Targeting MgrA-mediated virulence regulation in Staphylococcus aureus

Article abstract of DOI:10.1016/j.chembiol.2011.05.014

Increasing antibiotic resistance in human pathogens necessitates the development of new approaches against infections. Targeting virulence regulation at the transcriptional level represents a promising strategy yet to be explored. A global transcriptional regulator, MgrA in Staphylococcus aureus, was identified previously as a key virulence determinant. We have performed a fluorescence anisotropy (FA)-based high-throughput screen that identified 5, 5-methylenedisalicylic acid (MDSA), which blocks the DNA binding of MgrA. MDSA represses the expression of α-toxin that is up-regulated by MgrA and activates the transcription of protein A, a gene down-regulated by MgrA. MDSA alters bacterial antibiotic susceptibilities via an MgrA-dependent pathway. A mouse model of infection indicated that MDSA could attenuate S. aureus virulence. This work is a rare demonstration of utilizing small molecules to block protein-DNA interaction, thus tuning important biological regulation at the transcriptional level.

Full text of DOI:10.1016/j.chembiol.2011.05.014

Chemistry & Biology
Article
Targeting MgrA-Mediated Virulence Regulation
in Staphylococcus aureus
Fei Sun,1 Lu Zhou,1 Bing-Chuan Zhao,2 Xin Deng,1 Hoonsik Cho,4 Chengqi Yi,1 Xing Jian,1 Chun-Xiao Song,1
1,
Chi-Hao Luan,3 Taeok Bae,4 Zigang Li,2, and Chuan He
*
*
1Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL 60637, USA
2Key Lab of Chemical Genomics, School of Chemical Biology & Biotechnology, Shenzhen Graduate School, Peking University,
Shenzhen 518055, China
3High Throughput Analysis Laboratory, Northwestern University, Evanston, IL 60208, USA
4Department of Microbiology and Immunology, Indiana University School of Medicine-Northwest, Gary, IN 46408, USA
*Correspondence: lizg@szpku.edu.cn (Z.L.), chuanhe@uchicago.edu (C.H.)
DOI 10.1016/j.chembiol.2011.05.014
SUMMARY
has long been considered as the last resort therapy for MRSAs
(Fridkin et al., 2005), vancomycin-resistant strains with interme-
Increasing antibiotic resistance in human pathogens diate (VISAs) or full resistance (VRSAs) to vancomycin and other
glycopeptide inhibitors of cell wall synthesis have caused infec-
necessitates the development of new approaches
tions for which traditional antibiotic treatment have been in vain
(Chang et al., 2003; Walsh, 1993; Weigel et al., 2003).
Virulence suppression represents an alternative approach to
against infections. Targeting virulence regulation at
the transcriptional level represents a promising stra-
tegy yet to be explored. A global transcriptional regu-
lator, MgrA in Staphylococcus aureus, was identified
previously as a key virulence determinant. We have
performed a fluorescence anisotropy (FA)–based
combating infections (Balaban et al., 1998; Gresham et al.,
2000; Ji et al., 1997). The mode of action of conventional antibi-
otics largely relies on targeting essential cellular functions, such
as DNA replication, protein synthesis, and cell wall synthesis.
high-throughput screen that identified 5, 5-methyle-
nedisalicylic acid (MDSA), which blocks the DNA
binding of MgrA. MDSA represses the expression
Because of bactericidal (bacterial-killing) or bacteriostatic
(growth-inhibitory) effects, overuse of these antibiotics imposes
selective pressures on bacteria and thus leads to the emergence
of a-toxin that is up-regulated by MgrA and activates of drug-resistant strains. Given that most virulence factors are
nonessential for bacteria, in principle, antimicrobial agents that
are designed for inhibiting microbial virulence without inhibiting
the transcription of protein A, a gene down-regulated
by MgrA. MDSA alters bacterial antibiotic suscepti-
bilities via an MgrA-dependent pathway. A mouse
model of infection indicated that MDSA could atten-
uate S. aureus virulence. This work is a rare demon-
stration of utilizing small molecules to block
protein-DNA interaction, thus tuning important bio-
logical regulation at the transcriptional level.
growth presumably exert less selective pressure for the genera-
tion of resistance (Clatworthy et al., 2007). The expression of
virulence factors is coordinately regulated by multiple two-
component systems and global transcription factors. MgrA in
particular plays a key role in the regulation of the expression of
major virulence factors in S. aureus (e.g., capsular polysaccha-
ride, protease, a-toxin, nuclease, and protein A) (Ingavale
et al., 2005; Luong et al., 2006; Truong-Bolduc et al., 2005).
