RESEARCH ARTICLE

Hibifolin, a Natural Sortase A Inhibitor, Attenuates the

Pathogenicity of Staphylococcus aureus and Enhances the
Antibacterial Activity of Cefotaxime
Wu Song,a Li Wang,a Yinghua Zhao,b Gongga Lanzi,c Xingye Wang,a Chi Zhang,a Jiyu Guan,d Wei Wang,a Xuerui Guo,a,e Ying Meng,a
Bingmei Wang,a Yicheng Zhaoa,b

a College of Clinical Medicine, Changchun University of Chinese Medicine, Changchun, China

b Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, Center for Pathogen Biology and Infectious Diseases, The First Hospital of Jilin
University, Changchun, China
c Tibet University Medical College, Tibet, China
d Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin University, Changchun, China
e School of Pharmacy, Jilin University, Changchun, China

ABSTRACT This study aimed to identify hibifolin as a sortase A (SrtA) inhibitor and to
determine whether it could attenuate the virulence of methicillin-resistant Staphylococcus
aureus (MRSA). We employed a fluorescence resonance energy transfer (FRET) assay to
screen a library of natural molecules to identify compounds that inhibit SrtA activity.
Fluorescence quenching assay and molecular docking were performed to verify the direct
binding interaction between SrtA and hibifolin. The pneumonia model was established
using C57BL/6J mice by MRAS nasal administration for evaluating the effect of hibifolin
on the pathogenicity of MRSA. Herein, we found that hibifolin was able to inhibit SrtA
activity with an IC50 of 31.20 m g/mL. Further assays showed that the capacity of adhesion
of bacteria to the host cells and biofilm formation was decreased in hibifolin-treated
USA300. Results obtained from fluorescence quenching assay and molecular docking

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indicated that hibifolin was capable of targeting SrtA protein directly. This interaction
was further confirmed by the finding that the inhibition activities of hibifolin on mutant
SrtA were substantially reduced after mutating the binding sites (TRP-194, ALA-104, THR-
180, ARG-197, ASN-114). The in vivo study showed that hibifolin in combination with
cefotaxime protected mice from USA300 infection-induced pneumonia, which was more
potent than cefotaxime alone, and no significant cytotoxicity of hibifolin was observed.
Taken together, we identified that hibifolin attenuated the pathogenicity of S. aureus by
directly targeting SrtA, which may be utilized in the future as adjuvant therapy for S. aur-
eus infections.
IMPORTANCE We identified hibifolin as a sortase A (SrtA) inhibitor by screening the nat-
ural compounds library, which effectively inhibited the activity of SrtA with an IC50 value
of 31.20 m g/mL. Hibifolin attenuated the pathogenic behavior of Staphylococcus aureus,
including adhesion, invasion, and biofilm formation. Binding assays showed that hibifolin
bound to SrtA protein directly. Hibifolin improved the survival of pneumonia induced by Editor Kunyan Zhang, University of Calgary
S. aureus USA300 in mice and alleviated the pathological damage. Moreover, hibifolin Copyright © 2022 Song et al. This is an open-
access article distributed under the terms of
showed a synergistic antibacterial effect with cefotaxime in USA300-infected mice. the Creative Commons Attribution 4.0
International license.
KEYWORDS hibifolin, methicillin-resistant Staphylococcus aureus, sortase A,
Address correspondence to Yicheng Zhao,
pneumonia, cefotaxime yichengzhao@live.cn.
The authors declare no conflict of interest.

S
Received 15 March 2022
taphylococcus aureus, a typical Gram-positive bacterium, is a dreadful pathogen Accepted 12 July 2022
infecting multiple tissues and organs in various vertebrate species, ranging from Published 1 August 2022
humans to livestock (1). Antibiotic resistance has long been a worldwide health problem

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Hibifolin Is an Inhibitor of SrtA Microbiology Spectrum

due to the overuse and misuse of antibiotics (2). Methicillin-resistant Staphylococcus aureus
(MRSA) and even vancomycin-resistant S. aureus (VRSA) have already threatened human
health. MRSA has gained over 60% of all isolated S. aureus, and the incidence of MRAS has
risen to 49% in the United States (3). Novel pathogen-specific therapeutic strategies for
the prevention or treatment of serious bacterial infections have emerged, such as mono-
clonal antibodies (MAbs), nanomaterials, phages, and anti-virulence treatments (4–6).
Therefore, innovative anti-infective agents are urgently needed to overcome the antibiotic
resistance problem.
At present, the antivirulence treatment strategy has been an alternative approach
that focuses on interfering with bacterial virulence factors instead of inhibiting bacte-
rial growth or killing bacteria to treat infection (7). Virulence factors contain a variety of
functions, including the direct effectors of bacterial pathogenicity and essential factors
involved in host infection, host colonization, invasion of host tissues, adaptation to var-
ious host environments, destruction of host functions, and evasion of host defenses
(8). Antivirulence strategy holds tremendous promise to be the next generation candi-
date for MRSA infection therapies.
Sortase A (SrtA) is a transpeptidase found in Gram-positive bacteria that can anchor
surface proteins to the cell surface and recognize the C-terminal short peptide sequence
LPXTG of the substrate protein, which forms an amide bond with the N-terminal glycine
to the cell surface (9, 10). These surface proteins mediated by SrtA play an important role
in bacterial adhesion, colonization, biofilm formation, and immune evasion (11, 12).
Moreover, as a bacterial membrane enzyme, it is more likely to target intracellular pro-
teins, making it an ideal anti-virulence target (13). The development of SrtA inhibitors
has been an ideal strategy to combat antibiotic-resistant pathogen infections (14, 15).
Hibifolin, a flavonol glycoside, is isolated from the flowers of Abelmoschus manihot
(Linn.) Medicus (16). The biological consequences and applications of hibifolin have
been studied, including preventing Alzheimer’s disease (17), myocardial and cerebral
ischemic injury (16), and possessing anti-inflammatory activities (18). In this study, we
demonstrated that hibifolin attenuated the pathogenicity of S. aureus by directly tar-
geting SrtA, which may be utilized in the future as adjuvant therapy for S. aureus
infections.

