Punicalagin, an Inhibitor of Sortase A, Is a Promising Therapeutic Drug to Combat Methicillin-Resistant Staphylococcus aureus Infections

ABSTRACT

Antimicrobial resistance (AMR) poses a major threat to human health globally. Staphylococcus aureus is recognized as a cause of disease worldwide, especially methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA). The enzyme sortase A (SrtA), present on the cell surface of S. aureus, plays a key role in bacterial virulence without affecting the bacterial viability, and SrtA-deficient S. aureus strains do not affect the growth of bacteria. Here, we found that punicalagin, a natural compound, was able to inhibit SrtA activity with a very low half maximal inhibitory concentration (IC₅₀) value of 4.23 μg/mL, and punicalagin is a reversible inhibitor of SrtA. Moreover, punicalagin has no distinct cytotoxicity toward A549, HEK293T, or HepG2 cells at a much higher concentration than the IC₅₀ detected by MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assays. In addition, punicalagin visibly attenuated the virulence-related phenotype of SrtA in vitro by decreasing adhesion of S. aureus to fibrinogen, reducing the ability of protein A (SpA) displayed on the surface of the bacteria and biofilm formation. Fluorescence quenching elucidated the interaction between punicalagin and SrtA. Molecular docking further implied that the inhibitory activity lay in the bond between punicalagin and SrtA residues LYS190, TYR187, ALA104, and GLU106. In In vivo studies, we surprisingly found that punicalagin had a more effective curative effect combined with cefotaxime when mice were infected with pneumonia caused by MRSA. Essentially, punicalagin, a therapeutic compound targeting SrtA, demonstrates great potential for combating MRSA infections.

INTRODUCTION

Gram-positive bacteria are a common cause of nosocomial bloodstream and other infections, and infections caused by methicillin-resistant Staphylococcus aureus (MRSA), in particular, have long been a major challenge for clinicians (1). The Gram-positive bacterium S. aureus is an isolated human bacterial pathogen in a variety of human infections, ranging from relatively mild superficial skin to deep tissue infections (2). Conventional antibiotics or other antimicrobial agents used to be considered promising ways to reduce the infection of S. aureus bacteria (3). However, inappropriate overprescription and irrational use of antibiotics in treating infectious diseases caused favorable conditions, exposure, and spread of resistant strains of different pathogens (4, 5). Thus, antibiotic treatment is often ineffective owing to the development of antibiotic-resistant strains, such as MRSA and vancomycin-resistant S. aureus (VRSA). The spread of MRSA infections has fallen, but the consequences of MRSA infection are still serious (6). Therefore, new treatment strategies that mitigate drug resistance while reducing bacterial infection are critically needed.

The virulence factors secreted by S. aureus are closely related to the pathogenicity of S. aureus, including surface proteins and secreted toxins and enzymes that facilitate bacterial adherence, tissue invasion and destruction, and host defense evasion (7, 8). The proposed antivirulence strategy provides a new idea for reducing the generation of bacterial resistance to conventional antibiotics. Unlike the traditional antibiotic treatment strategy, the antivirulence strategy does not produce survival pressure on S. aureus (9). As targeted inhibitors, antivirulence drugs directly act on the virulence factors of S. aureus, thus reducing the pathogenicity of S. aureus, which means disarming the pathogen rather than destroying it (10).

We have a fairly good structural and biological understanding of the sortase A (SrtA) enzyme involved in S. aureus virulence. SrtA, which catalyzes the cell wall sorting reaction, recognizes surface proteins containing LPXTG motifs and cleaves them between threonine and glycine residues, thereby anchoring the surface proteins to the cell wall of S. aureus (11, 12). These surface proteins, such as the fibrinogen-binding clumping factors ClfA and ClfB and the fibronectin (Fn)-binding proteins FnbA and FnbB, play essential roles in host invasion, biofilm formation, and immune evasion (13–16). Moreover, the widespread existence of SrtA in Gram-positive bacteria with low GC content implies that SrtA inhibitors can be developed against a variety of Gram-positive bacterial infections.

Punicalagin is a polyphenol ingredient isolated from pomegranate (Punica granatum L.) or the leaves of Terminalia catappa L. (17). Punicalagin has various bioactivities, including influenza neuraminidase inhibition (18), PTP1B inhibition, implicated in Alzheimer’s disease (19), and antioxidant and anti-inflammatory effects (20). Here, we reveal the antivirulence effect of punicalagin for the first time. As a virulence inhibitor of S. aureus SrtA, punicalagin significantly reduced the activity of SrtA and related phenotypes in vitro. In addition, in vivo punicalagin also had a significant therapeutic effect on pneumonia infection induced by S. aureus in mice. In conclusion, these results indicated that punicalagin is a promising therapeutic drug to combat MRSA infections by targeting SrtA, especially combined with cefotaxime.

RESULTS

Punicalagin inhibits the activity of SrtA.

We screened a natural compound library using the fluorescence resonance energy transfer (FRET)-based cleavage of LPXTG, and punicalagin was identified as a SrtA inhibitor. According to previous studies in our laboratory (21), some drugs will affect the AgrA protein; we want to explore whether this drug also affects AgrA, so cellular thermal shift assays (CETSA) were further used to assess whether punicalagin was an inhibitor of AgrA protein. (Fig. 1A). With increasing temperature, the thermal stability of AgrA did not change, as shown in Fig. S2 in the supplemental material, indicating no direct interaction between punicalagin and AgrA.

