Identification of autoinducing thiodepsipeptides from staphylococci enabled by native chemical ligation

Article abstract of DOI:10.1038/s41557-019-0256-3

Staphylococci secrete autoinducing peptides (AIPs) as signalling molecules to regulate population-wide behaviour. AIPs from non-Staphylococcusaureus staphylococci have received attention as potential antivirulence agents to inhibit quorum sensing and viru

Full text of DOI:10.1038/s41557-019-0256-3

Articles
https://doi.org/10.1038/s41557-019-0256-3
Identification of autoinducing thiodepsipeptides
from staphylococci enabled by native
chemical ligation
Bengt H. Glessꢀ ꢀ1, Martin S. Bojer2, Pai Peng2, Mara Baldry2, Hanne Ingmer2 and
Christian A. Olsenꢀ ꢀ1*
Staphylococci secrete autoinducing peptides (AIPs) as signalling molecules to regulate population-wide behaviour. AIPs from
non-Staphylococcus aureus staphylococci have received attention as potential antivirulence agents to inhibit quorum sensing
and virulence gene expression in the human pathogen Staphylococcus aureus. However, only a limited number of AIP structures
from non-S. aureus staphylococci have been identified to date, as the minute amounts secreted in complex media render it dif-
ficult. Here, we report a method for the identification of AIPs by exploiting their thiolactone functionality for chemoselective
trapping and enrichment of the compounds from the bacterial supernatant. Standard liquid chromatography mass spectrom-
etry analysis, guided by genome sequencing data, then readily provides the AIP identities. Using this approach, we confirm
the identity of fiveꢀknown AIPs and identify the AIPs of elevenꢀnon-S. aureus species, and we expect that the method should be
extendable to AIP-expressing Gram-positive bacteria beyond the Staphylococcus genus.
ell-to-cell communication in staphylococci is mediated by 6species have been characterized thus far. Herein, we present an
secreted peptide signalling molecules—the so-called auto- alternative approach that allows rapid identification of AIPs using
C
inducing peptides (AIPs)1. These peptides are 7–12amino standard laboratory equipment, enabled by trapping the peptides
acids long, contain a carboxy (C)-terminal cyclic thiolactone (or through their thiolactone functionality and performing subsequent
lactone) and are part of the quorum-sensing machinery, which is genome sequence-guided liquid chromatography mass spectrom-
expressed by the chromosomal locus known as the accessory gene etry (LC-MS) analysis (Fig. 1).
regulator (agr)2–4. The agr system regulates group behaviour, such
as virulence gene expression and biofilm formation, in response Results and discussion
to cell density via a regulatory RNAIII. Once the AIP concentra- Development of native chemical ligation (NCL) trapping.
tion reaches a certain threshold, the cognate AIP activates its trans- C-terminal thiolactones are reactive intermediates in NCL reac-
membrane receptor AgrC, resulting in upregulated expression of all tions27, and AIPs readily react with thiols to form linear C-terminal
quorum-sensing-controlled virulence factors2–4. The agr has mostly thioesters28. We therefore envisioned that an acid-labile Rink-amide
been characterized in Staphylococcus aureus but is also present in resin29 loaded with unprotected cysteine would covalently trap AIPs
the coagulase-negative staphylococci, and more than one specificity from the bacterial supernatant through NCL reaction. This allows
group may be present in a given species, forming unique AIP–AgrC enrichment through extensive washing, cleavage from the solid sup-
pairs, such as for S. aureus where four agr groups exist (agr-I–IV)2.
port, and reconstitution of the resulting cysteine-modified AIP for
AIPs potently activate their cognate AgrC receptor and often LC-MS analysis. AIP identification is then guided by the genomic
inhibit AgrC receptors of other staphylococcal species or sub- sequence of the AIP precursor peptide AgrD, which has a con-
groups within their own species5,6. This bacterial cross-talk is served overall structure in all staphylococci3. The AIP-containing
not fully understood from a biological standpoint but offers a AgrD sequence is flanked by a C-terminal recognition sequence
platform for the development of quorum-sensing inhibitors. The and an amino (N)-terminal leader peptide, which can be identi-
AIPs of S. aureus agr-I–IV (1–4) have been investigated exten- fied by highly conserved residues (shown in blue boxes in Fig. 2b).
