Antibacterial mechanism of high-mobility group nucleosomal-binding domain 2 on the Gram-negative bacteria Escherichia coli

Antibacterial mechanism of high-mobility group nucleosomal-binding domain 2 on the Gram-negative bacteria Escherichia coli 高迁移率族蛋白N2（HMGN2）对革兰氏阴性大肠埃希菌的抗菌机制研究 1 2 first-author 1 2 2 2 2 2 2 2 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:xiaohuer1000@126.comNing_Huang741@163.com †E-mail:xiaohuer1000@126.comNing_Huang741@163.com xiaohuer1000@126.com 1 2 3 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:xiaohuer1000@126.comNing_Huang741@163.com †E-mail:xiaohuer1000@126.comNing_Huang741@163.com xiaohuer1000@126.com Research Unit of Infection and Immunity, Sichuan University, Chengdu 610041, China Research Unit of Infection and Immunity, Sichuan University, Chengdu 610041, China Department of Pathophysiology, West China College of Basic and Forensic Medicine, Sichuan University, Chengdu 610041, China Department of Pathophysiology, West China College of Basic and Forensic Medicine, Sichuan University, Chengdu 610041, China Sichuan University “985 Project Science and Technology Innovation Platform for Novel Drug Development”, Sichuan University, Chengdu 610041, China Sichuan University “985 Project Science and Technology Innovation Platform for Novel Drug Development”, Sichuan University, Chengdu 610041, China Objective: 2 To investigate the antibacterial mechanism ofhigh-mobility group nucleosomal-binding domain 2 (HMGN2) on Escherichia coli K12, focusing on the antibacterial and antibiofilm formation effects. Its chemotactic activity on human neutrophils was also investigated. Methods: 2 Human tissue-derived HMGN2 (tHMGN2) was extracted from fresh uterus fiber cystadenoma and purified by HP1100 reversed-phase high-performance liquid chromatography (RP-HPLC). Recombinant human HMGN2 (rHMGN2) was generated in E. coli DE3 carrying PET-32a-c(+)-HMGN2. Antibacterial activity of HMGN2 was determined using an agarose diffusion assay and minimum inhibitory concentration (MIC) of HMGN2 was determined by the microdilution broth method. Bacterial membrane permeability assay and DNA binding assay were performed. The antibiofilm effect of HMGN2 was investigated using a crystal violet assay and electron microscopy scanning. The activating effect and chemotactic activity of HMGN2 on neutrophils were determined using a nitroblue tetrazolium (NBT) reduction assay and Transwell chamber cell migration assay, respectively. Results: 2 HMGN2 showed a relatively high potency against Gram-negative bacteria E. coli and the MIC of HMGN2 was 16.25 μg/ml. Elevated bacterial membrane permeability was observed in HMGN2-treated E. coli K12. HMGN2 could also bind the bacterial plasmid and genomic DNA in a dose-dependent manner. The antibiofilm effect of HMGN2 on E.coli K12 was confirmed by crystal violet staining and scanning electron microscopy. However, the activating effects and chemotactic effects of HMGN2 on human neutrophils were not observed. Conclusions: 2 As an antimicrobial peptide (AMP), HMGN2 possessed a good capacity for antibacterial and antibiofilm activities on E. coli K12. This capacity might be associated with disruption of the bacterial membrane and combination of DNA, which might affect the growth and viability of E.coli . 目的 2 报道高迁移率族蛋白N2（HMGN2）对大肠埃希菌（ Escherichia coli ）K12的抗菌功能，并对其抗菌机制进行探讨，同时检验HMGN2对中性粒细胞是否具有趋化活性。 创新点 2 从分子水平上探讨了HMGN2对大肠埃希菌的抗菌机制。 方法 2 用反相高效液相色谱法从人类子宫纤维囊腺瘤中提取组织细胞的HMGN2分子（tHMGN2）。诱导重组表达质粒PET-32a-c(+)-HMGN2表达重组蛋白HMGN2（rHMGN2）。用琼脂糖凝胶弥散法对HMGN2的抗菌活性进行检测，并用微量肉汤稀释法测定HMGN2的最小抑菌浓度（MIC）。通过膜通透性实验和凝胶阻滞实验检测HMGN2 对细菌菌膜和核酸的作用。通过结晶紫实验和电镜扫描验证HMGN2的抗生物被膜形成作用。通过氮蓝四唑（NBT）法和Transwell趋化法分别验证HMGN2的活化效应和对中性粒细胞的趋化活性。 结果 2 我们分离纯化获得了高质量的天然和重组HMGN2分子，同时验证了HMGN2对革兰氏阴性大肠埃希菌具有较强的抗菌活性，MIC为16.25 µg/ml。细菌膜通透性实验发现HMGN2使大肠埃希菌膜渗透性明显增大。