Antibacterial Flavonoids from Medicinal Plants Covalently Inactivate Type III Protein Secretion Substrates

Article abstract of DOI:10.1021/jacs.5b11575

Traditional Chinese Medicines (TCMs) have been historically used to treat bacterial infections. However, the molecules responsible for these anti-infective properties and their potential mechanisms of action have remained elusive. Using a high-throughput

Full text of DOI:10.1021/jacs.5b11575

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Article
Anti-bacterial flavonoids from medicinal plants
covalently inactivate type III protein secretion substrates
Lun K. Tsou, Maria Lara-Tejero, Jordan RoseFigura, Zhenrun J. Zhang, Yen-
Chih Wang, Jacob S. Yount, Matthew Lefebre, Paul D. Dossa, Junya Kato,
Fulan Guan, Wing Lam, Yung-Chi Cheng, Jorge E. Galan, and Howard C Hang
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b11575 • Publication Date (Web): 05 Feb 2016
Downloaded from http://pubs.acs.org on February 5, 2016
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Anti-bacterial flavonoids from medicinal plants covalently
inactivate type III protein secretion substrates
Lun K. Tsou†,#, María LaraꢀTejero‡, Jordan RoseFigura†, Zhenrun J. Zhang†, YenꢀChih Wang†,
Jacob S. Yount†, Matthew Lefebre‡, Paul D. Dossa†, Junya Kato‡, Fulan Guan§, Wing Lam§,
YungꢀChi Cheng§, Jorge E. Galán*,‡, Howard C. Hang*,†
†Laboratory of Chemical Biology and Microbial Pathogenesis, The Rockefeller University, New
York, NY 10065, USA
‡Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT
06536, USA
§Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520,
USA
#Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes,
Zhunan Town, Miaoli County 35053, Taiwan, R.O.C.
Correspondence and requests for materials should be addressed to J. E. G. (jorge.galan@yale.edu)
and H.C.H. (hhang@mail.rockefeller.edu)
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ABSTRACT
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Traditional Chinese Medicines (TCMs) have been historically used to treat bacterial infections.
However, the molecules responsible for these antiꢀinfective properties and their potential
mechanisms of action have remained elusive. Using a highꢀthroughput assay for type III
protein secretion in Salmonella enterica serovar Typhimurium, we discovered that several TCMs
can attenuate this key virulence pathway without affecting bacterial growth. Amongst the
active TCMs, we discovered that baicalein, a specific flavonoid from Scutellaria baicalensis,
targets S. Typhimurium pathogenicity islandꢀ1 (SPIꢀ1) type III secretion system (T3SS) effectors
and translocases to inhibit bacterial invasion of epithelial cells. Structurally related flavonoids
present in other TCMs, such as quercetin also inactivated the SPIꢀ1 T3SS and attenuated S.
Typhimurium invasion. Our results demonstrate that specific plant metabolites from TCMs can
directly interfere with key bacterial virulence pathways and reveals a previously unappreciated
mechanism of action for antiꢀinfective medicinal plants.
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INTRODUCTION
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Medicinal plants have provided fertile ground for modern drug discovery1. Nonetheless,
these plantꢀderived extracts remain a poorly characterized reservoir of antiꢀinfective agents2,3.
For example, a variety of TCMs have been reported to have protective effects against bacterial
and viral infections, but the specific molecules responsible for these antimicrobial activities and
their mechanism(s) of action are largely unknown4. The molecules and mechanisms of TCMs
used to treat infectious gastroenteritis have been particularly elusive, since plant extracts and
metabolites attenuate infection but do not exhibit significant bactericidal activity5. These
observations suggest that specific compounds from TCMs may target virulence factors rather
than the growth of enteric pathogens.
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To explore the antiꢀinfective activity of TCMs on enteric pathogens, we focused on T3SSs as
these protein translocation needleꢀlike complexes are key virulence mechanisms for
Gramꢀnegative bacterial pathogens responsible for gastroenteritis. T3SSs are multiprotein
machines evolved to inject bacterial effector proteins into host cells to modulate key pathways
for infection. Salmonella enterica serovar Typhimurium (S. Typhimurium) encodes two of
these protein secretion systems within its pathogenicity islands to mediate bacterial entry into
nonꢀphagocytic cells as well as intracellular survival and replication6. Combined, these T3SSs
deliver up to 50 bacterial effector proteins that can target a diverse array of cellular activities
such as cytoskeleton dynamics, membrane trafficking and transcription6. Given the essential
and conserved roles of T3SSs amongst many enteric bacterial pathogens7 and the
uncharacterized antiꢀinfective activity of medicinal plants4, we screened a collection of TCMs
and discovered that specific flavonoids from TCMs, such as baicalein and quercetin, can
covalently label protein substrates of the SPIꢀ1 T3SS and inhibit S. Typhimurium invasion of
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host cells. Our results suggest that specific flavonoids from TCMs inhibit bacterial virulence
rather than targeting growth and reveals a key mechanism by which select TCMs can attenuate
bacterial pathogenesis.
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RESULTS
High-throughput screen of TCMs reveals specific flavonoid inhibitors of S. Typhimurium
type III protein secretion. To evaluate the activity of TCMs on type III protein secretion in S.
Typhimurium, we screened 120 TCMs with previously suggested antiꢀinfective activity4 using a
highꢀthroughput fluorescence previously developed by our laboratory (Figure 1A)8. The type III
protein secretion assay employs carboxypeptidase G2 (CPG2) as an enzyme reporter that it is
fused to the Cꢀterminus of a known S. Typhimurium T3SS protein effector (SopE2).
SopE2ꢀCPG2ꢀHA is secreted and specifically reports on type III protein secretion through
cleavage of fluorogenic substrates (GluꢀCyFur), which provides a sensitive method for
monitoring the inhibitory activity of small molecules targeted at T3SSs (Figure 1A)8. Our
screen of antiꢀinfective TCMs at 5 mg/mL revealed 30 medicinal plant extracts with significant S.