sively for this purpose7–16, and more recently, AIPs of non-S. aureus However, the cleavage site between the leader peptide and the thio-
staphylococci have received considerable attention17–21. A limiting lactone-forming cysteine cannot be predicted based on the genome.
factor for these studies has been the challenging identification of Therefore, examination of extracted ion chromatograms (EICs) for
new AIP molecules.
all seven possible linear AIP-Cys analogues is necessary (Fig. 2c).
First, we successfully tested our hypothesis with synthetic AIPs
The low concentrations of secreted AIPs and the complex nature
of the growth medium represent a significant obstacle. The AIPs and high-swelling polyethylene glycol polyacrylamide (PEGA)
known to date have been identified through activity-guided high- resin loaded with Rink-amide linker and cysteine (Cys-Rink-PEGA
performance liquid chromatography (HPLC) purifications1,5,22,23 or resin; 5) (see Supplementary Fig. 1). Next, we performed NCL
enabled by advanced mass spectrometry20,24–26. Even though the first trapping with lyophilized AIP-II (2)-containing bacterial superna-
AIPs were discovered more than 20years ago, only 11AIPs from tant that was reconstituted in phosphate buffer (0.1M, pH=7.4)
1Center for Biopharmaceuticals, Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen,
Copenhagen, Denmark. 2Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen,
Denmark. *e-mail: cao@sund.ku.dk
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H
S
containing 20% N,N-dimethylformamide (DMF) and tris(2-car-
boxyethyl)phosphine (TCEP; 30mM) (Fig. 2a). The resin was then
washed extensively, and resin-bound AIP-II (6) was released using
trifluoroacetic acid (TFA) to give linear AIP-II with a C-terminal
cysteine amide residue (7). Examination of the sequence of AgrD-II
provided 7 possible AIPs ranging from 6–12amino acids (Fig. 2b).
We displayed the m/z [M+H]+ values corresponding to the 7 pos-
sible linear peptides containing a C-terminal cysteine amide as
individual EICs and found a strong signal for the expected m/z
[M+H]+ =999.4 (Fig. 2c). The applicability of our NCL trapping
strategy was confirmed in a control experiment, where synthetic
AIP-II (2) was added to fresh tryptic soy broth (TSB) medium—
the medium used to grow S. aureus—and trapped using the same
method, giving identical results (Fig. 2c). The protocol was further
validated by confirming the identity of the remaining AIPs from
S. aureus (1, 3 and 4) (Table 1 and Supplementary Figs. 2–5).
S
H
HS
C
H2N
H2N
C
C
NCL
Acidic cleavage
m/z
Supernatant
Sequencing
G
Sequence analysis
LC-MS
C
DE
AIP precursor peptide
Bacteria
AIP + Cys-NH2
Fig. 1 | Overview of the reported workflow. Supernatants of bacterial
overnight cultures contain minute amounts of secreted AIPs. The AIPs contain
a thiolactone moiety, which enables chemoselective reaction with resin-bound
cysteine through NCL, and thereby covalent trapping of the AIPs as linear
peptide derivatives. The trapped AIPs may be released from the solid support
using TFA and subsequently analysed by LC-MS. The LC-MS analysis is guided
by the amino acid sequence of the AIP precursor peptide, which is obtained by
genomic sequencing. The identity of the excreted AIP is thus confirmed by an
m/z signal corresponding to the mass of the AIPꢀ+ꢀcysteine amide.
