The small molecule ZY-214-4 may reduce the virulence of Staphylococcus aureus by inhibiting pigment production

In recent years, clinical Staphylococcus aureus isolates have become highly resistant to antibiotics, which has raised concerns about the ability to control infections by these organisms. The aim of this study was to clarify the effect of a new small molecule, ZY-214-4 (C19H11BrNO4), on S. aureus pigment production. At the concentration of 4 μg/mL, ZY-214-4 exerted a significant inhibitory effect on S. aureus pigment synthesis, without affecting its growth or inducing a toxic effect on the silkworm. An oxidant sensitivity test and a whole-blood killing test indicated that the S. aureus survival rate decreased significantly with ZY-214-4 treatment. Additionally, ZY-214-4 administration significantly reduced the expression of a pigment synthesis-related gene (crtM) and the superoxide dismutase genes (sodA) as determined by real-time quantitative polymerase chain reaction (RT-qPCR) analysis. ZY-214-4 treatment also improved the survival rate of S. aureus-infected silkworm larvae. The small molecule ZY-214-4 has potential for the prevention of S. aureus infections by reducing the virulence associated with this bacterium.


Background
The skin and nasopharynx of approximately 20 to 30% of the world's population [1,2] are continuously colonized by the Staphylococcus aureus. This bacterium is an opportunistic pathogen that can cause superficial skin diseases and numerous fatal diseases such as bacteremia and infective endocarditis, and also causing osteoarticular, pleuropulmonary, and device-related infections [3][4][5][6]. Vancomycin, a glycopeptide antibiotic that can inhibit cell wall biosynthesis, is the first-choice treatment for methicillin-resistant S. aureus (MRSA) infections [7,8]; however, moderate or complete resistance to this antibiotic has become widespread among S. aureus strains [8,9]. Importantly, although significantly fewer antibiotics have been identified or synthesized this century compared with the last century [10], the prescription of antibiotics for the treatment of infections over the years has led to the emergence of drug-resistant S. aureus strains [11]. Eliminating bacterial virulence factors is increasingly used as a means of combating antibiotic resistance [12], and represents a strategy that avoids the emergence of drug resistance induced by bacterial stress [12,13].
Notably, the success of S. aureus as a pathogen also lies in its ability to reduce oxidative stress [14]. Superoxide dismutase (SOD) is a key detoxifying enzyme [14][15][16] that converts reactive oxygen species (ROS) into less harmful products, thereby allowing bacteria that infect the body to escape the body's immune system and survive [12,14]. Pigments produced by pathogenic microbes are known to be important virulence factors [17]. S. aureus defective in pigment production exhibit reduced infectivity and increased vulnerability to neutrophils [18], and cannot infect the mice in the mouse model [3,19]. For example, S. aureus mutants with defective carotenoid biosynthesis are more likely to be killed by oxidants, show impaired neutrophil survival and lower pathogenicity [20]. Pigment biosynthesis is mediated by proteins encoded by a five-gene cluster (crtM, crtN, crtP, crtQ, and crtO) [21], which represents a potential new target for antibacterial therapy.
ZY-214-4, molecular formula C 19 H 10 BrNO 4 , contains a chromone ring and an N-phenyl-substituted maleimide. Chromone and its derivatives are widely distributed in naturally occurring products and pharmaceuticals as key scaffolds, and chromone derivatives have been shown to exert antimicrobial activities against Penicillium spp., Escherichia coli, and Shigella flexneri [22][23][24]. Maleimide motifs are prevalent in many natural products and drug candidates, and possess a broad spectrum of biological properties, including antitumor and antibacterial activities [25][26][27]. However, no studies have reported on the antibacterial activity of chromone-maleimide hybrids in inhibiting golden pigment production in S. aureus. In this study, we sought to clarify whether subinhibitory concentrations of ZY-214-4 can inhibit pigment production in clinical S. aureus strains.

Results
The effect of subinhibitory concentrations of ZY-214-4 on the growth of S. aureus strains The minimum inhibitory concentration (MIC) of ZY-214-4 was 64 μg/mL against S. aureus strains SA21, SA882, and SA923, and 256 μg/mL against strains SA2698 and SA2956. To verify whether ZY-214-4 reduced the virulence of S. aureus by reducing the expression of virulence genes rather than the number of S. aureus cells, we generated a growth curve for these clinical isolates of S. aureus at a series of subinhibitory concentration (Additional Figure 1). We found that the number of bacteria in the late logarithmic growth period remained constant at the subinhibitory concentration of 4 μg/mL of ZY-214-4 ( Fig. 1). Therefore, this concentration was used for subsequent experiments.