Typhimurium pathogenicity islandꢀ1 (SPIꢀ1) T3SS inhibitory activity (Figure 1B and Table S1).
None of these TCMs had significant inhibitory activity on S. Typhimurium growth in culture
medium (Table S1). Of the most active TCMs (Figure S1A), we focused on Scutellaria
baicalensis (Huángqín) extracts (Figure 1B) as this medicinal plant is a key component of
“Scutellaria and Coptis Decoction”, an herbal remedy that has been historically used to treat
infectious gastroenteritis4,9. In accordance to the reported isolation of flavonoids from the
medicinal plant Scutellaria baicalensis10, our analysis of the major metabolites from Scutellaria
baicalensis and other active TCMs demonstrated that only specific flavonoids could inhibit the
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SPIꢀ1 T3SS reporter levels in the S. Typhimurium culture supernatant (Figure S1B). Notably,
baicalein (Figure 1C) from Scutellaria baicalensis reduced the levels of the SPIꢀ1 T3SS reporter
(Figure 1D) as well as endogenously expressed SPIꢀ1 T3SS substrates (SipA, SipB, SopB, SipC
and SipD) (Figure 1E and Table S2) in S. Typhimurium culture supernatant in doseꢀdependent
manner, without affecting the bacterial growth (Figure S1C) or the recombinant CPG2 reporter
activity (Figure S1D). Although baicaleinꢀtreated S. Typhimurium showed reduced levels of
secreted SPIꢀ1 T3SS reporter, the expression level of the reporter was unaffected (Figure 1C). In
contrast, chrysin and baicalin, a deoxygenated and a glycosylated derivative, found in Scutellaria
baicalensis extracts were both inactive (Figure S1A). These results demonstrated that a specific
flavonoid from the TCM Scutellaria baicalensis, baicalein, does not interfere with bacterial
growth but can target secreted SPIꢀ1 T3SS substrates.
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To understand structural features and scope of flavonoid activity on SPIꢀ1 T3SS, we
evaluated additional baicalein derivatives and other naturally occurring flavonoids. The analysis
of baicalein derivatives showed that the 1,2ꢀcatechol moiety and flavone scaffold are important
for full activity, since methylated or truncated forms of baicalein were inactive (Figure S2). Our
analysis of other naturally occurring flavonoid subꢀclasses at 25 ꢁM revealed that only flavones
and flavonols with 1,2ꢀcatechol functionality exhibited inhibitory activity greater than 50 percent
(Figure 2A). IC50 measurements of the active flavonoids showed that quercetin had similar
activity to that of baicalein (Figure 2B). Interestingly, quercetin is reported in most of the active
TCMs we evaluated (Figure S3) and is also a major component of many human dietary
supplements. Like baicalein, quercetin reduced the levels of SP1ꢀT3SS reporter and
endogenously expressed bacterial effectors in S. Typhimurium media (Figures S4A, B), but had
no effect on bacterial growth (Figure S4C). Based on the analysis from SopE2ꢀCPG2ꢀHA
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fluorescence reporter assay and the secretion of endogenous SPIꢀ1 T3SS substrates, the active
flavonoids have comparable or better activity than the previously reported synthetic T3SS
inhibitors, INPꢀ000711 and TTS2912 (Figures S4D, E). These results demonstrate that in
addition to baicalein, other naturally occurring flavonoids with specific 1,2ꢀcatechol motifs, such
as quercetin, can also antagonize SPIꢀ1 T3SS substrates.
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Flavonoids from medicinal plants inhibit S. Typhimurium invasion of epithelial cells. We
then tested the ability of the active flavonoids to inhibit S. Typhimurium invasion of cultured
epithelial cells, a phenotype strictly dependent on the activity of the SPIꢀ1 T3SS13. Bacteria
grown in the presence of baicalein or quercetin showed significant reduction in invasion of
cultured HeLa cells (Figure 3A). In contrast, bacteria grown in the presence of chrysin, an
inactive flavonoid (Figure S2), were able to enter cultured cells comparable to untreated bacteria
(Figure 3A). The inhibitory effect of quercetin at 100 ꢁM was almost complete since the
internalization levels of quercetinꢀtreated bacteria phenocopied the levels of a nonꢀinvasive S.
Typhimurium invA mutant, which lacks an essential component of the SPIꢀ1 T3SS14 (Figures
3B). The inhibition of S. Typhimurium invasion could also be readily observed by
immunofluorescence analysis of intracellular bacteria (Figure 3C). Preincubation of the bacteria
with active flavonoids was important, as treatment of HeLa cells with quercetin prior to or during
bacterial infection alone did not significantly affect S. Typhimurium invasion (Figure 3B). These
results demonstrate that active flavonoids, baicalein and quercetin, do not affect bacterial growth
and but can inhibit the SPIꢀ1 T3SSꢀdependent S. Typhimurium invasion of host cells.
Flavonoids from medicinal plants target S. Typhimurium type III secretion substrates. The
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observation that inhibition of bacterial invasion by quercetin required preincubation with S.
Typhimurium suggested that these flavonoids may block the synthesis or assembly of the SPIꢀ1
T3SS needle complex rather than protein secretion itself. Indeed, before the bacteria encounters
mammalian cells, the SPIꢀ1 T3SS is deployed and ready for protein delivery but contact with
mammalian cells is required for its full activation15. However, we found that S. Typhimurium
grown in the presence of baicalein or quercetin had wild type levels of SPIꢀ1 T3SS effectors
(SipB, SptP, SipC, SipA, SipD) needle components (InvG, PrgH, PrgK, PrgI) and fully
assembled needle complexes (Figures S5A, B). Since the assembly of the needle complex
requires type III protein secretion function, these results suggested that baicalein and quercetin
must inhibit S. Typhimurium invasion by a mechanism that did not involve complete block of
protein secretion complex. Consistent with this hypothesis, we observed that growth of S.