Identified AIPs. Next, we focused on the identification of new
non-S. aureus staphylococcal AIPs from a collection of diverse
Staphylococcus species (Table 1, Supplementary Tables 1–3 and
Supplementary Figs. 6–17). First, the structure of the recently
identified AIP of the coagulase-negative strain S. saprophyticus
a
SH
S
SH
O
O
H
TCEP (30 mM)
20% DMF in
phosphate buffer
(pH = 7.4, 0.1 M)
O
N
G
V
N
A
C
S
L
F
H2N
N
H
HS
S
F
L
Resin-bound AIP-II (6)
TFA cleavage
SH
H
N
+
H2N
37 °C, 16 hours
S
G
V
N
A
C
S
H2N
O
SH
AIP-II (2)
Cys-Rink resin (5)
O
NH2
G
V
N
A
C
S
S
L
F
H2N
N
Supernatant
containing AIP-II (2)
H
O
AIP-II + C-terminal cysteine amide (7)
b
Conserved thiolactone
S.aureus AgrD-II
N-terminal leader peptide
AIP-containing
sequence
Recognition sequence
Variable exotail length
c
Synthetic AIP-II control
Supernatant
EIC for [M + H]+
m/z = 999.4
EIC for [M + H]+
m/z = 999.4
0
1
2
3
4
0
1
2
3
4
Time (min)
Time (min)
999.4
999.4
m/z at
t = 1.40–1.90 min
0
1
2
3
4
5
6
7
8
Time (min)
EICs for [M + H]+ of 7 possible AIPs + Cys-NH2
900
1,000
1,100 900
1,000
1,100
m/z
m/z
Fig. 2 | NCL trapping and sequence-guided identification of AIP-II (2). a, Spent medium (supernatant) containing AIP-II (2) was lyophilized and
reconstituted in trapping buffer, to which Cys-Rink-PEGA resin (5) was also added. After incubation at 37 °C overnight, the resin was washed, and trapped
molecules were released by treatment with TFA and collected for LC-MS analysis. b, The AIP-II (2) precursor AgrD-II consists of three segments: an
N-terminal leader peptide; an AIP-containing sequence; and a C-terminal recognition sequence. The segments can be identified by the highly conserved
residues highlighted in blue boxes. The unpredictable length of the exotail of the AIP is defined by the seven-amino-acid sequence before the conserved
thiolactone moiety, giving rise to seven possible AIP structures. c, LC-MS analysis of the cleaved AIP-II-Cys-NH2 conjugate (7) through examination of the
EICs for m/zꢀ=ꢀ[Mꢀ+ꢀH]+ of the seven possible linear AIP-II derivatives containing the C-terminal cysteine amide confirmed the identity of AIP-II (2). This
was further validated by performing a control experiment with synthetic AIP-II (2).
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Table 1 | Identified AIPs
AIP Structure
Staphylococcus species
Sequence
AIP Structure
Staphylococcus species
Sequence
S. hyicus b
KINP-[CTVFF]
Shy-AIP (12)
S. aureus agr- Ia
YST-[CDFIM]
AIP-I (1)
S. aureus agr -IIa
GVNA-[CSSLF]
AIP-II ( 2)
S. chromogenesb
SINP-[CTGFF]
Sch-AIP (13)
S. aureus agr -III a
IN-[CDFLL]
S. argenteusb
YST-[CDFIM]
AIP-III ( 3)
AIP-I (1)
S. aureus agr -IVa
YST-[CYFIM]
AIP-IV ( 4)
S. schweitzerib
YST-[CYFIM]
AIP-IV ( 4)
S. saprophyticus a
INP-[CFGYT]
Ssa-AIP (8)
S. warneri b
YSP-[CTNFF]
Swa-AIP (14)
S. lugdunensis agr -IIb
DM-[CNGYF]
S. vitulinus b
VIRG-[ CTAFL]
Svi-AIP (15)
Slu-AIP-II ( 9)
b
S. schleiferi
S. hominis b
KYPF-[ CIGYF]
Ssc-AIP (10)
TYST-[CYGYF]
Sho-AIP (16)
S. simulans b
KYNP-[ CLGFL]
Ssi-AIP (11 )
S. haemolyticusb
SFTP-[CTTYF]
Sha-AIP (17)
aKnown AIP identity confirmed in this study. bAIP characterized for the first time from the mentioned species.
