The bacterial strains used in the study were SA75 and Newman. SA75 is a clinical S. aureus isolated from a patient with skin suppurative infection, which had remarkable hemolytic activity and pigment formation. The S. aureus ATCC 25904 Newman has the same phenotypic characteristics as SA75. All strains were incubated in trypticase soy broth (TSB) medium at 37 °C with shaking at 220 rpm. SYG-180-2-2 was synthesized by the School of Pharmacy, Wenzhou Medical University (Sheng et al., 2021). The details of the molecular structure, synthesis process and characterization of SYG-180-2-2 were shown in our previous study (Rao et al., 2021).

SYG-180-2-2 was prepared in dimethyl sulfoxide (DMSO, Biosharp, Beijing, China) at a concentration of 20 mg/mL. The broth microdilution method based on CLSI guidelines was used to determine the minimal inhibitory concentration (MIC) (CLSI, 2019). Specific methods for MIC values and growth curves of SYG-180-2-2 against S. aureus strains were described in our previous study (Rao et al., 2021).

The hemolytic activity of strains treated with SYG-180-2-2 was measured using sterile defibrillation rabbit blood. S. aureus strains were grown in TSB with or without 4 μg/mL SYG-180-2-2. After 16 h of incubation, cultures were adjusted to the same optical density (OD₆₀₀) and then centrifuged at 8000 rpm for 5 min at room temperature. Next, 200 µL of supernatant was added to 800 µL of phosphate-buffered saline (PBS) with a final concentration of 2.5% sterile defibrillation rabbit blood. Blood with Triton X-100 and PBS were used as positive (100% hemolysis) and negative (0% hemolysis) controls, respectively. Subsequently, the samples were incubated at 37°C for 1 h. Then, the mixtures were centrifuged at 8,000 rpm for 5 min and the absorbance of supernatants was measured at 600 nm. Hemolysis (%) = [(absorbance of the treated sample – absorbance of negative control)/(absorbance of positive control – absorbance of the negative control)] × 100%. All assays were performed in triplicate.

The S. aureus SA75 and Newman were cultured in TSB with or without SYG-180-2-2 (4 µg/mL) for 24 h and cultures were normalized to a same optical density at OD₆₀₀. Then, the supernatant was collected and filtered with a 0.22-µm filter. The α-toxin level was quantified by Staphylococcal α-Toxin ELISA kit (Chenglin Biological Technology Co., LTD, Beijing, China) following the manufacturer’s instructions. The test was performed independently in triplicate.

The measurement of pigment production was assessed as previously described with some modifications (Chen et al., 2016). In brief, the S. aureus SA75 and Newman were cultured in 4 mL of TSB medium with or without SYG-180-2-2 at 37°C for 48 h, with shaking at 220 r.p.m. 3 mL of bacteria cultures were centrifuged, washed twice with PBS and then resuspended in 1 mL of methanol for extraction of the pigment. Subsequently, the absorbance was measured at 450 nm for determination of the pigment concentration. Pigment inhibition (%) = [(OD₄₅₀ of control sample – OD₄₅₀ of the treated sample)/OD₄₅₀ of control sample] × 100%. The experiments were done in triplicate.

SA75 and Newman were grown in TSB with or without SYG-180-2-2 (4 µg/mL). After 2 days, the bacteria were washed twice with PBS and diluted to a concentration of 1 × 10⁷ CFU/mL. 250 μL of bacterial suspension was added in a 2-mL Eppendorf tube. Then, hydrogen peroxide (H₂O₂) was added to a 1 mM final concentration and the tubes were incubated at 37°C for 1 h with shaking at 220 r.p.m. The reaction was stopped by the addition of 1,000 U/mL of exogenous catalase (Sigma-Aldrich). Then, the cells were serially diluted with PBS and spread on the TSA plates. After incubating at 37°C for 24 h, viable cells were counted to assess whether SYG-180-2-2 affected the sensitivity of S. aureus to H₂O₂. All tests were run in triplicate.

