Juglone Inhibits Listeria monocytogenes ATCC 19115 by Targeting Cell Membrane and Protein

Foodborne disease caused by Listeria monocytogenes is a major global food safety problem. A potential solution is the antimicrobial development of the highly bioactive natural product juglone, yet few studies exist on its antibacterial mechanism against L. monocytogenes. Thus, we aimed to elucidate the antibacterial mechanism of action of juglone against L. monocytogenes by determining the resultant cell morphology, membrane permeability, membrane integrity, and proteome changes. The minimum inhibitory concentration of juglone against L. monocytogenes was 50 μg/mL, and L. monocytogenes treated with juglone had longer lag phases compared to controls. Juglone induced L. monocytogenes cell dysfunction, leakage of potassium ions, and membrane potential hyperpolarization. Confocal laser scanning microscopy and field-emission-gun scanning electron microscope assays revealed clear membrane damage due to juglone treatment. Fourier transform infrared analyses showed that L. monocytogenes responded to juglone by some conformational and compositional changes in the molecular makeup of the cell membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that juglone either destroyed proteins or inhibited proteins synthesis in L. monocytogenes. Therefore, our findings established juglone as a natural antibacterial agent with potential to control foodborne L. monocytogenes infections.


Introduction
Listeria monocytogenes is a Gram-positive, non-spore-forming bacterium that can acclimatize to various environments, including extreme conditions such as low pH (<4.4), water activity (<0.94), and temperature (4/45 • C) [1]. Thus, L. monocytogenes generally contaminate meat, vegetables, fish, processed and ready-to-eat food, and dairy products [2,3]. As a human and animal pathogen, L. monocytogenes is especially harmful to pregnant women, newborns, the elderly, and people with low immune function, causing life-threatening infections such as sepsis, encephalitis, and meningitis [4]. At these risks, the pathogen mortality rate can be as high as 20-30% [5]. Despite the current use of beta lactams alone or coupled with aminoglycosides to treat L. monocytogenes infections [6,7]. These therapies are becoming increasingly ineffective due to antimicrobial resistance [8,9], necessitating the development of new and effective antimicrobial agents against L. monocytogenes. Thus, we investigated the antimicrobial effects of juglone against L. monocytogenes in this study.
Many natural bioactive compounds are potential antibacterials with use that does not engender antimicrobial resistance [10]. One of these is juglone, also called 5-Hydroxy-1,4naphthoquinone (C 10 H 6 O 3 ), which is yielded by the walnut green husk. Not only is it lowly toxic and affordable [11,12], it also has good anticancer, antifungal, and antibacterial activities. Juglone is regarded as important drug candidate for the treatment of cancer, which had been proven to inhibit prostate cancer [13], cervical cancer [11], breast cancer [14], etc. Moreover, the antifungal activity of juglone was founded against Candida albicans, Penicillium spp., Aspergilluus spp., Hansenula spp., and Saccharomyces carlsbergensis. [15,16]. Gumus et al. (2020) reported that juglone is effective in clearing biofilm infections caused by Candida albicans. Additionally, there are some reports on juglone inhibiting bacteria [15].  analyzed differential proteins of Escherichia coli treated with juglone based on label-free quantitative proteomics, and the study suggested that juglone effectively restricted energy generation and blocked RNA formation and ribosome assembly [17]. Relatedly, juglone lysed Staphylococcus aureus by inhibiting pathways involved in protein synthesis, the tricarboxylic acid cycle, and nucleic acid synthesis [18].
Despite this proven antibacterial effect, there exist few studies on the inhibition of pathogens by juglone, and little is known about its antibacterial effect on L. monocytogenes. Thus, in this study, the inhibition zones, the minimum inhibitory concentrations (MIC), and growth-curve analyses of juglone against L. monocytogenes were determined to assess its antimicrobial activity. Then, membrane permeability was also analyzed by the leakage of potassium ions and membrane potential changes. The membrane integrity was further evaluated by confocal laser scanning microscopy (CLSM), field-emission-gun scanning electron microscope (FEG-SEM) assays, and Fourier transform infrared (FT-IR) analyses. Finally, we explored the effects of juglone on the pathogenicity of L. monocytogenes by analyzing changes in cellular proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Our findings established that juglone is a potential antibacterial agent for controlling foodborne L. monocytogenes infections.