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Our previous study identified that the natural compound scutellarin has anti-SrtA
activity (19). As a follow-up study, hibifolin was identified as a novel natural compound
targeting SrtA by fluorescence resonant energy transfer (FRET) screening from our nat-
ural compound library. We determined that hibifolin disrupted the biological function
of SrtA and displayed a protective effect against MRSA-induced lethal pneumonia,
which may provide a new candidate for effective control of MRSA infection.

RESULTS
Hibifolin inhibited the activity of SrtA. We first identified SrtA inhibitors from the
natural compound using detection of the fluorescence intensity of the fluorescent sub-
strate peptide Abz-LPATG-Dap (Dnp)-NH2 (Fig. 1A, left), which identified that the activity of
hibifolin remarkably inhibited the activity of SrtA with an IC50 of 31.20 m g/mL (Fig. 1C).
Subsequently, CETSA assays were further used to assess whether hibifolin was also an in-
hibitor of ClpP, MgrA or AgrAc and if there was a direct interaction between them (Fig. 1A,
right). As shown in Fig. S2 in Supplemental File 1, the thermal stability of ClpP, MgrA, or
AgrAc did not change with the increase in temperature, demonstrating that hibifolin was
not an inhibitor of these three proteins. Next, we evaluated the antibacterial activity of
hibifolin on S. aureus USA300. The MIC of hibifolin against USA300 was 512 m g/mL, along
with the growth curve data (Fig. 1D), indicated that hibifolin barely influenced S. aureus
USA300 growth. To assess the cytotoxicity, we performed MTT assays on A549, HepG2,
and HEK-293T cell lines. As shown in Fig. 1E to G, there was no significant difference in cell
viability among the groups, indicating hibifolin was almost noncytotoxic, at least to these
cells. Hibifolin was able to inhibit the activity of SrtA and neither affected bacterial growth

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Hibifolin Is an Inhibitor of SrtA Microbiology Spectrum

FIG 1 The inhibitory effect of the hibifolin on SrtA activity. (A) FRET and CETSA were used to screen inhibitors from natural
small molecule compounds. (B) The chemical structure of hibifolin. (C) The effect of hibifolin on SrtA activity with IC50 of
31.20 m g/mL. (D) Growth curves of S. aureus USA300 with or without hibifolin treatment (256 m g/mL). The viability of HepG2
(E), HEK-293T (F), and A549 (G) cells viability measured by MTT assay after being exposed to various concentrations of
hibifolin (0 to 256 m g/mL).

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nor had cytotoxicity. These characteristics suggested that hibifolin has potential for devel-
opment as a SrtA inhibitor.
Hibifolin inhibited the adhesion of S. aureus to fibrinogen. The biological conse-
quence of SrtA was evaluated under the intervention of hibifolin by the following
experiments. An adhesion assay was performed to access the ability of hibifolin-treated
S. aureus to adhere to fibrinogen. The result showed that the adhesion decreased to
26.85 6 3.35% in the presence of 256 m g/mL of hibifolin compared to that in the
USA300 group (Fig. 2A). The anchoring of surface proteins with LPXTG motifs to the
cell wall was regulated by SrtA, leading to the formation of biofilms. We employed a
crystal violet staining assay to assess biofilm formation. The results showed that hibifo-
lin significantly inhibited biofilm formation, even at a low dose of 32 m g/mL, compared
with that in the USA300 group (Fig. 2B). Surface protein A (SpA) served as a virulence
factor that specifically combines with Fc region of IgG, thereby increasing the odds of
immune evasion by interfering with opsonophagocytic clearance. To further detect the
effect of hibifolin on the S. aureus USA300 surface protein, the fluorescence intensity of
IgG labeling with FITC was assayed by flow cytometry to reflect the effect of hibifolin
on the level of SpA. As shown in Fig. 2C, compared with the control group, the fluores-
cence was decreased in the hibifolin-treated group. In addition, when A549 cells were
infected by S. aureus pretreated with hibifolin, the number of bacteria in the cells
decreased significantly compared to the vehicle group (4.77 6 0.67 versus 7.93 6 0.36,
P , 0.05), indicating that hibifolin could reduce the invasion of S. aureus on A549 cells

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FIG 2 Effects of hibifolin on the sortase A-related virulence factors. (A) Multiple concentrations of hibifolin suppressed
the adherence of S. aureus USA 300 to fibrinogen protein. (B) Biofilm formation is observed and quantified by staining
with crystal violet. Hibifolin suppresses the S. aureus USA300 biofilm formation. (C) S. aureus surface protein (SpA) level is
measured by flow cytometry using FITC-conjugated rabbit IgG. DsrtA and USA300 represent a positive-control group and
a negative-control group, respectively. (D) Different concentrations of hibifolin-treated S. aureus significantly inhibited
bacterial invasion of A549 cells. (E) Live/dead staining confirmed that hibifolin reduced damage to A549 cells by S.
aureus, and consistent results were obtained from LDH release assays (F).