(Ssa-AIP; 8)26 was confirmed, and the predicted AIP of the second strains (that is, S. vitulinus36 (Svi-AIP; 15), the antimicrobial
specificity group of the human pathogen S. lugdunensis agr-II peptide-producing strain S. hominis37 (Sho-AIP; 16) and the clini-
(Slu-AIP-II; 9)30 was identified to be seven amino acids in length. cally relevant strain S. haemolyticus38 (Sha-AIP; 17)) were found
The AIP of the agr-I-inhibiting S. schleiferi strain (Ssc-AIP; 10) was to be nine amino acids in length, revealing a total of seven new
then identified as a nonapeptide, in contrast with the previously AIPs that were different from the often previously predicted
predicted octapeptide (Ssc-AIP8, YPF-[CIGYF]; 10a)18,19, thus cor- octapeptide structures3.
recting its structure and highlighting the importance of direct AIP
identification from the biological source.
Quorum-sensing modulation. All newly identified AIPs were syn-
We further identified the AIPs of three animal pathogens— thesized using a previously reported on-resin cyclization–cleavage
namely S. simulans31 (Ssi-AIP; 11), S. hyicus32 (Shy-AIP; 12) and protocol19 in acceptable-to-good yields (Fig. 3).
S. chromogenes31 (Sch-AIP; 13)—as being nonapeptides, and
The new AIPs were then tested for their potential as quorum-
classified the recently described33 S. schweitzeri and S. argenteus as sensing modulators of all four S. aureus agr groups (Table 2).
strains that express AIP-IV (4) and AIP-I (1), respectively. Applying Activation assays were performed in case there was no detectable
the protocol further confirmed the predicted AIP34 of the human inhibition at a peptide concentration of 10μM. The trends of pre-
pathogen S. warneri35 (Swa-AIP; 14) to be the octapeptide struc- viously reported half-maximal inhibitory concentration (IC50) and
ture. Finally, the structures of the AIPs of three coagulase-negative half-maximal effective concentration (EC50) values, employing
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shown to control biofilm formation, virulence and carbohydrate
metabolism41–44, and an AIP of L. monocytogenes was recently
identified directly in spent medium by advanced LC-MS/mass
spectrometry to be the thiolactone-containing hexapeptide Lmo-AIP
(20; Fig. 4a)26. To further test our protocol, three different isolates
of L. monocytogenes were therefore grown in either TSB or brain
heart infusion media (a commonly used medium for the growth of
Listeria strains). NCL trapping experiments were then performed
on the lyophilized and reconstituted supernatants, as outlined
above. Surprisingly, no signal for the linear heptapeptide 21 (m/z
[M+H]+ =819.3) or other masses corresponding to potential AIP
sequences, could be detected in any of these experiments, includ-
ing the mass resulting from trapping the pentapeptide thiolactone
previously described44 (see Supplementary Fig. 18). However, this
thiolactone would be expected to undergo spontaneous S–N shift
under our trapping conditions to furnish the corresponding cyste-
ine-containing homodetic pentapeptide, which would not be prone
to undergoing NCL with resin (5).