Bacterial Strains and Growth Conditions
L. monocytogenes ATCC 19,115 was purchased from the American Type Culture Collection (Manassas, VA, USA) and cryopreserved at −80 • C [20]. Bacteria were cultured on trypticase soy agar (TSA; Land Bridge, Beijing, China) at 37 • C for 30 h. A single colony was incubated with shaking at 130 rpm for 18-24 h at 37 • C in 30 mL of sterile tryptic soy broth (TSB; Land Bridge, Beijing, China). The cell pellet was obtained by centrifugation (8000× g, 10 min, 4 • C) and rinsed twice with sterile TSB. Then, the pellet was resuspended in TSB, diluted to an OD 600nm = 0.5 (~10 8 CFU/mL), and used in subsequent assays.

Antimicrobial Susceptibility Testing
The inhibition effect of juglone against L. monocytogenes was tested by Oxford cup method [21], with some modifications. Briefly, L. monocytogenes (200 µL) cells were plated on TSA. An Oxford cup (Φ7.8 × 6 × 10 mm) loaded with 200 µL juglone of 250, 500, 1000, 2000, and 4000 µg/mL was placed on the surface of the TSA plate. Absolute ethanol and ampicillin Na (50 µg/mL) were used as negative and positive controls, respectively. The plates were incubated at 37 • C for 24 h. The diameters of the inhibition zones were measured by a cursor caliper.

Determination of Minimum Inhibitory Concentration (MIC)
The minimal inhibitory concentration of juglone against L. monocytogenes was determined using the broth dilution method based on the Clinical and Laboratory Standards Institute guidelines [22]. Briefly, a 100-fold dilution of L. monocytogenes in TSB was made, and seven test tubes were inoculated with 5 mL of it. To these, juglone solution was added to create a dilution series with final juglone concentrations of 400, 200, 100, 50, 25, 12.5, and 6.25 µg/mL. The blank control, negative controls and positive controls were TSB-mediumonly, juglone-free bacteria cultures containing 2% ethanol (v/v), and juglone-free bacteria cultures with ampicillin Na (50 µg/mL). All test tubes were mixed and incubated at 37 • C Foods 2022, 11, 2558 3 of 15 for 24 h. The MIC was defined as the lowest concentration of juglone that inhibited visually observable bacterial growth.

Growth Curves
Growth curves were constructed, as described by Shi et al. [23]. Briefly, L. monocytogenes was cultured overnight at 37 • C. The cells were rinsed with sterile TSB then diluted to an OD 600nm = 0.5 (~108 CFU/mL). A 100-fold dilution of this was used to make a microdilution series as follows. Bacteria cultures (125 µL) were mixed with 125 µL juglone solutions of various concentrations in a 100-well microtiter plate. This created a dilution series with final juglone concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, and 0.39 µg/mL. Juglone-free cultures containing 2% ethanol (v/v) were used as controls. The samples were incubated for 24 h at 37 • C, with OD 600nm measurements automatically taken after every hour using a Bioscreen C system (Labsystems, Helsinki, Finland).

Potassium Ion Leakage
To determine whether juglone resulted in the release of potassium ions from bacterial cells and into the media, a previously described method was used [24]. Briefly, L. monocytogenes was cultured overnight at 37 • C, after which cells were rinsed and resuspended at a concentration of OD 600nm = 0.5 in physiological saline. To these cultures (5 mL), juglone was added at final concentrations 0, 100, and 200 µg/mL. These were then incubated at 37 • C, after which cells were separated from supernatants by centrifugation (8000× g, 10 min) followed by filtration of supernatants through 0.22 µm pore-size PVDF membranes. The concentration of potassium ions in the cell-free supernatant was then determined by atomic absorption spectrophotometry (ZEEnit700P, Jena, Germany). In addition, KCl solutions of different concentrations were used as the standard curve.

