0 2 71 -4
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Fig. 4 E f f e c t s o f a n a l o g s o n D SF - i n d u c ed v i ru le n c e p h e n o ty p e s o f P. aeruginosa. a E f fe c t o f D S F a n d C 2 3 a l o n e o r i n c o m b i n a ti o n o n a t t a c h m e nt o f P.
aeruginosa s t ra i n s t o C F B E e p i t h e l ia l c e l l s . F o r t h e s e e x p e r im e n t s , c o m p o u n d s ( 0 . 5 µM ) w e r e a d d e d t o t h e c o - c u l t u r e a t 1 h a n d b a c t e r i a l a t t a c h m e n t t o t h e
C F BE e p i th e l i a l c e l l s w a s m ea s u re d a f t e r 2 4 h ( S e e M et h o d s) . A p-v a l u e o f < 0 .0 5 w a s c o n s i d e r e d s t a t i s t ic a l l y s i gn i fic a n t a n d i s d e s i gn a t e d i n t h e fig ur e s
w i t h a n a s t er i s k . D o u b l e a s t e r i s k s i n d i c a t e p-v a l u e s o f < 0 . 0 1. b E f f e ct o f 0 .5 µM D S F a n d C 2 3 a l o n e o r i n c o m b i n a t i o n o n a t t a c h m e n t o f P. aeruginosa t o a
g l a ss s u r f a c e a s a s s es s e d b y c r y s t a l v i o le t s t a in i n g. B i o fil m b i o m a s s i s m e a s u r e d a s a r a t i o o f a b s o r b an c e a t 5 5 0 a n d 6 0 0 n m . V a l u e s g i v en a r e t h e m e a n
a n d s ta n d a r d d e v ia t i o n o f t h r e e t e c h n ic a l m e a s u r e m e n t s. A p-v a l u e o f < 0 . 05 w a s c o n s i d e r e d s t a t i s t i c a ll y s i gn i fic a n t a n d i s d e s i gn a t e d i n t h e fig ur e s w i t h a n
a s te r i s k . D o u b l e a s t er i s k s i n d i c a t e p-v a l u e s o f < 0 . 0 1. c E ff e c t o f a d m i n i s t r a t i o n o f C 2 3 o n P. aeruginosa m o u s e a i r w a y i n f e c t i o n . C 5 7 B L/ 6 m i c e w e r e i nf e c t e d
7
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a n d b a c t e ri al l o a d s w e re d e t e r m in e d i n l u n g h o m o ge n a t es. E a c h d a t a p o i n t s h o w s t h e r e s u l t s f r o m a n i n d i v i d u a l m o u s e . A p-v a l u e o f < 0 . 05 w a s c o ns id e r e d
s ta t i s ti c a ll y s i g n ific a n t a n d i s d e s i g n a t e d i n t h e fig u r es w it h a n a s t e r i s k . D o u b l e a s t e r i s k s i n d i c a t e p-v a l u e s o f < 0 . 01
concentrations of C23 as low as 0.5 µM in the presence of DSF led vs. DSF plus C23) with all 21 of these genes altered in the same
to reduced biofilm and the development of small microcolonies. direction. The expression level (fold change in expression) of
Addition of C23 alone also caused a reduction of biofilm and the these genes was reduced in DSF plus C23 treatment when
formation of microcolonies (Supplementary Fig. 6).
compared with the DSF treatment alone (Supplementary Table 3).
To determine whether C23 modulates bacterial behavior Further, some genes significantly regulated by DSF showed no
during infections, C57BL/6 mice were inoculated intranasally significant alteration in the presence of DSF and C23. Examples
with suspensions of P. aeruginosa supplemented with C23, DSF, of this group of genes include popD (PA1709) and pmr operon
or C23 with DSF. As a control, bacterial inocula were suspended genes (PA4774, PA4776, PA4777). Also, various genes showed a
in PBS alone. At 24 h post infection, the P. aeruginosa PAO1 significant alteration in expression in response to DSF plus C23,
bacterial load was quite considerable in the control animals. but not to DSF alone (Fig. 5a, b). Selected genes were examined
However, in the presence of C23, the number of bacteria was using qRT–PCR to confirm the alteration in expression as
considerably reduced. With DSF alone, higher numbers of revealed by the microarray (Fig. 5b). Altogether, the combined
bacteria were seen after 24 h than in the control (Fig. 4c), in results of the experiments described above show that C23 not
agreement with previous trends seen in other in vivo models14. only can inhibit DSF-activated responses in vitro and in vivo, but
With C23 in combination with DSF, the bacterial load was also has a broader effect on gene expression independent of DSF-
reduced when compared to DSF alone (Fig. 4c). Additionally, C23 regulated responses.
alone appeared to slightly reduce the bacterial load (Supplemen-
tary Fig. 7). Thus, C23 reduced the bacterial load in this mouse separate experiment on bacteria grown in artificial sputum
model in the presence or absence of DSF. medium in the presence or absence of the compound at 50 μM.