FIG 1.

Punicalagin inhibits the activity of SrtA. (A) Screening of SrtA and AgrA inhibitors from hundreds of natural compounds by FRET and CETSA. When inhibition activity was greater than 60%, the compound was considered a potential SrtA inhibitor. (B) The structure chart of punicalagin with its PubChem CID and molecular weight and the High Performance Liquid Chromatography (HPLC) of punicalagin is provided in Fig. S1. (C) A FRET assay was used to determine that punicalagin is an inhibitor of SrtA with an IC₅₀ of 4.23 μg/mL. (D) The growth curve of S. aureus USA300 with or without punicalagin (32 μg/mL). (E to G) Effects of punicalagin on the cytotoxicity of A549 (E), HEK293T (F), and HepG2 cells (G) determined by MTT assays.

We determined that the half maximal inhibitory concentration (IC₅₀) of punicalagin was 4.23 μg/mL (Fig. 1C). It is worth noting that the MIC of punicalagin against the S. aureus strain was greater than 512 μg/mL, demonstrating that it did not have antibacterial activity. In addition, the growth of S. aureus USA300 was not notably affected by punicalagin (32 μg/mL), which indicated that punicalagin did not produce survival pressure on S. aureus (Fig. 1D). In addition, we performed [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] MTT assays on A549 (Fig. 1E), HEK293T (Fig. 1F), and HepG2 (Fig. 1G) cells to determine the cytotoxicity. Data showed that punicalagin had no influence on cell viability under the 32 μg/mL concentration compared to the dimethyl sulfoxide (DMSO) group (P > 0.05). Punicalagin’s significant inhibition of SrtA activity and its advantages did not affect bacteria. The absence of toxicity suggested its potential for further development as an anti-S. aureus infection drug.

Punicalagin influences SrtA-related phenotypes of S. aureus.

Fibronectin (Fn) is regarded as a bridge between the bacterial adhesion FnBP and the mammalian cell integrin, playing a critical role in the process of phagocytosis (22). To determine if punicalagin could reduce the adherence of S. aureus to Fn, we employed Fn-binding assays. As shown in Fig. 2A, punicalagin inhibited the adhesion of S. aureus to fibrinogen in a dose-dependent manner. As expected, the ΔsrtA group showed the lowest adhesion rate, which was only 14.88 ± 0.71%. The result showed that the adhesion decreased to 16.14 ± 1.35% in 32 μg/mL of punicalagin compared with the USA300 group. These results indicated that the inhibition of SrtA by punicalagin reduced the adhesion of S. aureus to fibrinogen.

FIG 2.

The effect of punicalagin on SrtA-related phenotypes of S. aureus. (A) Effect of punicalagin on the adhesion of S. aureus USA300 to fibrinogen. (B) Effect of punicalagin on the internalization of S. aureus into A549 cells. (C) Effect of punicalagin on S. aureus USA300 biofilm formation. (D) Effect of S. aureus surface protein (SpA) stained with FITC-labeled rabbit IgG detected by flow cytometry analysis. (E) Live/dead reagent-A549 cells were observed with fluorescent imaging. (F) The LDH release by A549 cells treated with punicalagin. *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with the untreated group. The experiment was repeated at least three times.

To investigate the effect of punicalagin on A549 cells that were infected by S. aureus, the bacteria were pretreated with different concentrations of punicalagin and then used to infect A549 cells. The number of surviving colonies in A549 cells was counted. As shown in Fig. 2B, the number of S. aureus bacteria entering the cells in the ΔsrtA group was the lowest compared with the DMSO group, and punicalagin decreased the quantity of bacteria in a dose-dependent manner, indicating that punicalagin weakened the S. aureus invasion.

In addition, to examine whether punicalagin prevents biofilm formation, an S. aureus biofilm assay was performed. It was visibly observed that punicalagin reduced biofilm formation in a dose-dependent manner by crystal violet staining assay; however, the ΔsrtA group formed very few biofilms (Fig. 2C). The inhibition of biofilm formation by 32 μg/mL punicalagin was similar to that observed in the ΔsrtA group. These results suggested that punicalagin reduced biofilm formation by inhibiting SrtA activity.

Staphylococcal protein A (SpA) is an important virulence factor of S. aureus. SpA has an LPXTG motif and is a substrate for SrtA. SpA binds immunoglobulins via their Fcγ domain and the Fab heavy chains of V_H3 clan antibodies, interfering with opsonophagocytosis of bacteria and the development of adaptive B cell responses (23, 24). After punicalagin combined with fluorescein isothiocyanate (FITC)-labeled IgG, the level of SpA anchored on the bacterial surface was further quantified using flow cytometry. As shown in Fig. 2D, the fluorescence intensity of the ΔsrtA was weak, only 824.30 ± 35.92 a.u., indicating that SpA did not anchor on the cell wall when SrtA was absent. In addition, the DMSO group showed a strong fluorescence intensity, and the median value was 6,691.00 ± 1,112.00. However, the fluorescence intensity decreased when S. aureus was treated with 32 μg/mL of punicalagin, and the median value was 3,632.00 ± 1,35.10 (P < 0.001). These data revealed that punicalagin reduced the anchoring of SpA to the bacterial cell wall by inhibiting SrtA.