HMGN2分子与大肠埃希菌K12染色体DNA和质粒DNA的结合均呈浓度依赖效应。银染和扫描电镜结果显示，HMGN2与大肠埃希菌共培养可干扰细菌生物被膜形成，并破坏已形成的早期和成熟生物被膜。然而HMGN2对中性粒细胞没有活化作用和趋化作用。 结论 2 作为抗菌肽，HMGN2对大肠埃希菌有良好的抗菌活性。该活性可能通过影响细胞膜的通透性和干扰细菌DNA转录以及干扰生物被膜而发挥作用。 High-mobility group nucleosomal-binding domain 2 (HMGN2) Bioactivity Membrane permeability Biofilm Chemotactic activity 高迁移率族蛋白N2（HMGN2） 膜通透性 生物被膜 趋化作用 Introduction 1 Antimicrobial peptides (AMPs) are produced by a wide variety of organisms in order to protect themselves from infection (Zanetti, 2004 2004 ; Zhang et al., 2015 2015 ). They often possess a broad-spectrum, non-specific activity against bacteria, viruses, fungi, etc. Primary mechanisms involved in the antimicrobial properties of AMPs are inhibiting cell-wall synthesis, altering cytoplasmic membrane, activating the autolysin, inhibiting nucleic acid and protein synthesis, and suppressing enzymatic activity (Brogden, 2005 2005 ), which differ from that of currently available therapeutic antibiotics, making them non-susceptible to the conventional multidrug resistance mechanism. In addition, some AMPs possess multiple immunoregulatory functions which are manifested as altering mammalian cell membrane properties or binding to its receptors to affect various cellular processes (Lai and Gallo, 2009 2009 ). Additionally, it has been proved that the secondary metabolites of AMPs are free amino acids which result in fewer side effects than conventional antibiotics. For these reasons, AMPs are promising as novel natural antibiotics. High-mobility group nucleosomal-binding domain 2 (HMGN2), a member of HMG proteins’ superfamily, is defined as an AMP by Reddy et al. ( 2004 2004 ). It is a non-histone nuclear protein that binds nucleosomes and regulates chromosome architecture and gene transcription, playing a vital role in processes of embryonic development and tumorigenesis (Reeves, 2010 2010 ). Physical and chemical analyses suggest that HMGN2 is a relatively small, cationic, and amphipathic molecule, with an α-helical structure and containing 89 amino acid residues (9.26 kD, < 10 kD) (Feng et al., 2005 2005 ). Our previous studies suggest that HMGN2 positively modulates the nuclear factor κB (NF-κB) signaling in the nucleus, and promotes lipopolysaccharide (LPS)-induced β-defensin expression in human lung epithelial cell A549 and mice (Deng et al., 2011 2011 ; 2012 2012 ). In some cases, HMGN2 could be released into the extracellular medium from interleukin 2 (IL-2) stimulated human mononuclear leukocytes (Feng et al., 2005 2005 ). And we also showed that HMGN2 is a novel antimicrobial molecule, having a potent antimicrobial activity against Escherichia coli ML-35p, hepatitis B virus in vitro (Feng et al., 2005 2005 ; 2009 2009 ). The antimicrobial effects of HMGN2 on Klebsiella pneumoniae internalization and invasion in bladder epithelial cells (Wu et al., 2011 2011 ; Cao et al., 2011 2011 ), and in E. coli and Pseudomonas aeruginosa have been reported (Feng et al., 2005 2005 ). However, the antibacterial mechanism of HMGN2 has not been fully elucidated. Bacterial biofilms play a critical role in the resistance to antibiotic chemotherapy and human immune defenses (Høiby et al., 2010 2010 ). Therefore, we evaluated the antibacterial and antibiofilm effects and the relative mechanisms of HMGN2 on E. coli K12. In addition, based on the multiple biological functions of AMPs, the activating effects and chemotactic activity of HMGN2 on human neutrophils were investigated. Materials and methods 1 Bacterial strains and cultures 2 E . coli (ATCC25922 and K12), P . aeruginosa (ATCC27853 and PAO1), Acinetobacter baumannii (clinical isolate), K . pneumoniae (clinical isolate), Staphylococcus aureus (ATCC25923), Streptococcus faecalis (clinical isolate), and Canidia albicans (ATCC90028) were used in this study. The clinical