Table 2 | IC50 and eC50 values (nM) for quorum-sensing
modulation measured in a β-lactamase assay using S. aureus
AgrC-I–IV reporter strains
AIP
AgrC-I
AgrC-IIa
AgrC-IIIa
AgrC-IVa
AIP-I (1)
5.4ꢀ ꢀ0.5b
110ꢀ ꢀ12
120ꢀ ꢀ15
59ꢀ ꢀ2b
700ꢀ ꢀ126
3.7ꢀ ꢀ0.5b
150ꢀ ꢀ63
47ꢀ ꢀ9
80ꢀ ꢀ11
40ꢀ ꢀ15
11ꢀ ꢀ1b
790ꢀ ꢀ66b
230ꢀ ꢀ19
>1,000
AIP-II (2)
AIP-III (3)
AIP-IV (4)
Ssa-AIP (8)
Slu-AIP-II (9)
Ssc-AIP (10)
Ssi-AIP (11)
Shy-AIP (12)
Sch-AIP (13)
Swa-AIP (14)
Svi-AIP (15)
Sho-AIP (16)
Sha-AIP (17)
9.8ꢀ ꢀ0.2c
2.6ꢀ ꢀ0.2b
d
d
d
360ꢀ ꢀ30
>1,000
2.8ꢀ ꢀ0.8
8.6ꢀ ꢀ0.5
3.3ꢀ ꢀ0.5
15ꢀ ꢀ1
–
–
–
d
d
d
–
–
–
86ꢀ ꢀ6
80ꢀ ꢀ16c
31ꢀ ꢀ6b
23ꢀ ꢀ2
50ꢀ ꢀ11
280ꢀ ꢀ66
180ꢀ ꢀ33
350ꢀ ꢀ85
>1,000
350ꢀ ꢀ90
200ꢀ ꢀ13
1,000ꢀ ꢀ105
800ꢀ ꢀ116
580ꢀ ꢀ61
4.0ꢀ ꢀ0.8
60ꢀ ꢀ13
100ꢀ ꢀ10
190ꢀ ꢀ15
134ꢀ ꢀ8
460ꢀ ꢀ59
690ꢀ ꢀ46
220ꢀ ꢀ46c
340ꢀ ꢀ91
1.6ꢀ ꢀ0.2c
To examine whether the hexapeptide Lmo-AIP (20) would be
trapped using our protocol, and to investigate the detection limit
in the two different media, we synthesized Lmo-AIP (20) and
performed NCL trapping experiments using the synthetic AIP
in growth media at varying concentrations (Fig. 4 and Supple-
mentary Fig. 18). We were pleased to find that, regardless of the
medium, concentrations as low as 0.063μM still allowed the detec-
tion of the linear heptapeptide 21 or its disulfide 22, corresponding
to a concentration of Lmo-AIP (20) of 12.5nM in the supernatant
before lyophilization and reconstitution in a trapping experiment
(Fig. 4a,b and Supplementary Fig. 18). This should be seen in
the light that S. aureus AIPs are estimated to reach a concentration
of around 1μM in the supernatant45. We further tested synthetic
Lmo-AIP (20), as well as the supernatants of our three L. mono-
cytogenes isolates, for their ability to inhibit or activate S. aureus
AgrC-I–IV. None of the supernatants showed measurable inhi-
bition, and Lmo-AIP (20) only partially inhibited AgrC-III at the
d
–
54ꢀ ꢀ5b
d
d
340ꢀ ꢀ25
3.5ꢀ ꢀ0.8
–
–
AIP-III
34ꢀ ꢀ1
10ꢀ ꢀ0.2
D4A (3a);
IN-[CAFLL]
Ssc-AIP8 (10a);
3.3ꢀ ꢀ0.7
62ꢀ ꢀ4
22ꢀ ꢀ4
29ꢀ ꢀ5b
YPF-[CIGYF]
aP3-fused β-lactamase reporter strains expressing AgrC-II–IV were constructed (see
Supplementary Information). bEC50 values. cOnly partial inhibition observed. All inhibition assays
were performed in the presence of 100ꢀnM cognate AIP and the results represent meansꢀ ꢀs.e.m.
of at least duplicate determinations performed in biological triplicate. dNo inhibition recorded at
the highest AIP concentration tested.
similar assays, are in agreement with our results7,10. Several newly highest tested concentration (Fig. 4c and Supplementary Fig. 18).