In the live/dead assay, uninfected cells retained green fluorescence, and inversely, injured cells retained red fluorescence (Fig. 2E). Simultaneously, the lactate dehydrogenase (LDH) assay showed that treatment with punicalagin could significantly decrease the release of LDH into the supernatants in a dose-independent manner compared with the DMSO group, indicating less cell death (Fig. 2F). These results showed that punicalagin had a protective effect on SrtA-mediated A549 cell injury.

In summary, these results revealed that punicalagin effectively inhibited the SrtA-related virulence phenotypes. Thus, we have a deeper understanding of the punicalagin biological function.

Punicalagin directly targets SrtA.

First, the expression level of SrtA was detected to test whether punicalagin affects the expression of SrtA by Western blot analysis. As shown in Fig. 3A, the expression level of SrtA in the punicalagin treatment group was similar to that in the DMSO group, indicating that punicalagin did not impact the expression of SrtA protein. Subsequently, a fluorescence quenching assay was employed to detect the binding affinity between punicalagin and SrtA. As Fig. 3B shows, the punicalagin suppressed the fluorescence intensity of SrtA in a dose-dependent manner. The binding constant (K_A) value was 6.26 × 10⁵ L/mol. The results showed that there was a strong interaction between punicalagin and SrtA. To further analyze the action between punicalagin and SrtA, we performed the reversible inhibition assay of SrtA. The activity of SrtA recovered to 84.52 ± 3.55% compared with the DMSO group, indicating that punicalagin was a reversible inhibitor of SrtA and was not covalently bound to the active site of SrtA (Fig. 3C). Molecular docking and site-directed mutation elucidated that the binding mechanism between punicalagin and SrtA. We found four key residues between punicalagin and SrtA (LYS190, TYR187, ALA104, and GLU106) (Fig. 3D), and the total binding free energy (ΔG_bind) was calculated to be −8.4 kcal/mol. The results of molecular docking guided us to perform point mutagenesis to assess the effect of the punicalagin on the transpeptidase activity of the mutant proteins. As shown in Fig. 3E, compared with the wild-type (WT) group, 32 μg/mL of punicalagin decreased the transpeptidase activity of the mutant proteins (A104G-srtA, E106A-srtA, Y187A-srtA, and K190A-srtA) to different degrees. These results indicated that E106, K190, Y187, and A104 were the potential amino acid sites for the binding of SrtA and punicalagin.

FIG 3.

Punicalagin combined to SrtA of S. aureus USA300. (A) Western blot assay to detect the expression of StrA in the S. aureus USA300 with different concentrations of punicalagin (0 to 32 μg/mL) (B) Binding affinity between punicalagin and SrtA determined by fluorescence quenching. The K_A was calculated by plotting the Stern-Volmer SrtA quenching. (C) Reversible inhibitory effect of punicalagin on SrtA. (D) The 3D structural determination of a SrtA with punicalagin complex by a molecular modeling method. (E) The inhibitory activity of WT SrtA and SrtA mutants (A104G-srtA, E106A-srtA, Y187A-srtA, and K109A-srtA) determined by FRET. ***, P < 0.001, calculated using one-way analysis of variance (ANOVA). (F to I) Binding affinity between punicalagin and SrtA mutants determined by fluorescence quenching. (J and K) The 3D structural determination of a SrtA with berberine chloride (J) or thiadiazole complex (K) by a molecular docking method.

To test the affinity of the hypothesized binding site to punicalagin which was made from docking experiments, we performed the fluorescence quenching assay. The K_A value between punicalagin and SrtA-A104G (Fig. 3F), SrtA-E106A (Fig. 3G), SrtA-Y187A (Fig. 3H), and SrtA-K190A (Fig. 3I) was 1.09 × 10⁵ L/mol, 1.11 × 10⁵ L/mol, 1.03 × 10⁵ L/mol, and 1.48 × 10⁵ L/mol, respectively. The K_A of SrtA mutants was significantly lower than that of SrtA, suggesting that A104, E106, Y187, and K190 are key amino acid residues for SrtA binding to punicalagin and that our hypothesis is right.

To compare punicalagin to previously published SrtA inhibitors, we chose two representative compounds for molecular docking. The first was a natural compound named berberine chloride (IC₅₀ = 23.44 μM), and the second was a synthetic compound named thiadiazole (IC₅₀ = 3.8 μM). Berberine chloride also exhibited potent inhibitory activity against S. aureus cell adhesion to Fn. The Fn-binding activity data highlight the potential of the treatment of S. aureus infections via inhibition of sortase activity (25). By molecular docking, we found that LYS69 and LEU110 were the two potential residues involved in the interactions between SrtA and berberine chloride (Fig. 3J), and the total binding free energy (ΔG_bind) was −6.9 kcal/mol. Thiadiazole also considered as a potent staphylococcal biofilm inhibitor (26). Molecular docking results revealed that LYS134, THR131, and PHE130 were the three potential residues involved in the interactions between SrtA and thiadiazole, with a ΔG_bind of −3 kcal/mol (Fig. 3K). The ΔG_bind of the SrtA-punicalagin complex was −8.4 kcal/mol. By comparing these three compounds, we found that punicalagin (IC₅₀ = 3.895 μM) had the strongest bond to SrtA. These results suggest that punicalagin is a direct inhibitor of SrtA and that there is a strong interaction between punicalagin and SrtA.