isolates were obtained from the Department of Pathophysiology in Medical West China Hospital of Sichuan University (Chengdu, China). Bacteria were cultured in a shaking lysogeny broth (LB) at 37 °C. After centrifugation, the bacterial concentration was adjusted in phosphate buffer saline (PBS) and determined by measuring absorbance at 600 nm. Human participants and sample collection 2 This study was subject to approval by the Ethics Committees of Sichuan University (No. JJ2014010) and was performed according to the Helsinki Declaration. All subjects provided informed written consent. Tumor tissue samples were collected during surgery resection from women with uterus fiber cystadenoma (aged from 30–40 years, without antibiotic or hormonal therapy prior to operation) in our hospital. The peripheral blood was collected from healthy adult volunteers (male, 24 years old) with heparin. The neutrophils were separated from the peripheral blood through a human neutrophils separation medium, then washed twice with sterile PBS, and resuspended in Roswell Park Memorial Institute 1640 (RPMI-1640) medium. Peptide purification and preparation 2 Human tissue-derived HMGN2 (tHMGN2) was extracted from fresh uterus fiber cystadenoma, and purified by HP1100 reversed-phase high-performance liquid chromatography (RP-HPLC, Hewlett-Packard, USA). Initially, the tumor tissues were washed with pre-cold PBS, finely chopped, and ground in liquid nitrogen. The tissues were homogenized in lysis buffer (5% (v/v) perchloric acid) and centrifuged at 4 °C. Supernatants were dialyzed in a 3500 molecular weight cut off tube (Spectrum Medical Industrial Co., USA) against water at 4 °C for 48 h, and then lyophilized and re-dissolved in 0.1% (v/v) trifluoroacetic acid. The peptide was purified by RP-HPLC on a Vydac 218TP C18 (0.46 cm×25 cm, 5 μm, 30 °C) equilibrated with 0.1% trifluoroacetic acid. The solution of 60% acetonitrile/0.1% trifluoroacetic acid was used as the eluant. The flow rate was 1 ml/min. Absorbance at 214 nm was measured, and the major peak in the chromatogram was identified. Recombinant human HMGN2 (rHMGN2) was generated in E. coli DE3 and purified by affinity chromatography and enzyme digestion methods. The transformed E. coli DE3 carrying PET-32a-c(+)-HMGN2 was cultured in Luria-Bertani containing 25 μg/ml ampicillin. Then 0.1 mmol/L isopropyl-β- D - thiogalactoside (IPTG) was added to induce protein expression at 30 °C. Bacterial cells were lysed by sonication and centrifuged at 4 °C. Then supernatants were purified using HisTrap™ Kit column (Pharmacia, Piscataway, USA). The eluents were dialyzed, purified by RP-HPLC, and cleaved by thrombin digestion. Protein concentration was determined using the bicinchoninic acid (BCA) kit (Thermo Scientific, Rockford, USA). The purity protein was confirmed by tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (tricine-SDS-PAGE), acid-urea-PAGE (AU-PAGE), and Western blotting with a primary anti-HMGN2 monoclonal antibody (Sigma, St. Louis, USA). Agarose radial diffusion antimicrobial assay 2 Agarose diffusion assay was used to evaluate the antibacterial activity of HMGN2 as previously described (Lehrer et al., 1991 1991 ). Plates were poured with 15 ml molten agarose gel containing 0.03% (0.3 g/L) soya peptone (Sigma, St. Louis, USA), 1% (0.01 g/ml) low electrolyte agarose (Sigma, St. Louis, USA), and 30 μl of E. coli suspension (0.5 McFarland scale, about 1×10 8 colony-forming unit (CFU)/ml). Subsequently, different concentrations of HMGN2 (5 μl) were added to 3-mm-diameter wells, which had been cut using sterile hole punchers and incubated at 37 °C for 3 h. Finally, the plates were overlaid with molten medium (50 °C) containing 1% low electrolyte agarose and 3% (0.03 g/ml) trypticase soybean and incubated at 37 °C overnight. The diameter of the zone of inhibition around the punched hole was used to assess the antibacterial potential against bacteria. Additionally, PBS was used as the negative control and lysozyme and human neutrophil peptide (HNP) were employed as the positive controls. Minimum inhibitory concentration assay 2 Minimum inhibitory concentrations (MICs) of HMGN2 were determined by the microdilution broth method following the guidelines of the Clinical Laboratory Standards Institute (Wiegand et al., 2008 2008 ). Mid-logarithmic bacterial cultures were diluted with sterile PBS to a final cell density of 1×10 6 CFU/ml, then 40 μl of the bacterial suspension was seeded in a 96-well plate. A volume of 10 μl of HMGN2 dilution with 10 graded serial concentrations within the 500–1 μg/ml range was added and the plate was incubated at 37 °C for 12 h. The lowest concentration of HMGN2 that can completely suppress the growth of the bacteria was identified as the MIC. Bacterial membrane permeability test 2 A bacterial membrane permeability assay was conducted as described previously (Wang et al., 2014 2014 ). A total of 200 μl bacterial suspension (1×10 8 CFU/ml) was incubated with HMGN2 or bovine serum albumin (BSA, 5 μg/ml) at 37 °C for 240 min. Thereafter, the bacterial suspensions were centrifuged (12 000 r/min) at 4 °C for 2 min. The absorbance of supernatants was determined by spectrophotometry at 260and 280 nm, respectively. An elevated absorbance indicates higher bacterial cell membrane permeability. DNA binding assay 2 E. coli K12 plasmid DNA was extracted using Eazy Nucleic Acid Isolation E.Z.N.A. Plasmid Miniprep Kit (Omega, Georgia, USA) following the manufacturer’s protocol. Genomic DNA was extracted by a cetyl triethyl ammonium bromide (CTAB)/phenol-chloroform DNA extraction method (Wang et al., 2013 2013 ). Overnight-cultured E. coli K12 was harvested and resuspended in a Tris-ethylene diamine tetraacetic acid (EDTA) buffer (pH 8.0). Then the bacterial suspension was incubated with 20 mg/ml protease K and 10% SDS at 37 °C for 60 min. The lysate was incubated with 5 mol/L NaCl and CTAB/NaCl solution for 30 min at 65 °C. Proteins were removed by addition of phenol/chloroform (1:1) twice. The supernatant was further precipitated by sodium acetate/alcohol (1:20), rinsed twice with 80% alcohol. The extracted DNA was dissolved in a Tris-EDTA buffer. The absorbances at 260 and 280 nm ( A 260 and A 280 ) were recorded. A 260 / A 280 and electrophoresis were used to analyze the DNA purity. For the DNA binding assay (Park et al., 1998 1998 ), 0.5 μg genomic DNA and 0.1 μg plasmid DNA were mixed with different amounts of HMGN2, rabbit neutrophil defensin, and BSA (as negative control) in a binding buffer (5% glycerol, 1 mmol/L EDTA, 1 mmol/L dithiothreitol (DTT), 20 mmol/L KCl, and 50 μg/ml BSA, 10 mmol/L Tris-HCl, pH=8.0), respectively and were incubated at room temperature for 3 h. Subsequently, 4 μl of native loading buffer (10% Ficoll 400, 50 mmol/L EDTA, 0.25% bromophenol blue, and 0.25% xylenecyanol, and 10 mmol/L Tris-HCl, pH=7.5) was added and an aliquot of 12 μl was applied to a 0.7% agarose gel electrophoresis, and finally imaged using the Molecular Image ® Gel Dox XR+gel imager (Bio-Rad, California, USA). Crystal violet staining 2 The effect of HMGN2 on biofilm formation was investigated using a crystal violet assay as previously described (Mitra et al., 2013 2013 ). E. coli K12 (2×10 6 CFU/ml) was mixed with 10 ml of tryptic soy broth (TSB) medium. A volume of 200 μl of the suspension was added to each well of a 96-well plate with or without HMGN2 (1× MIC) and the plate was placed in an incubator at 37 °C for 3 or 6 d with a fresh medium change every 24 h. Upon biofilm formation, HMGN2 was added to each well and inoculated for another 