The Natural Product Elegaphenone Potentiates Antibiotic Effects against Pseudomonas aeruginosa

Article abstract of DOI:10.1002/anie.201903472

Natural products represent a rich source of antibiotics that address versatile cellular targets. The deconvolution of their targets via chemical proteomics is often challenged by the introduction of large photocrosslinkers. Here we applied elegaphenone, a largely uncharacterized natural product antibiotic bearing a native benzophenone core scaffold, for affinity-based protein profiling (AfBPP) in Gram-positive and Gram-negative bacteria. This study utilizes the alkynylated natural product scaffold as a probe to uncover intriguing biological interactions with the transcriptional regulator AlgP. Furthermore, proteome profiling of a Pseudomonas aeruginosa AlgP transposon mutant provided unique insights into the mode of action. Elegaphenone enhanced the elimination of intracellular P. aeruginosa in macrophages exposed to sub-inhibitory concentrations of the fluoroquinolone antibiotic norfloxacin.

Full text of DOI:10.1002/anie.201903472

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Accepted Article
Title: The natural product elegaphenone potentiates antibiotic effects
against Pseudomonas aeruginosa
Authors: Weining Zhao, Ashley Renee Cross, Caillan Crowe-McAuliffe,
Angela Weigert-Munoz, Erika Elizabeth Csatary, Amy
Solinski, Joanna Krysiak, Joanna Goldberg, Daniel Wilson,
Eva Medina, William Wuest, and Stephan Axel Sieber
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201903472
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10.1002/anie.201903472
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The natural product elegaphenone potentiates antibiotic effects
against Pseudomonas aeruginosa
Weining Zhao1, Ashley R. Cross2,3,4, Caillan Crowe-McAuliffe5, Angela Weigert-Munoz1, Erika E.
Csatary6, Amy E. Solinski6, Joanna Krysiak1, Joanna B. Goldberg2,4,7, Daniel N. Wilson5, Eva Medina8,
William M. Wuest4,6,7, Stephan A. Sieber1,*
strategies for target deconvolution are applied. A common
method is to select for resistant strains and corresponding
sequencing to reveal mutations in the target gene.[4] However, this
classical method falls short when it comes to indirect resistance
mechanisms (e.g. drug efflux). Moreover, molecules that reduce
virulence, but do not kill bacteria are difficult to address via this
method. Thus, chemical proteomics, such as activity-based
protein profiling (ABPP), have been applied.[5] While some
antibiotics, including beta-lactams, covalently modify their target,
a large number of natural products bind reversibly.[6] For the latter
class, functionalization with a photocrosslinker is required in order
to withstand the conditions of mass-spectrometry (MS) based
proteomics referred to as affinity-based protein profiling
(AfBPP).[7] A major drawback of the corresponding photoprobes
is often a drop in biological activity when introducing these
modifications onto the parent scaffold. Inspired by a phenotypic
screen of synthetic compounds deliberately utilizing the
benzophenone photocrosslinker as an active moiety, we
searched for antibacterial natural products bearing this core
motif.[8] A large fraction of natural antibacterials exhibit phenolic
structures including the benzophenone motif-containing
elegaphenone (EL), a minimally characterized antibiotic produced
by the plant St. John’s Wort, used for diverse medical applications
(Figure 1A).[9] Here we show that EL not only kills Enterococcus
faecalis, but also attenuates virulence of Pseudomonas
aeruginosa. AfBPP utilizing the photoreactive core scaffold paired
with whole proteome studies provide unique insights into the
mode of action. Additionally, combination of EL with subinhibitory
concentrations of norfloxacin enhanced killing of intracellular P.
aeruginosa by macrophages.
Abstract: Natural products represent a rich source for antibiotics
addressing versatile cellular targets. The deconvolution of their
targets via chemical proteomics is often challenged by the introduction
of large photocrosslinkers. Here we select elegaphenone, a largely
uncharacterized natural product antibiotic bearing
a
native
benzophenone core scaffold, for affinity-based protein profiling
(AfBPP) in Gram-positive and Gram-negative bacteria. This study
utilizes the alkynylated natural product scaffold as a probe to uncover
intriguing biological interactions with the transcriptional regulator AlgP.
Furthermore, proteome profiling of a Pseudomonas aeruginosa AlgP
transposon mutant revealed unique insights into the mode of action.
Elegaphenone enhanced the killing of intracellular P. aeruginosa in
macrophages exposed to sub-inhibitory concentrations of the
fluoroquinolone antibiotic norfloxacin.
