Transcriptomic Analysis of the Molecular Mechanisms Underlying the

Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Transcriptomic Analysis of the Molecular Mechanisms Underlying the Antibacterial Activity of IONPs@pDA-Nisin Composites toward Alicyclobacillus acidoterrestris Zihan Song,†,‡,§ Chen Niu,∥ Hao Wu,† Jianping Wei,†,‡,§ Yuxiang Zhang,†,‡,§ and Tianli Yue*,†,‡,§,∥ †

College of Food Science and Engineering, Northwest A&F University, Yangling 712100, China Laboratory of Quality & Safety Risk Assessment for Agro-Products (Yangling), Ministry of Agriculture, Yangling 712100, China § National Engineering Research Center of Agriculture Integration Test (Yangling), Yangling 712100, China ∥ College of Food Science & Engineering, Northwest University Xian, Xian 710069, Shaanxi, PR China Downloaded by NOTTINGHAM TRENT UNIV at 02:47:38:354 on June 07, 2019 from https://pubs.acs.org/doi/10.1021/acsami.9b02990.

‡

ABSTRACT: A simple and no-drug resistance antibacterial method was developed by the synthesis of heat-stable and pHtolerant nisin-loaded iron oxide nanoparticles polydopamine (IONPs@pDA) composites. The composites had a crystal structure and diameters of 25 ± 3 nm, with a saturation magnetization (M s) of 43.7995 emu g −1 . Nisin was successfully conjugated onto the IONPs@pDA nanoparticles, as evinced by Fourier transform infrared spectroscopy and Xray photoelectron spectroscopy analyses. The novel synthesized material showed good performance in reducing Alicyclobacillus acidoterrestris, a common food spoilage bacterium that represents a significant problem for the food industry. Treatment of A. acidoterrestris cells with composites resulted in membrane damage, as observed by live/dead staining and scanning electron microscopy and transmission electron microscopy analyses. Further, the composites exhibited highly efficient antibacterial activity against cells in only 5 min. Transcriptomic sequencing of culture RNA pools after exposure to composites resulted in a total of 334 differentially expressed genes that were primarily associated with transcriptional regulation, energy metabolism, membrane transporters, membrane and cell wall syntheses, and cell motility. Thus, these results suggested that changes in transcriptional regulation caused by aggregated composites on target cells led to major changes in homeostasis that manifested by decreased energy metabolism, pore formation in the membrane, and repressed cell wall synthesis. Concomitantly, cell motility and sporulation activities were both repressed, and finally, intracellular substances flowed out of leaky cells. The proposed biocontrol method represents a novel means to control microorganisms without inducing drug resistance. Further, these results provide novel insights into the molecular mechanisms underlying the antibacterial activity of composites against microorganisms. KEYWORDS: nisin, IONPs@pDA nanoparticles, A. acidoterrestris, transcriptome analysis, antibacterial mechanism

1. INTRODUCTION Alicyclobacillus spp., the common contaminating bacteria in apple juice concentrate and related products, can metabolize 2methoxyphenol (guaiacol), 2,6-dibromophenol, and 2,6dichlorophenol, which are formed from ferulic acid via vanillin to induce a smoky, medicinal, antiseptic off-odor.1 Nisin is a natural food biopreservative that has been approved in nearly 50 countries and has been effective in controlling Alicyclobacillus genus. However, prolonged exposure of microorganisms to nisin results in drug resistance, representing a danger for food safety and human health.2 For example, nisin resistance has been observed in several bacterial species including Lactobacillus casei,3 Listeria monocytogenes,4 Streptococcus bovis,5 and Streptococcus thermophilus.6 To overcome the aforementioned drawbacks of nisin, polydopamine (pDA)−coated iron oxide nanoparticles (IONPs) were introduced to bioconjugate with nisin because of its ability to capture target bacteria, and they can also be recycled and © XXXX American Chemical Society

reused. Moreover, dopamine was proposed to act as a linker for immobilization between nisin and IONPs. Thus, we synthesized nisin-loaded IONPs@pDA composites that possessed efficient antibacterial activity and did not elicit drug resistance in cultures. The antibacterial mechanism of nisin has been studied worldwide. It is generally reported that nisin could insert into the bacterial membrane.7 Then, the pore formed in the cytoplasmic membrane results in the leakage of cellular content.8 RNA-sequencing (RNA-Seq) technology is a recently developed, powerful, and cost-efficient method to investigate gene expression levels.9 However, little study has been conducted to understand the mechanism of nisin toward Alicyclobacillus acidoterrestris at gene expression levels using Received: February 16, 2019 Accepted: May 22, 2019