identified AIPs (8, 9 and 14–17) exhibited weak-to-undetectable We conclude that Lmo-AIP (20) can be trapped and identified at
inhibition of the AgrC-I–IV activation in S. aureus. Medium-to- concentrations corresponding to just 12.5nM in bacterial super-
high potencies against AgrC-I–IV activation were exhibited by natants, and that if this AIP were expressed under our growth con-
Ssi-AIP (11), Shy-AIP (12) and Sch-AIP (13). Compounds 10–13 were ditions, it must have been secreted in a concentration below this
more potent than AIP-I–IV (1–4) against several subgroups, and very low threshold.
in some cases equipotent to the optimized, global, pan-inhibitor
AIP-III D4A (3a)12, rendering them promising candidates for
Cyclization
O
further quorum-sensing inhibitor development.
H
N
SPPSa
Nbzb
Interestingly, three tested AIPs (10, 10a and 16) were potent
activators of AgrC-IV, which is an intriguing discovery, keeping
in mind that the sequence similarity of AgrC-I and AgrC-IV is
87% and that their native AIPs are cross-activators2. To the best
of our knowledge, Ssc-AIP (10) and Sho-AIP (16) constitute the
first staphylococcal interspecies agonists discovered. Furthermore,
no compounds except AIP-III D4A (3a) exhibit potent inhibi-
tion against AgrC-IV. The S. aureus agr group IV is considered an
evolutionary divergence from group I39 and is known to have
early or hyperactive activation kinetics23. While a previous testing
of compound 10a was in agreement with the current results19, we
measured it to be an inhibitor of AgrC-IV in the original account18.
SH
O
TFAc
C
N
H2N
18
MeNbz
19
N
O
Cleavage
O
50% MeCN in
phosphate buffer
(pH = 6.8, 0.2 M)
S
Yield = 12–49%
after HPLC
C
H2N
50 °C, 2 h(d)
We suspect that this is due to the mentioned activation kinetics, Fig. 3 | Synthesis of AIPs. Reagents and conditions were as follows.
which makes the potency measurements highly sensitive to assay aAutomated fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide
conditions. Based on the collective evidence, we are confident that synthesis (SPPS) on MeDbz-Gly resin (18) using Fmoc-Xaa-OH
both 10 and 10a should be considered activators of the S. aureus agr (5ꢀequivalents), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
group IV.
hexafluorophosphate (HBTU) (5ꢀequivalents), i-Pr2EtN (10ꢀequivalents)
and DMF for 40ꢀmin. b4-Nitrophenolchloroformate (5ꢀequivalents) in
Investigation of Listeria monocytogenes. Having identified and CH2Cl2, then i-Pr2EtN (25ꢀequivalents) in DMF. cTFA–i-Pr3SiH–H2O for
characterized 16staphylococcal AIPs, we were interested in apply- 2ꢀhours. dDeprotected peptidyl-resin 19 was swollen in cyclization buffer for
ing the protocol to other Gram-positive bacteria known to have 2ꢀhours at 50ꢀ°C, furnishing the desired AIPs in 12–49% yield after HPLC
agr-like systems40. In L. monocytogenes, the agr system has been purification (length varying from hepta- to nona-petide).