Membrane Potential Determinations
Changes in membrane potential were determined as previously described [25], with some modifications. Briefly, L. monocytogenes was cultured for 18 h in 30 mL of TSB at 37 • C, harvested by centrifugation (8000× g, 10 min), rinsed twice, and resuspended at a concentration of OD 600nm = 0.5 in physiological saline. These cultures (125 µL, OD 600nm = 0.5) were supplemented with juglone (125 µL) to final concentrations of 0, 50, 100, and 200 µg/mL. The cultures were incubated at 37 • C for 30 min in black 96-well microtiter plates. Subsequently, cultures (250 µL) were mixed with the fluorescent probe bis-(1,3-dibutylbarbi-turic acid) trimethine oxonol (DiBAC4(3); Molecular Probes, Solarbio, Beijing, China) at the final concentration of one µM and transferred into individual wells of a black 96-well plate, then incubated for five minutes at 37 • C in the dark. After five minutes, fluorescence intensities were measured at excitation wavelength 492 nm and emission wavelength 515 nm with a multimode reader (Spectra Max M 2, Molecular Devices, San Jose, CA, USA). Background fluorescence caused by medium supplemented with juglone was also measured and used to correct fluorescence intensities.

CLSM Assay
The damage to L. monocytogenes cell membranes were observed using CLSM as described by Kang et al. [28], with some modifications. L. monocytogenes cells were collected per Section 2.2 and treated with juglone (0, 50, 100, and 200 µg/mL) at 37 • C for 3 h. Then, the cells were washed twice with sterile PBS, pH7.3, collected by centrifugation at 8000× g for 5 min, and resuspended in 200 µL working staining fluid (5 µM Syto-9 and 2 µg/mL PI). This was then mixed thoroughly at 37 • C in the dark for 15 min. Finally, the bacterial droplets were placed on a glass slide, covered with a coverslip, and scrutinized under CLSM (FV1200; Olympus, Tokyo, Japan) at 1000× magnification.

FT-IR Spectroscopy
Samples for FT-IR were prepared, according to the method of He et al. [29], with slight modifications. L. monocytogenes, prepared per Section 2.2, were incubated with juglone (50, 100, and 200 µg/mL) at 37 • C for 8 h, while control cells were treated with 2% ethanol (v/v) under the same conditions. The cell pellets were obtained after centrifugation (8000× g, 10 min) and rinsed twice with sterile PBS (pH 7.2) buffer. Samples were freeze-dried in vacuo for two days. Each sample (1.0 mg) was then mixed with dried potassium bromide powder (100 mg) and ground into a uniform powder for tableting. FT-IR spectras were obtained using a Bruker vertex70 FT-IR spectrophotometer (Bruker, Karlsruhe, Germany) at wavenumbers 400 cm −1~4 000 cm −1 with a resolution of 4 cm −1 for 32 scans. Finally, acquired spectras were smoothed, normalized, and baseline-corrected.

SDS-PAGE
The effects of juglone on intracellular soluble proteins of L. monocytogenes were analyzed using a modified SDS-PAGE method [30]. L. monocytogenes were cultivated in media supplemented with specific concentrations of juglone (MIC, 2 MIC, and 4 MIC) at 37 • C for 8 h. Cultures in juglone-free media supplemented with 2% ethanol (v/v) were used as control. Samples were centrifuged for 5 min (8000× g, 4 • C) after incubation. Supernatants were discarded, and cell pellets were then rinsed twice with PBS (pH 7.2). The lysate preparation (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Trion X-100, 1% sodium deoxycholate, 0.1% SDS, and protease and phosphatase inhibitor cocktail for bacterial cell extracts) was added into the cell pellet and incubated for 50 min on ice. The supernatant was collected by centrifugation (8000× g, 10 min, and 4 • C), and its protein concentration was determined using a BCA Protein Assay Kit (Solarbio, Beijing, China). Then, 10 µL SDS-PAGE loading buffer was added to 30 µL of samples. These mixtures were boiled for 8 min, cooled on ice, and then ran in SDS-PAGE gels. After electrophoresis, gels were stained with Coomassie brilliant blue R-250 and subsequently decolorized to visualize protein bands.

Statistical Analysis
All experiments were performed in triplicates. Data are expressed as means ± standard deviation. One-way analysis of variance (ANOVA) and Tukey's test multiple range tests (SPSS 23.0, SPSS Inc., Chicago, IL, USA) were used to evaluate the significant differences between means (p < 0.05).