MgrA is a member of the MarR (multiple antibiotic resistance
regulator)/SarA (staphylococcal accessory regulator A) family
INTRODUCTION
proteins and controls expression of ꢀ350 genes (Luong et al.,
Staphylococcus aureus is a common human pathogen that 2006). We have shown that MgrA is critical for S. aureus patho-
causes a wide variety of diseases, ranging from minor skin infec- genesis in vivo, and an mgrA mutant strain exhibits 1000–
tions to life-threatening endocarditis, pneumonia, blood infec- 10,000 fold virulence reduction in a mouse model of infection
tions, and toxic shock syndrome (Archer, 1998; Lowy, 1998). (Chen et al., 2006). We have also revealed that MgrA acts as
This pathogen can infect almost every tissue in the human a redox-switch via its sole and unique Cys12 to regulate gene
host. Its versatility is mostly due to the production of various viru- expression. Oxidation of this Cys leads to dissociation of the
lence factors, such as hemolysins, leukocidins, and immune oxidized MgrA from DNA and thus attenuation of the bacterial
modulators. More troublesome, strategies that fight staphylo- virulence. The regulation of MgrA could also be affected by envi-
coccal infection have deteriorated in the past several decades ronmental stimuli, as shown by a previous study in which the
owing to the emergence of multiple methicillin-resistant strains transcriptional expression of mgrA was down-regulated by salic-
(MRSAs) and more recent vancomycin-resistant strains. MRSAs, ylate (Riordan et al., 2007). Given the dramatic role MgrA plays in
which are isolated in up to 60% of community and 80% of S. aureus virulence, this transcription factor could be a feasible
hospital infections, have rendered the entire class of b-lactam target for developing antimicrobial drugs. Chemically inhibiting
antimicrobials obsolete as therapeutic agents (Centers for DNA binding of MgrA could potentially diminish S. aureus viru-
Disease Control and Prevention, 2007). Although vancomycin lence. As proof of principle, we demonstrate here that small
1032 Chemistry & Biology 18, 1032–1041, August 26, 2011 ª2011 Elsevier Ltd All rights reserved

Chemistry & Biology
Small Molecules Inhibit MgrA
Figure 1. High-Throughput Screening and
Hit Characterization Strategy
A
fluorescence anisotropy–based assay using
a fluorescein-labeled DNA recognized by MgrA
was used in HTS (Z0 factor of 0.81). Compounds
displaying R50% inhibition on the MgrA-DNA
binding were selected as positive hits. The 94 hits
were subjected to a fluorescence-based thermal
shift assay to further probe their direct interactions
with MgrA. Four top hits were evaluated in
in vitro experiments, such as EMSA, to confirm
their activity in disrupting MgrA-DNA interaction.
MDSA, the most effective inhibitor for MgrA, was
applied to a series of in vivo characterizations
including transcriptional expression analysis on
virulence factors, antibiotic resistance assays, and
a murine abscess infection model. See also Fig-
ure S1.
The other 95 hits with confident struc-
tural information and purity were picked
for the subsequent validation and
characterization.
Although FA has been widely used to
study the interaction of biomolecules
and proven to be an effective method
for HTS, several caveats must be ac-
counted for when using this type of
molecules identified from a high-throughput screen (HTS) are screen. First, the inherent fluorescent properties of test
able to disrupt the DNA binding of MgrA and that administration compounds could interfere with the FA readout and produce
of such a compound leads to a reduced staphylococcal infection false-positive results. Second, in the screen to disrupt protein-
in a murine abscess model of infection.
DNA interactions, some compounds could simply intercalate
into DNA and prevent the binding of MgrA. To rule out these
unwanted scenarios, initial screening hits were filtered by a
combination of cheminformatics (data analysis to exclude low-
confidence inhibitors and autofluorescent false-positive results),
RESULTS
Screening Inhibitors for MgrA
A
fluorescence anisotropy (FA)–based biochemical assay the secondary screen based on the fluorescence-based thermal
that monitors MgrA-DNA binding was optimized for HTS. The shift assay, and biochemical and phenotypical characterization
DNA probe (50-TAAACAACAAGTTGTCCAAA-30) containing an of top hits in S. aureus (Figure 1). The fluorescence-based
MgrA-binding box (AGTTGT) was synthesized and fluorescently thermal shift assay, which represents a general method for iden-
labeled with 6-carboxylfluorescein (Manna et al., 2004), a widely tification of inhibitors of target proteins from a number of hit
used fluorophore with excitation at 494 nm and emission at compounds, was used to verify the direct interaction between
521 nm (see Figure S1A available online). According to the Perrin MgrA and selected compounds (Lo et al., 2004). The basis of
equation, the FA value is proportional to the molecular volume this assay is that any protein of interest has a characteristic
(molecular weight) of fluorescently labeled biomolecules. In this melting temperature because of its intrinsic secondary and
case, the fluorescein-labeled DNA alone generates a small FA tertiary structure, which could be stabilized or destabilized
value (ꢀ0.8), whereas the formation of MgrA-DNA complex upon ligand binding. This effect is usually reflected by the shift
increases the FA value up to ꢀ2.8. The binding affinity of MgrA of the melting temperature of the target protein. Sypro orange,
to this particular DNA probe was determined using this method an environmentally sensitive fluorescent dye, could be used to
(Kd ꢀ0.14 mM) (Figure S1B). Translation of this assay into monitor the protein-unfolding transition. The 95 compounds ob-
a 96-well format plate yielded a Z0 factor of 0.81 with 20 nM tained from HTS were further tested using this method. Most
DNA and 400 nM MgrA, which is well above the value (0.5) sug- compounds were either able to increase or decrease the melting
gested for typical HTS assays (Figure S1C). A total of 88,564 temperature of MgrA (data not shown), indicating that these
compounds were screened in duplicate against the MgrA-DNA compounds disrupt MgrA-DNA interaction mostly by interacting
interaction using the NSRB library. Among the small molecules with the protein rather than intercalating into DNA. Of these, four
screened, 114 (0.13%) exhibited significant inhibition (R50%) prominent compounds are listed in Table 1. MDSA, reminiscent
on the DNA binding of MgrA. Of these, 19 hits are from natural of a dimerized salicylic acid, displayed the strongest inhibitory
product extracts and tentatively excluded from cherry picks effect on MgrA with an IC50 close to ꢀ8.0 mM, according to the
because of a lack of structural and composition information. FA assay and the electrophoretic mobility shift assay (EMSA)
Chemistry & Biology 18, 1032–1041, August 26, 2011 ª2011 Elsevier Ltd All rights reserved 1033

Chemistry & Biology
Small Molecules Inhibit MgrA
shown in the Northern blotting assays (Figure 2C), the expres-
sion level of hla in the wild-type Newman was greatly decreased
in the presence of 0.2 mM of MDSA. Because MgrA acts as an
activator of hla, mutation of mgrA led to a decreased hla expres-
sion as expected. The expression of hla in mgrA mutant was not
affected by the presence of MDSA, even up to 0.5 mM, strongly
suggesting that MDSA affects the expression of hla in an MgrA-
dependent manner. In addition to the strain Newman, the inhibi-
tion of MDSA on the expression of hla was also observed in the
MRSA strain USA300 according to western blot analysis (Fig-
ure S4). MgrA is known to act as a repressor of the spa gene
(Ingavale et al., 2005). As shown in Figure 2C, the treatment of
wild-type bacteria with MDSA resulted in the increased expres-
sion of spa, whereas in the mgrA mutant strain, the expression of
spa occurred at a higher level and was not affected by the pres-
ence of 0.5 mM of MDSA. Taken together, we have demon-
strated that MDSA exerts inhibitory effects on MgrA inside
bacteria, thus leading to the altered expression of the mgrA
regulon.