The effect of C23 alone on gene expression was examined in a
To verify that C23 functions by inhibiting DSF signaling RNA was extracted from these cultures and the level of expression
in vivo, we used microarray to compare the effects of adding of selected genes examined by qRT–PCR. Of the genes examined,
either DSF alone or DSF with C23 on P. aeruginosa gene gcdH (PA0447) and murA (PA4450) appeared to have elevated
expression during mouse lung infection relative to the gene expression as a response of C23 addition to P. aeruginosa PAO1,
expression of P. aeruginosa in the mouse lung with no compound. but showed little or no response to DSF (Supplementary Fig. 8).
RNA was derived from organisms isolated directly from the lung
homogenates of infected mice. The results showed that the
expression of 68 genes was significantly altered (>1.25-fold, padj
<
The DSF analog C23 improves antibiotic efficacy. Perception of
DSF by P. aeruginosa mediated by PA1396 leads to increased
expression of the pmrAB operon and the concomitant increased
level of resistance to the cationic antimicrobial peptides poly-
myxin B and E12,13. Further, DSF increases tolerance to poly-
myxin B in P. aeruginosa biofilms-airway epithelial cells co-
cultures14. These observations prompted us to examine the
influence of C23 on the sensitivity of P. aeruginosa to antibiotics.
As expected, addition of DSF led to pmrAB up-regulation and an
increased resistance to polymyxin B (Fig. 6a, b). By contrast,
addition of C23 plus DSF led to a marked reduction in pmrAB
expression and reduced resistance to polymyxin B. Although the
pmrAB expression level in response to C23 alone was close to
1 × 10−5) by the addition of DSF (Fig. 5a and Supplementary
Table 3). While many of these differentially expressed genes were
originally annotated as hypothetical proteins, several encode
factors required by P. aeruginosa for growth and/or host
colonization of the host; these genes included norB (PA0524),
popD (PA1709), sphA (PA2659) and narK (PA3876). Additional
regulated genes encoded factors that contribute to P. aeruginosa
virulence and biofilm formation such as the mexC (PA4599), pmr
operon (PA4774, PA4776, PA4777) and exoS (PA3841).
Addition of DSF and C23 significantly altered expression of 51
genes (Fig. 5a). Comparison with the effects of DSF alone showed
expression changes in 21 genes occurred in both treatments (DSF
6
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A R T I C L E
a
b
12.0
10.0
8.0
PAO1 + DSF
PA1 + DSF + C23
6.0
**
**
4.0
**
**
**
**
**
2.0
40
20
1
14
0.0
–2.0
–4.0
–6.0
–8.0
7
16
**
PAO1 + DSF: 68
PAO1 + DSF + C23: 51
Fig. 5 E f f e c t s o f D S F a n d C 2 3 o n P. aeruginosa g en e e x p r e s s i o n d u r i n g i n f e c t i o n . a V e n n d i a gr a m s s h o w i n g r e s u l t s o f c o m p a r at i v e t r a n s c r i p to m e p r ofil in g o f
t he e f f ec t s o f a d d i t i o n o f e i th e r D SF o r D SF w it h C 2 3 o n g en e e x p r e s s i o n i n w i l d - t y p e P. aeruginosa d u r i n g m o u s e l u n g i n f e c t i o n . T h e c o m p a r a t or i s P.
aeruginosa i n m o u s e l u n g w it h n o c o m p o u n d a d d i t io n . G e n e s s i g n ific a n t l y u p r e gu l a te d ( > 1 .2 5 - f o l d , pa d j < 1 × 1 0−5 ) a r e i n d i c a t e d i n r e d , t h o s e s i g ni fic a n t ly
d o w nr e g u l a te d a r e i n d i ca te d i n g re e n . T h e c o m p l e te s e t o f r e gu l a t e d g e n e s i s d e p i c t e d i n S u p p l e m en t a r y T a b l e 3 . b q R T–P C R a n a l y si s o f e x p r e s si on l ev e ls
o f s e l ec ted g e n e s i m p l i c a t ed i n v i ru le n c e a n d b i ofil m f o r m a t i o n i n r e s p o n s e t o a d d i t i o n o f D S F o r D S F a n d C 2 3 i n w i l d - t y p e P. aeruginosa d u r i n g m o us e l un g
i n f e c t i o n . T r a n s cr i pt l e v el s o f PA0524, PA1709, PA2659, PA3876, PA4774, PA4776, PA4777, a n d PA3841 w e r e e x a m i n e d . T h e q R T–P C R d a t a w e r e
n o rm a l i z e d t o proC a n d a r e p r e s e n t e d a s t h e f o ld c h a n g e w it h r e s p e c t t o t h e w i l d t y p e f o r e a c h g e n e . D a t a ( m e a n s s t a n d a r d d e v ia t i o n ) a r e f r o m o ne
r e p re s e nt a t i ve e xp er i m e n t ( n = 3 ) . A p-v a l u e o f < 0 . 0 5 w a s c o n s i d e r e d s t a t i s t i c a l l y s i gn ific a n t a n d i s d e s i gn a t e d i n t h e fig u r e s w i t h a n a s t e r i s k . D ou b le
a s te r i s k s i n d i ca te p-v a l u e s o f < 0 . 0 1
wild type, C23 did appear to slightly enhance the activity of Fig. 12). Altogether, these results demonstrate that C23 treatment
polymyxin B against PAO1 (Fig. 6a). We also examined the leads to reduced resistance of P. aeruginosa laboratory and
influence of DSF and C23 singly and in combination in resistance clinical strains to distinct antibiotics both in vitro and in vivo
to other classes of antibiotics used in treatment of P. aeruginosa. during infection, an effect manifested through a functional
Addition of DSF to the cultures led to increased resistance to PA1396 sensor kinase.