SA75 and Newman were grown in TSB with or without SYG-180-2-2. Overnight cultures were centrifuged at 1,2000 rpm for 1 min at room temperature and adjusted to a concentration of 1 × 10⁷ CFU/mL using sterile PBS. The bacterial suspensions were mixed gently with whole blood collected from healthy human volunteer at a ratio of 1:4 in 2-mL Eppendorf tubes (500μL). The tubes were incubated at 37°C for 6 h with shaking (220 r.p.m.) and bacterial viability was determined by plating dilutions on TSA plates. All experiments were run in triplicate.

S. aureus strains were cultured in TSB medium treated with either SYG-180-2-2 at the concentration of 4 μg/mL or without drug, and incubated at 37°C at 220 rpm. After 16 h, bacterial cells were centrifuged at 1,2000 rpm for 1 min at room temperature, resuspended in 80 μL of 20 mg/mL lysozyme and 4 μL of 1 mg/mL lysostaphin and incubated at 37°C for 1 h. Total RNA was isolated and quantified by a Nano-drop instrument (Thermo Fisher).

Then, total RNA (1 μg) was reverse transcribed into cDNA and the qPCR was performed. The primer pairs used in qPCR experiment are listed in Table 1 . Specific kits, instruments and parameters for RT-qPCR are shown in our previous studies (Rao et al., 2021). The gyrB gene was used as an internal reference to normalize the expressions of genes of interest and PCRs were performed in 20 μL reaction mixtures. The relative quantification method (2^-ΔΔCt) was used to analyze the transcription level of target genes. All analyses were conducted in triplicate.

The toxicities of the SYG-180-2-2 was tested by inoculating doses of 0.32 mg/kg to groups of 10 larvae. This therapeutic dose exceeded the dose used in this study. Sterile PBS was inoculated as a negative control. The larval mortality of the treatment group and the control group was observed for three consecutive days.

S. aureus strains were cultured in TSB with or without 4 μg/mL SYG-180-2-2 overnight at 37°C with shaking. Then bacteria were centrifuged at 1,2000 rpm for 1 min at room temperature, washed twice with PBS, and resuspended to 1 MacFarland standard (3.0 × 10⁸ CFU) in PBS. Groups of 10 larvae were inoculated with 10 μL of suspension, via last left forelimb using Hamilton syringe. In addition, one group of larvae was injected sterile PBS as a negative control. After the injection, each group of larvae was placed in a clean petri dish at 37°C. The mortality of larvae was observed every 1h for 12 times and then every 12 h to 3 days. The experiment was repeated more than three times.

Six-week-old female BALB/C mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). At the time of the experiment, they weighed approximately 20 ± 2 g. Briefly, the fur on the backs of mice was removed using depilatory cream (Reckitt Benckiser Plc.). There were two groups of eight mice each. The safety of the SYG-180-2-2 was tested by injecting 100 μL of 8 μg/mL SYG-180-2-2 on the skin of mice every 24 h for 3 days. Sterile saline was injected as a negative control. On the fourth day, the skins of the mice were observed and the skin of one mouse in each group was taken for pathological section.

The S. aureus strains were cultured for 3 h at 37 °C, and then the bacteria were harvested and washed twice with sterile PBS. The number of bacteria was determined by reading the literature and pre-experiments before the mice were infected (Chen et al., 2016; Haney et al., 2018). For each experimental group, seven mice were used. Mice were inoculated with 100 μL of 7.5 × 10⁶ CFU of S. aureus SA75 or 100 μL of 1.5 × 10⁷ CFU of S. aureus Newman subcutaneously, and at 1 h post-infection, saline containing equal concentration of DMSO (for controls) or 4 μg/mL SYG-180-2-2 was given directly into the subcutaneous space of the infected area every 24 h for 3 days. The area of skin abscesses was recorded daily using a caliper. The length (L) and width (W) of abscess were measured and calculated as follows: A =π (L×W)/2. All animals were killed 4 d after infection. Skin abscesses were excised and homogenized by an automatic tissue homogenizer. There were seven mice in each group, the skin abscesses of six mice were used for viable cell counting and one mouse skin abscess for histopathological analyses.