Table 1. The Structures of Representative MgrA Inhibitors
Revealed by FA-HTS and FA-Thermal Shift Assay
HTS Example Active
IC50 (mM)
Temperature (ꢁC)
8
7.7
30
28
2.7
3.6
32
3.5
Binding of MDSA to MgrA
Because we have previously solved the crystal structure of MgrA
(Chen et al., 2006), computational molecular docking (AutoDock
Vina, vina.scripps.edu) was applied to investigate the possible
mode of MDSA binding to MgrA. After running the program,
two possible binding sites (a and b) were revealed, and both
were equally energetically favored (Figure 3A). Interestingly,
both sites are close to the helix-turn-helix DNA-binding motif
that is highly conserved among the MarR/SarA family protein
(Alekshun et al., 2001), implying that MDSA interacts with the
DNA-binding domain of MgrA to interrupt its DNA binding. The
docking structure also reveals that MgrA bears a single Trp48
IC50 of each compound was determined by fluorescence anisotropy
assay and validated by EMSAs (Figure S3). The melting temperature shift
(DT) is presented as a mean value from two independent experiments
(Figure S2). See also Figures S2 and S3.
(Figure 2). In the thermal shift experiment, this compound was
capable of increasing the melting temperature of MgrA by
7.7ꢁC (Figure S2). The other three compounds 2, 3, and 4 fall
into the same class of molecule, 3-aryl-3-(2,5-dimethyl-1H-
pyrrol-1-yl) propanoic acid, and only differ at the aromatic
substituent at the 3 position. All three compounds exhibited
good inhibition activities on MgrA, with IC50 values ranging
from 28 mM to 32 mM. In the thermal shift assay, these three
compounds increased the melting temperature of MgrA by
approximately 2–3ꢁC (Figure S2), indicating that they might
interact with MgrA in a very similar manner. The activity of these
compounds to block DNA binding by MgrA was further
confirmed by EMSAs (Figures 2B; Figure S3). MgrA was shown
to be dissociated from its cognate DNA (hla promoter region) in
the presence of these small molecules. Consistent with the FA
and thermal shift assays, EMSA also showed that MDSA was
the most effective compound to disrupt the DNA binding of
MgrA, which prompted us to further investigate this compound.
˚
˚
residue that is located ꢀ20.3 A and ꢀ20.4 A away from the
a and b sites, respectively (Figure 3A). Because MDSA is a fluo-
rescent compound with excitation at 310 nm and emission at
421 nm, we envision that if MDSA does bind to either the a or
¨
b position, Forster resonance energy transfer (FRET) should
occur between the donor Trp48 (excitation at 278 nm and emis-
sion at 330 nm) and the acceptor MDSA. To test our hypothesis,
we measured the fluorescence of MgrA in the presence or
absence of MDSA. As shown in Figure 3B, MgrA (10 mM) dis-
played the maximum fluorescent signal at 330 nm in the absence
of MDSA. With the addition of MDSA, the peak at 330 nm, corre-
sponding to the emission of Trp48, was decreased, whereas the
emission of MDSA at 421 nm was elevated. Because the emis-
sion intensity at 330 nm depends on the molar ratio of MgrA-
MDSA versus MgrA, the binding affinity of MgrA to the small
MDSA Alters the Expression of Virulence Factors
in an MgrA-Dependent Manner
molecule could be estimated through this measurement (Kd
=
MgrA is a global transcriptional regulator that controls expres- ꢀ12.8 mM) (Figure 3C). We consider this value a more accurate
sion of a number of virulence factors, such as genes encoding determination of the binding affinity of MDSA to MgrA.
Far-UV (190–250 nm) circular dichroism (CD) spectroscopy is
a-toxin (hla) and protein A (spa) (Ingavale et al., 2005). MgrA
has been shown to positively regulate the expression of hla by widely used for monitoring protein secondary structure, whereas
binding to its promoter region (Ingavale et al., 2005). To test near-UV CD spectroscopy is used for comprehending tertiary
whether MDSA could abolish the binding of MgrA to the hla structure (Beychok, 1966; Greenfield, 2006). To investigate
promoter, EMSAs were performed in the presence of different whether the binding of MDSA to MgrA elicits a conformational
amounts of MDSA. As shown in Figure 2B, ꢀ8 mM of MDSA change, near-UV (250–320 nm) circular dichroism (CD) spectros-
could significantly attenuate the binding of MgrA to the hla pro- copy was used. As shown in Figure 3D, molar ellipticity of MgrA
moter. The presence of MDSA could also alter the expression (0.12 mM) was increased slightly at 262 nm but decreased at
level of both hla and spa inside S. aureus strain Newman. As 285 nm because of the presence of MDSA (0.3 mM), indicating
1034 Chemistry & Biology 18, 1032–1041, August 26, 2011 ª2011 Elsevier Ltd All rights reserved

Chemistry & Biology
Small Molecules Inhibit MgrA
Figure 2. MDSA Inhibits DNA Binding of MgrA
(A) An FA-based binding assay shows dissociation of
MgrA (1 mM) from DNA (0.01 mM) induced by MDSA (IC50
ꢀ8.0 mM).