Innovative antibacterial drugs are urgently needed to address
the current antibiotic crisis. Natural products have been a reliable
source and the majority of marketed drugs are based on this
class.[1] Looking at current modes of action, a limited number of
hot spot targets such as cell wall, nucleotide, and protein
biosynthesis have been evolutionarily selected. Furthermore, the
constant use of antibiotics has resulted in multiple resistance
mechanisms, calling for novel drugs that address unprecedented
and resistance-free pathways.[2] An intriguing new perspective for
developing next-generation antibiotics calls for the attenuation of
the production of toxins, which would decrease severity of
infections and minimize the development of resistance.[3] In order
to rapidly identify such innovative modes of action, several
EL was prepared following a route inspired from the olympicin
A synthesis (Scheme S1).[10] For the corresponding probes the
alkyne tag was directly appended to the monosubstituted
benzene ring in the para position. For this we devised a synthesis
of derivatives which bear an alkyne tag (ELP), exhibit a
methylated hydroxyl group (ELP2) or lack a hydroxyl group
(ELP5) on the adjacent benzene ring (Figure 1A). ELP was
synthesized by Friedel-Crafts acylation of p-bromobenzoic acid
chloride with trimethoxybenzene followed by Sonogashira
coupling with ethynyltrimethylsilane. Demethylation and
subsequent bisprotection of two hydroxyl groups allowed the
selective incorporation of a geranyl group in the ortho position.
Global deprotection under acidic conditions provided ELP in 42%
yield (Scheme 1). Probes bearing defunctionalized hydroxy
[*]
Corresponding author: SAS: stephan.sieber@tum.de
1 Department of Chemistry, Center for integrated Protein Science
Munich (CIPSM), Technische Universität München
Lichtenbergstraße 4, 85747 Garching, Germany
2 Division of Pulmonary, Allergy & Immunology, Cystic Fibrosis and
Sleep, Department of Pediatrics, Emory University School of
Medicine, Atlanta, GA USA
3 Microbiology and Molecular Genetics Program, Graduate Division
of Biological and Biomedical Sciences, Emory University, Atlanta,
GA USA
4 Emory+Children’s Center for Cystic Fibrosis and Airway Disease
Research, Emory University School of Medicine, Atlanta, GA USA
5 Institute for Biochemistry and Molecular Biology, University of
Hamburg, 20146 Hamburg, Germany
6 Department of Chemistry, Emory University, Atlanta GA USA
7 Emory Antibiotic Resistance Center, Emory University, Atlanta, GA
USA
groups were obtained via
a similar strategy with slight
modifications (Scheme S2, S3).
With the natural product and three probes in hand, we first
evaluated the biological activity against representative Gram-
positive and Gram-negative reference strains, i.e. E. faecalis and
P. aeruginosa, respectively. In line with previous reports, EL
exhibited antibiotic activity against E. faecalis as well as other
Gram-positive strains, including Staphylococcus aureus and
Listeria monocytogenes with MIC values ranging from 5 - 12.5
μM (Figure 1C).[9]
8 Helmholtz Center for Infection Research, Inhoffenstraße 7,
38124Braunschweig, Germany
Supporting information for this article is available on the WWW
under http://dx.doi.org/XXXXX
The mass spectrometry proteomics data have been deposited to the
ProteomeXchange Consortium via the PRIDE partner repository
with the dataset identifier PXD010476. See SI for details.
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Scheme 1: Synthesis of AfBPP Probe ELP. DCM: dichloromethane, RT: room temperature, THF: tetrahydrofuran, DMF: N,N-dimethylformamide,
CSA: camphorsulfonic acid.
ELP and ELP5 showed comparable potency while ELP2 was
completely inactive. None of the compounds inhibited P.
aeruginosa growth and even growth of a hyperpermeable strain
of Escherichia coli could only be affected by EL at high
concentrations. However, given the structural similarity of EL to
natural phenolic inhibitors of P. aeruginosa quorum sensing, we
tested the compounds against P. aeruginosa toxin production.[11]
We decided to monitor the levels of pyocyanin, a toxin important
In order to elucidate the corresponding cellular pathways
addressed by EL and analogs, we performed AfBPP utilizing the
alkynylated EL photoprobes. E. faecalis bacterial cells were
incubated with its closest derivative ELP, irradiated, lysed, and
clicked to biotin azide (Figure 2A). The probe-labeled proteome
was enriched on avidin beads and peptides were released for LC-
MS/MS analysis via tryptic digestion. MS analysis was performed
via isotopic dimethyl labeling[14] and the enrichment by ELP was
during lung infection in cystic fibrosis (CF) patients.[12] Satisfyingly, compared to cells treated with DMSO or the inactive probe ELP2.