Antimicrobial Effects of Juglone
The antibacterial activity of juglone was evaluated by inhibition zone and MIC. The diameter of the inhibition zone formed by juglone on the surface of L. monocytogenes was shown in Figure 1a. Low concentrations of juglone (250 µg/mL) inhibited the growth of L. monocytogenes, with inhibition zones of diameter 11.09 mm. This inhibitory effect increased with concentration, and the diameter of the inhibition zone was 20.27 mm at 4000 µg/mL of juglone. The inhibitory concentrations of juglone against L. monocytogenes are summarized in Figure 1b and Table 1 of juglone against L. monocytogenes was 50 µg/mL. Relatedly, Li et al. showed that the MIC of shikonin-another naphthoquinone compound-against L. monocytogenes was also 50 µg/mL, suggesting that compounds with similar structures have similar biological activities [31]. Previous studies have shown that carvacrol, thymol, clove oil, phenyl lactic acid, and olive oil polyphenol extract had respective MICs of 250, 250, 500, 1250, and 1250 µg/mL against L. monocytogenes [32][33][34][35][36]. Based on these previous reports, it appears that juglone has stronger antimicrobial activity against L. monocytogenes than other currently studied plant extracts. monocytogenes, with inhibition zones of diameter 11.09 mm. This inhibitory effect increased with concentration, and the diameter of the inhibition zone was 20.27 mm at 4000 μg/mL of juglone. The inhibitory concentrations of juglone against L. monocytogenes are summarized in Figure 1b and Table 1. Tubes 5-8 were turbid, and tubes 1-4 were clear, indicating that L. monocytogenes in tubes 1-4 have no visible growth. Thus, the MIC of juglone against L. monocytogenes was 50 μg/mL. Relatedly, Li et al. showed that the MIC of shikonin-another naphthoquinone compound-against L. monocytogenes was also 50 μg/mL, suggesting that compounds with similar structures have similar biological activities [31]. Previous studies have shown that carvacrol, thymol, clove oil, phenyl lactic acid, and olive oil polyphenol extract had respective MICs of 250, 250, 500, 1250, and 1250 μg/mL against L. monocytogenes [32][33][34][35][36]. Based on these previous reports, it appears that juglone has stronger antimicrobial activity against L. monocytogenes than other currently studied plant extracts. . Different letters denote significant differences (p < 0.05). Note: 1-10: 400 µg/mL, 200 µg/mL, 100 µg/mL, 50 µg/mL, 25 µg/mL, 12.5 µg/mL, 6.25 µg/mL, 2% ethanol, TSB, ampicillin Na (50 µg/mL).

Growth Curves
The growths of L. monocytogenes were completely inhibited by juglone at concentrations ranging from 50 µg/mL to 200 µg/mL, as per growth-curve analysis ( Figure 2). The lag phase of L. monocytogenes was prolonged by juglone at 6.25, 12.5, and 25 µg/mL. However, at concentrations of 6.25 µg/mL or below, juglone had no effect on the growth of L. monocytogenes. These results suggest that the inhibitory effects increased in a concentrationdependent manner. Growth-curve analysis not only showed the lag period was significantly prolonged at subinhibitory concentrations but also corroborated 50 µg/mL of juglone as its MIC against L. monocytogenes. Li et al. (2021) reported a similar effect of shikonin on the growth of L. monocytogenes [31], further supporting the hypothesis that compounds with similar structures have similar biological activities.

Growth Curves
The growths of L. monocytogenes were completely inhibited by juglone at con tions ranging from 50 μg/mL to 200 μg/mL, as per growth-curve analysis (Figure lag phase of L. monocytogenes was prolonged by juglone at 6.25, 12.5, and 25 μg/mL ever, at concentrations of 6.25 μg/mL or below, juglone had no effect on the grow monocytogenes. These results suggest that the inhibitory effects increased in a con tion-dependent manner. Growth-curve analysis not only showed the lag period w nificantly prolonged at subinhibitory concentrations but also corroborated 50 μg juglone as its MIC against L. monocytogenes. Li et al. (2021) reported a similar effec konin on the growth of L. monocytogenes [31], further supporting the hypothesis th pounds with similar structures have similar biological activities.