tobramycin and nalidixic acid but had no effect on carbapenem
resistance. The effect of DSF on increased tobramycin and nali-
dixic acid resistance was reversed by C23 (Supplementary Fig. 9).
To validate these results in other strains, we examined a panel
of P. aeruginosa clinical isolates and the data revealed that
addition of DSF led to a significant increase in the expression of
pmrAB in each of these strains (Fig. 6b). However, addition of
C23 reversed this DSF-induced effect in all strains, whereas C23
alone gave only modest or no increase in expression. In all of
these cases, changes in pmrAB expression were associated with a
concomitant alteration in resistance to polymyxin B, as seen with
PAO1 (Supplementary Fig. 9). Further, addition of C23 also
reversed DSF-induced tobramycin tolerance in the clinical
isolates, which decreased in all strains (Supplementary Fig. 10).
The ΔPA1396 mutant showed increased resistance to polymyxin
B, an effect that could not be reversed by addition of C23, as
expected. To determine whether PA1396 was also required for the
response of clinical isolates of P. aeruginosa to C23, we created
PA1396 gene disruption mutants in five strains, which were
selected because of their intrinsically higher level of pmrAB
expression and polymyxin resistance. None of the mutant strains
responded to C23, confirming the C23 antagonist effect on DSF is
mediated by PA1396 (Supplementary Fig. 11).
We also assessed the effect of C23 on the efficacy of tobramycin
treatment in the C57BL/6 mouse lung infection model. Mice were
infected with 1 × 107 CFU of PAO1 and treated by inhaling PBS
with or without C23. Tobramycin was administered at a
concentration of 30 mg/kg 1 h after infection, as previously
reported17,18. The lung bacterial load was determined 24 h post
infection. Control mice infected with P. aeruginosa and inhaled
PBS exhibited considerable colonization (Fig. 6c), which was
reduced by tobramycin treatment. However, adding C23 with
tobramycin resulted in a larger decrease of the bacterial load than
that seen with tobramycin alone (Fig. 6c). The kinetics of
clearance of ΔPA1396 from mice not treated with antibiotics was
similar to that of mice infected with PAO1 (Supplementary
Discussion
This study provides a detailed characterization of the membrane
topology of PA1396, including the identification of its input
domain, which has considerable sequence similarity with the
input domain of RpfC, the sensor histidine kinase for DSF-
mediated cell-to-cell signalling in Xanthomonas campestris pv.
campestris12,19. However, significant differences between PA1396
and RpfC also exist. RpfC contains a short N-terminal peri-
plasmic region implicated in DSF binding20, suggesting DSF is
captured in the periplasmic space. Residue E19, which is con-
served in all homologs with the RpfC-related input domain, is
among various key residues for DSF binding in RpfC12,20. In P.
aeruginosa PA1396, the corresponding residue is E8, also in the
N-terminal periplasmic region. However, deletion of a significant
part of the N-terminal region of PA1396 has no effect on DSF
sensing by P. aeruginosa. Unlike RpfC, our results indicate the
DSF binding capability of PA1396 requires residues in TMHs IV
and V. This suggests that DSF likely interacts with residues in
these TMHs at the membrane level.
The PA1396 recognition of DSF shares features with the CqsS
system of Vibrio cholerae, which detects the quorum-sensing
signal CAI-1 (S-3-hydroxytridecanone). Both CqsS and PA1396
have complex membrane spanning domains with multiple TMHs
and periplasmic and cytoplasmic loops of limited size and both
recognize ligands with alkyl chains of medium length and an
amphipathic nature. Nevertheless, substantial differences between
the systems occur. In CqsS, conserved residues in the first three
(of six) TMHs are obligatory for CAI-1 ligand binding and
subsequent signal transduction. Further, residues in the fourth
transmembrane helix enable CqsS to discriminate between CAI-1
and amino-CAI-1 and residues in the fifth TMH have roles in
restricting ligand head group size and tail length3,21. In contrast,
PA1396 truncation experiments suggest that the first three TMHs
of the protein are dispensable for DSF perception, but whether
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7

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N A T U R E C O M MU N IC A T IO N S | h t t p s : / / d o i . o r g / 1 0 .1 0 3 8 / s 4 14 6 7 - 0 1 9 -1 0 2 71 -4
Fig. 6 E ff e c t s o f D S F a n d C 2 3 o n a n t i b i o t i c r e s i s t a n c e o f P. aeruginosa
s t r a i n s . a E f fe c t o f a d d i t i o n o f D S F , C 2 3 , a n d D S F t o g e t h e r w i t h C 2 3 o n
t o l e r a n c e o f P. aeruginosa t o p o l y m y x i n s . T i m e - c o u r s es o f k i l l i n g o f P.