Typhimurium in the presence of quercetin led to reduced levels of some but not all SPIꢀ1 T3SS
secreted proteins in culture supernatants (Figure S5C). For example, the levels of secreted SipD
were unaffected by addition of quercetin (Figure S5C). Incubation of baicalein or quercetin with
S. Typhimurium resulted in the formation of a brown precipitate in the growth media that was
not observed with inactive controls (Figure S5D). Western blot analyses of this precipitate
material revealed the presence of wild type levels of SPIꢀ1 T3SS secreted proteins (Figure S5C).
These results indicated that active flavonoids do not inhibit type III protein secretion directly, but
may affect the folding, stability or activity of endogenous SPIꢀ1 T3SS protein substrates as well
as effectorꢀenzyme reporters such as SopE2ꢀCPG2.
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Although the active flavonoids do not appear to inhibit type III secretion per se, baicalein
and quercetin exhibited a strong inhibitory effect on S. Typhimurium invasion (Figure 3), a
phenotype strictly dependent on SPIꢀ1 T3SS effector function. This observation combined with
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the fact that the active flavonoids specifically affected the behavior of type III secreted proteins
indicated that these compounds might directly target protein effectors or the protein translocases,
which are type III secreted proteins required for the passage of effectors through the eukaryotic
cell membrane16. Based on the reactive 1,2ꢀcatechol motifs present in the active flavonoids
(Figure 2), we hypothesized that these compounds may covalently modify SPIꢀ1 T3SS
substrates, which could result in their functional inactivation.
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To test this hypothesis, we synthesized alkyneꢀflavonoid probes for activityꢀbased protein
profiling studies using click chemistry17,18 (Figures 4A and S6). As the phenolic hydroxyl groups
appeared to be important for baicalein activity (Figure S2), an alkyne tag was installed on the
Cꢀring of baicalein (alkꢀbaicalein) which retained activity compared to inactive
alkꢀMe3ꢀbaicalein probe (Figure 4B). These alkyneꢀflavonoid probes were then incubated with S.
Typhimurium under the same conditions of T3SS inhibition and assayed for protein labeling in
the cell lysate. Reaction of the S. Typhimurium cell lysates with azideꢀrhodamine (azꢀrho) dye
using click chemistry showed that alkꢀbaicalein could covalently modify bacterial proteins
compared to the inactive probes (Figures 4C and S7A). To determine whether quercetin could
also covalently modify S. Typhimurium proteome in a similar manner to baicalein, we performed
competition assays with the alkꢀbaicalein probe. Addition of either baicalein or quercetin, but not
inactive chrysin, reduced the labeling of alkꢀbaicalein in a doseꢀdependent manner (Figure S7B).
To identify the protein targets of the alkꢀbaicalein probe, we performed chemical proteomics
using a cleavable azideꢀfunctionalized biotinylated affinity tag followed by gelꢀbased protein
identification19. Compared to the control samples, alkꢀbaicalein preferentially labeled 16 S.
Typhimurium proteins with more than 5ꢀfold enrichment, including SPIꢀ1 T3SS protein
translocases (SipC and SipB) and effectors (SipA, SopB and SopE2) as well as the secreted
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needleꢀassembly regulatory protein InvJ (Figure 5A and Table S3). To validate the covalent
labeling of these SPIꢀ1 T3SS substrates, we focused on the most prominent alkꢀbaicalein labeled
protein SipA, an injected S. Typhimurium protein effector involved actin remodeling and
membrane ruffling required for bacterial invasion of epithelial cells20. Indeed, expression and
affinity enrichment of Cꢀterminal HAꢀtagged SipA (SipAꢀHA) in S. Typhimurium (Table S4)
confirmed that alkꢀbaicalein covalently labels this SPIꢀ1 T3SS substrate, as judged by inꢀgel
fluorescence detection (Figure 5B). Alkꢀbaicalein also labeled SipAꢀHA in SPIꢀ1 T3SS mutant
strain (invA) (Figure 5B), demonstrating flavonoid labeling of SPIꢀ1 T3SS protein substrates
does not require protein secretion and can occur within S. Typhimurium. One conserved feature
of SPIꢀ1 T3SS substrates is a hydrophobic Nꢀterminal domain that interacts with chaperones
involved in protein secretion21. We hypothesized that the active flavonoids may bind this
conserved hydrophobic motif within SipA and react with nucleophilic amino acids, as previously
suggested for αꢀsynuclein22. Analysis of an Nꢀterminal SipAꢀHA construct (1ꢀ270) expressed
in S. Typhimurium demonstrated that a truncated mutant contain this conserved hydrophobic
motif is sufficient for covalent flavonoid labeling (Figure 5C). Although alkꢀbaicalein also
covalently labeled other S. Typhimurium proteins (Figure 5A and Table S3), these proteins are
not associated with SPIꢀ1 T3SSꢀdependent invasion of host cells and are unlikely to be
functionally relevant. These results demonstrate that active flavonoids can covalently modify a
conserved chaperoneꢀbinding motif S. Typhimurium SPIꢀ1 T3SS substrates and may inactivate
these secreted effectors and translocases to prevent bacterial invasion.
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To independently validate the covalent interaction of specific flavonoids with SPIꢀ1 T3SS
substrates, we evaluated the binding of these compounds with SipAꢀglutathioneꢀSꢀtransferase
(GST) fusions in bacteria and in vitro using the intrinsic UVꢀabsorption properties of flavonoids
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at 370 nm (Figure 6). The SipAꢀGST constructs were also expressed in E. coli to evaluate
whether flavonoid covalent modification was specific to incubation with S. Typhimurium (Figure
6A). Indeed, expression of SipAꢀGST in E. coli incubated with baicalein or quercetin yielded
altered UVꢀabsorbance profiles from 350 – 400 nm of purified SipAꢀGST compared to untreated
or inactive flavonoid (chrysin)ꢀtreated samples (Figures 6B and S8). We then evaluated different
SipAꢀGST constructs to determine the domain of flavonoid labeling in E. coli, which
demonstrated that active flavonoids (quercetin) primarily associates with the Nꢀterminal
chaperone binding domain of SipA, independent of signal sequence (residues 1ꢀ27) and did not
occur within the Cꢀterminal actin binding domain of SipA (Figure 6C), which are consistent with
our analysis of alkꢀbaicalein labeling of SipAꢀHA constructs in S. Typhimurium (Figure 5).