Leakage of Intracellular Potassium Ions
The permeability of the cell membrane could be identified by quantifying the a of potassium ions release [29,37]. The amounts of extracellular potassium ions of L cytogenes with different treatments are displayed in Figure 3. Compared to the unt with-juglone group, the amount of potassium ions released was significantly eleva the juglone-treatment group. Concretely, the potassium ions leakage in 100, 200, a μg/mL juglone treatment increased by 218%, 230%, and 264%, respectively, comp the untreated-with-juglone group. Furthermore, the amount of potassium ions with different concentrations of juglone have a sharp rise in 30 min, indicating that has a rapid destruction of the permeability of the cell membrane. This sharp rise c ascribed to either elevated membrane permeability or depolarization of cell mem

Leakage of Intracellular Potassium Ions
The permeability of the cell membrane could be identified by quantifying the amount of potassium ions release [29,37]. The amounts of extracellular potassium ions of L. monocytogenes with different treatments are displayed in Figure 3. Compared to the untreated-with-juglone group, the amount of potassium ions released was significantly elevated for the juglonetreatment group. Concretely, the potassium ions leakage in 100, 200, and 400 µg/mL juglone treatment increased by 218%, 230%, and 264%, respectively, compared to the untreatedwith-juglone group. Furthermore, the amount of potassium ions treated with different concentrations of juglone have a sharp rise in 30 min, indicating that juglone has a rapid destruction of the permeability of the cell membrane. This sharp rise could be ascribed to either elevated membrane permeability or depolarization of cell membrane induced damage on potassium channels [26,38]. However, different antimicrobial agents have different effects on the release of potassium ions from bacterium. Fang et al. (2022) reported that phenyl lactic acid promoted the release of potassium ions from Vibrio parahaemolyticus within five minutes [39]. On the other hand, β-type oligomeric procyanidins from a lotus seedpod could promote the release of potassium ions from enterotoxigenic E. coli in a longer process of about 6 h [40]. These differences in duration might be due to the different modes of action of the various antibacterial agents.
Foods 2022, 11, x FOR PEER REVIEW reported that phenyl lactic acid promoted the release of potassium ions from Vibr haemolyticus within five minutes [39]. On the other hand, -type oligomeric procy from a lotus seedpod could promote the release of potassium ions from enteroto E. coli in a longer process of about 6 h [40]. These differences in duration might be the different modes of action of the various antibacterial agents.

Membrane Potential
In analyses of changes in membrane potential, L. monocytogenes that had bee vated in media supplemented with 50~400 μg/mL of juglone had a noticeable decr fluorescence in comparison with the untreated-with-juglone group (Figure 4). Mo the higher the concentration of juglone is, the lower the resultant fluorescence. Mem potential depolarization and hyperpolarization are hallmarks of membrane dama DiBAC4(3) is a lipophilic anionic fluorescent dye commonly used to detect cell mem potential [41]. It enters depolarized cells and increases fluoresce when bound to in lular proteins but leaves hyperpolarized cells with decreased fluorescence [19]. Th results indicate that L. monocytogenes treated with juglone displayed membrane po hyperpolarization. This phenomenon is mainly caused by change of pH and excita ion motion, especially the diffusion of potassium ions or potassium ions with severa ions [27]. Similar research reported that alkyl ferulate esters can hyperpolarize membranes of L. monocytogenes [42]. Relatedly, citral caused cell membrane hyper zation in Yersinia enterocolitica and Cronobacter sakazakii [23,43].

Membrane Potential
In analyses of changes in membrane potential, L. monocytogenes that had been cultivated in media supplemented with 50~400 µg/mL of juglone had a noticeable decrease in fluorescence in comparison with the untreated-with-juglone group (Figure 4). Moreover, the higher the concentration of juglone is, the lower the resultant fluorescence. Membrane potential depolarization and hyperpolarization are hallmarks of membrane damage [27]. DiBAC4(3) is a lipophilic anionic fluorescent dye commonly used to detect cell membrane potential [41]. It enters depolarized cells and increases fluoresce when bound to intracellular proteins but leaves hyperpolarized cells with decreased fluorescence [19]. Thus, our results indicate that L. monocytogenes treated with juglone displayed membrane potential hyperpolarization. This phenomenon is mainly caused by change of pH and excitation of ion motion, especially the diffusion of potassium ions or potassium ions with several other ions [27]. Similar research reported that alkyl ferulate esters can hyperpolarize the cell membranes of L. monocytogenes [42]. Relatedly, citral caused cell membrane hyperpolarization in Yersinia enterocolitica and Cronobacter sakazakii [23,43].