(B) EMSA confirming that MDSA induces dissociation of
MgrA (0.5 mM) from its cognate DNA. 32P-labeled DNA
PCR-amplified from the hla promoter region was used.
(C) Northern blot analysis showing that MDSA alters the
expression of virulence factors (spa and hla) regulated by
MgrA; 5 mg of total cellular RNA was used, and the
ethidium bromide–stained gel of the loaded RNA sample is
shown below.
See also Figure S4.
rial resistance to multiple antibiotics such as
tetracycline, norfloxacin, and ciprofloxacin
(Kaatz et al., 2005; Truong-Bolduc et al., 2005;
Truong-Bolduc et al., 2006). MgrA is also shown
to affect bacterial resistance to vancomycin
(Chen et al., 2006; Trotonda et al., 2009;
Truong-Bolduc and Hooper, 2007). Mutation of
mgrA leads to increased resistance of the
bacterium to these antibiotics. Therefore,
MDSA should alter staphylococcal resistance
that the binding of MDSA could affect the tertiary structure to these antibiotics if it does exert inhibitory effects on MgrA
of MgrA.
inside S. aureus. As shown by the plate sensitivity assay in Fig-
ure 5, in the absence of MDSA, the mgrA mutant strain displayed
stronger resistance to vancomycin (1.2 mg/ml), compared to the
SAR Studies of MDSA
Multiple MarR/SarA family proteins, including the Escherichia wild-type Newman, as expected. The phenotype can be com-
coli MarR, E. coli EmrR, Methanobacterium thermoautotrophi- plemented with pYJ335-mgrA, which restores vancomycin
cum MTH313, and Salmonella typhimurium SlyA, have been susceptibility to the wild-type level. In the presence of MDSA
shown to contain potential salicylate-binding sites; however, (0.2 mM), both the wild-type and complementary strain exhibited
salicylate associates with these proteins and attenuates their an increased vancomycin resistance that is comparable to the
DNA binding only at very high concentrations (e.g., at mM level mgrA mutant strain, implying that MgrA is inhibited in both
for MarR) (Martin and Rosner, 1995; Perera and Grove, 2010). strains. Similar effects were observed on the plates containing
Because MDSA appears to be a dimerized salicylate, it is impor- fluoroquinolones (norfloxacin [0.8 mg/ml] and ciprofloxacin
tant to ask whether salicylate itself could disrupt the MgrA-DNA [0.3 mg/ml]); the wild-type Newman was more susceptible to
interaction. Unlike MDSA that can abolish the MgrA-DNA binding fluoroquinolones than the mgrA mutant in the absence of
at ꢀ8 mM, up to 250 mM of salicylate showed almost no effect on MDSA, whereas the growth inhibition of the wild-type Newman
the DNA binding of MgrA, as shown in Figure 4, indicating that caused by norfloxacin or ciprofloxacin was alleviated on the
dual phenolic moiety is imperative for the function of MDSA. plate containing 0.2 mM of MDSA. The complementary strain
We tested other modifications. The treatment with 250 mM of carrying pYJ335-mgrA showed hypersensitivity toward both
3, 30-dimethylenebenzoic acid 6, which lacks the OH group at norfloxacin and ciprofloxacin, probably because of the elevated
aromatic ring, was unable to dissociate MgrA from DNA, sug- level of exogenous mgrA under this particular condition.
gesting that the hydroxyl group is required for the binding. The Measurements of minimum inhibition concentration (MIC) for
COOH group is also required for binding; the dimethyl ester of these antibiotics (vancomycin, norfloxacin, and ciprofloxacin)
MDSA cannot disrupt the binding of MgrA to DNA. The confor- further confirmed our observation from the plate sensitivity
mation of MDSA is crucial to its interaction with MgrA. As shown assays. As shown in Table S1, the wild-type Newman showed
in Figure 4, Olsalazine 8, an anti-inflammatory medicine that is greater resistance toward vancomycin, norfloxacin, and cipro-
structurally analogous to MDSA but has a rigid trans configura- floxacin in the presence of MDSA (0.2 mM) than its absence,
tion, could not affect the DNA binding of MgrA at 250 mM level. whereas the antibiotic resistance of its mgrA mutant was not
Collectively, all the structural features of MDSA as well as its flex- affected by MDSA. In addition to the strain Newman, the mgrA
ible conformation are required for its ability to disrupt MgrA-DNA mutant of USA300 also showed increased resistance toward
interaction.
vancomycin in comparison to the wild-type USA300. Similar to
Newman, MDSA (0.2 mM) increased the vancomycin resistance
of the wild-type USA300, but not its mgrA mutant (Table S1).
Although the resistance of USA300 toward ciprofloxacin or nor-
MDSA Changes the Susceptibility of S. aureus
to Antibiotics
MgrA negatively regulates the expression of efflux pumps in- floxacin appears to be unaffected by the mutation of mgrA, both
cluding Tet38, NorA, NorB, and NorC, which account for bacte- of which showed equal resistance (Table S1), the presence of
Chemistry & Biology 18, 1032–1041, August 26, 2011 ª2011 Elsevier Ltd All rights reserved 1035

Chemistry & Biology
Small Molecules Inhibit MgrA
Figure 3. Binding Assays of MDSA to MgrA
(A) Docking models of MDSA binding to MgrA. Molecular
docking experiment indicates two plausible binding sites
(a and b) for MDSA. The distance between a site and Trp48
˚
˚
is 20.3 A. The distance between b site and Trp48 is 20.4 A.