aeruginosa b y 4 μg / m l p o l y m y x i n B w e r e e s t a b l i s h e d f o r b a c t e r i a s u s pe nd e d
i n s o d i u m p h o s p h a t e b u f f e r . B a c t er i a f o r t h e s e e x p e r i m en t s w e r e g r o w n i n
B M2 m e d i u m w i t h g l u c o s e s u p p l e m en t e d w i t h 2 m M M g2 +. D S F a nd C 23
w e r e a d d e d t o t h e s e c u l t u r e s t o a fin a l c o n c e n t ra t i o n o f 5 0 μM . V a lu e s a r e
t h e a v e r a g e o f t h r ee t e c h n i c a l r e p e a t s ( m e a n s t a n d a r d d ev i a t i o n) . T he
o b s e r v e d v a l u e s o f a d d i t i o n o f D S F a n d C 2 3 w e r e s i gn ific a n t l y d if f e r e n t
f r o m t h e a d d i t i o n o f D S F a l o n e (p < 0 .0 5 , A N O V A ) . b E f fe c t o f a dd it i o n o f
D S F , C 2 3 , a n d D S F w i t h C 2 3 o n a n t i b i o t i c r e s i s t a n c e g e n e e x p r e s s i on i n
c l i n i c a l i s o l a t e s o f P. aeruginosa a s c o m p a r e d t o t h e l a b o r a t o r y s t r a i n P A O 1 .
T r a n s c r i p t l e v e l s o f PA4775 i n P A O 1 , t h e i s o ge n i c ΔPA1396, a n d a s e le c t i on
o f c l i n i c a l i s o l a t e s o f P. aeruginosa ( C F 3 , C F 9 , C F 1 4, C F 2 1 , a n d C F 2 2 ) w e r e
e x a m i n e d u s i n g q R T–P C R a s d e s c r i b e d i n M e t h o d s . T h e q R T –P CR d at a
w e r e n o r m a l i z ed t o proC a n d a r e p r es e n t e d a s t h e f o l d c h a n g e r e la t i v e t o
t h e w i l d t y p e f o r e a c h g e n e i n e a c h s t r a i n w i t h n o a d d i t i o n . D a t a ( m e a ns
s t a n d a r d d e v ia t i o n ) a r e f r o m o n e r e p r e s e n t a t i v e e x p e r i m e n t ( n = 3 ). A p-
v a l u e o f < 0 . 05 w a s c o n s i d e r e d s t a t i s t i c a l l y s i gn ific a n t a n d i s d e s ig n at e d i n
t h e fig u r e s w i t h a n a s t e r i s k . D o u b l e a s t e r i s k s i n d i c a t e p-v a l u e s o f < 0 . 0 1 .
c E ff e c t o f a d m i n i s t r at i o n o f C 2 3 o n P. aeruginosa c l e a r a n c e b y t o br a m y c i n
t r e a tm e n t i n t h e m o u s e a i r w a y . C 5 7 B L/ 6 m i c e w e r e i n f e c t e d i nt r a n as a l ly
a
100
90
80
70
60
50
40
30
20
10
0
PAO1
PAO1 + DSF
PAO1 + C23
PAO1 + DSF + C23
0
1
2
3
5
10
20
Treatment time (min)
b
14.0
12.0
10.0
8.0
DSF
C23
DSF + C23
6.0
7
4.0
**
**
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**
**
*
w i t h o u t 5 0 μM C 2 3 . T o b r a m y c i n w a s a d m i n i s t er e d a t a c o n c e nt r at io n o f
3 0 m g / k g 1 h a f t e r i n f e c t i o n . A f t e r 2 4 h o f i n f e c t i o n , t h e m i c e w e r e
h a r v e s t e d , a n d b a c t e r i a l l o a d s w e r e d e t e r m i n ed i n l u n g h o m o g en at e s . E ac h
d a t a p o i n t s h o w s t h e r e s u l t s f r o m a n i n d i v i d u a l m o u s e . A p-v a l ue o f < 0 . 0 5
w a s c o n s i d e r e d s t a t i s t i c a ll y s i gn ific a n t a n d i s d e s i gn a t e d i n t h e fig ur e s w i t h
a n a s t e r i s k . D o u b l e a s t e r i s k s i n d i c a t e p-v a l u e s o f < 0 . 01
2.0
0.0
PAO1 PA1396 CF3
CF9
CF14
CF21
CF22
c
1.E+07
1.E+06
1.E+05
1.E+04
1.E+03
1.E+02
1.E+01
1.E+00
**
PA1396 does not respond to cis-2-decenoic acid, a molecule
produced by P. aeruginosa and which is implicated in biofilm
dispersal12,13,20
.