(Fig. 2D). Live/dead staining and LDH release assays further revealed that the killing
ability of hibifolin-treated S. aureus on A549 cells was significantly attenuated (Fig. 2E
and F). These results demonstrated that hibifolin inhibited the SrtA-related virulence
phenotypes, including adhesion, invasion, and biofilm-forming functions of MRSA in
vitro.
Hibifolin directly targeted SrtA. To verify that hibifolin could directly bind to SrtA,
Western blot and fluorescence quenching assays were carried out. The results showed
that the expression of SrtA protein after treatment with different concentrations of
hibifolin (0 to 256 m g/mL) was comparable to that of the untreated group, indicating
that hibifolin did not affect the expression of SrtA (Fig. 3A). Fluorescence quenching
analysis was employed to detect the binding affinity between hibifolin and SrtA. As
shown in Fig. 3B, Hibifolin suppressed the fluorescence intensity of SrtA in a dose-de-
pendent manner. We assessed the binding constants KA for hibifolin binding to SrtA by
using fluorescence quenching experiments. The KA value was 1.72  104 L/mol, reveal-
ing a direct binding interaction between SrtA and hibifolin.

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FIG 3 Determination of the binding sites for the hibifolin on SrtA. (A) Expression of different concentrations of
hibifolin on SrtA expression by Western blot, which shows no significant effect. (B) The substrate-binding affinity of
hibifolin was measured by SrtA fluorescence quenching. SrtA was treated with a 10-fold IC50 of hibifolin and then
diluted the concentration. The group without hibifolin is assumed as 100% activity and the binding affinity is
indicated by KA. (C) Reversible inhibitory effect of hibifolin on SrtA. (D) Molecular modeling predicted the interaction
between hibifolin and SrtA. (E) Five mutants TRP-194, ALA-104, THR-180, ASN-114, and ARG-197 showed increasing
resistance to hibifolin inhibition. Relative activities of recombinant SrtA were quantified via FRET assays.

To further analyze whether the inhibition of SrtA by hibifolin was reversible, SrtA
was mixed with a 10-fold IC50 concentration of hibifolin and cultured with fluorescent
peptide substrates. The recovery rate was 83.99 6 2.06% compared with the control

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group (Fig. 3C), demonstrating that hibifolin acted as a reversible inhibitor of SrtA,
which bonded noncovalently to the active site of SrtA.
Furthermore, molecular docking elucidated the binding mode between hibifolin
and SrtA using Autodock vina 1.1.2 according to a previously documented procedure
(20). The 3D structures were downloaded from the Protein Data Bank. We drew the 2D
structure via the ChemBioDraw Ultra 14.0 (Fig. 3D). Discovery studio was used to ana-
lyze the interaction of docking. The molecular docking simulation indicated that hibifo-
lin was bound to the binding pocket of SrtA, depending mostly on hydrogen bonding,
and electrostatic interactions. In addition, it is well known that hydrogen bonding
interactions play a crucial role in protein-ligand interaction and make an important
contribution to the total binding affinity. In the present study, we analyzed the hydro-
gen bond networks and there were 5 hydrogen residues around the SrtA binding sides:
THR-180, ASN-114, ALA-104, AGR-197, and TRP-194. Specifically, AGR-197 served as an
important site in the SrtA active pocket while the side chain NH of AGR-197 formed
two moderate frequency electrostatic and a hydrogen bond (Table S4 in Supplemental
File 1). The molecular docking score was 26.8 kcal/mol. Following this lead, the activity
of hibifolin on the mutated SrtA (TRP-194, ALA-104, THR-180, ARG-197, and ASN-114)
was significantly lower than that of the wild-type (WT) group, and in particular, the
ARG-197 mutant USA300 almost completely lost its activity, confirming the pivotal role
of ARG-197 in the binding process of SrtA and hibifolin. In summary, these results veri-
fied the direct binding interaction between SrtA and hibifolin.
Hibifolin reduced lung damage induced by S. aureus. The finding that hibifolin
inhibited SrtA in vitro prompted us to evaluate the therapeutic effect of hibifolin in

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FIG 4 Protective effect of hibifolin and cefotaxime combination against lethal pneumonia in MRSA-infected mice. (A)
Flow chart of MRSA-induced mouse pneumonia infection model. (B) Survival rate of C57BL/6J mice treated with
hibifolin (100 mg/kg/d) or combination (hibifolin [100 mg/kg/d] 1 cefotaxime [40 mg/kg/d]) which infected with a

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lethal dose of S. aureus USA300. (C) CFU number in lung tissues. (D) Gross pathological changes in the lungs were
observed by HE staining. Scale bar, 1 cm and 100 m m, respectively. (E–G) The levels of inflammatory cytokines (IFN-g;
IL-6; TNF-a) in the alveolar lavage fluid of each group of mice were determined by ELISA. **, P , 0.01; ***, P , 0.001
compared with the USA300 group; #, P , 0.05, comparisons were considered statistically significant.