Punicalagin protected mice from MRSA-induced lethal pneumonia.

In order to increase the clinical application value of punicalagin, we screened several antibiotics to determine which had a synergistic effect with punicalagin using a checkboard assay (Table S3). The results showed that punicalagin had a better synergistic effect combined with cefotaxime and ceftriaxone sodium with a fractional inhibitory concentration index (FICI) value of 0.28125 and 0.3125, respectively (Fig. S3). As a FICI of ≤0.5 indicates synergism, 0.5 < FICI ≤ 1 indicates additivity, 1 < FICI ≤ 2 indicates irrelevance, and a FICI of >2 indicates antagonism; thus, we chose cefotaxime as the antibiotic to perform the following experiments.

As shown in Fig. 4B, no death occurred in the control group, while the survival rate was 50% in the ΔsrtA group, and the survival rate of mice infected with S. aureus USA300 was 20% within 96 h, indicating that the infection model of S. aureus was successfully established. The survival rate within 96 h of punicalagin and cefotaxime alone was 40% and 60%, respectively. When punicalagin and cefotaxime were combined together, the survival rate of mice could be significantly increased to 70% (n = 10). The mortality rate of mice in the treatment group was significantly lower than that in the control group at 24 h, 36 h, 48 h, 60 h, and 96 h after infection, indicating significant synergistic effects in the in vivo study (**, P < 0.01; ***, indicates P < 0.001 compared with the USA300 group). The combination of punicalagin and cefotaxime significantly improved survival in mice infected with pneumonia.

FIG 4.

Punicalagin protected mice from S. aureus infection. (A) Schematic diagram of a survival and mouse pneumonia model. (B) Effect of punicalagin on survival after 96 h in C57BL/6J mice (n = 10). **, P < 0.01; ***, P < 0.001 compared with the USA300 group. (C) The bacterial load of lung tissue (n = 5). (D) The histopathology of lung tissue detected by HE staining (n = 5). Scale bars, 1 cm and 100 μm. (E to G) ELISA for the secretion level of inflammatory factors (IFN-γ, IL-6, and TNF-α) in the alveolar lavage fluid of each group of mice (n = 3).

After mice were infected with S. aureus for 48 h, the lung tissue was ground, and the colony number was determined. As shown in Fig. 4C, the number of bacterial colonies in the lung tissues of mice in the infected group was 9.90 ± 0.73 log₁₀ CFU/g, and the number of viable bacterial colonies in the lung tissues was reduced to 6.74 ± 0.28 log₁₀ CFU/g by treatment with punicalagin (n = 5). The number of viable bacterial colonies in the cefotaxime treatment group was 5.19 ± 0.50 log₁₀ CFU/g, while the number of viable bacterial colonies in the punicalagin and cefotaxime combined group was only 3.92 ± 0.27 log₁₀ CFU/g, indicating that the combination of punicalagin and cefotaxime could significantly reduce the number of bacterial colonies in the lung tissue. Punicalagin combined with cefotaxime had certain therapeutic effects on MRSA strain USA300 pneumonia.

The results showed that the lung tissues in the combined group of punicalagin and cefotaxime were pink and spongy with less local infection, while the lung tissues of untreated mice were dark red with more serious infection (n = 5). Hematoxylin and eosin (HE) staining and pathological tissue analysis results showed that mostly the alveolar cavities of the lung tissues in the infection group were infiltrated by inflammatory cells, while the infiltration of inflammatory cells was significantly reduced in the combined group of punicalagin and cefotaxime, and the therapeutic effect was significantly better than that of the drug alone (Fig. 4D).

We further evaluated the effects of the combination of punicalagin and cefotaxime on inflammatory cytokines (gamma interferon [IFN-γ], interleukin-6 [IL-6], and tumor necrosis factor alpha [TNF-α]) during alveolar lavage in mice (n = 3). As shown in Fig. 4E to G, the levels of inflammatory cytokines in the infection control group were significantly increased. However, the levels of cytokines in the ΔsrtA group were significantly reduced, indicating that SrtA is an important virulence factor in S. aureus. In addition, the concentrations of IFN-γ, IL-6, and TNF-α in the infected mice were significantly decreased in the cefotaxime treatment group and the combined treatment group.

In another related development, punicalagin potentiates the effect of oxacillin on MRSA by reducing the transcription of mecA (a gene marker for methicillin resistance), causing a reduced level of penicillin binding protein 2a (PBP2a) (27). In combination with our findings, punicalagin holds promise as an adjuvant therapeutic agent in combination with antibiotics. In summary, punicalagin had a more effective curative effect combined with cefotaxime when mice were infected with lethal pneumonia caused by MRSA. There still is a lot of potential in exploring the combined use of antivirulence drugs and antibiotics (Fig. 5).

FIG 5.

Punicalagin reduces the formation of biofilm of S. aureus and attenuates the adhesion and invasion to the host and the anchoring of surface proteins by inhibiting SrtA.