FEG-SEM-Based Observations of Cell Morphology
The antibacterial mode of action of juglone was further understood through the effect of juglone on the morphological changes of L. monocytogenes by FEG-SEM. As shown in Figure 5, L. monocytogenes cells cultivated in media supplemented with juglone (50 μg/mL, Figure 5B) had continuous pits on the bacterial surface and were more wrinkled and deformed compared to the controls, which were plump, short-rod-shaped, and smooth-surfaced ( Figure 5A). Those from media supplemented with juglone (100 μg/mL) had deeper cell pits and some ruptured cell membranes ( Figure 5C). Those from media supplemented with juglone (200 μg/mL) had completely destroyed membranes, lacked their original rod-shaped morphology, leaked cell contents, and generally were fragmented cells (Figure 5D). This revealed concentration-dependent damage of the L. monocytogenes membrane by juglone. Moreover, these findings show that juglone ruptured the cell membrane in an irreversible manner, resulting in a bactericidal effect. Linalool and noni fruit extract had the same effect on L. monocytogenes [44,45]. However, alkyl ferulate esters achieves their bacteriostatic effect by making holes in the surface of the cell membrane of L. monocytogenes [42]. Citral treatments led to Yersinia enterocolitica cell membrane atrophy and sagging but no membrane rupture nor the formation of pores [43]. Therefore, different natural compounds have different modes of action on different pathogens. Altogether, cell membrane damage is one of the targets of juglone against L. monocytogenes.

FEG-SEM-Based Observations of Cell Morphology
The antibacterial mode of action of juglone was further understood through the effect of juglone on the morphological changes of L. monocytogenes by FEG-SEM. As shown in Figure 5, L. monocytogenes cells cultivated in media supplemented with juglone (50 µg/mL, Figure 5B) had continuous pits on the bacterial surface and were more wrinkled and deformed compared to the controls, which were plump, short-rod-shaped, and smoothsurfaced ( Figure 5A). Those from media supplemented with juglone (100 µg/mL) had deeper cell pits and some ruptured cell membranes ( Figure 5C). Those from media supplemented with juglone (200 µg/mL) had completely destroyed membranes, lacked their original rod-shaped morphology, leaked cell contents, and generally were fragmented cells ( Figure 5D). This revealed concentration-dependent damage of the L. monocytogenes membrane by juglone. Moreover, these findings show that juglone ruptured the cell membrane in an irreversible manner, resulting in a bactericidal effect. Linalool and noni fruit extract had the same effect on L. monocytogenes [44,45]. However, alkyl ferulate esters achieves their bacteriostatic effect by making holes in the surface of the cell membrane of L. monocytogenes [42]. Citral treatments led to Yersinia enterocolitica cell membrane atrophy and sagging but no membrane rupture nor the formation of pores [43]. Therefore, different natural compounds have different modes of action on different pathogens. Altogether, cell membrane damage is one of the targets of juglone against L. monocytogenes.

CLSM-Based Observations of Cell Membrane Injury
Cell membrane damage by juglone was confirmed by CLSM integrated with the fluorescent probes SYTO 9 and PI. SYTO 9 has membrane permeability and penetrates cells freely, resulting in emission of green fluorescence. PI only penetrates damaged cell membranes, resulting in emission of red fluorescence. There was a transition from green to red fluorescence as a function of the concentration of juglone treatment ( Figure 6). L. monocytogenes cells in the juglone-free group emitted distinct green fluorescence only, indicating that they had intact cell membranes. After treatment with juglone (50 μg/mL), the green-fluorescence intensity diminished and small amounts of red fluorescence were observed, indicating cytoplasmic membrane injury in a small proportion of cells. After treatment with juglone (100 μg/mL), the green-fluorescence intensity further decreased, while the red-fluorescence intensity was further increased, indicating a larger proportion of cells had lost their membrane integrity. After treatment with juglone (200 μg/mL), the treated L. monocytogenes cells only emitted red fluorescence. This demonstrated that juglone exerted the membrane damage in a concentration-dependent manner. Relatedly, juglone damaged the cell membranes of Candida albicans and Pseudomonas syringae in a concentration-dependent manner [12,15]. Altogether, these findings support the previous hypothesis that juglone damaged the cell membranes of L. monocytogenes.