A

DOI: 10.1021/acsami.9b02990 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

stirred for 2 h in the sterile phosphate buffer with pH adjusted to values between 2 and 11. Following treatment with different pHadjusted PBS at 4 °C, the composites were magnetically separated and washed three times with PBS (pH 6.86). To evaluate the stability of the composites over a range of temperature, they were again suspended in PBS (pH 6.86) and then incubated at 40, 60, 80, and 100 °C for 10, 20, and 30 min. The suspensions were then immediately transferred to ice water. Following each of the aforementioned treatments, the composites were added into DSM 3922 cell cultures in the AAM broth (106 CFU/mL) with gentle stirring for 1 h. An estimation of viable cells was then evaluated by counting the colony-forming units (CFU) after culture dilution plating. 2.5. Fluorescent-Based Analysis of Cellular Viability. 2.5.1. Abundance of Bacterial Cells Retaining Membrane Integrity. A live/dead cell staining assay (BacLight bacterial viability kits 7012, Molecular Probes, USA) was used to evaluate the extent of membrane integrity of treated cells using previously described methods with slight modification.13 Briefly, live and killed cell cultures were adjusted to 106 CFU/mL and then diluted to achieve five proportions of live cell suspensions (0, 10, 50, 90, and 100%) as standard solutions. 2× working stain solutions were obtained by mixing SYTO 9 and propidium iodide (PI) in a 1:1 ratio. Bacterial cells (106 CFU/mL) were then incubated with nisin-loaded IONPs@pDA composites for 5, 30, and 120 min with gentle stirring. The cells were magnetically extracted and washed three times with 0.85% NaCl, followed by addition of 100 μL of each bacterial cell suspension and 100 μL of 2× working stain into a 96-well flat-bottom microplate. Fluorescence was then measured in each well using a fluorescence microplate reader (Infinite M200 PRO; Tecan), and the excitation/emission maxima are 485/530 nm for SYTO 9 and 485/630 nm for PI. 2.5.2. Laser Scanning Fluorescence Microscopy. To evaluate the live/dead ratio after treatment with composites, the cellular suspensions (106 CFU/mL) were treated in the same manner as in the membrane integrity assays. Live/dead staining assays (BacLight bacterial viability kits 7012, Molecular Probes, USA) were conducted following the manufacturer instructions. Briefly, equal volumes of SYTO 9 and PI were mixed thoroughly in a 1.5 mL microcentrifuge tube. A 3 μL/mL solution of the dye was then added to bacterial cells and thoroughly mixed for 15 min in the dark at room temperature. Fluorescence images were then obtained via confocal laser scanning fluorescence microscopy (CLSM, Leica TCS SP8 Germany). The excitation/emission wavelengths used were 480/500 nm for the SYTO 9 dye and 490/635 nm for PI. 2.6. Scanning Electron Microscopy and TEM. Changes in cell morphologies as a result of exposure to nisin-loaded IONPs@pDA composites were evaluated with scanning electron microscopy (SEM) (S-4800, Hitachi, Japan). Prior to observations, the cells were fixed with 2.5% glutaraldehyde at 4 °C overnight. After washing three times with PBS buffer (0.1 M, pH 7.2), the cells were progressively dehydrated with 30, 50, 70, 90, and 100% ethanol for 10 min. Samples were then completely dried with CO2 for 3 h and coated with gold by sputtering (10 mM). Pretreatment of cells for TEM was according to the steps described above. After fixation with osmic acid and repeated dehydration with ethanol, the precipitates were permeated using white resin and then embedded in a drying oven at 60 °C for 48 h. Sections of approximately 70 nm were then prepared for observation on a JEM-1230 transmission electron microscope (JEOL, Japan). 2.7. Transcriptomic Analysis. To evaluate transcriptional changes of cultures in response to nanoparticle exposure, log-phase A. acidoterrestris DSM 3922 cells were treated with nisin-loaded IONPs@pDA composites for 0, 0.5, 2, and 5 min with gentle stirring. Each treatment was repeated in triplicate. Total culture RNA was then extracted using Magen HiPure Universal RNA kits. RNA quantities were assessed with agarose gel electrophoresis, a NanoDrop 2000 spectrophotometer (Thermo Scientific, United States), and an Agilent 2100 bioanalyzer (Agilent Technologies, United States). cDNA libraries were then constructed and sequenced on an Illumina HiSeq 2500 platform at the Gene Denovo Biotechnology Co. sequencing center (Guangzhou, China). High-quality RNA-Seq data

transcriptomics. Moreover, transcriptomic analysis could help evaluate the interactions between IONPs@pDA nanoparticles that are bioconjugated with nisin and A. acidoterrestris to better understand the molecular mechanisms underlying these interactions. From the above observations, evaluating the composite response in A. acidoterrestris via gene expression could provide novel insights into the mechanism underlying the antibacterial activity of nisin-loaded IONPs@pDA. Furthermore, the present study first investigated and provided results of the time-dependent changes in vital genes and proteins involved in the resistance of A. acidoterrestris to nisin-loaded IONP@pDA stress.