DISCUSSION

S. aureus has been included as one of the six high-priority threatening pathogens collectively referred to as the “ESKAPE” pathogens: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanni, Pseudomonas aeruginosa, and Enterobacter, in order of the number of deaths they cause; each was responsible for more than 250,000 deaths associated with antimicrobial resistance (AMR) in 2019 (28, 29). Currently, the declining performance and increasing disease burden of traditional antimicrobial agents to confront bacterium-mediated disease make the development of alternative therapeutic strategies a priority (30). The widespread prevalence and spread of MRSA in the world requires us to further search for new strategies to combat S. aureus infection.

The scope of screening for SrtA inhibitors was wide, involving natural, synthetic, and high-throughput screening methodologies (31, 32). To our knowledge, many natural compounds have been investigated as inhibitors of SrtA based on its functions (33). Natural compounds have been used as medicines throughout human history. Many natural compounds are used as SrtA inhibitors, such as salvianolic acid A (33), chalcone (16), and flavonoids (34). It is worth mentioning that many natural compounds are still unidentified, and enormous opportunities for drug discovery are still open.

We identified punicalagin from a screen of 116 natural compounds, and it inhibits SrtA with an IC₅₀ value of 4.23 μg/mL. Moreover, we elucidated the SrtA-related phenotypes of S. aureus in vitro, including protein A (SpA) displayed in intact bacteria, adhesion of S. aureus to fibrinogen, formation of biofilm, and A549 cell invasion. Then we further analyzed the direct interaction between punicalagin and SrtA by molecular docking and fluorescence quenching assays and found that E106, K190, Y187, and A104 were the potential sites for SrtA and punicalagin. Finally, we observed that the combination of punicalagin and cefotaxime had a better therapeutic effect on S. aureus-induced pneumonia in mice. In conclusion, our findings provide new strategies to fight against S. aureus infection.

In our previous work, we identified scutellarin (21) as a SrtA inhibitor after screening a collection of 72 natural compounds that included flavonoids, terpenoids, alkaloids, and quinones. In the present study, 44 natural compounds were updated, among which 2 were identified as potential SrtA inhibitors. Punicalagin was selected for subsequent studies because of its high SrtA inhibitory activity at low doses during initial screening. Reviewing previous studies, the overall hit rate was 2.59%. We provide a representative list of the present study in Table S1, which contains 44 natural compounds with their SrtA relative inhibition rate.

Punicalagin, isolated from pomegranate fruit pith and carpellary membrane, has a wide range of biological and pharmacological activities (35). Punicalagin is an active compound found in the pomegranate rind. A previous study reported that the expression of alpha-toxin and enterotoxins in S. aureus is sensitive to the action of punicalagin (36). Moreover, punicalagin had the ability to induce morphological damage to the cell membrane and inhibit biofilm formation remarkably (37). In addition, activity of the virulence of pathogens and their anti-quorum-sensing (anti-QS) potential have been rarely reported (38). Thus, we wonder if punicalagin could affect the biological activity of S. aureus and the infections caused by MRSA via targeting SrtA.

FRET has been widely used to screen for SrtA inhibitors, which can be used to identify and cleave the LPXTG motif (39). There are different methods for initial screening of SrtA inhibitors. Volynets et al. (40) screened the inhibitors by quantifying the fluorescence intensity upon the substrate cleavage using a sortase A activity assay kit. In addition, Zhang et al. (41) used in silico screening for compounds that bind the active site of sortase and experimental validation to identify SrtA inhibitors. In Maresso et al.’s study (42), purified S. aureus SrtA was incubated with compound in Me₂SO, followed by addition of fluorescent substrate in reaction buffer for 24 h. Fluorescence was measured using an EnVision plate reader.

Compared with other screening methods, our screening method as an initial screening method is relatively more accurate and faster. In our study, the FRET method was used to screen SrtA inhibitors from natural compounds. We also use multiple approaches to verify SrtA inhibitors. Many researchers now use FRET as an effective initial screening method. Zhang et al. (16) found that chalcone attenuated S. aureus virulence by targeting SrtA and alpha-hemolysin (Hla) using FRET as the initial screening method. Mu et al. (33) identified that salvianolic acid was an SrtA inhibitor using the same method. In Lin Wang et al.’s (43) study, the activity of chlorogenic acid against SrtAΔN₅₉ was determined using a FRET assay.

Multitarget and multipharmacological effects of natural compounds can affect the virulence of S. aureus in different ways. In this study, we solely focused on the inhibition of SrtA by punicalagin in a low-dose range of 0 to 32 μg/mL, and the growth of S. aureus was not affected in this dose range. We verified that punicalagin was an SrtA inhibitor by associated virulence phenotypes and protein-drug interaction mechanisms. Interestingly, there are also reports of punicalagin suppressing mecA-mediated methicillin resistance and damaging the staphylococcal cell membrane at high concentrations (27, 37). This further suggests that punicalagin plays an anti-infection role against S. aureus at both low and high concentrations, making it a candidate for an anti-infective agent.

In this study, we first found the antivirulence effect of punicalagin. As a virulence inhibitor of S. aureus SrtA, punicalagin can significantly reduce the activity of SrtA and related phenotypes in vitro and has a significant therapeutic effect on pneumonia infection induced by S. aureus in mice in vivo. Our novel findings may represent new insights into treating MRSA infection.

MATERIALS AND METHODS

Bacterial strains and chemicals.