(B) FRET generated between Trp48, the only Trp in the
MgrA sequence, and the bound MDSA. Excitation was at
278 nm. Emission of Trp48 in MgrA was at 330 nm.
Emission of MDSA was at 421 nm. The fluorescence of the
Apo-MgrA (1 mM) was shown in black. FRET experiments
were shown in colored bands.
(C) A binding curve of MDSA to MgrA obtained from the
FRET experiment; 1 mM MgrA was used. The concentra-
tion of MDSA varies from 64 mM to 2 mM. Shown are the
mean and standard deviation of three independent ex-
periments.
(D) The near-UV (250–320 nm) CD spectrum of MgrA
(0.12 mM) in the absence or presence of MDSA (0.3 mM).
Apo-MgrA is shown in yellow. The MgrA-MDSA complex
is shown in pink.
See also Figure S7.
MDSA (0.2 mM) could still induce increased fluoroquinolone from mice coadministered with the compound. The number of
resistance of not only the wild-type USA300 but also its mgrA colony-forming units (CFUs) in the corresponding organs
mutant, implying the existence of a possible mgrA-independent including livers and kidneys was only ꢀ10% of that from mice
pathway affected by MDSA in USA300. Overall, the intrinsic without treatment (Figure 6). Collectively, these data demon-
resistance of the mgrA mutant of either the strain Newman or strate that MDSA is able to prevent S. aureus infection in the
USA300 to antibiotics was less affected by MDSA, indicating mouse. Future modifications may improve the in vivo potency.
that MDSA affects S. aureus antibiotic resistance via an mgrA-
dependent pathway. Together, the data suggest that MDSA
inhibits MgrA inside S. aureus, thereby affecting functions regu-
lated by MgrA.
DISCUSSION
A major challenge facing contemporary medicine is the develop-
ment of new antibiotics against human pathogens that are
resilient to most current antimicrobial therapies (Borlee et al.,
2010; Clatworthy et al., 2007; Park et al., 2007; Rainey and
MDSA Attenuates S. aureus Virulence in the Mouse
Model of Abscess Formation
Because most virulence factors play nonessential roles in the Young, 2004; Skaar, 2010; Stringer et al., 2010; Swoboda
in vitro growth of S. aureus, we envision that the small molecule et al., 2010; Wright et al., 2005). It has been well documented
designed for virulence suppression should not affect the viability that S. aureus has developed multiple resistance mechanisms
of S. aureus under normal conditions, yet it should suppress against almost all known antibiotics, a problem that urgently
in vivo pathogenesis of S. aureus. As shown in Figures S5 and requires identification of new therapeutic targets and develop-
S6, S. aureus displayed no growth defect in the presence of up ment of alternative strategies for combating S. aureus (Fridkin
to 10 mM of MDSA. We next employed a mouse model of et al., 2005; Lowy, 1998; Walsh, 1993; Weigel et al., 2003).
abscess formation to test the potential virulence suppression MgrA, a member of the MarR/SarA family transcriptional regula-
function of MDSA. The diacid form of MDSA could suffer from tors that controls the expression of many virulence determinants,
an absorption/distribution (AD) problem once entering the is essential for staphylococcal virulence in animal model exper-
mouse body. Specifically, the negative charge may prevent the iments. Therefore, inhibition of the function of MgrA is of great
molecule from efficiently crossing the tissue-blood barrier to therapeutic potential.
reach infection sites. Previous pharmacokinetic (PK) studies on
Virulence regulators are promising targets for developing
methyl-salicylate (esterified salicylate) have revealed that methyl novel antibiotics. However, targeting virulence regulation has
salicylate maintains an extremely high permeability through not been fully exploited, and only a limited number of examples
blood-brain barrier and blood-tissue barrier while undergoing are present so far (Gauthier et al., 2005; Hung et al., 2005). In
rapid and efficient hydrolysis inside animal tissues (Finkel et al., Vibrio cholerae, an inhibitory small molecule (e.g., virstatin) ob-
2009). The liver was shown to be the major site of methyl salicy- tained through HTS has been found to disrupt the dimerization
late hydrolysis (Davison et al., 1961). Because MDSA has high and function of ToxT, a virulence transcriptional regulator,
structural similarity to salicylate, we synthesized the dimethyl thereby preventing the expression of several critical virulence
form 7 of MDSA and envisioned that hydrolysis of 7 in the liver factors in V. cholerae (Shakhnovich et al., 2007). To our knowl-
or kidney would lead to accumulation of MDSA, which in turn edge, the strategy of using a small molecule to target transcrip-
could potentiate its antistaphylococcal activity inside these tional regulators has yet to be applied to S. aureus. Our study
organs. To test this idea, we simultaneously injected S. aureus represents an early success that demonstrates virulence sup-
and 7 into mice via the retro-orbital route. After 4 days, we pression in S. aureus by targeting the MgrA regulator with small
observed attenuated abscess formation in kidneys and livers molecules.
1036 Chemistry & Biology 18, 1032–1041, August 26, 2011 ª2011 Elsevier Ltd All rights reserved

Chemistry & Biology
Small Molecules Inhibit MgrA
directly perturbs the DNA-binding domain of MgrA. Our compu-
tational docking experiment has indicated that MgrA bears two
possible binding sites for MDSA around its DNA-binding lobe
(the HTH motif). This hypothesis was supported by the FRET
experiment that showed close proximity of the MgrA-bound
MDSA to Trp48 nearby. Potentially, MDSA binds the DNA-
binding HTH motif of MgrA, which alters its conformation, thus
disrupting DNA binding of MgrA.