EL and its corresponding probes revealed strong inhibition of
pyocyanin production, with IC50 values ranging between 5.5 ± 1.1
and 12.6 ± 1.1 μM (Figure 1B). In addition, EL and ELP5 also
reduced pyoverdine levels, another P. aeruginosa toxin, albeit at
much higher concentrations (Figure S1A). Interestingly, the
benzophenone core scaffold by itself neither inhibited E. faecalis
growth, nor reduced P. aeruginosa pyocyanin production,
emphasizing the importance of the natural product substitution
pattern for activity (Figure S1B).
Fumarate reductase (Frd), an enzyme involved in important
cellular pathways such as anaerobic respiration,[15] was among
the most significant hits (Figure S2). In addition, proteins
associated with protein synthesis pathways were significantly
enriched including elongation factor Tu (EF-Tu) and 30S
ribosomal protein S2 (RpsB). In order to gain more direct insights
into target pathways, we performed whole proteome analysis of
ELP treated E. faecalis at sub-MIC concentrations. Interestingly,
proteins belonging to the V-type ATP synthase and V-type
ATPase complexes, directly associated with Frd-mediated
respiration, were consistently up-regulated (Figure S3, S4). By
contrast, no direct link to ribosome function could be established
which was further corroborated by a lack of activity of EL and
derivatives in translation inhibition assays using reconstituted
extracts from Enterococcus rivorum,[16] a close homolog of E.
faecalis that displays a comparable MIC value (12.5 μM) (Figure
S5).
We next focused on targets of ELP5 in clinically relevant Gram-
negative P. aeruginosa via the above described LC-MS/MS
platform. Again, 30S ribosomal protein S2 was strongly enriched,
accompanied by the transcriptional regulator AlgP and a probable
iron sulfur protein. Competition experiments with an excess of EL
in presence of the probe confirmed these proteins as specific
targets of the natural product (Figure 2B, 2C, S6). Unlike the study
in E. faecalis, EF-Tu could not be detected as significantly
enriched. While the function of the iron sulfur protein is largely
unexplored, AlgP is known as a histone-like protein that may
directly or indirectly regulate alginate gene expression.[17]
Recombinant AlgP was also labeled by the probe suggesting
direct binding (Figure S7). To further understand AlgP function in
more detail, we investigated toxin expression in algP transposon
mutants. Two mutants (PW9843 and PW9844) were available in
the PAO1 transposon mutant library[18] and confirmed by PCR for
the correct insertion (Figure S8A). These insertions occur in a
region containing multiple KPPA amino acid repeats that are
known to facilitate DNA binding and play an important role for
AlgP function.[17a] Of note, while
a strong attenuation of
pyoverdine and a moderate reduction of pyocyanin levels were
observed for PW9844, no significant effect was obtained for
PW9843, suggesting that this latter insertion may not fully
compromise protein function (Figure S8B, S8C). In addition,
dose-response studies with ELP5 revealed a concentration-
dependent decrease of pyocyanin levels for PW9843 and, to a
lesser extent, also for PW9844 (Figure S8D). In case of
pyoverdine, PW9843 exhibited a similar behavior as wild type in
response to ELP5 while PW8944 showed almost undetectable
Figure 1: A) Structures of elegaphenone (EL) and derivatives. B) The
amount of extracellular pyocyanin in P. aeruginosa PAO1 upon treatment
with elegaphenone and derivatives in a dose-dependent manner. The data
is based on two biological experiments with technical triplicates. C) MIC
values of elegaphenone derivatives in different pathogenic bacterial
strains. Lack of growth inhibition is highlighted in grey. E. coli BW25113
ΔbamBΔtolC is a hyperpermeable E. coli strain.[13]
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expression level in presence or absence of the compound (Figure
S8E). Overall, these results indicate that the transposon insertion
within PW9844 may completely compromise AlgP function and
corresponding toxin expression, more prominently for pyoverdine,
compared to the PW9843 mutant. In order to clarify how this is
linked to the ELP5 phenotype we selected PW9844 for whole
proteome analysis and comparison with compound treated cells.
Thus, proteomes of P. aeruginosa PAO1 treated with ELP5 and
the PW9844 AlgP transposon mutant were analyzed via label free
LC-MS/MS, revealing a striking overall similarity of regulated
proteins (Figure S9, S10). Interestingly, pyoverdine biosynthesis
proteins as well as MexGHI-OpmD, a known transporter of
phenazines, were strongly downregulated in both samples.[19] By
contrast, no pronounced changes for pyocyanin biosynthesis
enzymes were observed, suggesting that the lower extracellular
levels of this toxin may stem from reduced MexGHI mediated
transport. Taken together, our proteomic profiles confirm a strong
link between compound treatment and impairment of AlgP
function. However, the differences in pyoverdine levels of
PW9844 and compound-treated wild type cells point towards a
more complex regulatory function which needs to be explored in
future studies.