We demonstrate that DSF and cis-unsaturated fatty acid ana-
logs enhance the tolerance of P. aeruginosa to a range of anti-
biotics, including polymyxins, tobramycin, and nalidixic acid; this
action requires the sensor kinase PA1396. In contrast, Deng
et al.24, reported that both DSF and its analogs promote sensi-
tivity of several Gram-positive pathogens including Bacillus cer-
eus and Staphylococcus aureus to various antibiotics, including
kanamycin and gentamicin. The molecular mechanisms under-
lying these effects in B. cereus are unclear, but it is intriguing that
cis- and trans-2-unsaturated fatty acids are equally effective in
inducing a response.
PAO1
PAO1 +
Tobramycin
PAO1 + C23 +
Tobramycin
THMs I–III of PA1396 have roles in detection of other ligands is
unknown.
Previous work on interspecies signaling in P. aeruginosa
demonstrated that the cis-2-unsaturated fatty acids DSF and
BDSF trigger pmr gene expression, but weak or no activity was
observed with the trans-derivatives and related saturated fatty
acids. Our analysis provides detailed structural requirements for
interspecies signaling. All trans-unsaturated fatty derivatives,
irrespective of chain length, had little or no signaling activity on
pmr gene expression. Similarly, any derivative lacking a free
carboxylic acid group (through esterification, conversion to a
hydroxamic acid or substitution with a 2-amino pyridine moiety)
was inactive. The signaling activity was not significantly affected
by the position of the methyl group (at position 11 in DSF) but
was partially reduced by introducing a second double bond.
Notably, a derivative of DSF with a second double bond (cis, cis-
11-methyldodeca-2,5-dienoic acid) occurs naturally in various
Xanthomonas species, but also has reduced bioactivity compared
to DSF to trigger DSF-regulated genes22. Further, interspecies
signaling in P. aeruginosa depends on the chain length of the cis-
unsaturated fatty acid; molecules with chain lengths longer than
14 (this work) and less than 1212,13,23 are inactive. In particular,
We reasoned that if DSF signaling activates expression of genes
involved in antibiotic tolerance, structural analogs of the molecule
could act as antagonists. Accordingly, we show that various
effects of DSF on P. aeruginosa, including induction of pmr gene
expression and persistence in the mouse model is antagonized by
the C23 analog. Antagonism of DSF action may have an appli-
cation to control of P. aeruginosa in multispecies infections with
pathogens that produce DSF family signals. An example is
infection associated with cystic fibrosis (CF), where P. aeruginosa
can occur together with S. maltophilia and Burkholderia species,
both of which produce DSF and BDSF. The detection of DSF and
BDSF signals at physiologically relevant levels in the sputum of
CF patients supports the contention that interspecies signaling
may occur in these infections, where it may affect the antibiotic
sensitivity of P. aeruginosa as suggested by in vitro experiments.
Intriguingly, C23 has additional modulating effects on P. aer-
uginosa that cannot be attributed to its role as a DSF antagonist.
For example, C23 affects gcdH and murA expression, two genes
that do not respond to DSF. C23 also affects biofilm formation,
both in co-culture with lung epithelial cells and in ‘complex’
8
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(2 0 1 9 ) 1 0 : 2 3 3 4 | h t t p s : / / d o i . o r g / 1 0 . 1 0 3 8 / s 4 1 4 6 7 - 0 1 9 - 1 0 2 7 1 - 4 | w ww .n a t u r e . c o m / n a t u re c o m m u n i c a t i o n s

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medium, in a DSF-independent manner and contributes to a Truncation and mutagenesis of PA1396 gene. To construct strains harboring
truncated PA1396 alleles, we employed plasmid pME6032. We amplified PA1396
alleles with deleted for amino acids 1–35, 1–40, 1–82, 1–104, 1–114, 1–136, and
1–143, respectively. These amplified fragments were ligated into pME6032 using
more rapid clearance of P. aeruginosa from the lungs of mice.
More detailed molecular knowledge of the activities of PA1396
may afford insight into these different actions of C23. For
example, can PA1396 sense other environmental signals for
biofilm formation (in addition to DSF) perhaps through TMHs
I–III? Is transduction of these signals also blocked by C23? Can
C23 alone cause reduced auto-phosphorylation of PA1396
in vivo? Work has indicated the two-component regulator
PA1397 is involved in signal transduction beyond PA139614.
However, the findings are not consistent with a simple model in
which DSF-induced phosphorylation of PA1397 directly activates
pmr gene expression, indicating that other regulatory elements
are involved. Future work will be directed at identifying these
components.
In conclusion, pharmacological inhibition of DSF-mediated
interspecies signaling in P. aeruginosa using a DSF analog
potential has identified a potential lead compound to develop
molecules that could be used as antibiotic adjuvants improving
the efficacy of existing antibiotics and help control diseases
caused by this human pathogen.
appropriate restriction sites. Primer sequences and restriction sites are provided in
Supplementary Table 5. The DNA fragments encoding the proteins with alterations
in Y116, L117, T121, L123, L128, T135, P136, W138, A140, Q142, M144, L148,
M149, V154, I155, and F157 were synthesized by Gene Oracle (Santa Clara, USA)
in pGOv4 and sub-cloned pME6032. All constructed plasmids were transformed
into ΔPA1396 selecting for GmR.