2. MATERIALS AND METHODS 2.1. Materials. Nisin (10 000 IU/mL) was purchased from SigmaAldrich (USA), while other common reagents were of analytical grade and purchased from a local supplier including dopamine, phosphatebuffered saline (PBS), ethanol, osmic acid, and white resin. A. acidoterrestris DSM 3922 culture was supplied by the China Center of Industrial Culture Collection. 2.2. pDA Encapsulation and Nisin Immobilization. IONPs were synthesized using previously described methods.10,11 To encapsulate the IONPs with pDA, 10 mg of IONPs were mixed with 4 mg/mL dopamine solution (10 mM PBS, pH 8.5).12 Continuous stirring of the solution was then conducted for 24 h at room temperature, followed by magnetic separation of the pDAcoated IONPs (IONPs@pDA) and thorough rinsing with Milli-Q deionized water. Bioconjugation of nisin to IONPs@pDA was conducted by mixing 10 mg of nanoparticles in a 10 mg nisin solution (10 mM PBS, pH 6.86) with continuous stirring for 1 h at room temperature. The nisin-loaded IONPs@pDA composites were then magnetically extracted from the dispersion and washed three times with Milli-Q deionized water. The loading amount of nisin in the composite was measured using the Bradford method. The standard solutions of nisin (0−12.0 mg/mL) were used for the calibration curve. 2.3. Material Characterization. Characterization of the surface morphologies and microstructures of the IONPs, IONPs@pDA, and nisin-loaded IONPs@pDA composites were performed by transmission electron microscopy (TEM), vibrating sample magnetometry (VSM), and X-ray diffraction (XRD) analysis. TEM analysis was conducted with a FEI Tecnai G2 F20 transmission electron microscope that was operated at 200 kV voltage. Magnetization of the samples was subsequently measured at room temperature using VSM (VSM-7307, Lake Shore, USA). In addition, XRD was conducted using a Bruker D8 ADVANCE X-ray diffractometer. Fourier transform infrared (FT-IR) spectroscopy and X-ray photoelectron spectroscopy (XPS) were used to assess the chemical composition of the nisin-loaded IONPs@pDA. FT-IR spectroscopy was conducted on a Bruker Vetex70 FT-IR spectrometer, while XPS analysis was conducted with a PE PHI-5400 spectrometer with a monochromatized Al Kα source. 2.4. Antibacterial Activity of Nisin-Loaded IONPs@pDA Composites. 2.4.1. Bacterial Strains. The A. acidoterrestris DSM 3922 strain used in this study was provided by the German Resource Centre for Biological Material. Cultures were grown at 45 °C for 18 h in Alicyclobacillus spp. medium (AAM) that contained yeast extract (2.0 g), glucose (2.0 g), (NH4)2SO4 (0.4 g), MgSO4·7H2O (1.0 g), CaCl2 (0.38 g), KH2PO4 (1.2 g), MnSO4 (0.5 g), and distilled water to 1 L with pH adjusted to 4.2. 2.4.2. pH- and Temperature-Dependence of Antibacterial Activity. Optimal antibacterial treatment times were first assessed for the cultures. Solutions of log-phase (106 CFU/mL) DSM 3922 cells were cultivated in the AAM broth and 8 mg of nisin-loaded IONPs@pDA composites for different lengths of time (15, 30, 45, 60, and 120 min). To evaluate the pH-dependent stability of the composites, nisin-loaded IONPs@pDA composites were gently B


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Figure 1. Characterization of IONPs@pDA-nisin composites. (A) TEM micrograph. (B) Selected area electron diffraction observations. (C) Single crystallite lattice fringe observations. (D) Hysteresis loops of the IONPs, IONPs@pDA, and IONPs@pDA-nisin composites at room temperature. (E) XRD analysis of the IONPs, IONPs@pDA, IONPs@pDA-nisin composites, and Fe3O4 nanoparticles at room temperature. (F) FT-IR transmittance spectra of the IONPs, IONPs@pDA, and IONPs@pDA-nisin composites. (i.e., clean reads) was obtained by removing low-quality reads that contained (1) adapters, (2) greater than 10% ambiguous nucleotides (N), and (3) greater than 50% low quality (Q-value ≤ 20) bases. Expression levels were determined using several methods including g quantification of gene expression level (HTSeq v0.6.1 software package, https://htseq.readthedocs.io/en/release_0.11.1/index.html) and identification of differentially expressed genes (DEGs) using previously described methods.14,15 DEGs were defined based on the following criteria: |log 2| > 2 and p < 0.05. RNA-Seq data was deposited in the National Center for Biotechnology Information database under accession PRJNA508544. 2.8. Statistical Analyses. All experimental data described here comprises triplicate experiments. Differences in data distributions were analyzed via one-way analysis of variance (ANOVA) in the DPS software package (Data Processing System, China). In addition, Duncan’s multiple range tests were used to evaluate differences in the mean values of treatment groups (p < 0.05).

magnetization value (Ms) for each was 48.8, 45.9, and 43.8 emu g−1, respectively (Figure 1D). Ms values decreased with dopamine and nisin modifications, likely due to the increased size of the particles. The decrease also indicated that the pDA coatings and nisin modifications were successful. In addition, the S-shaped magnetic hysteresis loops of the three curves exhibited zero remanence and coercivity, indicating that the composites inherited superparamagnetic characteristics. XRD analyses of the IONPs, IONPs@pDA, and IONPs@ pDA-nisin composites (Figure 1E) indicated that all samples exhibited the same diffraction peaks at 2θ of 30.2°, 35.6°, 43.2°, 57.1°, and 62.7°, which corresponded to signals of the (220), (311), (400), (511), and (440) of crystalline planes, respectively (JCPDS card no. 19-0629). These results thus suggested that the dopamine and nisin modifications did not obviously affect the crystal structures of the IONPs. FT-IR analyses were used to verify the covalent attachment of dopamine and nisin to the IONPs (Figure 1F). The FT-IR spectra for all IONP-based nanomaterials exhibited a characteristic absorption band at 580 cm−1 corresponding to Fe−O stretching vibrations associated with the IONPs. Other bands at 1036 and 1489 cm−1 were attributable to the C−O and benzene groups within the pDA-coated nanoparticles, respectively.16 In addition, the C−N stretching vibrations observed at 1200 cm−1 confirmed the presence of new Schiff bases that were formed during dopamine and nisin modifications.17 Further, the spectra of nisin and IONPs@ pDA-nisin composites displayed characteristic bands at 2978