All data was analyzed using GraphPad Prism (version 8.0) and presented as mean ± SD (standard deviation). Two-tailed t-tests were performed for the experiments in this study. The difference was considered to be statistically significant when a p value less than 0.05.

The MIC values of SYG-180-2-2 against SA75 and Newman were > 128 μg/mL. At 4 μg/mL of SYG-180-2-2, the OD of bacteria at the late logarithmic growth period was consistent ( Figure 1 ). Our prior growth curve experimental data showed that SYG-180-2-2 reduced amounts of JP5023 and JP4856 growing at 8 μg/mL (Rao et al., 2021). However, SYG-180-2-2 did not reduce the number of SA75 and Newman at 8 μg/mL ( Supplementary Figure 1 ). Here, we choose a lower concentration of 4 μg/mL to conduct the following studies.

The effect of SYG-180-2-2 on hemolysis activity of S. aureus supernatants was determined by comparing the hemolysis percentage of treated and untreated groups. As shown in Figure 2A , the hemolysis activity of the untreated group was significantly higher than that of the treated with SYG-180-2-2. For SA75, the hemolysis rate of without SYG-180-2-2 treated group was 92.53 ± 1.32%, and after being treated, its rate decreased by 80.70 - 85.97%. For Newman, the hemolysis rate of without SYG-180-2-2 treated group was 97.80 ± 2.19%, and after being treated, its rate decreased by 50.00 - 56.14% ( Figure 2B ).

Then, an ELISA kit was used to quantitatively detect the level of alpha-hemolysin between untreated strains and treated strains to explore whether SYG-180-2-2 could reduce its hemolysis activity by reducing α-toxin. For SA75, the α-hemolysin production of the untreated group was 28.84-37.63 pg/mL, and after being treated with SYG-180-2-2, its production was 27.87-33.40 pg/mL. For Newman, the α-hemolysin production of the untreated group was 31.77-35.35 pg/mL, and after being treated with SYG-180-2-2, its production was 32.10-35.03 pg/mL. These results showed that there was no significance difference between the treated and untreated groups ( Supplementary Figure 2 ).

SYG-180-2-2 has potent activity against S. aureus staphyloxanthin formation in vitro according to the qualitative and quantitative experiments of staphyloxanthin synthesis. As shown in Figure 3A , the staphyloxanthin formation of the untreated group was yellowish, while that of the treated with SYG-180-2-2 group was colorless. After being treated with SYG-180-2-2, staphyloxanthin inhibition rates of SA75 and Newman were 96.04 ± 0.57% and 96.26 ± 2.31%, respectively ( Figure 3B ).

Because SYG-180-2-2 inhibited the production of S. aureus pigment ( Figure 3A ), and once the pigment was blocked, it may increase the sensitivity of bacteria to oxidative killing, so we compared the sensitivity of SYG-180-2-2-treated and untreated S. aureus to H₂O₂. Survival of the SYG-180-2-2-treated SA75 was ~9 times lower than that of the untreated SA75 (2.00% vs. 18.50%), notably, survival of the SYG-180-2-2-treated Newman was 850 times lower than that of the untreated Newman (0.02% vs. 17.00%; Figure 4A ). Subsequently, we investigated whether SYG-180-2-2 sensitizes S. aureus to human whole blood. As shown in Figure 4B , the survival of SYG-180-2-2-treated Newman was ~7 times lower than that of the untreated Newman (1.50% vs. 10.00%), however, the survival of SYG-180-2-2-treated SA75 had no significance change compared with the untreated SA75 (2.75% vs. 4.25%). The human whole-blood killing assay suggested that a part of S. aureus was sensitive to human blood immune clearance after SYG-180-2-2 treatment.