2 d. After incubation, plates were then washed twice with PBS to remove the non-adherent bacteria. Adherent bacterial cells were stained with 25 μl of 1% crystal violet for 15 min; excess crystal violet was then rinsed twice with PBS. Subsequently, the dye bound to the adherent bacterial cells was re-dissolved with 200 μl of 95% ethanol. The optical density (OD) of each well was measured at 595 nm. Scanning electron microscopy assay 2 Bacterial incubation and HMGN2 treatment were carried out using the same methods previously described. After HMGN2 treatment, the filter membranes were fixed with 3% glutaraldehyde and 1% osmic acid in 0.2 mol/L PBS for 4 h at 4 °C and dehydrated in a series of ethanol solutions with graded concentration (30%–100%). Thereafter, samples were dried in a critical point dryer, and sputter-coated with gold and imaged using a JSM-7500F electron microscope (JEOL, Japan) at 20 kV. Nitroblue tetrazolium reduction assay 2 The activating effect of HMGN2 on neutrophils was determined using an nitroblue tetrazolium (NBT) reduction assay as described before (Park et al., 1968 1968 ). A total of 5×10 5 neutrophils were incubated in a freshly prepared solution containing PBS, 0.2% NBT (Sigma, Shanghai, China), and 1% human albumin as well as various concentrations of HMGN2 or LPS (Sigma, Shanghai, China) at 37 °C for 30 min. Then, the cells were collected and stained with Wright’s dye. Finally, at least 100 cells counted in 6 high power fields per glass slide were observed under the light microscope, and the NBT-positive cells (with blue-black granules of formazan in cytoplasm) were calculated. Transwell chamber cell migration assay 2 Transwell chamber cell migration assay was conducted as has been described before (Degryse et al., 1999 1999 ). Shortly, Transwell chambers were used with microporous filters (3 μm pore size). The human neutrophils were seeded in the upper well of the Transwell chamber with about 2×10 5 cells/well. The HMGN2 was diluted in a serum-free DMEM medium and was added to the lower well of the Transwell chamber. Thirty minutes later, the filters were rinsed twice with PBS, fixed with methanol, and stained with a solution of Giemsa. Finally, the filters were washed and the migrating cells in 5 fields per filter were observed under a light microscope (×200). Casein was used as a positive control and the serum-free DMEM medium was taken as the negative control. Statistical analysis 2 Data were expressed as mean±standard deviation (SD) of triplicate experiments. The differences among groups were analyzed by one-way analysis of variance (ANOVA) using the SPSS 17.0 software (SPSS Inc., Chicago, USA). Differences with P < 0.05 were considered to be statistically significant. Results 1 Peptide isolation and purification 2 Typical human tHMGN2 was observed in Figs. 1a–1d 1a–1d . The retention time of the tHMGN2 was about 17.5 min (Fig. 1a 1a ). Then this peak was collected, and identified by tricine-SDS-PAGE. The molecular weight of this protein was about 14 kD (Fig. 1b 1b ). Meanwhile, the protein was identified as HMGN2 by Western blotting (Fig. 1c 1c ). Finally, AU-PAGE assay suggested that HMGN2 was a cationic molecule (Fig. 1d 1d ). (a, e) RH-HPLC chromatogram profiles of HMGN2 from the human uterine fibroid cystadenoma and E. coli DE3 carrying PET-32a-c(+)-HMGN2; (b, f) Identification of the molecular weight via SDS-PAGE assay; (c, g) Western blotting assay for the identification of HMGN2; (d, h) Identification of molecular charge through AU-PAGE assay. MW: molecular weight Similarly, the rHMGN2 was observed in Figs. 1e–1h 1e–1h . The crude peptide was successively purified by affinity chromatography and RP-HPLC. The major peak was collected and cleaved by thrombin, which was further purified by RP-HPLC. The