3. RESULTS AND DISCUSSION 3.1. Characterization. The morphology and size of nisinloaded IONPs@pDA composites were observed using TEM, which indicated that the quasi-spherical nanoparticles had an average diameter of 25 ± 3 nm (Figure 1A). Different ring diameters corresponded to different IONP lattice planes (Figure 1B). In addition, adjacent two-dimensional lattice fringes were observed on the surface of the composites (Figure 1C). Interplanar spacing was approximately 0.206, 0.258, and 0.302 nm corresponding to the (400), (311), and (220) lattice planes (JCPDS card no. 19-0629). The magnetic characteristics of the IONPs, IONPs@pDA, and IONPs@pDA-nisin composites were evaluated using VSM. The saturation C


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Figure 2. (A) XPS spectrum for the IONPs@pDA-nisin catalyst and the corresponding spectra for (B) Fe 2p, (C) N 1s, (D) C 1s, and (E) O 1s.

cm−1 for methylene groups, also providing evidence for the presence of nisin layers on [email protected] XPS spectral analysis of the IONPs@pDA-nisin composites indicated the presence of Fe 2p, O 1s, N 1s, and C 1s signatures (Figure 2A). Within the Fe 2p spectrum (Figure 2B), two absorption peaks corresponding to Fe 2p1/2 at 712.0 eV and Fe 2p3/2 at 725.2 eV were observed, indicating that the IONPs were present as Fe3O4.19 The spectra of the N 1s regions exhibited three absorption peaks (Figure 2C) at 399.9, 401.0, and 402.5 eV corresponding to the presence of benzenoid amine (−NH−), iminium, and radical cation amine groups, respectively.20 These three components were clearly attributable to the chemisorbed pDA and nisin. C 1s peaks were observed at 284.8, 285.8, and 288.7 eV (Figure 2D) and could be deconvoluted to C−C,21 C−O/C−N,22 and carboxylate carbon (−COOH),23 respectively. Last, the O 1s region spectra exhibited two peaks at 531.7 and 530.3 eV (Figure 2E), indicating the presence of CO and Fe−O in IONPs@pDA-nisin composites.19 Collectively, the XPS fitting results were consistent with the FT-IR results and provided further evidence for the coexistence of dopamine and nisin on the nanoparticles. The loading amount of nisin in the composite was 0.40 mg/ mg.

3.2. Antibacterial Activity of Nanoparticles toward Vegetative Cells. No significant differences in the antimicrobial activity of IONPs@pDA-nisin composites were observed among treatment time groups (p < 0.05; Table 1). Consequently, the shortest treatment time (15 min) was chosen for further experimentation. Solutions of IONP@pDAnisin composites at all pH values tested between 2 and 11 resulted in significant reductions of cell counts (p < 0.05; Table 2). However, the highest cell count reductions were Table 1. Abundances of A. acidoterrestris Vegetative Cells (log CFU/mL) after Exposure to IONPs@pDA-Nisin Composites for Different Lengths of Timea time (min) control 15 30 45 60 120

log CFU/mL 4.92 1.24 1.24 1.24 1.24 1.24

± ± ± ± ± ±

0.01a 0.05b 0.04b 0.05b 0.00b 0.02b

a Statistical significance was assessed by ANOVA (n = 3) at the 95% confidence level. The same letters indicate the lack of statistical significance between groups.

D


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ACS Applied Materials & Interfaces Table 2. Abundances of A. acidoterrestris Vegetative Cells (log CFU/mL) after Exposure to IONPs@pDA-Nisin Composites at Different Media pH Valuesa pH

log CFU/mL

control 2 3 4 5 6 7 8 9 10 11

4.82 2.89 1.69 1.38 1.36 0.80 0.80 1.75 2.04 2.54 2.35

± ± ± ± ± ± ± ± ± ± ±

0.02a 0.05b 0.49de 0.25e 0.60e 0.17f 0.17f 0.41de 0.04cd 0.09bc 0.17bc

a

Statistical significance was assessed by ANOVA (n = 3) at the 95% confidence level. The same letters indicate the lack of statistical significance between groups.

observed between pH 6.0 and 7.0, whereas the lowest cell count reductions were observed at the lowest and highest pH values of 2.0, 10.0, and 11.0. In addition, the composites showed better activity at treatments with pH of 3.0, 4.0, 5.0, and 8.0. The composites also retained high antimicrobial activity after treatment at different temperatures over different time lengths (Table 3). Accordingly, cell counts of controls were significantly different than within all treatments (p < 0.05). However, the duration of incubation was not significantly associated with differences in antimicrobial activity when incubated at the same temperature (p > 0.05). The number of viable cells was statistically different between treatments at lower (45, 60 °C) and higher temperatures (80, 100 °C) when incubating for 10 and 20 min. However, cell counts were significantly different between the 45 °C treatment and all other temperatures (60, 80, 100 °C) when incubated for 30 min. The activity of nisin has been previously shown to be diminished at extreme pH values.24,25 However, nisin showed good pH- and temperature-related stability in our experiments after covalent immobilization onto IONPs@pDA nanoparticles, indicating that it could be a promising means to control Alicyclobacillus spoilage in acidic juices. 3.3. Fluorescence-Based Live/Dead Assays. To evaluate the effects of nanoparticles on cells, cellular membrane integrity was measured (Figure 3A). A strong correlation (R2 = 0.99) was observed between the green fluorescence intensity and the fraction of live bacterial cells in the culture. Exposure to the composites caused a 72% reduction in cellular fluorescence after treatment for 5 min. However, no significant differences were observed among the three different treatment