vivo against MRSA-induced pneumonia in mice. First, the checkerboard assays were
used to screen for antibiotics synergistic with hibifolin. The results showed that the
fractional inhibitory concentration index (FICI) of cefotaxime combined with hibifolin
was 0.312 (FICI , 0.5 indicates synergy), which exhibited significant synergistic anti-
MRSA effects in vitro (Table S2 in Supplemental File 1). Therefore, the therapeutic effect
of the combination of hibifolin and cefotaxime on MRSA-induced pneumonia in mice
could be further evaluated. In survival experiments, the survival rate of the infected
mice was only 10% after 72 h of infection with S. aureus (2  108 CFU), while the sur-
vival rate of the cefotaxime-treated group was 60%. The combination of hibifolin and
cefotaxime increased survival by 20% compared to cefotaxime alone. There was no sig-
nificant difference between cefotaxime treatment and the combination treatment (P =
0.27; Fig. 4B). In addition, compared with the infected mice group, the combination of
hibifolin and cefotaxime markedly reduced the number of colonies in lung tissue. The
difference between cefotaxime treatment and the combination treatment groups was
statistically significant (P , 0.05, Fig. 4C). These data revealed that hibifolin in combina-
tion with cefotaxime protected mice from USA300 infection-induced pneumonia,
which was more potent than cefotaxime alone. In terms of appearance and pathologi-
cal changes, USA300-infected mice showed inelastic lungs with alveolar infiltration and

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severe damage observed under microscopy. In comparison, the lung tissues of the
hibifolin in combination with the cefotaxime-treated group appeared to have a
smooth, pinkish appearance with no obvious pathological changes. We then con-
firmed that hibifolin had a therapeutic effect on MRSA-infected pneumonia (Fig.4D). In
addition, the levels of interferon (IFN)-g, interleukin (IL)-6, and tumor necrosis factor
(TNF)-a in the lung perfusate were significantly lower in the coadministered group
(Fig. 4E to G). In conclusion, hibifolin reduced the virulence of S. aureus in vivo and in
vitro, providing significant protection against pneumonia caused by S. aureus.

DISCUSSION
Staphylococcus aureus is a prominent cause of infection in health care settings, with
multidrug resistance to almost all known antibiotics (21). Therefore, to cope with the
challenges brought about by the rapid emergence of drug resistance, developing
novel anti-MRSA-infected therapy must be prioritized. Sortase A is essential for the
pathogenicity of S. aureus. SrtA catalyzes the covalently binding of various surface viru-
lence factor proteins to bacterial cell walls and plays a key role in the pathogenesis of
Gram-positive bacteria, such as adhesion to host tissues, evasion of host defenses, and
biofilm formation (12). Knockout of srtA inhibits the assembly and display of functional
surface adhesins in the cell wall envelope and the ability to produce an acute infection
(22). Therefore, interference with surface protein anchoring by targeting SrtA is a
promising anti-virulence strategy to attenuate the pathogenicity of S. aureus.
At the present stage, available SrtA inhibitors include natural products, synthetic small
molecule compounds, and designed peptide-analogs (23). Natural products provide an im-
portant source of SrtA inhibitors, and although this process is time-consuming, it may lead
to the discovery of unexpected lead compounds. In addition, it has been suggested that
the low incidence of infectious diseases in wild plants is because of the production of nat-
ural antimicrobial substances (24). These antimicrobial molecules have weak antimicrobial
activity and may act in synergy with their production of natural anti-virulence substances
that resist bacterial adhesion and invasion. For example, plants of the genus Berberis pro-
duce the low potency antibacterial substance berberine and 59-methoxyhydnocarpin, of
which 59-methoxyhydnocarpin potentiates the antibacterial activity of berberine (25). This
may partly explain the considerable amount of SrtA inhibitors in natural products.

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Flavonoids are classified into 12 major subclasses based on chemical structures, such as
anthocyanidins, flavanols, flavonols, flavones, flavanones, flavanonols, isoflavones, and
others (26). In addition, each class of flavonoids can form glycosides (a genin with a flavo-
noid structure, containing a glycosidic moiety). The sugars can be joined to the flavonoid
by tan O and by a C (among others), forming O-glycosides and C-glycosides (27). In our
previous series of studies, we focused on SrtA and ClpP virulence targets. Until now, we
have found that scutellarin (19) targets both SrtA and ClpP, quercetin (28), and myricetin
(29) target ClpP. It is worth noting that although SrtA and ClpP have great differences in
structure and biological functions, the inhibitors of these two targets screened from
numerous natural compounds with different molecular structures belong to flavonoids.
Although flavonoid inhibitors of these virulence targets have anti-infective effects, they
also have their unique pharmacological effects, which provide more options for infected
patients suffering from an underlying disease. What is noteworthy is that we have focused
on the pan assay interference compounds (PAINS) (30), and we found that there was no
PAINS group in hibifolin by searching the database (http://www.swissadme.ch/index.php).
PAINS is usually a type of compound with a high hit rate that disturbs screening or detec-
tion methods. Its low potency is also an important factor influencing drug development.
With our accumulation of anti-virulence studies on many natural compounds, it
appears that flavonoids and flavonol glycosides differ in their sensitivity to SrtA and ClpP.
By listing a dozen of flavonoids and flavonol glycosides with their SrtA or ClpP relative inhi-
bition rate, respectively (Table S5 in Supplemental File 1), we found that quercetin had the
activity of inhibiting ClpP, while hibifolin, glycosylated quercetin, showed SrtA inhibiting
activity. In addition, we enumerated two pairs of representative molecular structures that

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FIG 5 Hibifolin reduced the formation of biofilm of S. aureus and attenuated the adhesion and invasion to the host and the
anchoring of surface proteins by inhibiting SrtA.

conform to glycosylation of flavonoids, “hibifolin-quercetin” and “scutellarein-scutellarin”.