To monitor conformational changes due to flavonoid binding interactions, we performed
circular dichroism (CD) experiments on SipAꢀ1ꢀ271ꢀGST constructs from E. coli incubated with
quercetin or baicalein. In agreement with the published crystal structure of SipA21,23, the CD
results showed that SipAꢀ1ꢀ271ꢀGST retained an αꢀhelical character with negative bands at 222
nm and 208 nm and a positive band at 193 nm23 (Figure 6D). Treatment with baicalein or
quercetin induced significant CD spectra changes at the characteristic αꢀhelical bands, which
suggests association with the active flavonoids altered the secondary structure of SipA
Nꢀterminal domain (Figure 6D). Incubation of purified SipA Nꢀterminal domain (1ꢀ271)ꢀGST
fusion with quercetin in vitro did not enhance UVꢀabsorbance at 370 nm or alter the CD spectra
(Figure 6D). These results suggest that the active flavonoids baicalein and quercetin bind to the
Nꢀterminal domain (1ꢀ271) of SipA inside bacteria, but not in vitro and affect the secondary
structure of this chaperoneꢀbinding domain in SPIꢀ1 T3SS substrates.
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DISCUSSION
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TCMs have been used to treat infections by enteric bacterial pathogens, but the molecular
mechanisms by which these medicinal plant extracts attenuate bacterial pathogenesis have been
unclear. By focusing on type III protein secretion responsible for bacterial virulence, we have
discovered specific compounds within TCM extracts and their mechanism of action that provides
an explanation for some of the previously proposed antiꢀinfective properties of these ancient
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medicines4,24
.
While flavonoids exhibit diverse pharmacological activities in mammalian
cells25, are potential antiviral agents26 and have minimal activity on bacteria growth as
antibiotics27,28, we demonstrated that these plantꢀderived metabolites can attenuate infection by
targeting virulence factors such as protein substrates delivered by T3SSs. Our subsequent
chemical proteomic studies demonstrated that the most active flavonoids, contained reactive 1,2
catechol motifs that covalently modify secreted bacterial effectors and translocases required
bacterial invasion of host cells. The active flavonoid covalent modification of conserved
hydrophobic domains of T3SS substrates and may interfere with the folding, secretion or activity
of these key virulence factors. Our observations may explain some of the previously reported
antiꢀinfective properties of TCM extracts that produce active flavonoids4,24. As activated
phenolic chemotypes present in baicalein and quercetin are also found in previous reported T3SS
inhibitors (INPꢀ000711 and TTS2912) and other active plant metabolites from TCMs associated
with protein aggregation29, the covalent modification of proteins described here may provide a
general mechanism for inactivating proteins. Since many plant bacterial pathogens use type III
secretion systems to cause disease and sense flavonoids30, it is possible that some of these
compounds may have evolved as a consequence hostꢀpathogen interactions. Moreover,
flavonoids have also been reported to target other antiꢀinfective properties such as expression of
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virulence genes31,32, toxin activity33ꢀ35, quorum sensing36ꢀ39 and biofilm formation40,41 in bacterial
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pathogens as well as attenuate destructive host inflammation42ꢀ44
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Given the broad
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pharmacological activities of flavonoids, these plant metabolites may affect microbial virulence
mechanisms, including type III protein secretion substrates shown here, in addition to host
pathways for their antiꢀinfective properties in vivo. With the resurgence in covalent drugs45, the
development of more potent and bioavailable flavonoid antagonists of T3SSs should yield new
antiꢀinfective agents to selectively combat bacterial pathogens.
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EXPERIMENTAL SECTION
High-throughput type III secretion assay for screening of extracts and compounds. S.
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Typhimurium containing SopE2ꢀCPG2ꢀHA plasmid was grown overnight at 37 C in LB
containing 50 µg/mL ampicillin and was diluted by 1:30. In each well of the sterile Nunc 96
deepꢀwell plates (2 mL), 400 µL of the diluted culture was then grown for 4 hours in the
presence of compounds or TCMs at indicated concentrations with DMSO or H2O as controls. All
added volumes of inhibitors or DMSO were <1% of the total volume in each experiment. After 4
hours, the OD600 was measured to confirm comparable bacterial growth. 250 µL of each sample
was transferred into a 96 well plate and bacterial cells were spun down at 5,250 g for 6 minutes.
20 ꢁL of supernatant from each sample was then transferred into a 384 well costar blackꢀbottom
plate. 80 ꢁL of CPG2 buffer (50 mM Tris, 0.1 mM ZnCl2, pH 7.4) containing 0.1% Brijꢀ97 to aid
in solubility of CyFur8 and 10 ꢁM GluꢀCyFur was added, and fluorescence readings (λex= 563
nm, λem= 610 nm with no cutoff) were started immediately and monitored as kinetic readings for
2 hours. Absorbance and fluorescence data were collected on SpectraMax M2 multiꢀdetection
reader (Molecular Devices). The percent inhibition by chemical inhibitors or TCMs was
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determined by calculating the rates of change in fluorescence of the CPG2ꢀexpressing strain in
the presence of compound in comparison to the DMSO or H2O control. IC50 values of the
inhibitors were then determined using KaleigaGraph version 4.1 and fitting to the exponential
decay equation: y = m1 + m2*exp(ꢀm3*x).
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Plasmids, bacterial strains, and growth conditions. S. Typhimurium strain expressing
SopE2ꢀCPG2ꢀHA fusion protein8 and the S. Typhimurium strains SB90546, SB30046, IR71547 and
the T3SSꢀdefective invA mutant14 have been previously described. All LB media used were made
from BD DifcoTM LB (LuriaꢀBertani) Broth Miller, which contains 10 g/L NaCl.