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a
Cys-Rink resin (5),
DMF/phosphate buffer/medium
(2:3:5 v/v, 10 ml),
O
SH
F
SH
O
S
S
O
O
S
V
F
TCEP (30 mM), 37 °C, 16 hours
NH2
NH2
+
A
C
M
F
V
A
C
F
M
F
V
H2N
N
H2N
N
M
H
H
A
C
F
Then TFA–H2O (97:3), 22 °C, 2 hours
Then LC-MS analysis
H2N
O
Lmo-AIP (20)
Varying concentrations
21 m/z [M + H]+ = 819.3
Disulfide 22 m/z [M + H]+ = 817.3
b
22
817.3
8 × 106
6 × 106
4 × 106
2 × 106
0
8 × 106
8 × 106
6 × 106
4 × 106
2 × 106
0
819.3
22
21
21
6 × 106
22
4 × 106
22
2 × 106
21
815 820 825
900
815 820 825
900
0
2.0
2.5
3.0
3.5
2.0
2.5
Time (min)
Lmo-AIP (20) (5.0 µM)
3.0
3.5
2.0
2.5
3.0
3.5
700
800
700
800
m/z
m/z display at
Time (min)
Time (min)
m/z
m/z display at
t = 2.40–2.80 min
L
mo-AIP (20) (2.0 µM)
Lmo-AIP (20) (0.063 µM)
t = 2.90–3.30 min
c
AgrC-I
AgrC-II
AgrC-III
AgrC-IV
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
–8 –7 –6 –5 –4 –3 –2 –1
–8 –7 –6 –5 –4 –3 –2 –1
–8 –7 –6 –5 –4 –3 –2 –1
–8 –7 –6 –5 –4 –3 –2 –1
log[[20] (mM)]
log[[20] (mM)]
log[[20] (mM)]
log[[20] (mM)]
Fig. 4 | Detection limit of NCL trapping for synthetic L. monocytogenes AIP (20). a, NCL trapping of synthetic Lmo-AIP (20) dissolved at varying
concentrations in 50% TSB medium (total volume,ꢀ10ꢀml). Although TCEP is present during trapping, some oxidation to form the cyclic disulfide occurs,
presumably during TFA cleavage. b, Display of overlaid EICs for the linear peptide 21 ([Mꢀ+ꢀH]+ m/zꢀ=ꢀ819.3) and its corresponding disulfide 22 ([Mꢀ+ꢀH]+
m/zꢀ=ꢀ817.3) after TFA cleavage and LC-MS analysis for three concentrations (2.0ꢀμM, 0.5ꢀμM and 0.063ꢀμM). This clearly shows a signal for 22 at the
lowest concentration (0.063ꢀμM). c, β-Lactamase assay inhibition curves of Lmo-AIP (20) against AgrC-I–IV show no or very weak inhibition. The error
bars represent the s.e.m. of two individual assays performed in biological triplicate.
Methods
Considering these results, we are confident that our protocol
will be useful for the investigation of quorum-sensing systems
of Gram-positive bacteria that rely on thiolactone-containing
signalling molecules beyond the staphylococci.
NCL trapping of AIPs from bacterial supernatants. Fmoc-Cys(StBu)-Rink-
PEGA resin (5) (50mg) was placed in a 10ml fritted polypropylene syringe,
swelled in DMF for 30min and washed with DMF (5×1min). Te resin was
treated with piperidine in DMF (1:4 v/v, 2.0ml) (1×2min and 1×20min) and
washed with DMF (3×1min), MeOH (3×1min) and H2O (3×1min). Te
resin was treated with a TCEP solution (0.5M, pH=7.00, 0.5ml) for 15min
and subsequently washed with H2O (3×1min), MeOH (3×1min) and DMF
(3×1min). Lyophilized supernatant (original volume 50ml) was dissolved in
10ml trapping bufer (phosphate bufer (0.1M, pH=7.4)–DMF–TCEP solution
(0.5M, pH=7.0)=7.4:2:0.6 v/v/v) and the solution was added to the resin. Te
syringe containing the trapping mixture was agitated at 37°C overnight. Te
trapping solution was removed from the resin and the resin was subsequently
washed with DMF (3×1min), MeOH (3×1min) and H2O (3×1min). Te resin
was then treated with a TCEP solution (0.5M, pH=7.00, 0.5ml) for 10min and
subsequently washed with H2O (3×2ml). A solution of DMF in H2O (10ml, 1:1
v/v) was added to the resin and the resin was agitated at 37°C. Afer 30min, the
resin was washed with DMF (3×1min), MeOH (3×1min) and DCM (3×1min)
and dried under suction for 15min. Te dried resin was treated with a cleavage
cocktail (1.5ml TFA–Milli-Q water, 97:3 v/v) for 2h at room temperature. Te
peptide containing cleavage solution was removed from the resin and collected,
and the resin was rinsed with neat TFA (1.0ml). Te combined TFA fractions were
evaporated under an N2 stream to near-dryness, redissolved in a solution of MeCN
in H2O (100μl, 1:1 v/v) and fltered (0.22μm). Te solution was analysed by LC-MS
as described.