ZY-214-4 inhibited pigment production
We undertook a quantitative and qualitative assessment of pigment synthesis in ZY-214-4-treated and untreated cells. ZY-214-4 treatment markedly inhibited golden pigment production. Compared with the golden pigmentation of untreated S. aureus, that of S. aureus treated with ZY-214-4 was white or light yellow (Fig. 2a). Quantitative analysis showed that pigment production was decreased by 38 (Fig. 2b).
The effect of ZY-214-4 on the susceptibly of S. aureus to human blood and H 2 O 2 As ZY-214-4 could inhibit pigment production in S. aureus, and because the pigment can shield S. aureus cells from host oxidants, we next compared the sensitivity of ZY-214-4-treated (4 μg/mL) and untreated S. aureus to H 2 O 2 and healthy human blood. The results of an H 2 O 2 sensitivity assay showed that ZY-214-4-treated cells were substantially more sensitive to H 2 O 2 than untreated control cells (Fig. 3a). Moreover, compared with untreated controls, both the number of colonies and the survival rate of clinical S. aureus strains were greatly decreased in the whole blood of healthy volunteers following ZY-214-4 treatment (Fig. 3b). Together, these results indicated that ZY-214-4 treatment reduced the resistance of S. aureus to human blood and H 2 O 2 .
Treatment with subinhibitory concentrations of ZY-214-4 downregulated the expression of the sod and crtM genes of S. aureus We observed that pigment synthesis was reduced in S. aureus and that the bacterium was more sensitive to H 2 O 2 and healthy blood following ZY-214-4 treatment. To further explore the mechanism underlying these effects of ZY-214-4 on S. aureus, we used RT-qPCR to measure the expression levels of crtM, which is involved in antioxidant pigment synthesis, and that of sodA and sodM, which are coding for superoxide dismutase, the enzymes that scavenge oxygen free radicals and play a key role in the evasion of host defenses. We found that the expression of crtM and sodA were down-regulated in ZY-214-4-treated S. aureus cells when compared with that in controls, and 3 out 5 strains were significant for reduction in expression of sodM. (Fig. 4).

Analysis of the cytotoxicity of ZY-214-4
To evaluate the cytotoxicity of ZY-214-4, we injected silkworms with different concentrations of ZY-214-4 (2-8 μg/mL) and evaluated the effects after 24 h. No deaths were observed in either the treatment or corresponding concentration of DMSO control group (Data not shown).

ZY-214-4 reduced the virulence of S. aureus in infected silkworms
We found that, in vivo, the virulence of S. aureus was significantly lower with ZY-214-4 treatment (4 μg/mL) than without. As shown in Fig. 5, following S. aureus infection, mortality occurred later in ZY-214-4-treated silkworm larvae than in untreated animals. After 5 h, the mortality rate of untreated silkworm larvae was 100% for those infected with the S. aureus SA21 strain, 100% for those infected with the SA882 strain, 90% for those infected with the SA923 strain, 90% for those infected with the SA2698 strain, 100% for those infected with the SA2956 strain. The respective values for ZY-214-4treated silkworm larvae were 50, 20, 10, 30, and 30%. These results indicated that ZY-214-4 treatment can delay death in S. aureus-infected insects.