CLSM-Based Observations of Cell Membrane Injury
Cell membrane damage by juglone was confirmed by CLSM integrated with the fluorescent probes SYTO 9 and PI. SYTO 9 has membrane permeability and penetrates cells freely, resulting in emission of green fluorescence. PI only penetrates damaged cell membranes, resulting in emission of red fluorescence. There was a transition from green to red fluorescence as a function of the concentration of juglone treatment ( Figure 6). L. monocytogenes cells in the juglone-free group emitted distinct green fluorescence only, indicating that they had intact cell membranes. After treatment with juglone (50 µg/mL), the green-fluorescence intensity diminished and small amounts of red fluorescence were observed, indicating cytoplasmic membrane injury in a small proportion of cells. After treatment with juglone (100 µg/mL), the green-fluorescence intensity further decreased, while the red-fluorescence intensity was further increased, indicating a larger proportion of cells had lost their membrane integrity. After treatment with juglone (200 µg/mL), the treated L. monocytogenes cells only emitted red fluorescence. This demonstrated that juglone exerted the membrane damage in a concentration-dependent manner. Relatedly, juglone damaged the cell membranes of Candida albicans and Pseudomonas syringae in a concentration-dependent manner [12,15]. Altogether, these findings support the previous hypothesis that juglone damaged the cell membranes of L. monocytogenes.

FT-IR Analysis
FT-IR spectroscopy was widely used to explored the antimicrobial mode of action and monitor the structural changes in bacterial cell membranes under various forms of stress [46,47]. The FT-IR spectra of bacterial cells are generally distinguished into five regions: lipids (3000-2800 cm −1 ), protein/amides I and II (1700-1500 cm −1 , phospholipids/DNA/RNA (1500-1185 cm −1 ), polysaccharides (1185-900 cm −1 ), and the fingerprint region (900-600 cm −1 ) [46]. The FT-IR analysis of juglone-treated L. monocytogenes at peaks of 2928 cm −1 (representing the CH2 antisymmetric stretching of lipids) had band intensities that increased with increasing juglone concentration, which indicated an increase in the saturated lipid concentration of the membrane (Figure 7). The enhanced peak intensity of another fatty-acid-associated band (CH2 bending) at around 1455 cm −1 further supported the results. Increases in saturated lipid concentration are often explained as an increase in rigidity and permeability of the bacterial cell membrane [29,48]. These increments occur when the cellular membrane is damaged, leading to cell destruction and death [49,50]. Similar results were observed for S. aureus that were subjected to an ultrasound coupled

FT-IR Analysis
FT-IR spectroscopy was widely used to explored the antimicrobial mode of action and monitor the structural changes in bacterial cell membranes under various forms of stress [46,47]. The FT-IR spectra of bacterial cells are generally distinguished into five regions: lipids (3000-2800 cm −1 ), protein/amides I and II (1700-1500 cm −1 , phospholipids/DNA/RNA (1500-1185 cm −1 ), polysaccharides (1185-900 cm −1 ), and the fingerprint region (900-600 cm −1 ) [46]. The FT-IR analysis of juglone-treated L. monocytogenes at peaks of 2928 cm −1 (representing the CH 2 antisymmetric stretching of lipids) had band intensities that increased with increasing juglone concentration, which indicated an increase in the saturated lipid concentration of the membrane (Figure 7). The enhanced peak intensity of another fatty-acid-associated band (CH 2 bending) at around 1455 cm −1 further supported the results. Increases in saturated lipid concentration are often explained as an increase in rigidity and permeability of the bacterial cell membrane [29,48]. These increments occur when the cellular membrane is damaged, leading to cell destruction and death [49,50]. Similar results were observed for S. aureus that were subjected to an ultrasound coupled with thyme essential oil nanoemulsions [29]. With increasing con-centrations of juglone, L. monocytogenes cells showed a corresponding increase in spectral intensities in regions 1653 cm −1 (dominated by amide I; C-O hydrogen-bonded stretching vibrations), reflecting the proteinaceous contents of the cells [51]. This might be attributed to the denaturation of proteins in the cell walls and cell membranes after treatment with juglone. Furthermore, juglone treatment caused changes in bands at 1397 cm −1 (attributed to C=O symmetric stretching of COO-group in amino acids, fatty acids), 1235 cm −1 (attributed to phosphate group (P=O) asymmetric stretching of the phosphodiester bond of nucleic acid), and 1072 cm −1 (attributed to symmetric stretching of P=O phospholipids in cell membranes) [47,52]. These changes demonstrated that juglone resulted in either L. monocytogenes cellular membrane damage or some conformational and compositional changes in the molecular makeup of the cell membrane.
Foods 2022, 11, x FOR PEER REVIEW with thyme essential oil nanoemulsions [29]. With increasing concentrations of jug monocytogenes cells showed a corresponding increase in spectral intensities in region cm −1 (dominated by amide I; C-O hydrogen-bonded stretching vibrations), reflect proteinaceous contents of the cells [51]. This might be attributed to the denatura proteins in the cell walls and cell membranes after treatment with juglone. Furthe juglone treatment caused changes in bands at 1397 cm −1 (attributed to C=O sym stretching of COO-group in amino acids, fatty acids), 1235 cm −1 (attributed to pho group (P=O) asymmetric stretching of the phosphodiester bond of nucleic acid), an cm −1 (attributed to symmetric stretching of P=O phospholipids in cell membranes) These changes demonstrated that juglone resulted in either L. monocytogenes cellula brane damage or some conformational and compositional changes in the mo makeup of the cell membrane.