Figure 3. Effects of IONPs@pDA-nisin composites on the membrane integrity of A. acidoterrestris DSM 3922. (A) Bars indicate standard deviation for triplicate measurements and ** indicates p ≤ 0.01. Confocal fluorescent images of live and dead A. acidoterrestris (106 CFU/mL) cultures for the control group (B1) and those treated with 10 mg/mL of nisin-loaded IONPs@pDA composites for 5 (B2), 30 (B3), and 120 min (B4). The scale bar is 25 μm.

Table 3. Abundances of A. acidoterrestris Vegetative Cells (log CFU/mL) after Exposure to IONPs@pDA-Nisin Composites for Different Lengths of Timea temperature (°C) 0 10 min 20 min 30 min

45 5.58 1.67 1.63 1.61

± ± ± ±

0.08a 0.01def 0.12ef 0.18f

60

80

100

1.71 ± 0.07cdef 1.80 ± 0.09bcde 1.80 ± 0.07bcd

1.92 ± 0.06b 1.88 ± 0.03bc 1.86 ± 0.10bc

1.85 ± 0.05bcd 1.86 ± 0.11bc 1.82 ± 0.12bcd

a

Statistical significance was assessed by ANOVA (n = 3) at the 95% confidence level. The same letters indicate the lack of statistical significance between groups. E


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ACS Applied Materials & Interfaces times (p > 0.05). Importantly, the composites exhibited a high antibacterial efficiency for A. acidoterrestris compared to other natural antibacterial compounds, including papain and bromelain treatment for 24 h, commercial rosemary extract treatment for 4−8 h, and heat treatment with saponin extracts for 3−5 d.26,27 CLSM was also utilized to evaluate the antimicrobial effects of IONPs@pDA-nisin composites on A. acidoterrestris via fluorescence measurements. Specifically, SYTO 9 and PI were used to stain cellular DNA. The SYTO 9 green fluorescence stain interacts with both intact and damaged membranes in live and dead cells, respectively, thereby labeling all bacterial cells. In contrast, the PI red fluorescence stain only penetrates bacteria with damaged membranes (i.e., dead cells), resulting in reduced SYTO 9 stain fluorescence. Few dead cells were observed in the control (Figure 3B1), and the cells were uniformly dispersed. In contrast, introduction of the composites resulted in considerable antibacterial activity [Figure 3(B2,B3)], even with treatment for only 5 min. After treatment for 30 and 120 min [Figure 3(B3,B4)], strong red fluorescence and weak green fluorescence were observed, further indicating the ability of the composites to kill bacterial cells, rather than diminish growth. 3.4. Disruption of A. acidoterrestris Cellular Integrity by Nisin-Loaded IONPs@pDA Composites. Nisin results in bacterial cell death via pore formation in the cell wall and membrane.28 Consequently, SEM was used to investigate the morphological changes associated with A. acidoterrestris exposure to composites. Untreated cells exhibited typical membrane structure integrity with smooth surfaces (Figure 4A). After treatment for only 5 min, ubiquitous pore formation occurred in addition to the presence of rough cell surfaces (Figure 4B), thus indicating efficient antibacterial activity by the composites. These results were consistent with those for the live/dead assays. Remarkably, the cell walls became wrinkled, damaged, and fractured after treatment for 30 min (Figure 4C). After exposure for 120 min, cellular structures were totally destroyed with significant disorganization of A. acidoterrestris cells (Figure 4D). To better understand the effects of the composites on cellular physiology, intracellular ultrastructure was observed using TEM. Untreated cells were characterized by a clear cell wall and a uniform cytoplasm (Figure 4E). In contrast, the treated cells exhibited gradual reduction of cytoplasm substance organization in a time-dependent manner. These remarkable deformations were observed after treatment with composites for only 5 min (Figure 4F). Moreover, the cells became entirely leaky after treatment for 30 and 120 min (Figure 4G,H). Overall, these results indicated excellent antibacterial efficiency of the nisin-loaded IONPs@pDA composites, owing to the rapid conjugation of cells to adhesive IONPs@ pDA nanoparticles. Thus, the enrichment of bacterial conjugation by NPs improves the efficiency of the antibacterial activities of the composites. 3.5. Transcriptomic Analysis of A. acidoterrestris after Composite Exposure. The potential mechanism of antibacterial activity of nisin-loaded IONPs@pDA composites on A. acidoterrestris was investigated using global transcriptome RNA sequencing. The live/dead assays and cellular integrity investigations indicated that the antibacterial activity of the composites was achieved after only 5 min. In addition, RNA

Figure 4. SEM (A−D) and TEM (E−H) micrographs of A. acidoterrestris (106 CFU/mL) treated with nisin-loaded IONPs@ pDA composites (10 mg/mL) for 0 (A,E), 5 (B,F), 30 (C,G), and 120 min (D,H). The red arrows indicate pore formation, fractures, and destruction of A. acidoterrestris cells.