Profiling of secreted proteins in bacterial culture supernatants. 1:30 dilutions of overnight
cultures of S. Typhimurium were grown in LB for 4 h in the presence of compounds at indicated
concentrations. Secreted proteins from 1 mL culture were precipitated overnight with a final
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concentration of 10% TCA at 4 C. Secreted effectors were pelleted at 14,000 rpm for 30
minutes and washed with 250 µL iceꢀchilled acetone. This procedure was repeated 2 times and
the precipitates were allowed to dry for 15 minutes before the addition of 4% SDS. Secreted
proteins were then separated by a 4ꢀ20% SDS PAGE and stained with coomassie blue.
Cell culture, bacterial infection, flow cytometry and immunofluorescence. HeLa cells were
cultured in 12ꢀwell plates in DMEM supplemented with 10% FBS (Gemini BioꢀProducts) at 37
oC in a humidified incubator with an atmosphere of 5% CO2. S. Typhimurium strains IR715,
SB300, and the invA deletion mutant were grown overnight in LB at 37 oC in a shaker set at 250
rpm. Bacteria were diluted 1/33 in LB containing DMSO or inhibitors, and were grown for an
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additional 4 hours. Optical density readings were used to determine an MOI of 10 and bacteria
was added to cells in a total volume of 500 uL DMEM/10% FBS containing inhibitors or DMSO
as a control. Plates containing cells and bacteria were then centrifuged at room temperature for
5 min at 1000 x g. Plates were then transferred back to the 37 oC incubator for 30 min to allow
infection to proceed. Cells were then washed three times with room temperature PBS
containing 100 ug/mL gentamycin and were then incubated with DMEM/10% FBS containing
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100 ug/mL gentamycin and inhibitors or DMSO as a control at 37 C for an additional 30 min.
Cells were then washed an additional three times with room temperature PBS containing 100
ug/mL gentamycin to remove any remaining extracellular bacteria. Cells were trypsinized and
then fixed with ice cold 3.7% paraformaldehyde in PBS for 10 minutes followed by
permeabilization with ice cold 0.2% saponin in PBS for 10 min. Cells were then blocked with
ice cold 2% FBS in PBS for 10 min. All antibody stainings and washes were performed with
iceꢀcold 0.2% saponin in PBS. Cells were stained for bacterial antigens with antiꢀSalmonella
rabbit serum (Biodesign International, 1/250 dilution) for 1 h and washed three times. Goat
antiꢀrabbit secondary antibody conjugated to AlexaFluor 488 (Invitrogen) was used at a 1/1000
dilution for 30 min. Cells were then washed 3 times. Flow cytometry was performed using a
Becton Dickinson LSRII machine and FlowJo software was used for analysis. 30,000 cells
were analyzed for each sample. Cellular debris was eliminated from analysis of all samples
based on forward and side scatter measurements and never constituted more than 10% of
collected events. Gates drawn to illustrate infected cells were based on a value close to 0% for
nonꢀinfected samples. For the CFUꢀbased infection studies, a protocol was used based on a
previous report13. For microscopy, HeLa cells were grown on glass coverslips. Infections and
staining were performed using the same protocol as described above for flow cytometry. For the
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imaging the extracellular S. Typhimurium, the scrappedꢀharvest HeLa cells were not
permeablized and there is no gentamycin added. Cells were treated with TOPROꢀ3 (1/1000
dilution, Invitrogen) as a final step to stain nuclei and were mounted using Prolong Gold
Antifade Reagent (Invitrogen).
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Analysis of endogenously expressed T3SS needle components and substrates. S.
Typhimurium supernatants and cell lysates were prepared as previously described and analyzed
by western blot by pooling specific antibodies for T3SS substrates48. Needle complexes were
purified from S. Typhimurium strain SB905 as previously described49, and subsequently
analyzed by western blot using a previously described antibody specific for the needle complex
components PrgH, PrgK, and InvG50. Purified needle complexes were applied to glow
discharged carbon coated Cuꢀgrids and stained with 2% phosphotungstic acid, pH 7.0 and
imaged by electron microscopy. Images were acquired using a Tecnai Biotwin TEM (FEI
Company, Hillsboro) at 80 kV using Morada Soft Imaging system and 6 M pixel CCD camera
(Olympus, Munster, Germany).
Coomassie staining of secreted proteins. 1:30 dilutions of overnight cultures of S. typhimurium
were grown in LB for 4 h in the absence or presence of compounds at indicated concentrations.
Secreted proteins from 1 mL culture were precipitated overnight with a final concentration of
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10% TCA at 4 C. Secreted effectors were pelleted at 14,000 rpm for 30 minutes and washed
with 250 µL iceꢀchilled acetone. This procedure was repeated 2 times and the precipitates were
allowed to dry for 15 minutes before the addition of Laemmli buffer. Secreted proteins were
then separated by 4ꢀ20% SDS-PAGE and stained with SimplyBlue™ SafeStain (Invitrogen).
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Gels were imaged with ChemiDoc™ XRS+ System and Image Lab™ Software (BioꢀRad).
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Preparation of bacterial total cell lysates. S. Typhimurium strain 14028 WT and ∆invA mutant
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carrying pBADꢀSipAꢀHA or pBADꢀSipA(1ꢀ270)ꢀHA were cultured in the absence or presence of
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0.2% arabinose and labeled with alkꢀbaicalein or DMSO control in 4 mL LB at 37 C with 220
rpm shaking for 4 hr. S. Typhimurium cells were pelleted at 15000 g for 1 min, and pellets were
lysed with 200 ꢁL CelLytic B 2X Cell Lysis Reagent (Sigma) containing 1X EDTAꢀfree protease
inhibitor cocktail (Roche), 0.5 mg/ml lysozyme (in ddH2O) (Sigma), and 0.2 ꢁL S16 benzonase
(Sigma). After reꢀsuspension, pellets were sonicated for 10 sec for 3 times, then were incubated
on ice for 30 min. Cell lysates were centrifuged at 15000 g for 1 min to remove cell debris and
supernatants were collected. Protein concentration was estimated by BCA assay with BCA
Protein Assay Kit (Thermo).