We focused on the following three indexes: inhibition rates of SrtA and ClpP, molecular
docking data, and Gaussian data. First, the results of molecular docking in Fig. S3 in
Supplemental File 1 showed that the flavonoid and the corresponding flavonoid glycoside
both bind to the same site, the biding pocket, of SrtA or ClpP. Exceptionally, scutellarin
prefers to bind the inactive site, which may also affect the polymerization of the ClpP pro-
tein which in turn affects its activity. From the Gaussian data and molecular data, we con-
cluded that SrtA and ClpP affinity (docking score) increased with the increase of Gaussian
DG, molecular volume, and steric hindrance energy after glycosylation, which seemed to
imply that the glycosylation of flavonoids enhanced the affinity of flavonoids to both SrtA
and ClpP (Table S6 in Supplemental File 1). The experimental data suggested that the sec-
ond pair of data conforms to the rule we obtained from the virtual experiments above: gly-
cosylation enhances the sensitivity to ClpP and SrtA. However, we did not find such a rule

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from the first pair. Although we have attempted to analyze the effect of glycosylation of
flavonoids on the sensitivity of SrtA and ClpP, the evidence is far from adequate. We can
only draw a vague conclusion from our existing data that glycosylation of flavonoids
appears to enhance their sensitivity to ClpP and SrtA. As more evidence accumulates in
the future, the structure-activity relationship will become clear.
Studies have demonstrated that hibifolin has neuroprotective effects, as evidenced
by protection against neuronal cell damage caused by amyloid b -protein as well as
potential antidepressant effects (17). Our findings herein expand the anti-infective
application of hibifolin. Natural compounds tend to be multitargeted compared to syn-
thetic compounds by virtual screening. We, therefore, speculate that hibifolin may
have multiple protective effects against severe S. aureus infections due to its pharma-
cological activity in addition to its anti-infective activity. Hibifolin reduced the virulence
of S. aureus by inhibiting SrtA activity. We identified hibifolin from hundreds of natural
compounds as a candidate capable of inhibiting SrtA activity with the IC50 of 31.20 m g/
mL using an established screening method for SrtA inhibitors. We consider that hibifo-
lin reduced the virulence of S. aureus by inhibiting SrtA activity, as SrtA is one of the
most essential virulence factors in S. aureus. As shown in Fig. 5, hibifolin prevented
SrtA from covalently anchoring surface proteins to the cell wall. Therefore, these sur-
face proteins cannot play a role in bacterial adhesion and invasion of host tissues, bio-
film formation, and immune evasion through inhibition and phagocytosis. Hibifolin
visibly attenuated the virulence-related phenotype of SrtA by decreasing the adhesion
of S. aureus to fibrinogen, reducing the ability of SpA displayed on the surface of the
bacteria and biofilm formation. The results of fluorescence quenching and molecular

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Hibifolin Is an Inhibitor of SrtA Microbiology Spectrum

docking revealed the interaction between SrtA and hibifolin. The in vivo results indi-
cated that hibifolin could protect mice from lethal pneumonia brought on by MRSA.
The fluorescence intensity of fluorescence substrate Abz-LPETG-Dnp-NH2 was detected
using FRET technology. FRET technology is a powerful tool for detecting nanometre-scale
distances and changes in nanometre-scale distances between biological macromolecules
in vivo, which is widely used to detect whether there is a direct interaction between two
protein molecules in each cell (31). In Shen et al. (32) study, FRET technology was used to
screen for compounds that promote the formation of A1R and D1R heterodimers. For
another, the initial DNA melting screen was performed using a FRET DNA melting-based
assay (33).
We agree with Cascioferro et al. (34), that the future of antivirulence agents should
be in combination with antimicrobial or immune-activating drugs, which may not be
strong enough on their own. In this study, we combined hibifolin with cefotaxime, the
preferred first-line antibiotic for MRSA infection (35). Data from in vivo study showed
that hibifolin exhibited a significant protective effect against USA300-induced lethal
pneumonia, as evidenced by increased survival, improved pathological manifestations,
and reduced levels of the inflammatory cytokine.
The available evidence is not enough to clarify the structure-activity relationship of
flavonoid inhibition on virulence targets of S. aureus. We also look forward to the sub-
sequent work on the structural optimization of hibifolin as a leading compound and
the elucidation of the structure-activity relationship. In addition, there is a need for
pharmacokinetic and safety information on hibifolin.
As a SrtA inhibitor of S. aureus, hibifolin significantly attenuates the pathogenicity
of S. aureus and enhances the antibacterial activity of cefotaxime. Due to the properties
of reducing the virulence and producing less selective pressure on MRSA, we should
expect hibifolin to be applied as an adjuvant therapeutic agent for MRSA infections,
which may help to reduce the use of antibiotics and delay the development of antibi-
otics resistance.