quantities decreased considerably after 5 min. Consequently, transcriptomic analyses were conducted for treatment times of 30 s (T1), 2 min (T2), and 5 min (T3; Table 4). After quality filtering raw reads, over 9.80 ×108 clean reads with Q30 values for over 96.4% of the sequence were obtained for all samples (Table 4). Further, over 93.3% of the clean reads were mapped to reference the sequences. A total of 334 DEGs were obtained after comparing the treatment and control samples (Figure 5B). There were 119 DEGs when comparing the control (CK) against the T1 group, with 14 upand 105 down-regulated; 95 DEGs were observed when comparing the CK and T2 groups, with 11 up- and 84 downregulated; and 120 DEGs were observed when comparing the CK and T3 groups, with 45 up- and 75 down-regulated. 3.6. Genes Involved in the Response to Composite Exposure. A total of 65 DEGs were associated with nucleotide processes including metabolism, ribosome bioF


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membrane to the exterior. Furthermore, the ulaB gene (N007_RS41445) that encodes the phosphor transferase system (PTS) ascorbate transporter subunit IIB was repressed by 4.58-fold at 5 min. The PTS system is involved in sugar transporters found within the inner membrane.39 The downregulation of sugar transporters probably affected the cell wall biosynthesis by the effect of sugar intermediates. Another explanation for sugar metabolism was probably that it supported the energy for cells resisting the composites.8 Two-component systems are important parts of cellular responses to peptides.38 Two DEGs encoding two-component systems were up-regulated, while five DEGs were downregulated (Figure 7A). Two-component systems are bacterial signal transduction systems that respond to environmental stimuli and generally consist of a response regulator protein and a histidine protein kinase that is a member of the intramembrane-sensing histidine kinase group.40 The torS (N007_RS27510) and N007_RS24325 genes encoding histidine kinases were down-regulated by 2.34-fold and 1.38fold at 5 min, respectively, which possibly resulted from composite exposure stress. In addition, genes associated with MFS transporters and amino acid metabolism were significantly expressed in cells after exposure to nisin-loaded IONPs@pDA composites (Figure 7A). MFS transporters belong to the secondary membrane transport protein superfamily and are involved in transporting small solutes into and out of cells including amino acids, simple monosaccharides, and peptides, among other compounds.41 Ten DEGs encoding MFS transporters were observed that could alter amino acid metabolism. In addition, 28 DEGs encoding amino acid metabolism were significantly expressed, indicating that the disordered transporter systems of the cells (ABC transporters systems, two-component systems, and MFS transporters) resulted in disturbance of the amino acid metabolism. Considering these results together, composite effects on the disordered transporter system led to changes in cell homeostasis followed by alteration of cell membrane compositions. 3.8. Changes in Cellular Membranes and Cell Walls. The proposed mechanism for the antibacterial activity of nisin involves two mechanisms. Nisin is first suggested to bind to lipid II and interrupt the biosynthesis of peptidoglycan, which is an important component of the cell wall. In addition, the formation of nisin−lipid II aggregates leads to pore formation.42,43 Consequently, DEGs primarily related to the cell wall synthesis and cell membrane integrity were analyzed (Figure 7B). Among the 36 DEGs in this category, six were related to the cell wall synthesis, whereas 22 were related to the cell membrane synthesis and integrity, and eight were related to lipid metabolism. The galK (N007_RS29165) and f rvX (N007_RS39555) genes were differentially expressed in the treatment samples and encoded galactokinase and endoglucanase, respectively. Importantly, these enzymes are associated with amino sugar and nucleotide sugar metabolisms in addition to starch and sucrose metabolisms. These metabolisms are closely associated with defining the cell wall architecture.14 N007_RS0122935, which encoded a murein L,D-transpeptidase that may be related to peptidoglycan biosynthesis,44 was also down-regulated by 2.43-fold after 5 min of composite exposure. Nisin causes the leakage of intracellular metabolites and, particularly, phosphoenolpyruvate in L. monocytogenes.45 Consistent with this observation, the PDRP1 (N007_RS40195) gene encoding a

Table 4. Summary of RNA-Seq Data Generated for Control and Treatment (30 s, 2 min, 5 min) Samples parameter raw reads clean reads total mapped (%) error rate (%) Q20 (%) Q30 (%)54 GC content (%)

control (CK)

2 min (T2)

5 min (T3)

1 134 162 700 1 083 829 118 93.3

1 838 598 700 1 784 601 456 96.4

30 s (T1)