Conclusion
The data presented here provide a readily applicable protocol
that exploits the chemoselective reaction between thiolactones
and a resin-bound cysteine, which allows trapping of AIPs
from bacterial supernatants via NCL. The developed method
facilitated rapid doubling of the number of known staphylococcal
AIPs, and thus provides the means to start mapping of the staphy-
lococcal AIP signalling molecule landscape. The Staphylococcus
genus currently includes 52 species and 28 subspecies46, where
several species have more than one specificity group, such as
S. aureus agr-I–IV. Thus, a conservative estimate of the poten-
tial number of staphylococcal AIPs is likely to comprise more
than 100 peptides still to be discovered. We furthermore expect
the methodology to be broadly applicable to other Gram-positive
bacteria that use thiolactone-containing auto inducers and
therefore to enable the achievement of a better understand-
ing of quorum sensing in general. Finally, access to new AIPs
paves the way to detailed studies of quorum sensing and its
impact on important pathogen properties such as biofilm
formation, colonization, virulence and interactions with com-
mensal staphylococci.
Sequence-guided LC-MS analysis. The filtered TFA cleavage solution was
analysed using a Waters Acquity ultra-HPLC system equipped with a Phenomenex
Kinetex column (1.7μm, 100Å, 50×2.10mm) applying a gradient with eluent C
(0.1% HCOOH in water) and eluent D (0.1% HCOOH in MeCN) rising linearly
from 0–95% of D over 10.0min at a flow rate of 0.6mlmin−1 and an injection
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Nature Chemistry
Articles
volume of 40μl. The total ion chromatograms were analysed by displaying EICs of
m/z [M+H]+ values of the possible linear peptides with an additional C-terminal
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Reporting Summary. Further information on research design is available in the
Nature Research Reporting Summary linked to this article.
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Data availability
Primary sequencing data are deposited at the National Centre for Biotechnology
Information (NCBI GenBank). All other data generated and analysed during this
study are available in the article and its Supplementary Information. Further details
are available from the corresponding author on request.
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Received: 23 September 2018; Accepted: 14 March 2019;
Published: xx xx xxxx
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Acknowledgements
Competing interests
We thank P. Martín-Gago for fruitful input and T.W. Muir for encouraging comments.
P. S. Andersen is acknowledged for providing bacterial strains. This work was supported
by the Carlsberg Foundation (2013-01-0333 to C.A.O.) and University of Copenhagen
(PhD fellowship to B.H.G.).
The authors declare no competing interests.
Additional information
Supplementary information is available for this paper at https://doi.org/10.1038/
s41557-019-0256-3.
Author contributions
Reprints and permissions information is available at www.nature.com/reprints.
Correspondence and requests for materials should be addressed to C.A.O.
B.H.G. and C.A.O. conceptualized the study. B.H.G., M.S.B., P.P. and M.B. performed
the experiments. B.H.G. and C.A.O. wrote the original draft of the manuscript. B.H.G.,
M.S.B., H.I. and C.A.O. reviewed and edited the final manuscript. C.A.O. acquired
funding. H.I. and C.A.O. provided resources and materials. H.I. and C.A.O. supervised
the study.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in
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© The Author(s), under exclusive licence to Springer Nature Limited 2019
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Corresponding author(s):
Christian Adam Olsen
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The study included the trapping and identification of 16 autoinducing peptides from staphylococci supernatants, of which 5 were previously
identified and 11 were unknown. This represents a respectable sample size in comparison to the number of previously identified autoinducing
peptides. 16 synthetic autoinducing peptides were assayed for the ability to modulate quorum sensing in Staphylococcus aureus agr-I-IV.
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