Through the hemolytic activity assay, we found that SYG-180-2-2 greatly reduced the hemolytic activity of S. aureus, so we analyzed the hemolysis-related genes hla and hlb from the transcriptional level. We found that there was no significance difference in the expression of hla, while the expression of hlb decreased by 21.77-fold in SA75 and 2.06-fold in Newman, respectively ( Figure 5 ). We conducted RT-qPCR to explore the correlation between the expression of hlb and saeRS. We found that the expression of saeRS in strains treated with SYG-180-2-2 was significantly decreased ( Figure 5 ). After SYG-180-2-2 treatment, the expression of saeR decreased 4.41-fold in SA75 and 2.95-fold in Newman, respectively. In order to further study the mechanism of SYG-180-2-2 on staphyloxanthin, we performed qPCR experiments of staphyloxanthin synthesis-associated genes and confirmed that the expression levels of crtN, crtM and sigB were significantly decreased by 3.96-fold, 3.80-fold and 1.14-fold in SA75 and 1.76-fold, 1.81-fold and 8.08-fold in Newman, respectively. ( Figure 5 ). Further, qPCR confirmed that SYG-180-2-2 treatment also downregulated the expression of oxidative stress related genes such as sodA, sodM and katA, being 1.53-fold, 6.67-fold and 1.21-fold in SA75 and 1.25-fold, 2.00-fold and 1.60-fold in Newman, respectively ( Figure 5 ).

There was no death of larvae in the high-dose SYG-180-2-2 group (0.32 mg/kg) and the control group (inoculated with PBS) for 3 days, indicating that SYG-180-2-2 was not toxic within the tested dose. We found that the virulence of S. aureus treated with SYG-180-2-2 in larvae was significantly lower than that of the untreated S. aureus. After 72 hours, the death rates of SA75 and Newman treated with SYG-180-2-2 were reduced by 30% and 60%, respectively ( Figure 6 ).

As shown in Figure 7A , as in the control group, the skin of mice injected with SYG-180-2-2 was intact and there was no abscess formation. As depicted in Figure 7B , as in the control group, after three injections of 100 μL of 8 μg/mL SYG-180-2-2, the epidermis was intact, the dermis was not obvious damaged, and there was no obvious inflammatory cell infiltration. The result showed that SYG-180-2-2 had no damage to the skin of mice.

We used a mouse skin abscess model to further explore the effects of SYG-180-2-2 on skin abscess formation and invasiveness of S. aureus in vivo. We injected the bacterial suspension subcutaneously into the back of mice, and the amount of injection was the same in each mouse. The sizes of abscess formed after infection were different, we tried to choose the same place and the area of abscess formation was easy to observe. As shown in Figure 8A , treatment with SYG-180-2-2 significantly restrained the size of abscess after 1 day of infection and significantly reduced the formation of yellow eschar on day 4 (blue circles). Figures 8B, C plotted changes in abscess area of SA75 and Newman before and after treatment with SYG-180-2-2, respectively. Abscesses in all groups were most pronounced on day 1 after infection, and then began to decrease and heal gradually. Notably, the abscess sizes of SA75 wild-type group were significantly larger than those of SA75-treated group on the first day post-infection (107.13 mm² vs. 53.01 mm²). Mice were sacrificed 4 days after infection and bacterial load in skin was determined. After treatment, the loads of SA75 and Newman were decreased by ~0.7 log¹⁰ CFU/abscess and ~0.5 log¹⁰ CFU/abscess, respectively ( Figures 8D, E ). As depicted in Figure 8F , compared with the treated group, the skin sections of the untreated group showed more severe skin lesions and inflammatory cell infiltration. In particular, in the SA75-untreated group, the epidermis was completely destroyed, the dermis was severely damaged and immune cells were infiltrated, suggesting severe infection and deep inflammation. In the SA75-treated group, the epidermis was intact and the infiltration of subcutaneous immune cells was largely controlled. The Newman group also had a large skin recovery after treatment. These results showed that SYG-180-2-2 could weaken the virulence of S. aureus.