This study was conducted under the approval of Biosafety Committee of Changchun University of Chinese Medicine. S. aureus strain USA 300 and the ΔsrtA strain, obtained from our lab, were used in the present study and cultured in brain heart infusion (BHI) broth (Hopebio, Qingdao, China) at 37°C. Punicalagin was purchased from Weikeqi Biology (purity, >98%; Chengdu, China). The fluorescent peptide Abz-LPATG-Dap (Dnp)-NH₂ was synthesized by LifeTein LLC. Cefoxitin, oxacillin sodium, penicillinG Na, and vancomycin were purchased from Yuanye Co., Ltd. (Shanghai, China). Cefepime and cefotaxime were purchased from Solarbio Biotechnology Co., Ltd. (Beijing, China). Ceftriaxone sodium, ceftiofur sodium, and ceftaroline fosamil were purchased from Dalian Meilun Biotechnology Co., Ltd. (Dalian, China).

Cloning, expression, and purification of SrtA.

The gene encoding SrtA (lacking the N-terminal transmembrane domain [N_1–59]) was amplified from S. aureus genomic DNA using the primers srtA-F and srtA-R by PCR. The resulting amplified fragment was digested with BamHI and NdeI and ligated into the same sites of pET28a, yielding pET28a-SrtA. Site-directed mutagenesis to introduce the substitutions LYS190, TYR187, ALA104, and GLU106 into SrtA was performed using the QuikChange site-directed mutagenesis kit (Agilent, UK). All primers are listed in Table S2. The pET28a-srtA plasmid was transformed into Escherichia coli BL21(DE3) by the heat shock method and incubated in LB medium (kanamycin, 50 μg/mL) at 37°C and 220 rpm until the optical density at 600 nm (OD₆₀₀) reached 0.8. Then 0.5 mM IPTG (isopropyl-β-d-thiogalactopyranoside) was added and cultured overnight at 16°C and 180 rpm. The supernatant was collected by centrifugation at 4°C for 1 h at 12,000 rpm. Subsequently, the mutant SrtA were carried out by Ni-NTA (Beyotime Biotechnology, Shanghai, China).

SrtA activity assay.

A fluorescence resonance energy transfer (FRET) assay was used to measure the activity of SrtA. Briefly, 100 μL of a mixture consisting of the SrtA reaction buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10 mM CaCl₂), purified SrtA protein (4 μM), and different concentrations of punicalagin (0 to 256 μg/mL) were added in the 96-well plates (Corning, USA) and incubated. Then, the fluorescent peptide substrate Abz-LPATG-Dap (Dnp)-NH₂ (10 μM) was added and incubated for 1 h at 37°C. The fluorescence intensity was read using emission and excitation wavelengths of 420 and 309 nm with a microplate reader (MultiSkan Go; Thermo Scientific, USA). When inhibitory activity was greater than 60%, the compound was considered a potential inhibitor. Subsequently, a gradient concentration inhibition curve was performed, and the IC₅₀ was calculated. The experiment was repeated at least three times.

Growth curve.

The overnight cultured S. aureus USA300 and ΔsrtA strains were transformed into tryptic soy broth (TSB) medium at a ratio of 1:100 and cultured until the OD₆₀₀ reached 0.3. Experiments were divided into three groups: the control group with added DMSO, the drug group with added punicalagin (32 μg/mL), and the ΔsrtA group. The bacterial solution was collected at different time points to determine the absorbance at 600 nm. The growth curves were plotted with the time and the corresponding OD values.

Determination of the MIC.

The MIC of punicalagin was measured using the broth microdilution method recommended by the Clinical and Laboratory Standards Institute (CLSI) (44, 45). Briefly, 5 × 10⁴ CFU/mL S. aureus USA300 were inoculated in 100 μL of cation-adjusted Mueller-Hinton broth (CAMHB) medium in a 96-well plate, followed by the addition of serial dilutions of punicalagin (0 to 512 μg/mL). Then the 96-well plate was incubated in an incubator at 37°C for 18 h, and the absorbance at 600 nm was measured using a microplate reader (Thermo Fisher, USA).

Fibrinogen binding assay.

S. aureus USA300 and ΔsrtA strains were cultured in BHI broth until the exponential stage, at which point different concentrations of punicalagin were added. All samples were cultured at 37°C for 2 h until the OD₆₀₀ reached 0.5. The 96-well plates were coated with 100 μL fibrinogen (20 μg/mL) at 4°C overnight. The plates were washed with phosphate-buffered saline (PBS) and sealed with bovine serum albumin (BSA) at 37°C for 2 h. After washing with PBS, a 100-μL cell suspension was added and incubated at 37°C for 2 h. Subsequently, after removing the suspension, the plates were washed three times with PBS; the adherent bacterial cells were then fixed with 4% formaldehyde for 30 min and incubated with 40 μL crystal violet (12.5 g/L) dying for 20 min. Then, the excess dye was washed away with PBS, 200 μL anhydrous ethanol was added, and 100 μL was transferred to a new 96-well plate to detect the OD₅₇₀ with a microplate reader.

Invasion assay.