Protein-DNA interaction is central to transcriptional regulation
in biology. Despite a growing number of examples of small
molecules targeting transcription (Chau et al., 2005; Darnell,
2002; Dervan et al., 2005; Kiessling et al., 2007; Lauth et al.,
2007; Lu et al., 2005; Rishi et al., 2005; Vassilev et al., 2004),
the process of identifying effective small molecules that exhibit
activity inside cells is still challenging. In very few examples
have small molecules been identified that effectively disrupt
protein-DNA interactions. We show here that MDSA is able to
disrupt MgrA-DNA interaction and further attenuate staphylo-
coccal virulence expression. The MarR/SarA family proteins
present excellent targets because they have long been estab-
lished as transcription regulators responsive to environmental
stimuli such as small molecule ligands, ROS, and pH (Perera
and Grove, 2010; Wilkinson and Grove, 2006), of which the
intrinsic conformational flexibility and the existence of ligand-
binding pocket provide the opportunity to identify inhibitors
that can bind these proteins and disrupt regulatory function. It
has been recognized that most MarR/SarA proteins comply
with a two-state model (Perera and Grove, 2010): the apo-
protein compatible with DNA binding and the modified form
incompatible with DNA binding owing to ligand binding, or post-
translational modifications (PTS), such as oxidation and phos-
phorylation (Didier et al., 2010; Truong-Bolduc et al., 2008;
Truong-Bolduc and Hooper, 2010). Given that MgrA is sub-
jected to multiple PTSs, such as oxidation of Cys12 and phos-
Figure 4. Structure and Activity Relationship (SAR) Studies of MDSA
Four derivatives (salicylic acid 5, 3, 3-methylenedibenzoic acid 6, methyl 5,
5-methylenedisalicylate 7, and Olsalazine 8) of MDSA are unable to disrupt the
MgrA-DNA interaction at concentrations of up to 250 mM.
Structurally, MDSA is a reminiscent of salicylic acid, a preva- phorylation at Ser/Thr (Chen et al., 2006; Truong-Bolduc et al.,
lent plant hormone that has been shown to affect S. aureus in 2008), the binding of MDSA to MgrA might trigger a similar
many aspects (Riordan et al., 2007). Previous studies have conformational change and preconfigure the protein into the
showed that salicylate induces increased staphylococcal resis- second state that is incompatible with DNA binding. Future
tance to multiple antimicrobials, such as the DNA topoisomer- study will focus on structurally elucidating the mode of action
ase inhibitor fluoroquinolones (ciprofloxacin and norfloxacin), of MDSA to gain further insight into tuning the function of
the protein synthesis inhibitor fusidic acid, and the DNA-interca- MgrA with small molecules.
lating dye ethidium (Gustafson et al., 1999; Price et al., 2002).
Unlike salicylate which reduces the growth of S. aureus at SIGNIFICANCE
2 mM (Riordan et al., 2007), even 10 mM of MDSA does not
inhibit growth of different S. aureus strains tested (Figure S5). MgrA is a global transcriptional regulator essential for the
Although salicylate has been shown to down-regulate the pathogenesis of S. aureus. It provides a potential drug target
expression of transcription factors mgrA and sarR while up- for combating staphylococcal infections. In this study, we
regulating sarA transcription (Gustafson et al., 1999), our identified a small-molecule MDSA that inhibits DNA binding
EMSA indicated that a high concentration (250 mM) of salicylate of MgrA through a high-throughput screening. MDSA alters
has little effect on the DNA-binding activity of MgrA, which the transcriptional expression of virulence factors, including
excludes the possibility that salicylate alters mgrA transcription hla and spa, both of which are known to be regulated by
through directly interfering its autoregulation (Ingavale et al., MgrA. MDSA also affects bacterial antibiotic resistance
2003).
toward vancomycin, norfloxacin, and ciprofloxacin via an
The small-molecule MDSA might inhibit the DNA binding of mgrA-dependent pathway. Simultaneous administration of
MgrA via two possible modes. Because MgrA functions as esterified MDSA with S. aureus could attenuate S. aureus
a dimer, MDSA might disrupt the dimerization of MgrA. However, virulence, as demonstrated in a mouse model of infection.
our gel filtration analysis showed that even 1 mM of MDSA was This study demonstrates that small molecules can be devel-
unable to change the dimeric status of MgrA (Figure S7), which oped to disrupt protein-DNA interactions of transcriptional
excludes this possibility. The other possibility is that MDSA regulators as a strategy to combat bacterial infection.
Chemistry & Biology 18, 1032–1041, August 26, 2011 ª2011 Elsevier Ltd All rights reserved 1037

Chemistry & Biology
Small Molecules Inhibit MgrA
Figure 5. Effects of MDSA on Staphylo-
coccal Antibiotic Susceptibility toward Van-
comycin, Norfloxacin, and Ciprofloxacin
The antibiotic resistance levels were tested in the
presence or absence of MDSA (200 mM) by a plate
sensitivity assay; 10 ml of mid-log culture was
diluted 1000-fold in PBS before plating in dupli-
cate. Van, vancomycin (1.2 mg/ml); Nor, nor-
floxacin (0.8 mg/ml); Cip, ciprofloxacin (0.3 mg/ml).
See also Table S1.