1 015 930 200 980 437 553 95.4

1 251 536 900 1 213 686 004 95.7

0.01 99.0 96.6 54.1

0.02 99.0 96.7 54.2

0.02 98.9 96.4 54.2

0.01 99.0 96.7 54.2

genesis, and transcriptional regulation. Sixty-five DEGs were generally down-regulated (Figure 6A), indicating a negative effect of the composites on the translational activities of cells. Notably, 78.6% (22 out of 28) of the transcriptional regulation DEGs were down-regulated. In particular, PadR family transcriptional regulators (N007_RS25880 and N007_RS36230) were down-regulated by 1.48- and 3.31-fold after 5 min. PadR family regulators are located upstream of genes that code for membrane proteins. Thus, it is likely that the biosynthesis of membrane proteins was inhibited after exposure to the composites.29 A total of 48 DEGs associated with energy metabolism were also observed, which included components of nitrogen metabolism, sulfur metabolism, inositol phosphate metabolism, oxidative phosphorylation, and the citrate cycle (TCA cycle) (Figure 6B). These DEGs were generally down-regulated, with 66.7% (34 out of 48) being transcribed at lower levels. These results indicated that a decrease in the energy metabolism or energy conservation led to nutrient limitation as a response to the environment stress.30 Several oxidoreductases were also variably up- or down-regulated in samples including af r (N007_RS24445), cbaA (N007_RS24560), sll1783 (N007_RS25800), SH2032 (N007_RS25355), Mf lv_1521 (N007_RS38830), and gno (N007_RS31205) genes (Figure 7B). A similar regulation of these oxidoreductases has been observed as a stress response in Saccharomyces cerevisiae, Mycobacterium tuberculosis, Ruminococcus gauvreauii, and Veronica Guariglia-Oropeza strains.31−34 3.7. Membrane Transport. A total of 39 DEGs were observed that encoded transporter modules comprising ATPbinding cassette (ABC) transporters, major facilitator superfamily (MFS) transporters, and two-component regulatory systems (Figure 7A). ABC transporter systems are efflux systems that transport molecules into and out of cells including ions, sugars, and other organic compounds.35 In addition, ABC transporters respond to environmental stressors by effluxing harmful molecules outside of cells.36 Accordingly, previous research has demonstrated that ABC transporters can act as resistance factors against antibacterial peptides.37 A total of 10 DEGs annotated as ABC-transporter components were upregulated, whereas eight DEGs were down-regulated. Yur genes homologs are involved in preventing cell damage due to harmful compounds.38 Notably, yurO (N007_RS24380) and yurM (N007_RS24390) genes were up-regulated by 1.77-fold and 8.55-fold at 5 min, respectively. These results indicated that ABC transporters were likely involved in transporting nisin-loaded IONPs@pDA composites from the cytoplasmic G


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Figure 5. Overview of transcriptomic profiles of A. acidoterrestris DSM 3922 after IONPs@pDA-nisin composites treatment. (A) Circos graphic of overall transcriptional changes after treatments for different lengths of time. The first outer circle represents the complete genome of strain DSM 3922. The second, third, and fourth inner circles indicate DEGs in strains treated with the composites for 30 s, 2 min, and 5 min, respectively. DEGs were defined based on the following criteria: |log 2| > 2 and p < 0.05. Red and green bars indicate up-regulated and down-regulated genes, respectively. (B) Abundances of up- and down-regulated DEGs. The CK, T1, T2, and T3 groups correspond to cells with composite treatment for 0 s, 30 s, 2 min, and 5 min, respectively.

stimulation of a cell wall stress response.45 Expression of the tcyA (N007_RS41620) gene associated with the cell wall stress emerged after treatment with composites, indicating a response in cell wall alteration due to stress induced by the composites.

phosphoenolpyruvate synthase regulatory protein was upregulated in the treated cells. The amino acid ABC transporter corresponding to an amino acid-binding protein related to the cell envelope synthesis has previously been associated with H


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Figure 6. Analysis of DEGs associated with (A) nucleotide and transcriptional regulation and (B) energy metabolism following treatment with IONPs@pDA-nisin for different times (30 s, 2 min and 5 min). Red indicates up-regulated genes, while blue indicates down-regulated genes.

Function. A number of novel transcriptional responses were observed here for Alicyclobacillus, including those of genes encoded in transcriptional regulation, cell motility, sporulation, and ribosomal functions (Figure 7C). The yybA (N007_RS32110), yhjH (N007_RS34070), and marR (N007_RS42940) genes that encoded MarR family transcriptional regulators were down-regulated after treatment with the composites. MarR is a repressor to the marRAB operon and regulates the response to environmental stresses.48 Moreover, marR was suggested to play a key role in regulating the resistance of Escherichia coli to multiple antibiotics.49 Repression of the MarR family transcriptional regulator likely suggested that a drug resistance signal was not induced during exposure to the composites. In contrast, the kstR2 (N007_RS25425) and yxbF (N007_RS28890) genes that encoded TetR family transcriptional regulators were upregulated by 3.35-fold after 5 min, and their expression was only present after 30 s of composite treatment. TetR family transcriptional regulators typically control the gene expression as a response to external stresses.50 Consequently, the up-

A total of 22 DEGs involved in the cell membrane stress response were observed. Among these, nine were encoded membrane proteins and two were encoded integral membrane components. These results were consistent with the aforementioned inhibition of membrane proteins via PadR family transcriptional regulation. Lipid II is a critical component of the mechanisms underlying antibacterial activity and is transported across the membrane by lipid II flippase.46 The N007_RS36235 gene encoding lipid II flippase was significantly up-regulated in the 30 s and 2 min composite treatments and then sharply down-regulated after 5 min of treatment. These contrasting regulation trends were potentially due to the involvement of lipid II in the two antibacterial mechanisms described above. Fatty acids are involved in responses to external stressors via alteration of membrane fluidity or permeability.14,46,47 Accordingly, four DEGs were observed (N007_RS25355, N007_RS29345, N007_RS33, N007_RS34205900, and N007_RS34205), which encoded in fatty acid metabolism, degradation, and biosynthesis. 3.9. Gene Expression Involved in Transcriptional Regulation, Cell Motility, Sporulation, and Ribosomal I