MATERIALS AND METHODS

Strains and reagents. Staphylococcus aureus USA300 was purchased from ATCC (Manassas, VA,
USA). Staphylococcus aureus USA300 SrtA deletion strain (DsrtA) and pET28a-SrtA plasmid were estab-

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lished in our laboratory. Bovine fibrinogen was purchased from Yuanye Biology (Shanghai, China). A flu-
orescent substrate peptide Abz-LPATG-Dap (Dnp)-NH2 was purchased from LifeTein, LLC (Beijing, China).
Hibifolin (purity . 98%) was purchased from Pufei De Biotech Co., Ltd. (Chengdu, China), and the graph
of HPLC identification results is presented in Fig. S1 Conventional antibiotics were purchased from
Sangon Biotech (Shanghai, China).
Construction of SrtA and its mutant plasmids. Construction of SrtA and its mutant plasmids was
performed as in our previous work (19). The DNA sequence encoding the SrtA residues Gln-60-Lys-206
was amplified by PCR, using S. aureus USA300 genomic DNA as a template. The pET-28a vector was
cloned after PCR products were digested in XhoI and BamHI. The pET-28a-SrtA DN59 plasmid was gener-
ated after sequence confirmation by DNA sequencing. Then, the Mut Express II Fast Mutagenesis kit V2
(Vazyme Biotech, Nanjing, China) was used for site-directed mutagenesis of TRP-194, ALA-104, THR-180,
ARG-197, and ASN-114. The mutant primer pairs used to create the mutants are shown in Table S1 in
Supplemental File 1.
Expression and purification of SrtA and its mutant protein. The recombinant pET28a-SrtA plas-
mid and its mutant plasmids were transferred into E. coli expression host BL21(DE3) by heat shock
method, respectively (36). The bacteria were then cultured until the optical density at 600 nm (OD600)
reached 0.8 and continued to culture with 0.5 mM IPTG at 16°C for 8 h. Based on the recombinant
pET28a-SrtA plasmid containing 6  His tags, therefore the induced protein was purified by Ni-NTA
(Beyotime Biotechnology, Shanghai, China). Subsequently, the nonspecific protein was eluted with
10 mM imidazole, and then the purified protein was eluted with 100 mM imidazole.
SrtA inhibitor screening. Fluorescence resonant energy transfer (FRET) was used to screen the
effect of the SrtA inhibitor (22). Purified SrtA (4 m M) and hibifolin with different concentrations were
added into a 100 m L reaction system, incubating at 37°C for 1 h. A fluorescent substrate peptide Abz-
LPATG-Dap (Dnp)-NH2 (10 m M) (Beijing, China) was added to initiate the reaction. After incubating at
37°C for 20 min, the fluorescent microplate reader was measured with the excitation wavelength at
309 nm and the emission wavelength at 420 nm. Subsequently, a gradient concentration inhibition
curve was performed and IC50 was calculated. When the inhibitory activity was greater than 60%, the
compound was considered a potential inhibitor. The representative list of our nature compounds library
has been compiled in Table S3 in Supplemental File 1 along with the results of the initial screening.

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Hibifolin Is an Inhibitor of SrtA Microbiology Spectrum

Reversible inhibition experiment of SrtA. A reversible assay was performed in accordance with
the previous study (19). Ten-fold IC50 of hibifolin was added to 100 m L of the purified SrtA (4 m M) at 37°C
for 1 h. Then, the above reaction solution was diluted at 1:100. Subsequently, the substrate peptide
(10 m M) was added and incubated at 37°C for 20 min. The fluorescence intensity was read under the ex-
citation wavelength was 309 nm, and the emission wavelength was 420 nm.
Growth assays. Growth assays were carried out with reference to a previous literature report (37).
The overnight cultured S. aureus USA300 was diluted at 1:100 and transferred into fresh BHI broth with
or without 128 m g/mL hibifolin, culturing until the OD600 was 0.3. The bacterial solution was divided into
the control group (WT 1DMSO), drug group (WT 1 hibifolin), and DsrtA group. The growth was
recorded at 1 h intervals within 24 h. A UV spectrophotometer measured the growth curve at an absorb-
ance of 600 nm.
Checkerboard studies. The checkerboard microtiter assay (38) was applied for determining the
interaction of combinations of hibifolin with Cefotaxime, Doxycycline, Ceftriaxone Sodium, Vancomycin,
Oxacillin sodium, Ceftiofur Sodium, Cefepime, Ceftaroline fosamil, relatively. One hundred microliters of
CAMHB medium were added to a 96-well plate, followed by a fold dilution of hibifolin and antibiotics.
MRSA USA300 (105 CFU/well) was added to each well and the MIC was calculated as wells with the low-
est concentrations of drugs with no visible growth after incubation at 37°C for 16 h. The fractional inhibi-
tory concentration index (FICI) of each combination were calculated according to the following formula:
FICI = FICantibiotic 1 FIChibifolin = MICcombine-antibiotic/MICantibiotic 1 MICcombine-hibifolin/MIChibifolin. FICI # 0.5 indi-
cates synergism; 0.5 , FICI # 1 indicates additivity; 1 , FICI # 2 indicates irrelevance; FICI . 2 indicates
antagonism.
Cell viability. Cell viability was carried out as previously described (39). Human hepatocellular carci-
noma (HepG2), human kidney epithelial cell line (293T), and human non-small cell lung cancer (A549)
cells of 2  104 cells/well were seeded in 96-well plates for 24 h with 5% CO2, relatively. Different con-
centrations of hibifolin (0 to 256 m g/mL) were then added and maintained at 37°C for 24 h. Then,
0.5 mg/mL of MTT solution was incubated in 96-well plates after discarding the medium. The plates
were set in the cell incubator for 4 h and the crystals were dissolved with DMSO. The absorption at
490 nm was measured with a microplate reader.
Fibronectin-binding assay. A fibronectin-binding assay was completed as described but with slight
revision (40). Bovine blood fibrinogen (Fg) was added into 96-well plates at 20 m L/mL per well followed
by cultured overnight at 4°C. After being washed by PBS, 100 m L of 1% BSA was added into each well of
96-well plates and blocked the medium for 2 h. The cultures of S. aureus USA300 in the BHI medium and
placed on a shaker at 200 rpm for 24 h, and different concentrations of hibifolin were added, among
which the USA300, DsrtA, and BHI medium represented negative, positive, and control groups. The bac-
terial culture was cultured at 37°C until OD600 = 0.5. After incubating, the unbound bacteria were gently
washed using PBS and fixed with 4% formaldehyde for 30 min. After dying with crystal violet dye for
20 min, the absorbance at 595 nm was measured by a microplate reader.
Biofilm formation. The 96-well plates were added 100 m L of 20% freeze-dried rabbit blood at 4°C
for 24 h. Staphylococcus aureus USA300 or DsrtA were diluted at a ratio of 1:100 and cultured in BHI
broth supplementing with 0.5% glucose and 3% NaCl. Then, the solution in 96-well plates was carefully