retention time of rHMGN2 was about 21 min (Fig. 1e 1e ). Then this peak was collected, and identified by SDS-PAGE. The molecular weight of this protein was about 14 kD (Fig. 1f 1f ). Then, the protein was identified as HMGN2 by Western blotting (Fig. 1g 1g ). The AU-PAGE result showed that HMGN2 was an amphipathic molecule (Fig. 1h 1h ). Antimicrobial effects and MIC of HMGN2 2 As shown in Table 1 1 , tHMGN2 and rHMGN2 both possessed powerful antimicrobial activity on E. coli , and lower potency on A. baumannii . However, antimicrobial effects of HMGN2 on P. aeruginosa , K. pneumoniae , S. aureus , S. faecalis , and C. albicans were not observed in the present study. Both tHMGN2 and rHMGN2 could obviously inhibit the growth of E. coli K12 (Fig. 2 2 ), and the MIC of HMGN2 against E. coli K12 was 16.25 μg/ml as obtained through the broth microdilution method. Representative inhibitory zones of HMGN2 for E. coli K12 strain in agarose radial diffusion plate The antimicrobial activities of HMGN2 were expressed as the inhibitory-zone diameters ( d ). +++: d ≥8 mm; ++: 8 mm > d > 5 mm; +: 5 mm≥ d ≥3 mm; −: no antimicrobial activity, d < 3 mm; ND: not done. Vehicle: PBS; tHMGN2: tissue-derived HMGN2; rHMGN2: recombinant HMGN2; HNP: human neutrophil peptide 2 Group 2 E. coli 2 P. aeruginosa 2 A. baumannii clinical isolate 2 K. pneumonia clinical isolate 2 S. aureus 25923 2 S. faecalis clinical isolate 2 C. albicans 90028 25922 Clinical isolate 27853 POA1 Vehicle − − − − − − − − − tHMGN2 +++ +++ ND ND + − ND ND ND rHMGN2 +++ +++ − − + − − − − HNP +++ +++ − − + − − − − Bacterial cell membrane permeability assay 2 To evaluate whether HMGN2 disrupted the bacterial membrane integrity, the amounts of bacterial nucleic acids and proteins released from HMGN2 or vehicle-treated bacterial cells were measured at 260 and 280 nm by ultraviolet (UV) spectrophotometry. After being incubated with HMGN2 for 4 h, the bacterial membrane permeability significantly increased, which manifested as elevated OD values at 260 and 280 nm (Fig. 3 3 ). This result suggests that the antibacterial effect of HMGN2 is involved in bacterial membrane damage. Bacteria were incubated with PBS (Cont), BSA, and HMGN2 (1/4, 1/2, and 1× MIC) for 4 h at 37 °C. Then the OD values at 260 and 280 nm of supernatants were measured. Data are presented as mean±SD, n =9. * P < 0.05, ** P < 0.01 vs. control Binding assay between HMGN2 and bacterial genomic/plasmid DNAs 2 To further explore the mechanisms of the antibacterial effect, the genomic and plasmid DNAs binding properties of HMGN2 were examined. As shown in Fig. 4 4 , HMGN2 suppressed the migration of bacterial genomic and plasmid DNAs above the weight ratios of 1:10 (Fig. 4a 4a ) and 1:8 (Fig. 4b 4b ), respectively. In contrast, the rabbit defensin and BSA did not suppress the migration of DNA, which indicated that rabbit defensin and BSA could not bind to the bacterial genomic and plasmid DNAs. (a) Genomic DNA; (b) Plasmid DNA. 1: negative control (BSA, 5 μg/ml); 2–5: the ratio of DNA:rabbit defensin (1:1, 1:10, 1:20, 1:50); 6–12: the ratio of DNA:HMGN2 (1:1, 1:2, 1:4, 1:8, 1:10, 1:20, 1:50) Disruption of E .coli K12 biofilm by HMGN2 2 Fig. 5 5 depicts the changes of biofilm formation in HMGN2 (1× MIC)-treated E. coli K12 strain. Firstly, crystal violet staining was used to evaluate the effect of HMGN2 on the biofilm of E. coli K12. After 3 (Fig. 5a 5a ) and 6 d (Fig. 5b 5b ) exposure to PBS, BSA, and HMGN2, the K12 strain still produced moderate biofilm. However, only HMGN2 could obviously disrupt the biofilm formation and damage the formed biofilm (Figs. 5c–5j 5c–5j ). A similar result was obtained from the scanning electron microscopy observation. The control membrane showed a well-developed biofilm growth of E . coli K12 (Figs. 5d and 5h 