Chemical proteomic analysis with alkyne-flavonoid probes. S. Typhimurium strain (IR715)
were labeled with alkꢀbaicalein, alkꢀMe3ꢀbaicalein, alkꢀoroxylin A or DMSO control in LB at 37
oC for 4 hr. S. Typhimurium pellets were lysed with 0.1% SDS buffer (50 mM triethanolamine
(TEA), 150 mM NaCl, 0.1% SDS, 1X EDTAꢀfree protease inhibitor cocktail (Roche), 5 mM
PMSF from a 250 mM stock in EtOH, 0.1 ꢁL S16 benzonase (Sigma)) by sonicating pellet for 10
sec, then incubating on ice for 10 min. After which 5 ꢁL of 10 µg/mL lysozyme (in ddH2O)
(Sigma) was added to each sample and incubated on ice for 30 min. SDS buffer (12% w/v in
50 mM TEA, 150 mM NaCl) was then added to bring final SDS concentration to 4%. Cells were
sonicated once more for 5 sec. Cell lysates were centrifuged at 1000 g for 5 min to remove cell
debris and supernatant was collected. From these alkyneꢀflavonoid or DMSOꢀtreated samples,
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50 ꢁg of total cell lysates was suspended in 44.5 ꢁL of 4% SDS buffer. CuAAC reactants 1 ꢁL
of 5 mM azꢀrho (100 ꢁM final concentration), 1 ꢁL of freshly prepared 50 mM
tris(2ꢀcarboxyethyl)phosphine hydrochloride (TCEP) (final concentration 1 mM), 2.5 ꢁL 2 mM
tris[(1ꢀbenzylꢀ1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methyl]amine (TBTA) (final concentration 100 ꢁM) and 1
ꢁL 50 mM CuSO4·5H2O (final concentration 1 mM), were then added for a final volume of 50
ꢁL. CuAAC reactions were allowed to proceed for 1 h at room temperature and a
chloroformꢀmethanol precipitation of the proteins was performed before SDSꢀPAGE. Inꢀgel
fluorescence scanning was performed using a Typhoon 9400 imager (Amersham Biosciences).
For proteomic experiments, cell lysates (5ꢀ10 mg) were brought up to a volume of 8.9 mL
with a final SDS concentration of 2%. To this, 200 ꢁL of 5 mM azidoꢀazoꢀbiotin19; 200 ꢁL of 50
mM TCEP; 500 ꢁL of 2 mM TBTA; and 200 ꢁL of 50 mM CuSO4 were added, and samples
were incubated for 1.5 h at room temperature. After incubation, proteins were precipitated by
chloroform/methanol precipitation as described: 4x MeOH, 1.5X chloroform, and 3X ddH2O
were added to reaction, and samples were vortexed vigorously. Samples were centrifuged at
5200 g for 30 min, and the aqueous layer was removed. Samples were washed two times with 4X
methanol, by vortexing vigorously, centrifuging at 5200 g for 30 min, then decanting off the
supernatant each time. Protein pellets were airꢀdried, then resuspended in 1 mL 4% SDS buffer
and 20 ꢁL 0.5 M EDTA pH 8. Protein concentration was estimated by BCA assay.
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Cell lysates (1ꢀ5 mg) after CuAAC reaction were added to PBSꢀwashed highꢀcapacity
streptavidin agarose beads (Thermo scientific) (beads in a 50% Brijꢀ97 buffer slurry, 100 ꢁL per
5 mg protein). Beads were incubated with cell lysates for 1 h at room temperature with rocking.
Beads were then washed 2X with 10 mL of 0.1% SDS in PBS, 3X with 10 mL PBS, 2X with 10
mL 50 mM ABC, then incubated for 30 min with freshly made 8 M urea solution (500 ꢁL 8M
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urea, 25 ꢁL 200 mM TCEP, 25 ꢁL 400 mM iodoacetamide). Beads were washed 2X with 10 mL
50 mM ABC, then transferred to a dolphin tube with 1 mL ABC. Proteins were eluted off beads
in two steps with a total of 400 ꢁL freshly prepared elution buffer (0.5% SDS, 25 mM Na2S2O4
in 50 mM ABC) by rocking for 1 h at room temperature, then for 30 m. Eluant was concentrated
using a Microcon YMꢀ10 centrifugal filter (Millipore) as per manufacturers protocol. Microcon
filters were rinsed twice with 50 ꢁL 1% SDS/75 mM BME to collect all sample, and samples
were dried by vacuum at room temperature. Samples were resuspended in 25 ꢁL LDS buffer (1X
LDS (Invitrogen), 5% BME), boiled for 5 min at 95 oC, spun at 1000 g for 1 min, then separated
by SDSꢀPAGE. Gels were stained with Coomassie blue and destained overnight (40% methanol,
10% acetic acid, 50% water). Each gel lane was cut into 8 slices. These slices were washed once
in 50 mM ABC, twice in 1:1 50 mM ABC to acetonitrile (ACN), and once in ACN. Slices were
dried by vacuum. 2 ꢁg of trypsin in 50 mM ABC was added to each gel slice, and slices were
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incubated overnight at 37 C. Proteins released from the gel were collected, and gel slices were
washed in 1:1 50 mM ABC:ACN + 0.1% TFA twice, collecting the supernatant each time. The
pooled supernatant was dried by vacuum then resuspended in 25 ꢁL 0.1% TFA in water for
analysis by LCMS/MS. Proteomics data analysis was performed at Rockefeller University
Proteomics Research Center using Mascot through Proteome Discoverer (Thermo Proteome
Discoverer version 1.3.0.339.).