Human non-small cell lung cancer (A549) cells were inoculated into 24-well cell culture plates at 2 × 10⁴ per well and cultured overnight at 37°C in a cell culture medium containing 5% CO₂. Different concentrations of punicalagin (0 to 32 μg/mL) were mixed with S. aureus USA300 and incubated until the OD₆₀₀ was 1.0. Subsequently, the bacteria were collected by centrifugation and resuspended with an equal volume of Dulbecco’s modified Eagle’s medium (DMEM). Then, 1 mL of the resuspended bacterial solution was added to the cell culture medium and incubated at 37°C for 2 h. After washing with PBS, 1 mL of DMEM containing gentamicin (300 μg/mL) was added. The cells were incubated at 37°C for 1 h and then rinsed three times with PBS. Then, they were lysed by 1% Triton X-100 for 30 min. The lysates were gradient-diluted with PBS, and the number of CFU was determined by manual counting.

Biofilm formation assay.

Overnight cultured S. aureus USA300 and ΔsrtA were diluted at 1:100 in BHI medium containing 3% NaCl and 0.5% glucose. Then 100 μL bacterial solution was added to each well of 96-well plate, which was already coated with 50 μL 20% freeze-dried rabbit plasma (HopeBio, Qingdao, China) at 4°C overnight. Subsequently, punicalagin was added along the concentration gradient and cultured at 37°C for 24 h. Then, 50 μL crystal violet (12.5 g/L) was added to each well for 20 min of staining. Sterile distilled water was used to wash away the excess dye. After drying, 200 μL anhydrous ethanol was added to each well to elute the binding dye, and 100 μL was transferred to a new 96-well plate. The absorbance of the plates was read at OD₅₇₀ with a microplate reader.

FITC-IgG binding to staphylococcal protein A (SpA).

Overnight cultures of S. aureus USA300 and ΔsrtA were diluted at 1:1,000 in TSB medium with different concentrations of punicalagin or DMSO, respectively. Bacteria were incubated at 37°C until the OD₆₀₀ values reached 1.0, followed by centrifuging and collecting. After being washed twice with PBS, bacteria were resuspended with 50 μL of FITC-labeled rabbit anti-goat immunoglobulin G (IgG) (Proteintech, Wuhan, China) and incubated at room temperature in the dark for 2 h. Bacteria were rewashed and stabilized with 4% formaldehyde. Fluorescence intensity was measured by flow cytometry (CytoFlex; Beckman Coulter, USA) to assess the amount of SpA.

Checkerboard assay.

The checkerboard method (45, 46) was used to screen antibiotics which had the synergistic effect with punicalagin and to analyze the combined effect of punicalagin and cefotaxime on clinically isolated MRSA strains. In the 96-well plates, 100 μL of different final concentrations of punicalagin were added to each well in a certain sequence. Then, different concentrations of antibiotics (cefeoxime, ceftriaxone sodium, ceftaroline fosamil, cefoxitin, oxacillin sodium, ceftiofur sodium, cefepime, vancomycin, and penicillinG Na) were added using the microdilution. The absorbance was measured at 600 nm after incubation at 37°C for 16 h with a microplate reader. The fractional inhibitory concentration index (FICI) was calculated according to the formula FICI = FIC_A + FIC_B = C_A/MIC_A + C_B/MIC_B.

FICI ≤ 0.5 indicates synergism; 0.5 < FICI ≤ 1 indicates additivity; 1 < FICI ≤ 2 indicates irrelevance; FICI > 2 indicates antagonism.

Cell viability.

A549, human hepatoma (HepG2), and human embryonic kidney (HEK293T) cells (stored in the lab) were seeded in 96-well plates at a density of 2 × 10⁴ cells per well in the incubator. After culturing until the bottom of the 96-well plate was lined with cells, different concentrations of punicalagin (0 to 32 μg/mL) were added and culturing continued at 37°C for 24 h. Then, 10 μL 0.5 mg/mL of MTT solution (Beyotime Biotechnology, Shanghai, China) was added and incubated for 4 h, followed by addition of 100 μL DMSO to dissolve the crystals. The absorption at 490 nm was measured with a microplate reader.

Live/dead and cytotoxicity assays.

Overnight-cultured S. aureus USA300 was transferred to fresh TSB diluted at 1:100 for further culturing until the OD₆₀₀ reached 0.5. After centrifuging and discarding of the supernatant, the bacteria were cleaned with PBS twice and suspended in a fresh DMEM (without fetal bovine serum [FBS]). Then, different concentrations of punicalagin (0 to 32 μg/mL) were added to the bacterial solution. A549 cells were cultured in DMEM (10% FBS) and placed in 24-well plates with 1 × 10⁵ cells in each well. The culture medium of 24-well plate cells was discarded and washed twice with PBS, and 500 μL bacterial solution was added as described above. After incubation for 5 h at 37°C, a Live & Dead Calcein AM, propidium iodide (PI) cytotoxicity assay kit (L6037M; US Everbright, Suzhou, China) was used according to the manufacturer’s instructions to detect the protective effect of punicalagin on cell injury, and it was observed under the microscope. The supernatant of the culture was collected, and the LDH levels in the supernatant were measured using an LDH cytotoxicity assay kit (C0016; Beyotime, Shanghai, China) according to the manufacturer’s instructions.

Cellular thermal shift assay (CETSA).