SDS-PAGE Analysis
Protein is closely related to the physiological functions of cells. The protein ba the marker and cells were shown in Figure 8. After cultivation in media supplem with juglone (2MIC and 4MIC), bacterial protein bands of L. monocytogenes were and even disappeared when compared to those of the control. This implied that j inhibited L. monocytogenes by affecting protein synthesis. This is similar to the effe Poly-lysine and lactic acid on L. monocytogenes [53,54]. Protein bands between 20-2 in L. monocytogenes treated with juglone at the MIC, 2MIC, and 4MIC were increase significantly than those of the control, as were those of 50-70 kDa and 25-40 kD cells treated with juglone at the MIC. Previous studies have found that bacteria ca thesize small proteins or molecular chaperones to strengthen resistance to e

SDS-PAGE Analysis
Protein is closely related to the physiological functions of cells. The protein bands of the marker and cells were shown in Figure 8. After cultivation in media supplemented with juglone (2MIC and 4MIC), bacterial protein bands of L. monocytogenes were fainter and even disappeared when compared to those of the control. This implied that juglone inhibited L. monocytogenes by affecting protein synthesis. This is similar to the effect of ε-Poly-lysine and lactic acid on L. monocytogenes [53,54]. Protein bands between 20-25 KDa in L. monocytogenes treated with juglone at the MIC, 2MIC, and 4MIC were increased more significantly than those of the control, as were those of 50-70 kDa and 25-40 kDa from cells treated with juglone at the MIC. Previous studies have found that bacteria can synthesize small proteins or molecular chaperones to strengthen resistance to external pressure and protect cells from protein denaturation when faced with external stress [55]. Thus, we hypothesize that L. monocytogenes synthesize some stress proteins to strengthen resistance to the effects of juglone. pressure and protect cells from protein denaturation when faced with external stress [55]. Thus, we hypothesize that L. monocytogenes synthesize some stress proteins to strengthen resistance to the effects of juglone.

Conclusions
In conclusion, the present research elucidated the antibacterial activity and mechanism of action of juglone against L. monocytogenes. The antibacterial mechanism of action of juglone against L. monocytogenes included increased cytomembrane permeability, cell membrane hyperpolarization, destroyed proteins or inhibited proteins synthesis, changes in membrane integrity, and, ultimately, complete disintegration of the cells. Overall, our findings show that juglone is a potential antibacterial agent, which is, thus, an alternative or supplemental strategy to mitigate the infections caused by L. monocytogenes. Further research should focus on the effect of juglone on the antimicrobial signaling pathways of L. monocytogenes by transcriptome and proteomics analyses.

Conclusions
In conclusion, the present research elucidated the antibacterial activity and mechanism of action of juglone against L. monocytogenes. The antibacterial mechanism of action of juglone against L. monocytogenes included increased cytomembrane permeability, cell membrane hyperpolarization, destroyed proteins or inhibited proteins synthesis, changes in membrane integrity, and, ultimately, complete disintegration of the cells. Overall, our findings show that juglone is a potential antibacterial agent, which is, thus, an alternative or supplemental strategy to mitigate the infections caused by L. monocytogenes. Further research should focus on the effect of juglone on the antimicrobial signaling pathways of L. monocytogenes by transcriptome and proteomics analyses.  Data Availability Statement: Data is contained within the article and is available at request from the corresponding author.