Construction of pBAD-SipA-HA and pBAD-SipA(1-270)-HA. SipAꢀHA were amplified from
genome of S. Typhimurium strain 14028 WT with primers ZZ91 and ZZ92. PCR product was
digested with EcoRI and XbaI and ligated to pBAD vector to yield pBADꢀSipAꢀHA.
pBADꢀSipA(1ꢀ270)ꢀHA were made through siteꢀdirected mutagenesis on pBADꢀSipAꢀHA with
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primers ZZ115 and ZZ116, and QuikChange II XL SiteꢀDirected Mutagenesis Kit (Agilent)
according to manufacturer’s manual.
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In-gel fluorescence analysis of SipA-HA and SipA(1-270)-HA. From the alkꢀbaicalein or
DMSOꢀtreated total cell lysates prepared as described above, 250 ꢁg of each total cell lysates
were incubated with 20 ꢁL PBSꢀTꢀwashed EZview™ Red AntiꢀHA Affinity Gel (Sigma) at 4 oC
for 1 hour with endꢀtoꢀend rotation. Samples were washed with 200 ꢁL PBSꢀT for 3 times, and
36 ꢁL of PBS was added to each sample. 4 ꢁL of click chemistry reagents were added to each
sample as a master mix (azꢀrho: 0.1 mM, 10 mM stock solution in DMSO;
tris(2ꢀcarboxyethyl)phosphine hydrochloride (TCEP): 1 mM, 50 mM freshly prepared stock
solution in ddH2O; tris[(1ꢀbenzylꢀ 1Hꢀ1,2,3ꢀtriazolꢀ4ꢀyl)methyl]amine (TBTA): (0.1 mM, 2 mM
stock in 4:1 tꢀbutanol: DMSO); CuSO4 (1 mM, 50 mM freshly prepared stock in ddH2O).
Samples were mixed well and incubated at room temperature for 1 h. After incubation, samples
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were washed with 200 ꢁL PBSꢀT for 3 times, and were boiled with Laemmli buffer 95 C for 5
min before being loaded onto a 4ꢀ20% gel (BioꢀRad) for SDSꢀPAGE. Inꢀgel fluorescence
scanning was performed using a Typhoon 9400 imager (Amersham Biosciences. AntiꢀHA
western blotting was performed in parallel to confirm proper expression of HAꢀtagged proteins,
with monoclonal antiꢀHAꢀHRP antibody HAꢀ7 (Sigma) at 1:5000 dilution.
Analysis of SipA-GST interaction with flavonoids. The SipAꢀglutathione Sꢀtransferase (GST)
fusion constructs21,51 were expressed in E. coli BL21 (DE3) containing and induced at OD 0.8
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with 1 mM IPTG for 16 hrs at 20 C. Flavonoids (100 ꢁM) were added 1 hour before IPTG
induction. Cells collected via centrifugation and lysed in Wash Buffer (1XPBS, 1 mM DTT, 1
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mM EDTA) with 0.1% TritonꢀX and a protease inhibitor cocktail (Roche). The clarified cell
lysates were placed on preꢀequilibrated 1 mL Glutathione columns (Qiagen) and washed with 10
column volumes of Wash Buffer. Protein was eluted with Elution Buffer (50 mM Tris pH 8.0, 0.4
M NaCl, 50 mM reduced glutathione, 0.1% Triton Xꢀ100, 1 mM DTT) and 1 mL fractions were
collected. All fractions were tested for protein purity via SDSꢀPAGE gels. Fractions containing
SipA were pooled, concentrated, and buffer exchanged into 1XPBS. The SipAꢀGST
constructed purified in this way were SipA 1ꢀ270, SipA 27ꢀ270, and SipA 497ꢀ669. The protein
samples were then characterized with UVꢀvisible (Molecular Devices SpectraMax M2) and
circular dichroism (Aviv model 202 CD spectrometer) spectroscopy to determine if any
structural changes occurred in the flavonoid bound proteins. Experiments were run at 0.25
mg/mL with proteins purified from cultures treated either with quercetin, baicalein, or chrysin. In
addition, SipA from untreated cultures was tested with the addition of 25 ꢁM and 50 ꢁM
exogenous quercetin. The spectra window was set between 180 and 275 nm. The CD spectra
of SipA were shown to be mostly αꢀhelical, which agrees with previous crystallography studies.51
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ASSOCIATED CONTENT
Supporting information. Supporting information is available free of charge on ACS
Publications website at DOI: TBD. Details on screening and characterization of TCMs and
flavonoids as well as synthesis, characterization and proteomic analysis of flavonoid probes are
included.
AUTHOR INFORMATION
Corresponding Authors
*Eꢀmail jorge.galan@yale.edu (J.E.G.) and hhang@mail.rockefeller.edu (H.C.H.).
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGEMENTS
We thank The Rockefeller Proteomics Resource Center for mass spectrometry analysis. We thank
the Stebbins laboratory (Rockefeller University) for SipAꢀGST plasmids. P.D. was graduate
student in Weill CornellꢀRockfellerꢀSloan Kettering TriꢀInstitutional Training Program in
Chemical Biology. J.R. was supported NIH F32 postdoctoral fellowship AI096890. YꢀC.C. is a
fellow of the National Foundation for Cancer Research. This work was supported by grants to
YꢀC.C. (NCI PO1CA154295), J.E.G. (NCCAM R01AT007671 and NIAID AI30492) and H.C.H
(NCCAM R01AT007671, Starr Cancer Consortium and Lerner Trust).