Synthesis of DSF analogs. The starting point for the synthesis of cis-2-dodecenoic
acid C2 (BDSF) and 11-methyldodec-2-enoic acid C1 (DSF) involved a Swern
oxidation of the starting alcohol with dimethyl sulfoxide (DMSO) and oxalyl
chloride at –78 °C to corresponding aldehydes decanal and 11-methyldecanal33. A
subsequent Wittig reaction of the purified aldehyde with the modified Horner-
Wadsworth-Emmons phosphonate salt ethyl [bis(2,2,2-trifluoroethoxy)-phosphi-
nyl]acetate in the presence of sodium hydride (NaH) in THF afforded both the cis-
and trans-α,β-unsaturated esters, ethyl dodec-2-enoate and ethyl 11-methyldodec-
2-enoate34. Hydrolysis of the cis-α,β-unsaturated esters with lithium hydroxide
(LiOH) in THF:MeOH:H2O (2:1:1 (v/v/v)) gave required products C2 and C135,36
.
Treatment of C1 with propylphosphonic anhydride and cyclopropylamine gave the
required amide analog37. DSF antagonist C23 was prepared by way of an EDCI-
mediated coupling of C1 with methanesulfonamide in the presence of dimethy-
laminopyridine and subsequent isolation of the desired cis-isomer by reverse phase
HPLC. All the chemicals and reagents were purchased from Sigma-Aldrich unless
otherwise stated. Additional synthetic procedures and analytical data used in this
study are delineated in Supplementary Note.
Methods
Bacterial strains and culture conditions. Pseudomonas aeruginosa PAO1 was
obtained from the Genetic Stock Center (http://www.pseudomonas.med.ecu.edu/)
(strain PAO0001). Other bacterial strains and plasmids used in this study are listed
in Supplementary Table 4. P. aeruginosa and other strains were routinely grown at
37 °C in Luria–Bertani (LB) medium while Xanthomonas campestris strains were
routinely grown at 30 °C in NYGB medium, which comprises Bacteriological
Peptone (Oxoid, Basingstoke, UK), 5 g/l; yeast extract (Difco), 3 g/l and glycerol,
20 g/l. The FABL medium consists of 97% FAB medium [(NH4)2SO4, 2 g/l;
Na2HPO4 2H2O, 6 g/l; KH2PO4, 3 g/l; NaCl, 3 g/l; MgCl2, 93 mg/l; CaCl2, 11 mg/l)
and 3% L medium (Bactotryptone, 10 g/l; yeast extract, 5 g/l; sodium chloride, 5 g/l;
and D-glucose 1 g/l). Cultures were also grown in artificial sputum medium which
comprises: 5 g mucin from pig stomach mucosa (Sigma), 4 g DNA (Fluka), 5.9 mg
diethylene triamine pentaacetic acid (Sigma), 5 g NaCl, 2.2 g KCl, 5 ml egg yolk
emulsion (Oxoid) and 5 g amino acids per 1 l water (pH 7.0)14,16. The antibiotics
used included tobramycin, polymyxin, kanamycin, rifampicin, gentamycin, spec-
tinomycin, nalidixic acid, carbapenem, and tetracycline at the indicated
concentrations.
DSF analog structure analysis. Nuclear magnetic resonance spectra 1H, 13C, 1H-
1H COSY, and DEPT were recorded on a Bruker Avance 400 NMR Spectrometer
(400 MHz for 1H) and a Bruker Avance 500 NMR Spectrometer (500 MHz for 1H
and 125 MHz for 13C) with trimethylsilylchloride as an internal standard in CDCl3.
1H-13C correlated HMBC and HMQC spectra were performed by Bruker Avance
500 spectrometer. Mass spectra ESI-MS and high-resolution mass spectra ESI-MS
were performed on a Waters/Micromass: LCT Premier Time of Flight and a
Quattro Micro triple quadrupole instruments, respectively. Infrared spectra were
measured using NaCl plates on a Perkin Elmer paragon 1000 Fourier-transform
infrared spectroscopy (FT-IR) spectrometer.
DSF bioassays. The original DSF bioassay is based on its ability to restore
endoglucanase production to rpfF mutant of Xcc as described in ref. 38. DSF activity
was expressed as the fold increases in endoglucanase activity over the control. We
also used the bioassay previously described13 that relies on DSF-dependent
induction of fluorescence (pmr-gfp) in P. aeruginosa PAO1.
DNA manipulation. Molecular biological methods such as isolation of plasmid and
chromosomal DNA, PCR, plasmid transformation, as well as restriction digestion
were carried out using standard protocols. PCR products were cleaned using the
Qiaquick PCR purification kit (Qiagen) and DNA fragments were recovered from
agarose gels using Qiaquick minielute gel purification kit (Qiagen). Oligonucleotide
primers were purchased from Sigma-Genosys. Primer sequences are provided in
Supplementary Table 5.