S11, S12). Thus, combining EL/ELP5 with an antibiotic can
reduce antibiotic doses for the treatment of infection.
Figure 3: A) Number of viable P. aeruginosa PAO1 cultured in the
presence or absence of 50 µM EL or ELP5 for 5 h. Cultures were
inoculated with 5 x 105 P. aeruginosa bacteria. B) Intracellular viable P.
aeruginosa in human THP-1 macrophages. The number of viable
intracellular P. aeruginosa was determined after lysing the infected THP-1
macrophages. Concentration of EL derivatives and norfloxacin was 50 µM
and 1.5 µg/mL, respectively. Each bar represents the mean ± SD of the
data from three independent experiments. **, p < 0.05; ***, p < 0.005; one-
way ANOVA with Tukey’s multiple comparison test.
In conclusion, we use here the intrinsic photoreactivity of a
selected antibacterial natural product with a previously unknown
mode of action. In P. aeruginosa the compounds appear to
mediate inhibition of toxin production via AlgP binding and
downregulation of MexGHI, which is responsible for phenazine
transport providing a rational for the observed reduction of
extracellular pyocyanin levels. Furthermore, combination of EL
with sub-inhibitory concentrations of norfloxacin facilitated the
killing of intracellular P. aeruginosa in macrophages. Given the
paucity of compounds active against Gram-negative strains, the
low toxicity and unique target profile of EL represents an intriguing
starting point for further development.
Acknowledgement
The work was funded by grants of the Deutsche
Forschungsgemeinschaft (SI-1096/10-1 to S.A.S. and WI3285/6-
1 to D.N.W.), by the National Science Foundation CHE1755698
(W.M.W.) and the National Institute of General Medical Sciences
GM119426 (W.M.W.). A.R.C. was supported in part by the
CF@LANTA RDP Fellowship Program from the Cystic Fibrosis
Foundation (MCCART15R0). We thank Dr. Klaus Richter for his
kind support for C. elegans experiments, Prof. Markus Gerhard
for the supply of S. aureus clinical isolates, and Sabine Lehne for
excellent technical support.
Figure 2: A) Overall scheme of gel-free affinity-based protein profiling
(AfBPP) employing isotope labeling. Avid: Avidin; CC: click chemistry; DM:
stable isotope dimethyl labeling. B) and C) Volcano plots of gel-free AfBPP
experiment in P. aeruginosa PAO1 treated with 50 μM ELP5 vs. a 10-fold
excess of EL or DMSO, respectively (both soluble fraction). Blue dots
depict targets that are enriched by ELP5 (criteria: log2-fold enrichment ≥ 2
and -log10(p-value) ≥ 2.5) and competed by EL (criteria: log2-fold
enrichment ≥ 2 and -log10(p-value) ≥ 2.5). Green squares denote targets
that are enriched by ELP5 but not competed by EL while black dots denote
background proteins. For a full list of proteins please refer to supporting
information.
Keywords: Chemical Proteomics • Antibiotics • Pseudomonas
aeruginosa • Virulence • Natural product
Given the importance of P. aeruginosa as a pathogen, we
selected this bacterium for further evaluation of the EL treatment
potential.[20] Because EL/ELP5 did not possess growth-inhibitory
activity on P. aeruginosa (Figure 3A), we investigated if these
compounds could be used to potentiate the activity of sub-
inhibitory concentrations of antibiotics and thereby facilitate
natural bacterial elimination by the host immune system. In vitro
infection assays with human THP-1 macrophages demonstrated
that the presence of EL or ELP5 enhanced the capacity of sub-
inhibitory concentrations of the marketed antibiotic drug
norfloxacin to kill intracellular P. aeruginosa (Figure 3B).
Furthermore, neither EL nor ELP5 were cytotoxic for THP-1
macrophages and we did not observe adverse effects in
Caenorhabditis elegans up to a concentration of 300 μM (Figure
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Entry for the Table of Contents (Please choose one layout)
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COMMUNICATION
Natural product elegaphenone kills
Gram-positive E. faecalis and
inhibits virulence of Gram-negative
Weining Zhao, Ashley R. Cross,
Caillan Crowe-McAuliffe, Angela
Weigert-Munoz, Erika E. Csatary,
Amy E. Solinski, Joanna Krysiak,
Joanna B. Goldberg, Daniel N.
Wilson, Eva Medina, William M.
Wuest, Stephan A. Sieber*
P.
aeruginosa.
Its
structure
intrinsic
was
benzophenone
utilized to exploit its cellular targets
via chemical proteomics.
Page No. – Page No.
The natural product elegaphenone
potentiates antibiotic effects
against Pseudomonas aeruginosa
This article is protected by copyright. All rights reserved.