E. coli BL21(DE3) containing pET28a-AgrA and 0.5 mM isopropyl-beta-d-thiogalactopyranoside (IPTG; Beyotime Biotechnology, Shanghai, China) was used to induce protein expression. The supernatant was gathered as the lysate after ultrasonication and centrifugation. The supernatant was incubated with punicalagin (128 μg/mL) at 37°C for 1 h after being centrifuged at 18,000 × g for 20 min at 4°C. The samples were heated to various temperatures at a temperature gradient of 25.0, 35.0, 39.7, 43.4, 47.3, and 50.0°C for 5 min. After that, samples were immediately placed in ice water and centrifuged at 18,000 × g for 20 min to yield a supernatant. After analysis 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 (NIH).

Western blot analysis.

S. aureus USA300 treated with different concentrations of punicalagin (0 to 32 μg/mL) was collected by centrifugation using PBS suspension. Total protein of S. aureus was extracted by 10 mg/mL lysozyme, 4 mg/mL lysostaphin, and 200 μL radioimmunoprecipitation assay (RIPA), which was followed by determining the protein concentration with the bicinchoninic acid method. Then, the same amount of total bacterial protein (20 μg) was isolated using 12% SDS-PAGE and transferred to the polyvinylidene difluoride (PVDF) membrane (Beyotime, Shanghai, China). The membrane was sealed with 5% BSA for 2 h and incubated with rabbit anti-SrtA polyclonal antibody (1:200, prepared by our laboratory) at room temperature. After washing with Tris-buffered saline with Tween 20 (TBST), horseradish peroxidase (HRP)-labeled goat anti-rabbit antibody (1:5,000; Beyotime) was incubated at room temperature for 2 h. Subsequently, ECL Plus (Beyotime) was used to expose and record the bands.

Fluorescence quenching assay.

The purified SrtA protein was diluted to 500 ng/mL with PBS. SrtA reaction buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 10 mM CaCl₂) was added and mixed with various punicalagin concentrations (0 to 4 μg/mL). After incubation at room temperature for 10 min, the fluorescence spectra of the mixed solution were measured using a fluorescence spectrophotometer (Thermo Scientific, USA). The excitation and emission slits were selected at 5 nm, and the excitation wavelength was 280 nm. The fluorescence emission spectra in the range of 280 to 400 nm were scanned, and the fluorescence intensity at 326 nm was recorded. The K_A value was calculated using the method of a previous study (47).

Reversible inhibition assay of SrtA.

To evaluate if punicalagin is a reversible inhibitor of SrtA, 100 μL of purified SrtA (4 μM) was incubated with punicalagin at a final concentration of 10-fold IC₅₀ for 1 h at 37°C. Then, 9.9 mL of SrtA reaction buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 10 mM CaCl₂) was added. Subsequently, 1 μL of the substrate peptide (10 μM) was added to the 190 μL diluted mixture. After incubation for 20 min, the fluorescence intensity at 309 nm excitation wavelength and 420 nm emission wavelength were measured with a microplate reader.

Molecular docking.

The SrtA structure was obtained from the PDB database (PDB code 1T2P), and the 3D structures of punicalagin were constructed with the ChemBio3D Ultra 12.0 software package. AutoDock Vina 1.1.2 software was used to simulate the optimal conformation for molecular docking.

Pneumonia model experiment.

All animal experiments in this study were strictly conducted following guidelines of the Experimental Animal Ethics Committee of Changchun University of Chinese Medicine. C57BL/6J female mice (6 to 8 weeks of age) were selected to establish a pneumonia infection model to study the therapeutic effect of punicalagin on acute pneumonia caused by MRSA.

There were 108 mice used in the experiment. For the survival assessment, mice were infected intranasally with 2 × 10⁸ CFU of S. aureus USA300, and then they stood for 30 s to ensure that each mouse inhaled the bacteria into the lungs (n = 10). Then, the S. aureus USA300 group was given punicalagin (100 mg/kg/day), cefotaxime (100 mg/kg/day), and punicalagin (100 mg/kg/day) combined with cefotaxime (100 mg/kg/day) as treatment. The survival rate within 96 h in each group was analyzed. The statistical significance of the treated and control groups was assessed using the log-rank tests for the survival curves. (**, P < 0.01; ***, P < 0.001 compared with the USA300 group).

For the bacterial load and histopathology of lung tissue, mice in each group were infected with 30 μL (1 × 10⁸ CFU) of S. aureus culture via intranasal drip for 2 days (n = 5). The mice were then euthanized by cervical dislocation, and the lungs were weighed and homogenized. Next, the homogenate was appropriately diluted and spread on BHI agar plates. After incubation at 37°C overnight, the number of colonies was counted. The left lung of mice in each group was taken and perfused and fixed in 10% formalin. Hematoxylin and eosin (HE) staining was performed, and histopathological changes in the lungs were observed under an optical microscope.

To determine the levels of IFN-γ, IL-6, and TNF-α, the trachea of anaesthetized mice was separated, and the distal trachea was ligated 48 h after administration. Then, alveolar fluid was taken from the alveoli twice with 0.5 mL of sterile PBS, three times in a row (n = 3). The numbers of cytokines were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits (eBioscience, San Diego, CA) according to the manufacturer’s protocols. All animal experiments were replicated at least twice.

Statistical analysis.

The statistical significance of the treated and control groups was assessed using the log-rank tests for the survival curves. The data were expressed as the mean ± standard deviation (SD) for each experimental group. The experimental data in this study were analyzed using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). Statistical significance was considered P < 0.05.