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discarded, and 100 m L bacterial solution per well with different concentrations of hibifolin (32 to
256 m g/mL) was added to each well, respectively. The 96-well plates were cultured at 37°C for 24 h, then
cell plates were washed three times with PBS to remove unbound bacteria. Then biofilm was evaluated
by 0.1% crystal violet staining for 20 min (41). Gently rinsed unbound dyes with sterile PBS buffer and
blown dried at room temperature. After that, 200 m L of 95% ethanol was added to each well. Relative
biofilm formation was determined by measuring the absorbance at 570 nm.
Surface protein analysis. The overnight cultures of S. aureus USA300 and DsrtA were diluted 1:100 in
BHI medium. The cultures were then incubated on a shaker to an OD600 of 0.3. Hibifolin was added with dif-
ferent concentrations until the OD600 was 1.0. The bacteria were collected at 5,000 rpm for 5 min and washed
with PBS twice a time. After incubating with 0.5% BSA for 20 min, and rewashing with PBS, the USA300 were
resuspended with 4% formaldehyde and collected with centrifugation (40). The USA300 were resuspended
with 50 m L FITC labeled rabbit IgG (1:100) and incubated in the dark at 37°C for 2 h. After washing twice to
remove the dye, the bacteria were resuspended with PBS. The fluorescence intensity was measured by flow
cytometry (Beckman Coulter, USA) to evaluate the amount of SpA.
Cell invasion assays. The invasion of A549 cells by S. aureus was detected as previously described
(40). A549 cells were incubated in 24-well plates at 2  104 per well overnight (37°C, 5% CO2). Overnight
cultured S. aureus USA300 and DsrtA were diluted 1:100 into a 1 mL system and hibifolin with a final con-
centration of 256,128,64,32 m g/mL was added. Then the bacteria solution was incubated until the OD600
reached 1.0 with a shaker rate of 200 rpm at 37°C. USA300 and DsrtA were set as negative and positive
controls. Then, 400 m L bacterial solution was added to 24-well plates and cultured in a 37°C biochemical
incubator for 2 h. After each well was washed three times with PBS, 1 mL of DMEM supplemented with
300 m g/mL gentamicin was added and incubated for 1 h at 37°C. The A549 cells on the coverslips were
then lysed with 1% Triton X-100 for 30 min and plated onto BHI broth agar plates for CFU.
Live/dead assays. The analysis of live/dead assays was performed using a previously described
method (42). A549 cells were cultured in DMEM, with 10% fetal bovine serum (FBS). After being digested
with 0.25% trypsin, the cells were seeded in 24-well plates at 1  105 per well and cultured at 37°C, 5%
CO2 for 24 h. Next, fresh DMEM with S. aureus and different concentrations of hibifolin (32 and 128 m g/
mL) was used to replace the culture medium and was incubated for 5 h at 37°C. Then the Live/Dead
(green/red) reagent (Beyotime, Shanghai, China) was used to assess the therapeutic effect of hibifolin on
A549 cells using a confocal laser scanning microscope.