Screen of MgrA Inhibitors
A high-throughput screen (HTS) of compound
libraries to identify MgrA inhibitors was performed
at The National Screening Laboratory for the
Regional Centers of Excellence in Biodefense
and Emerging Infectious Disease (NSRB) at Har-
vard Medical School. The MgrA protein (700 nM,
EXPERIMENTAL PROCEDURES
30 mL) was added to the black polystyrene 384-well plate, except column
24, to which 30 ml of empty control buffer (10 mM Tris [pH 7.4] and 25 mM
NaCl) was added. Test compounds (10 ng in 1 mL) were added into each
well except columns 23 (negative controls) and 24. The solution was incubated
at room temperature for 20 min. Then 20 mL of DNA solution (50 nM) was added
to all wells and the mixtures were incubated for another 20 min. The Envision
plate reader (475 nm/525 nm) was used to detect FA values. For hit selections,
we set up the cut-off range minus positive control (column 24) plus 50%
of dynamic range (column 23 minus 24). Ninety-five compounds out of
88,564 were cherry-picked for further validation and characterization.
Unless stated otherwise, all chemicals were purchased from Sigma-Aldrich,
and the restriction enzymes were purchased from New England Biolabs.
Because all assays were performed under physiological conditions (pH 7.4),
compound MDSA was always used in the form of disodium salt unless
otherwise noted. Other organic compounds were dissolved in DMSO for
biochemical and biological assays.
Ethics Statement
The animal study presented in this report (Approval ID: NW-20) was approved
by the Institutional Animal Care and Use Committee at Indiana University
School of Medicine-Northwest, which complies with the guidelines of the
National Institutes of Health.
Fluorescence-Based Thermal Shift Assays
The thermal shift assay was conducted as previously described (Lo et al.,
2004). The fluorescent dye Sypro orange (Sigma), an environmentally sensitive
fluorophore, was used to monitor the unfolding of MgrA. The basis of this fluo-
rescence-based thermal shift assay is that the protein unfolding exposes
Sypro orange to a hydrophobic environment, leading to increased fluores-
cence of Sypro orange. This assay was performed in the iCycler iQ Real
Time PCR Detection System (Bio-Rad). Solutions of 20 ml of MgrA (final
concentration, 5 mM), 50 ml of 5X Sypro orange, 2 ml of compound (20 ng),
and 28 ml of buffer (100 mM NaCl and 10 mM Tris [pH 7.4]) were added to
the wells of the 96-well iCycler iQ PCR plate. The plate was heated from
25ꢁC to 77ꢁC with a heating rate of 0.5ꢁC/min. The fluorescence intensity
was measured with Ex/Em: 490 nm/530 nm. Ninety-five compounds from
cherry picks were tested in duplicate. The data were processed as previously
described (Lo et al., 2004).
Bacterial Strains, Plasmids, and Culture Conditions
The bacterial strains used in this study are listed in Table S2. Unless otherwise
mentioned, S. aureus strain Newman is the strain used in the study. S. aureus
cells were grown in tryptic soy broth (TSB). For routine culture of mgrA trans-
poson mutants, erythromycin (10 mg/ml) was added, while chloramphenicol
(10 mg/ml) was added to mgrA mutant complemented with pYJ335-mgrA.
Expression and Purification of the MgrA Protein
The expression and purification of His6-MgrA have been described previously
(Chen et al., 2006). Briefly, E. coli (BL21star (DE3)) carrying pet28a::mgrA in LB
was grown at 37ꢁC. After reaching mid-log phase (OD600, ꢀ0.6), cells were
induced with 1 mM of IPTG for 4 hr at 30ꢁC. Cells were harvested and stored
at ꢂ78ꢁC before use. MgrA was purified by Ni-NTA column following the
manufacture’s manual (GE Healthcare). The purity of MgrA was determined
by SDS-PAGE. Protein was desalted to remove imidazole before subsequent
biochemical assays.
FRET Measurement of Small Molecule Binding to MgrA
Various amounts of MDSA ranging from 64 mM to 2 mM were added to the
buffer (100 mM NaCl and 10 mM Tris [pH 7.4]) containing 1 mM of MgrA. The
change of fluorescence was monitored at 330 nm (MgrA) and 421 nm
(MDSA), respectively. Excitation was set at 278 nm.
Oligonucleotide Synthesis
Circular Dichroism Spectrometry
The oligonucleotide (50-6-F-TAAACAACAAGTTGTCCAAA-30) containing
6-carboxylfluorescein was prepared by incorporating the 50-fluorescein phos-
phoramidite (6-FAM) (Glen Research, Inc.) at the 500 end during solid-phase
synthesis. The resulting oligonucleotide was purified with denaturing poly-
acrylamide gel electrophoresis. The labeled oligonucleotide was annealed
with its complementary strand and then used for FA-based binding assays.
The MgrA protein (0.12 mM) was incubated with 0.3 mM of MDSA in PBS buffer
at 25ꢁC for 10 min. Near-UV region (250–350 nm) CD spectrum was measured
at 25ꢁC by AVIV 202 CD Spectrometer (AVIV Biomedical Inc.). The protein
sample without the small molecule was also tested as a comparison.
Synthesis of Compound 7
Compound 7 was obtained according to literature methods (Cameron et al.,
2002; Guo et al., 2005). Briefly, MDSA (1 g) was dissolved in 20 ml of methanol
and then 1 ml of HCl (ꢀ10 M) was added. The mixture was stirred and refluxed
at 70ꢁC overnight. The resulting product was isolated with silica gel column.
FA–Based Binding Assays
To determine the binding affinity of MgrA to the labeled DNA, different amounts
of MgrA, varying from 0.0015 mM to 6.0 mM, were added into the 96-well format
plate containing 20 nM DNA, 10 mM Tris (pH, 7.4), and 25 mM NaCl. The
Envision plate reader (475 nm/525 nm) was used to measure FA values. The
Kd value was calculated using Microsoft Excel. The measurements were
done in triplicate.