Discussion
Multidrug-resistant strains of S. aureus are a leading cause of skin and soft tissue infection [28,29]. The ability of S. aureus to survive under diverse environmental pressures is an important determinant of its pathogenicity [21,30], highlighting the need for the development of alternative treatments. Many studies have shown that the S. aureus pigment is a key factor in its virulence [18,31,32]. The biosynthetic pathway of pigment is disrupted in a "deleted" crtM of S. aureus, resulting in the absence of pigmentation and enhanced susceptibility to killing by ROS [18]. One study reported that, in a mouse subcutaneous abscess model, S. aureus mutants with impaired carotenoid biosynthesis were more easily killed by oxidants and neutrophils and exhibited lower pathogenicity when compared with their wild-type counterparts [18].
In this study, we synthesized a new small-molecule compound-ZY-214-4-and selected five clinical S. aureus strains isolated from different sites of infection to investigate the effect of subinhibitory concentrations of ZY-214-4 on the virulence of this bacterium. Because we found that high concentrations of ZY-214-4 could inhibit S. aureus growth (Additional Figure 1), we selected a subinhibitory concentration (4 μg/mL) that did not affect the growth of the bacterium, thus excluding the possibility that any reduction in virulence could result from a reduction in the number of bacteria. The pigment of S. aureus has been reported to be an important virulence factor [33]. The pigment has antioxidant properties, and its many double bonds can react with ROS produced by neutrophils and macrophages, thereby protecting S. aureus against oxidative stress [34]. The first key step in pigment biosynthesis is catalyzed by dehydrosqualene synthase (also known as diphosphonene synthase or CrtM) [35]. Many related studies have found that there is a positive correlation between pigment production and crtM expression [35,36]. Here, we found that pigment production and crtM gene expression were significantly downregulated in S. aureus under the effect of ZY-214-4. We speculate that ZY-214-4 exerts its inhibitory effect on pigment production by reducing the expression of crtM.
To deal with ROS, bacteria have evolved complex oxidative stress response mechanisms [37]. Notably, S. aureus has developed several means of escaping the immune systems of its hosts [38,39], including phagocytemediated oxidative killing [40,41]. This resistance is mediated by SOD production [42][43][44]. The absence of sodA can reduce S. aureus virulence in a model of abscess or retroorbital infection [45,46]. SodM is as important as SodA [16]. SOD is a representative antioxidant enzyme that can eliminate ROS produced under oxidative stress. SOD may also help bacterial pathogens survive against oxidative outbreaks produced by inflammatory cells [47]. As sodA genes was downregulated in this study, the expression of sodM in more than half of S. aureus was also significantly down-regulated. We suggest that subbacteriostatic concentrations of ZY-214-4 can weaken the antioxidant defense of S. aureus by inhibiting sod expression. Insects possess both cellular and humoral immune response pathways, and the related literature reported that the virulence of the strain was weakened by drug action [48,49]. In our study, we found that ZY-214-4 could reduce the virulence of S. aureus in the silkworm. Under the same conditions, the survival time of treated animals was significantly different from that of untreated controls.
The use of mammals for drug development is expensive and ethically problematic [50]. The mechanisms involved in the absorption, distribution, metabolism, and excretion of chemicals are similar in silkworm larvae and mammals [51,52]. In this study, we found that ZY-214-4 was not cytotoxic within the concentration range tested, and may be beneficial for the treatment of S. aureus infection.

Conclusions
In summary, we found that treatment with a subinhibitory concentration of a new small molecule, ZY-214-4, can reduce the virulence of S. aureus by inhibiting pigment production. This study provides a basis for exploring potential drug targets and developing new drugs for the treatment of S. aureus infection. However, this study also had some limitations. For example, the level of protection that ZY-214-4 provides against mortality of silkworms is not impressive. Further investigations are needed to clarify the mechanisms underlying how ZY-214-4 regulates the expression of crtM and sod.

Bacterial strains
The strains used in this study are listed in Table 1. The five S. aureus strains-SA21, SA882, SA923, SA2698, and SA2956-were isolated from patients at the First Affiliated Hospital of Wenzhou Medical University. The S. aureus isolates and the medical records of the patients were obtained for research purposes with the approval of the Ethics Committee of The First Affiliated Hospital of Wenzhou Medical University. Written informed consent was obtained from all the patients.

Procedure for the synthesis of C 19 H 11 BrNO 4
ZY-214-4 ( Fig. 6) was synthesized by the School of Pharmacy, Wenzhou Medical University [53]. In step 1, chromone 1 (0.2 mmol, 1 equivalent) and maleimide 2 (0.5 mmol, 2.5 equivalent) were completely dissolved in 2 mL of 1,2-Dichloroethane (0.1 M DCE) in a 12-mL screwcap tube. In step 2, [Ru(p-methylbenzyl)Cl 2 ] 2 (0.01 mmol, 0.05 equivalent), AgNTf 2 (0.04 mmol, 0.2 equivalent), and AgOAc (0.6 mmol, 3 equivalent) were added to the reaction mixture at room temperature. For step 3, the mixture was placed on a heating mantle and the temperature was raised to 120°C for 0.5 h, with stirring. In step 4, when the reaction was completed, the entire reaction mixture was directly loaded into a silica gel column, followed by purification with petroleum ether/ EtOAc (step 5), yielding the desired product (product 3) with a yield of 75%. All the reagents used were of analytical grade (Additional Figure 2).

MIC determination
ZY-214-4 was dissolved in dimethyl sulfoxide (DMSO, BOYUN, SH, 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) [54]. The MIC was defined as the lowest concentration at which no visible bacterial growth was observed. To exclude the influence of the solvent, during the determination, we simultaneously tested the same volume of solvent as a control.

Growth assay
The S. aureus strains were grown in TSB (Becton, Dickinson and Company, NJ, USA) to an optical density (OD) of 0.3 at 600 nm, following which the cultures were aliquoted into five flasks. Different doses of ZY-214-4 were then added to the culture to final concentrations of 4 μg/mL,8 μg/mL and 16 μg/mL. An Erlenmeyer flask containing only TSB was used as a blank control. All the cultures were incubated at 37°C with shaking at 220 rpm. The OD 600 value was measured hourly for 24 h. The assay was performed in triplicate.