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Hibifolin Is an Inhibitor of SrtA Microbiology Spectrum

Cellular thermal shift assay (CETSA). E. coli BL21(DE3) containing pET28a-ClpP, MgrA, or AgrAc was
used to induce the relative expression of protein by adding 0.5 mM IPTG, and the supernatant of the
lysate was collected by centrifugation after ultrasonic fragmentation (43). The supernatant was incu-
bated with hibifolin at 37°C for 1 h through centrifugation at 18,000 g for 20 min at 4°C. The samples
were heated to various temperatures at a temperature gradient for 5 min, respectively. We then put
samples into ice water for 3 min immediately and gently centrifuged at 18,000g for 20 min to yield a su-
pernatant. After being analyzed by SDS-PAGE, the samples were incubated with Coomassie brilliant blue
G-250 stain, and the relative intensities of the indicated proteins were visualized using ImageJ software.
Western blot. To assess the effect of hibifolin on SrtA expression in S. aureus USA300, different con-
centrations of hibifolin (0 to 128 m g/mL) were added to S. aureus USA300 medium, shaking at 220 rpm
at 37°C until the OD600 value reached 2.0. The total proteins of S. aureus were isolated according to the
conventional method (44). The total proteins were separated by 12% SDS-PAGE, transferred to polyviny-
lidene fluoride (PVDF) membrane, blocked with 5% BSA for 2 h, and incubated with rabbit anti-SrtA pol-
yclonal antibody (1:3000, prepared in the laboratory) at room temperature. Following incubation with
HRP-labeled goat anti-rabbit IgG (1:10000) for 1 h at room temperature, the membrane was visualized
using a super ECL Plus (Beyotime, Beijing China) and the gray value of the target band was analyzed
using ImageJ software.
Fluorescence quenching. The binding constants of hibifolin with SrtA and the SrtA mutant were
measured by fluorescence quenching assay as previously described (40). The SrtA fluorescence emission
spectra were recorded in the absence or presence of different concentrations of hibifolin. The excitation
wavelength was set to 280 nm using a fluorescence spectrophotometer, and the emission spikes were
recorded at 280 to 400 nm. The widths of the excitation and emission slits were 5 and 10 nm, depending
on the width of the slit. The relative fluorescence intensity was plotted against different concentrations
of hibifolin based on fluorescence quenching to obtain data and KA values were calculated.
Molecular docking. Although the X-ray crystallography 3D structure (PDB accession no. 1T2P) was
in Protein Data Bank, the structure of SrtA was obtained. The 3D structures of hibifolin were constructed
by the ChemBio3D Ultra 12.0 software package. A standard docking procedure for a SrtA protein and
hibifolin was performed with AutoDock-Tools 1a.5.6 software. Finally, the discovery studio was used to
analyze the interaction of docking.
Murine pneumonia models. To investigate the therapeutic effect of hibifolin on acute pneumonia
caused by MRSA, the murine pneumonia models were established using C57BL/6J mice (18 to 22 g, 6 to
8 weeks old) (45). Mice were infected intranasally with 2  108 CFU of S. aureus USA300 suspension and
then held for 30 s to guarantee that each mouse would inhale bacteria into its lungs. After 1 h of infec-
tion, mice were injected subcutaneously with hibifolin (100 mg/kg/d) or combination (hibifolin [100 mg/
kg/d] 1 cefotaxime [40 mg/kg/d]). They were then reinjected in 12 h intervals, and the survival rate of
mice was recorded every 12 h for 96 h.
To further assess the therapeutic effect of hibifolin on pneumonia in mice, the bacterial load in the
lung tissues and histopathological changes in the lungs were analyzed. Mice in each group were allowed
to remain infected for up to 2 days by nasal drip injection of 30 m L (1  108 CFU) of S. aureus culture.
Then, mice were sacrificed using the cervical dislocation method, and the lungs were collected, weighed,

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and homogenized. The homogenate was then adequately diluted and spread on BHI agar plates. After
the plate was incubated overnight at 37°C, the number of colonies was counted. The left lung of each
group was collected, perfused, and fixed in 10% formalin. The pathological changes in the lung tissue
were observed under a light microscope after staining with routine hematoxylin and eosin (H&E). In
addition, alveolar lavage fluid was collected from each group of mice and changes in inflammatory fac-
tors (IFN-g, IL-6, and TNF-a) were measured by ELISA kit (Sbjbio, Nanjing, China).
Statistical analysis. The statistical significance of the treated and USA300 group was assessed using
the log-rank tests for the survival curves (**, P , 0.01; ***, P , 0.001, compared with the USA300 group). For
other assays, a Student's t test was used. P , 0.05 was considered statistically significant. The differences
were analyzed using GraphPad Prism 8.0 statistical software. The data are presented as the mean 6 SD.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only.
SUPPLEMENTAL FILE 1, PDF file, 1.4 MB.

ACKNOWLEDGMENTS
The animal experiments were conducted following the principles of the
Experimental Animal Ethics Committee of the Changchun University of Chinese
Medicine. The ethical approval number was 2021156.
Y.Z. designed the study. X.W. participated in the expression and purification of the
SrtA protein. B.W. and J.G. participated in the SrtA inhibitor screening and SrtA-related
assay. X.G. and Y.M. participated in the TSA and CETSA assay. W.W., Y.Z., and G.L.
participated in the WB and Urease activity assay. J.G., L.W., and X.G. participated in the
animal experiment and molecular docking. L.W. and X.W. conducted the analysis. W.S.
and L.W. drafted the manuscript. All authors read and approved the final manuscript.
We declare no conflict of interest.

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Hibifolin Is an Inhibitor of SrtA
This work was partly supported by the National Natural Science Foundation of China
(grant no. 81860567), Science and Technology Development Plan Project of Jilin
Province Science and Technology Department (grant no. 20200404085YY), “Xinglin
Scholar Project” of Changchun University of Chinese Medicine (grant no. QNKXJ2-
2021ZR05) and Technology Project of Education Department of Jilin Province (grant no.
JJKH20210952KJ).
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