Reconstitution and phosphorylation of PA1396-His in liposomes. Using a
variation in the method previously described39,40, E. coli strains containing pBAD/
Myc-His (PA1396-pBAD/Myc-His) were induced with 0.2% arabinose and purified
through nickel columns according to the manufacturer’s instructions (Qiagen).
Liposomes were reconstituted as previously described40–42. Briefly, 50 mg of E.
coli phospholipids (44 μl of 25 mg/ml; Avanti Polar Lipids) were evaporated and
then dissolved into 5 ml of potassium phosphate buffer containing 80 mg of N-
octyl-β-D-glucopyranoside. The solution was dialyzed overnight against potassium
phosphate buffer. The resulting liposome suspension was subjected to freeze–thaw
in liquid nitrogen. Liposome size was analyzed by dynamic light scattering.
Liposomes were stored at 4 °C. Liposomes were then destabilized by the addition of
26 mg of dodecylmaltoside, and 5 mg of PA1396-His (dual Myc- and His- tagged
protein) was added, followed by stirring at room temperature for 10 min.
Two-hundred-sixty milligrams of Biobeads (BioRad) were then added to
remove the detergent, and the resulting solution was allowed to incubate at 4 °C
overnight. The supernatant was then incubated with fresh Biobeads (BioRad) for
1 h in the morning. The resulting liposomes containing reconstituted PA1396-His
were frozen in liquid N2 and stored at −80 °C until used. The orientation of HKs in
the liposome system was established by other groups and can be confirmed from
the accessibility of ATP to the kinase site and anti-Myc antisera (Invitrogen
R950–25, 1:1000 dilution) to the C-terminal PA1396-MycTag without disruption
of the liposomes.
Twenty microliters of the liposomes containing PA1396-His were adjusted to
10 mM MgCl2 and 1 mM DTT, and various concentrations of agonist or
antagonist, frozen and thawed rapidly in liquid nitrogen, and kept at room
temperature for 1 h (this allows for the signals to be loaded within the liposomes).
[γ32P]dATP (0.625 μl) (110 TBq/mmol) was added to each reaction. To some
reactions, 10 μg of PA1396-His was added. At each time point (0, 10, 30, 60, or 120
min), 20 μl of SDS loading buffer was added. For all experiments involving PA1396
alone, a time point of 10 min was used. The samples were run on SDS/PAGE
without boiling and visualized using a Molecular Dynamics PhosphorImager.
These were quantified by ImageJ software.
Cloning the PA1396 gene. The DNA fragments encoding the full-length PA1396
protein or truncation of interest were synthesized by Gene Oracle (Santa Clara,
USA) in pGOv4 and sub-cloned into pET47b, pME6032 or pBAD/Myc-His before
transformation into E. coli BL21 (DE3). Genomic regions are described in Sup-
plementary Table 5. BL21 (DE3) cells were grown in LB media and induced with
0.25 mM IPTG; protein overexpression was carried out at 37 °C for 1 h. Purifica-
tion was achieved by Ni2+ affinity chromatography using the N-terminal His6 tag.
Construction of targeted PA1396–phoA and PA1396–lacZ fusions. Trans-
membrane domain (TMD) predictions of the P. aeruginosa PA1396 protein were
obtained using the TOPRED25, TMHMM26, DAS-TMFILTER27,28, SOSUI29 and
HMMTOP30 programs, each with their default settings.
Suitable fusion sites in the periplasmic and cytoplasmic loops were identified
using a consensus based on the TMS prediction data obtained (Supplementary
Fig. 1). The sites selected correspond to amino acids E4, N6, V37, E38, G66, A70,
G107, G109, A137, Q139, Q141, and L205. The DNA fragments encoding the
proteins of interest were synthesized by Gene Oracle (Santa Clara, USA) in pGOv4
and sub-cloned into topology reporter plasmids phoA (pRMCD28) and lacZ
(pRMCD70). Genomic regions are described in Supplementary Table 5.
The fusion junction of each construct was confirmed by sequencing. Alkaline
phosphatase assays were carried out according to Daniels et al.31. β-Gal assays were
carried out according to Baker et al.32, except that overnight cultures were sub-
cultured and grown to OD < 0.6 before activity assays. Samples were assayed in
triplicate over at least three independent experiments.
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9

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N A T U R E C O M MU N IC A T IO N S | h t t p s : / / d o i . o r g / 1 0 .1 0 3 8 / s 4 14 6 7 - 0 1 9 -1 0 2 71 -4
Biofilm assays. Biofilm was assessed by attachment to glass and was determined
by crystal violet staining. Log-phase-grown bacteria were diluted to OD600 nm =
0.02 in LB broth and 5 ml was incubated at 37 °C for 24 h in 14-ml glass tubes.