Electrophoretic Mobility Shift Assays
DNA probe was PCR amplified from the hla promoter region with primers listed
32
in Table S3. DNA was labeled with P at the 50 end using T4 polynucleotide
1038 Chemistry & Biology 18, 1032–1041, August 26, 2011 ª2011 Elsevier Ltd All rights reserved

Chemistry & Biology
Small Molecules Inhibit MgrA
dures (Lan et al., 2010). Primers used for amplification of DNA fragments in
Northern blotting are listed in Table S3.
Antibiotic Susceptibility Assays
Minimum inhibitory concentration (MIC) was determined by the microdilution
method in 96-well plates. Antibiotics were serially diluted twofold in 100 ml of
LB. Overnight cultures were diluted with 13 PBS to cell density of 107 CFU/ml.
To each well of the 96-well plate, aliquots of 5 ml were added for a final inoc-
ulum of approximately 5 3 104 CFU/well. After incubation at 37ꢁC for 24 hr,
the MICs were determined visually.
Plate Sensitivity Assays for Antibiotic Resistance
TSA plates were made with designated amounts of MDSA and the antibiotic
(vancomycin, ciprofloxacin, or norfloxacin). The overnight culture was diluted
by 100 fold into fresh TSB containing 0.2 mM of MDSA. After 3 hr at 37ꢁC, the
mid-log culture was diluted by 1000 fold in PBS. Aliquots (10 ml) of the diluted
cultures for each strain were spotted onto the solid media and grown at 37ꢁC
for 24 hr. Each experiment was repeated at least three times to ensure
consistency.
Mouse Models of Abscess Formation
This assay was performed as described previously (Chen et al., 2006) with
slight modifications. To determine the effect of MDSA on the virulence of
S. aureus, its esterified derivative 7 was used. After measuring the weight of
the mice, 1 3 107 CFU of the wild-type Newman strain in PBS mixed with/
without small molecules (0.3 mg, the final concentration is 10 mM in the cell
culture) were administered to 16 Balb/c mice (Harlan, USA) via retro-orbital
injection. The average body weight of mice is ꢀ20 g. Four days after the injec-
tion, the mice were sacrificed and their organs (kidney and liver) were har-
vested. The harvested organs were homogenized; then the CFU of bacteria
in the organs were measured using serial dilutions on TSA plates. A Student’s
t test was performed to evaluate the statistical significance of the data by using
Microsoft Excel. As a control experiment to ensure that the same dose of small
molecules did not affect bacterial fitness in vitro, the same cell cultures in PBS,
after being treated with these compounds for 1 hr at 37ꢁC, were plated on TSA.
No growth defects were observed compared to the culture not treated with
small molecules.
SUPPLEMENTAL INFORMATION
Figure 6. Coadministration of the Dimethyl Ester MDSA 7 Attenuates
Staphylococcal Virulence in a Murine Abscess Model
The test strains (1 3 107 CFU) mixed with and without compound 7 were
administered into 16 mice per strain via retro-orbital injection. Four days later,
after measuring weight losses, organs were harvested and bacterial CFU in the
organs was measured by the serial dilution method. In the graph, each dot
represents a mouse. The group not treated with small molecule is indicated as
‘‘WT.’’ The group treated with small molecule is indicated as ‘‘15 mg/kg.’’ The
statistical difference was determined by Student’s two-tailed t test. See also
Figures S5 and S6.
Supplemental Information includes seven figures, three tables, and Supple-
mental Experimental Procedures and can be found with this article online at
doi:10.1016/j.chembiol.2011.05.014.
ACKNOWLEDGMENTS
We thank S. Chiang and other members of The National Screening Laboratory
for the Regional Centers of Excellence in Biodefense and Emerging Infectious
Disease (NSRB) for technical assistance during high-throughput screening.
We thank Drs. O. Schneewind and D. Missiakas at the University of Chicago
for providing transposon mutants. This work was financially supported by
the National Institutes of Health and the National Institute of Allergy and Infec-
tious Diseases (grant AI074658 to C.H.) and a Burroughs Wellcome Fund
Investigator in the Pathogenesis of Infectious Disease Award (to C.H.). F.S.
is a Scholar of the Chicago Biomedical Consortium with support from The
Searle Funds at The Chicago Community Trust.
kinase (Promega). MgrA (0.4 mM) was incubated with various amounts of test
compounds and 2 ng of radioactive DNA probe in 25 ml of the binding buffer
(10 mM Tris-HCl [pH 7.4], 50 mM KCl, 5 mM MgCl2, 10% glycerol, and
3 mg/ml sheared salmon sperm DNA). After 10 min at room temperature, the
samples were analyzed by 8% native polyacrylamide gel electrophoresis
(100 V for pre-run, 85 V for 85 min for sample separation). The gels were dried
and subjected to autoradiography on a phosphor screen (BAS-IP, Fuji).
Received: March 10, 2011
Revised: May 2, 2011
RNA Isolation and Northern Blotting
To isolate the RNA for Northern blot analysis, all S. aureus strains were grown
at 37ꢁC overnight in tryptic soy broth (TSB), diluted 100-fold in fresh 10 ml TSB
containing various amounts of MDSA (0, 200 mM, 350 mM, and 500 mM) in
a 50-ml conical tube (BD Biosciences), and incubated at 37ꢁC with shaking
at 250 rpm for 2.5 hr (OD600, 0.8). Cells were harvested and disrupted mechan-
ically (Fast Prep FP120 instrument; Qbiogene, Heidelberg, Germany). The
RNeasy Mini Kit (QIAGEN) was used for the subsequent RNA purification.
RNA concentration and purity were determined by UV absorption at 260 and
280 nm. Northern blotting was performed following previously reported proce-
Accepted: May 26, 2011
Published: August 25, 2011
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