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Figure 7. Analysis of DEGs associated with (A) membrane transporters, (B) membrane and cell wall syntheses and oxidoreductase, and (C) transcriptional regulation, cell motility, sporulation, and ribosomal functions, following treatment with IONPs@pDA-nisin for different times (30 s, 2 min, and 5 min). Red indicates up-regulated genes, while blue indicates down-regulated genes.

regulated TetR family transcriptional regulators probably indicated a resistance to composites. Cellular motility and bacterial sporulation are survival strategies used to mitigate the environmental stress.51 The f liR (N007_RS31885), f liL (N007_RS31920), ylzI (N007_RS31925), and f liS (N007_RS37550) genes encoded flagellar assembly proteins. Only f liS genes were up-regulated in treated cells, whereas the other three were down-regulated (Figure 7C). Motility suppression has also been observed in Bacillus cereus strains in response to different stresses.51,52 Here, the IONPs@pDA exhibited a general tendency to adsorb cells, which could further decrease cell motility. The yyaC (N007_RS31505) and gerKA (N007_RS38510) genes that encoded the spore protease YyaC and spore germination

proteins, respectively, were both down-regulated after composite treatment, indicating efficient antibacterial activity against sporulation (Figure 7C). Thus, the nisin-loaded IONPs@pDA composites inhibited spore formation, which critically controlled spoilage by A. acidoterrestris. Three 50S ribosomal protein genes, rpmB (N007_RS27320), rpmH (N007_RS43595), and rpmF (N007_RS43475), exhibited 2.54-, 11.6-, and 6.09-fold down-regulations in expression after 5 min, respectively. Furthermore, the rpsT (N007_RS35675) gene encoding the 30S ribosomal protein exhibited 2.74-fold down-regulation. These results suggested that protein translation was highly repressed when cells were exposed to nisin-loaded IONPs@ pDA composites.53 Ribosomal activities can consume a J


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Figure 8. Model of A. acidoterrestris response to nisin-loaded IONPs@pDA composites. Red, green, and orange boxes correspond to up-regulated, down-regulated, and disordered genes, respectively.

considerable amount of cellular energy.54 Thus, the inhibition of ribosomal protein expression could be related to an energy shortage in the cells. Differences in transcriptional responses by A. acidoterrestris to antibacterial compounds compared to those that have been previously observed could be due to species-specific differences in these activities. In addition, some of these responses could be specific to the influence of IONPs@pDA nanoparticles on A. acidoterrestris. Thus, further investigations of the potential transcriptional responses of A. acidoterrestris to NPs are necessary. The antibacterial mechanism of novel synthesized composites was different from other nanocomposites such as cytolytic peptide melittin (MLT)-loaded zeolitic imidazolate framework-8 nanoparticles (MLT@ZIF-8 NPs) and silver-nanoparticle-decorated quercetin nanoparticles (QA NPs).9,55 The MLT@ZIF-8 NPs could induce A549 cell apoptosis by regulating the p53 pathway. The QA NPs showed efficient antibacterial activities against drug resistance bacteria by cell wall organization or biogenesis and cell wall macromolecule metabolic process. Therefore, the antibacterial mechanism of nanocomposites is different because of the different antibiotic active substance.

exhibited highly efficient antibacterial activity against A. acidoterrestris within only 5 min based on fluorescent-based cellular live/dead tests. SEM and TEM analyses of A. acidoterrestris treated with the composites indicated damage to their cellular integrity by membrane-associated changes. In addition, the mechanisms underlying the antibacterial activities of nisin-loaded IONPs@pDA composites were evaluated by considering cellular physiological changes on the gene level using mRNA-Seq characterization. DEGs mostly encoded in transcriptional regulation, energy metabolism, membrane transport, membrane and cell wall syntheses, and cellular motility. In addition, four previously unknown transcriptional responses to antibacterial compound exposure were observed for the first time and included genes for two transcriptional regulators and those associated with cellular motility, sporulation, and ribosome function. Additional validation studies should be conducted to further clarify the contribution of the IONPs@pDA nanoparticles to the antibacterial activity of the composites. Nevertheless, as shown in Figure 8, these results suggested that the composites adhered to cell surfaces, interacted with membranes, and caused a series of changes in transcriptional regulation and in energy metabolism. These processes likely then resulted in pore formation in the membrane and inhibition of cell wall synthesis and disordered membrane transporter function. In addition, cell motility and spore germination were repressed, and lastly, a loss of intracellular substances occurred. The results of this study describe an effective method to control bacteria and help mitigate the occurrence of drug-resistant bacteria. Further,

4. CONCLUSIONS Here, IONPs@pDA nanoparticles were synthesized and successfully conjugated to nisin. Moreover, their antibacterial activity, heat stability, and pH stability were characterized using TEM, VSM, XRD, FT-IR, and XPS. The composites K


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interactions between the composites and microorganisms, as detailed by the transcriptomic analyses, provide novel insights into the molecular mechanism underlying the antibacterial activity of nisin toward A. acidoterrestris.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +86-29-87092492. ORCID

Tianli Yue: 0000-0002-4768-5831 Funding

This work was financially supported by the National Natural Science Foundation of China (31671866, 31371814). Notes

The authors declare no competing financial interest.

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REFERENCES

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Transcriptomic Analysis of the Molecular Mechanisms Underlying the

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