After gently pouring off the media, bacterial pellicles were washed twice with water
and were then stained with 0.1% crystal violet. Tubes were washed and rinsed with
water until all unbound dye was removed. Bound crystal violet was eluted in
ethanol and measured at OD595. Three independent assays were carried out for
each strain. For P. aeruginosa biofilms developed on cystic fibrosis bronchial cells
(CFBE) cells using a co-culture model system as previously described14. In short,
epithelial cells were seeded at a concentration of 106 cells/well in 6-well tissue
culture plates or 2 × 105 cells/well in 24-well tissue culture plates and maintained in
minimal essential medium (MEM) with 10% fetal bovine serum, 2 mM l-gluta-
mine, 50 U/ml penicillin, and 50 μg/ml streptomycin. The cells were grown at 37 °C
and 5% CO2 until ~90% confluence before inoculation with bacteria. P. aeruginosa
was inoculated at a concentration of ~2 × 107 CFU/ml into 1.5 ml MEM/well
(without fetal bovine serum, penicillin, or streptomycin. After 1 h, the supernatant
was replaced with fresh MEM supplemented with 0.4% arginine, and the system
was incubated at 37 °C and 5% CO2 for 4 h. To determine the numbers of biofilm
CFU, we washed the cells two or three times with phosphate-buffered saline (PBS)
to remove planktonic bacteria and treated the cells with 0.1% Triton X-100 for 10
to 15 min to lyse the epithelial cells. For P. aeruginosa biofilms developed in μ-well
chambers, compounds (0.5 µM) were added to in ABTGC media and biofilms were
developed for 16 h in chambers (Ibidi) as previous described16. Confocal laser
scanning microscopy was used to image biofilms and biofilm biomass was quan-
respect to the wild type for each gene was seen regardless of normalized to 16s
rRNA and proC. Only proC normalization is reported.
Statistical analysis. Significant differences between the means the standard
deviation of two different groups were examined using a Student's t-test; for more
than two groups one-way ANOVA was used. To determine the differences between
infected mice group Mann–Whitney U-test was used. A p-value of <0.05 was
considered statistically significant and is designated in the figures with an asterisk.
Double asterisks indicate p-values of <0.01.
Reporting summary. Further information on research design is available in
the Nature Research Reporting Summary linked to this article.
Data availability
The microarray data have been submitted to the Gene Expression Omnibus (GEO)
database with accession code GSE110126 . Source data underlying Figs. 1c, d, 2b, c, 3a, b,
3d, 4a–c, 5b, 6a–c, Supplementary Figs. 1, 3–12, and Supplementary Tables 1, 2 are
provided as a Source Data file. All the data produced for this study are available from the
corresponding authors upon request.
Received: 4 August 2015 Accepted: 24 April 2019
tified using COMSTAT14
.
Polymyxin killing curves. Killing curves were carried out at 37 °C temperature as
previously described13,19. In short, mid-log-phase cultures (OD600 0.4–0.6) of
PAO1 wild-type strain, PAO1 supplemented with 50 µM DSF or the ΔPA1396
mutant grown in BM2 medium supplemented with 0.5 mM and 2 mM Mg2+ were
diluted 1:100 into 30 mM sodium phosphate buffer, pH 7.0, containing 4 μg/ml of
polymyxin B sulfate and 5 μg/ml polymyxin E sulfate (Sigma). Samples were
shaken gently, and aliquots removed at specified time intervals were assayed for
survivors by plating appropriate dilutions onto LB agar.
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mice (~8-weeks-old) were anesthetized and intranasally inoculated with 20 μl of the
bacterial suspension (1 × 107 CFU/mouse) in PBS with PAO1, PAO1/DSF, PAO1/
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PAO1/or selected DSF analog (1 × 107 CFU/mouse). Each mouse received was
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Gene expression-profiling experiments. For the in vivo RNA samples, infected
animals as described above were euthanized ~24 h post infection43,44. RNA was
isolated from airway homogenate pellets using Trizol according to the manufac-
turer's instructions. Samples were treated with DNAse, then rRNA was removed
from 1 to 5 μg of total RNA using the RiboZero kit for Gram-negative bacteria
(Epicenter). rRNA-depleted samples were concentrated by EtOH precipitation.
cDNA synthesis and hybridization to Affymetrix GeneChip P. aeruginosa genome
arrays were carried out by a commercial Affymetrix Genechip service supplier
(Conway Institute of Biomolecular & Biomedical Research, UCD, Ireland).
Microarray data analysis was performed using Biomedical Genomics Workbench
software (Qiagen).
Quantitative real-time PCR assays. P. aeruginosa strains (including mutants)
were inoculated at an OD600 of 0.1 in LB (with 50 µM DSF-like signal molecules if
used). Cultures were harvested during mid-log-phase (OD600 = 0.6) and RNA was
extracted using the High Pure RNA Isolation Kit (Roche). The expression of genes
was monitored by qRT–PCR, as described previously17,19 Similar fold changes with
17. Anderson, G. G., Moreau-Marquis, S., Stanton, B. A. & O’Toole, G. A. In
vitro analysis of tobramycin-treated Pseudomonas aeruginosa biofilms on
cystic fibrosis-derived airway epithelial cells. Infect. Immun. 76,
1423–1433 (2008).
10
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