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Figure 1. Baicalein from Scutellaria baicalensis (Huángqín) extract inhibits SPIꢀ1 T3SS. (A)
Scheme for SopE2ꢀCPG2ꢀHA:GluꢀCyFur reporter system for monitoring type III protein
secretion. SopE2ꢀCPG2ꢀHA (SPIꢀ1 T3SS) reporter encodes an enzyme activity of
carboxypeptidase G2 (CPG2) that when fused to the Cꢀterminus of a known S. Typhimurium
T3SS bacterial effector (SopE2) can be secreted and specifically report on type III protein
secretion through cleavage of fluorogenic substrates (GluꢀCyFur). (B) Screen of TCM activity at
5 mg/mL using SopE2ꢀCPG2ꢀHA:GluꢀCyFur reporter system. TCMs were incubated with
bacteria for 4 hr and growth media was collected and analyzed for CPG2 activity using
GluꢀCyFur. CyFur fluorescence was monitored at 610 nm with excitation at 563 nm. (C)
Structure of baicalein. (D) Doseꢀdependent activity of baicalein on SPIꢀ1 T3SS
(SopE2ꢀCPG2ꢀHA) reporter in S. Typhimurium growth media and cell lysate. (E)
Doseꢀdependent effect of baicalein on the levels of SPIꢀ1 T3SS secreted proteins (SipA, SipB,
SopB, SipC, and SipD) and flagella components levels in S. Typhimurium growth media. The
identities of the polypeptides were confirmed by inꢀgel digestion and LCꢀMS/MS analysis.
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Figure 2. Survey of naturally occuring flavonoids against type III protein secretion reporter.
(A) Structures of six major classes of flavonoid family: I, flavone (showing the positions of A, B,
and C ring), II, flavonol, III, flavanone, IV, isoflavone, V, flavanonol, and VI, proanthocyanidin.
Profile of T3SS inhibitory activity of 28 compounds representing 6 different classes of
flavonoids at 25 ꢁM using SopE2ꢀCPG2ꢀHA reporter system. Mean + s.d., n = 3. (B) Chemical
structures and IC50 values of most active T3SS inhibitors measured with the SopE2ꢀCPG2ꢀHA
reporter fluorescence assay. Mean + s.d., n = 3.
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Figure 3. Active flavonoids inhibit SPIꢀ1 T3SSꢀmediated bacterial invasion of epithelial cells.
(A) Left: Flow cytometry analysis of flavonoids at 100 ꢁM on S. Typhimurium invasion of HeLa
cells judged by antiꢀS. Typhimurium antibody staining. MOI = 10, 30 minute infection.
Experiment was done in triplicate and similar results were seen in two independent runs. Right:
Baicalein and quercetin blocked the invasion of S. Typhimurium into HeLa cells in a
doseꢀdependent manner (darker color: 100 ꢁM, lighter color: 50 ꢁM). Mean ± s.d., n = 3. The
values have been normalized to those of DMSO treated, which were considered 100 %. (B)
Growth of S. Typhimurium in quercetin abolishes bacterial invasion of cultured epithelial cells.
S. Typhimurium was grown in the presence of quercetin (labeled "wt grown in quercetin") or,
alternatively, quercetin was added shortly (30 min) before bacteria were added to cultured
epithelial cells (labeled "wt in quercetin). The ability of S. Typhimurium to enter into cultured
epithelial cells was evaluated by the gentamicin protection assay13 as described in the
Supplemental Methods. Values are the mean ± standard deviation of three independent
measurements and represent the % of the inoculum that survive the gentamicin treatment
because of their intracellular location. The values have been standardized to those of wild type,
which were considered 100 %. The indicated p values were determined by the standard Student
t test. * indicates p <0.001 (change panel C for the revised version, which includes *). (C)
Immunofluorescence analysis of intracellular S. Typhimurium in HeLa cells with 100 ꢁM
flavonoids. Scale bar = 10 ꢁm.
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Figure 4. Chemical proteomic analysis of alkꢀbaicalein labeled proteins in S. Typhimurium.
(A) Alkyneꢀflavonoid probes for activityꢀbased protein profiling studies. (B) Effect of
alkyneꢀflavonoid probes on the levels of SPIꢀ1 T3SS secreted proteins in S. Typhimurium culture
growth media. (C) Inꢀgel fluorescence analysis of alkyneꢀflavonoid probe labeling of S.
Typhimurium proteins following click chemistry reaction with azꢀrho fluorescence dye.
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Figure 5. Validation of alkꢀbaicalein labeled SPIꢀT3SS substrate, SipA. (A) Summary of S.
Typhimurium proteins that were labeled by alkyneꢀflavonoid probes. The total number of
peptideꢀsequence matches (PSM) between alkꢀbaicalein and the inactive probes,
alkꢀtrimethylbaicalein, alkꢀoroxylin A were compared to DMSO control. The graph shows the 16
proteins that were labeled by alkꢀbaicalein with more than 5ꢀfold PSM enrichment compared to
control samples. (B) Alkꢀbaicalein labeling of SipAꢀHA in wildꢀtype (wt) and T3SSꢀdeficient
(invA) S. Typhimurium. SipAꢀHA was immunopurified from alkꢀbaicalein treated S.
Typhimurium, reacted with azꢀrho and analyzed by inꢀgel fluorescence and antiꢀHA western blot.
Coomassie blue staining of bacterial culture supernatants was performed in parallel to assay on
protein secretion. (C) SipAꢀHA and SipA(1ꢀ270)ꢀHA was immunopurified from alkꢀbaicalein
treated S. Typhimurium and analyzed as described in (B).
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Figure 6. Binding and circular dichroism analysis (CD) of SipAꢀflavonoid interactions. (A) SipA
domains. (B) Spectra of SipAꢀGST fusion purified from the E. coli treated with flavonoids.
Quercetin and baicalein treated SipAꢀGST had a shoulder at the 370 nm region, while the
inactive flavonoid chrysin and untreated SipA did not show this spectral feature. (C) Spectra of
the Nꢀ and Cꢀterminal domains of SipA purified with and without quercetin. Quercetinꢀtreated
SipA Nꢀterminal construct exhibits a peak at 370 nm, while the signal sequence mutant (27ꢀ271)
and Cꢀterminal domainꢀGST fusion did not. (D) CD spectra of SipA untreated and expressed in
the presence of flavonoids. CD spectra of SipAꢀ1ꢀ271ꢀGST expressed without the presence of
flavonoids showed a characteristic αꢀhelical pattern. Treatment with 50 ꢁM quercetin and
baicalein induced significant spectra change at 208 nm and 222 nm. However, addition of
quercetin to purified SipAꢀ1ꢀ271ꢀGST in vitro did not induce changes in CD spectra.
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