Pigment extraction
To evaluate pigment production, the five S. aureus strains were inoculated into 10 mL of TSB with or without ZY-214-4 (4 μg/mL). After 12 h of incubation, the cultures were centrifuged at 10,000 rpm (enppendorf, F-34-6-38) for 10 min. The pellets were washed twice with  Oxidant susceptibility assay H 2 O 2 sensitivity assays were performed as previously described [56]. Control and ZY-214-4-treated (4 μg/mL) S. aureus were pelleted by centrifugation at 8000 rpm (enppendorf, F-34-6-38) for 10 min and resuspended in PBS containing 0.25% H 2 O 2 (The chemical reagent 30% hydrogen peroxide was diluted by aseptic PBS) at 37°C for 1 h. The cells were then serially diluted with PBS, spread on TSB agar plates, and incubated at 37°C for 12 h. The numbers of viable cells were counted after incubation to determine whether ZY-214-4 affected S. aureus susceptibility to H 2 O 2 .
Human whole-blood killing assay For the whole-blood killing assay, cultures of each strain treated or not with ZY-214-4 (4 μg/mL) were centrifuged and resuspended in sterile PBS to a final concentration of 1 × 10 7 CFU/mL. Whole blood from healthy human volunteers was collected into Vacutainer PT tubes (Becton, Dickinson and Company, NJ, USA). Aliquots (600 μL) of whole blood were transferred into 1.5-mL test tubes and mixed with 200 μL of bacterial samples to a final concentration of 2.5 × 10 6 CFU/mL as previously described [57]. The tubes were incubated at 37°C with shaking (250 rpm) for 1 h, following which dilutions were spread on Colombian blood plates to count the numbers of colonies.

RNA-seq and identification of differentially expressed genes
Bacteria were cultured for 12 h in TSB with or without ZY-214-4 (4 μg/mL) and then collected by centrifugation at 12,000×g for 1 min at 4°C. RNA was extracted using the QIAGEN RNeasy Maxi Kit (QIAGEN, BER, Germany) following the manufacturer's instructions. The RNA was sequenced using the Illumina HiSeq X platform with a paired-end read length of 150 bp. DEGseq software [58] was used to analyze the effect of ZY-214-4 on gene expression. Differences in gene expression were considered significant with |log2 (fold change)| > 1 and p < 0.005.

Quantitative real-time RT-PCR
S.aureus was cultured in the medium with and without ZY-214-4 (4 μg/mL). After 12 h, RNA was extracted as described above. The primer pairs used for qPCR are listed in Table 2. Total RNA was reverse transcribed using a Takara RNA PCR Kit (Takara, Tokyo, Japan). qPCRs were performed in 20-μL reaction mixtures using Luna Universal qPCR Master Mix (New England Biolabs, MA, USA). Each test was performed independently in triplicate.

Assessment of the toxicity of ZY-214-4 in the silkworm
The toxicity of ZY-214-4 against the silkworm was assessed as previously described, with slight modifications [59]. A disposable plastic syringe (Terumo, TY, Japan) was used to inject different concentrations (2-8 μg/mL) of ZY-214-4 (0.05 mL) into the body of  Table 2 Primers used for RT-qPCR The infection of silkworm larvae for the assessment of S. aureus virulence following ZY-214-4 treatment Staphylococcus aureus strains were cultured on Columbia blood agar plates at 37°C overnight. The next day, S. aureus was inoculated into TSB and grown to the logarithmic phase at 37°C with shaking (220 rpm). ZY-214-4 was added to a final concentration of 4 μg/mL. A bacterial solution without ZY-214-4 was used as control. After 12 h, the bacteria were collected by centrifugation at 8000 rpm for 5 min at 4°C, washed three times with phosphate-buffer saline (PBS), and diluted to 0.5 McFarland standard at 600 nm. The total colony units were further adjusted to obtain the required dose. For the infection of silkworm larvae, there were 10 larvae in each group, and the weight of each larva is 250 mg. Injection was performed as previously described [60] with slight modifications. In brief, a syringe was used to inject 50 μL of S. aureus into the last left forelimb of each larva. After the injection, the larvae were placed in an incubator at 37°C, and larval mortality was recorded. Larvae were considered to be dead when they did not respond to touch. Silkworm larvae that were not exposed to ZY-214-4 and those injected with phosphate-buffered saline (PBS) were used as controls.

Statistical analysis
GraphPad Prism 6 (GraphPad Software, version 6.00, https://www.graphpad.com/) was used to analyze the experimental data. A p-value < 0.05 was considered statistically significant. In addition to using log rank test analysis of survival rate of silkworm, all others used oneway analysis of variance.