5d and 5h ), whereas E . coli K12 treated with HMGN2 developed poor biofilm formation (Figs. 5e and 5i 5e and 5i ) compared to that of the control sample. HMGN2 also could damage the early-and maturely-formed biofilms (Figs. 5f and 5j 5f and 5j ). Taken together, these results demonstrated that HMGN2 disrupted the biofilm of E . coli K12. (a) Early and mature biofilm staining via crystal violet. (b) Early-and maturely-formed biofilm staining via crystal violet. Data are presented as mean±SD, n =9. * P < 0.05, ** P < 0.01, vs. control. (c–j) Representative images of scanning electron microscopy, original magnification ×5000. (c, g) Negative control of E . coli K12 biofilm; (d) Early biofilm (3 d); (h) Mature biofilm (6 d); (e) Early biofilm formation cultured in the presence of HMGN2; (i) Mature biofilm formation cultured in the presence of HMGN2; (f) Change of early-formed biofilm cultured in the presence of HMGN2; (j) Change of maturely-formed biofilm cultured in the presence of HMGN2 Nitroblue tetrazolium reduction assay and Transwell chamber migration assay 2 To demonstrate the activating effect and chemotactic activity of HMGN2 on human neutrophils in vitro, the NBT reduction assay and Transwell chamber migration assay were carried ou. As shown in Fig. 6a 6a , 100 μg/ml LPS could obviously activate the human neutrophils. However, various concentrations of HMGN2 did not show the ability to induce activation of neutrophils in vitro. The Transwell chamber assay (Figs. 6c–6e 6c–6e ) suggested that HMGN2 did not possess chemotactic activity on human neutrophils. Hence, HMGN2 was not considered as a neutrophil activator or chemokine. (a, b) Data are presented as mean±SD, n =9; (c–e) Representative images of Transwell assay, original magnification ×200. (a) The number of NBT-positive cells; (b) The number of migrated cells; (c) Negative control; (d) 100 μg/ml HMGN2; (e) 5 mg/ml casein (as positive control). White arrow: migrated cells; Black arrow: filter holes Discussion 1 As the incidence of antibiotic resistance in pathogenic bacteria rises worldwide, there is an urgent need to develop novel antimicrobial agents to fight various types of infectious disease (Stewart and Costerton, 2001 2001 ). AMPs, so-called “natural antibiotics”, have drawn the focus of pharmacists because of their potential for antibiotic resistance (Brogden, 2005 2005 ). The present study set out to probe the antibacterial mechanism of an AMP named HMGN2 on the E. coli K12 strain, focusing on the antibacterial and antibiofilm effects, as well as the activating effects and chemotactic effects on human neutrophils in vitro. In the present study, both tHMGN2 and rHMGN2 showed antibacterial activities against E. coli and A. baumannii . However, no effects on other Gram-negative bacteria (such as P. aeruginosa , K. pneumoniae ), Gram-positive bacteria (like S. aureus , S. faecalis ), or fungi (like C. albicans ) were observed. This result indicated that HMGN2 possessed relatively high potency against Gram-negative bacteria E. coli . The model organism E. coli K12 strain was used to evaluate the antibacterial activity of these two kinds of HMGN2. The result showed that the effectiveness of tHMGN2 and rHMGN2 was almost equivalent (Fig. 2 2 ) and the MICs of these two kinds of HMGN2 were 16.25 μg/ml for E. coli K12. Hence, rHMGN2 was used in the next assay. We further investigated the antibacterial mechanisms of HMGN2 on E. coli . AMPs possess unique properties and alternative modes of action different from the conventional antibiotics which penetrate into bacterial cells and interfere with intracellular targets (Liu et al., 2013 2013 ). Cationic and amphipathic AMPs that can bind to the surface of bacterial cells are composed of the negatively charged components in a non-specific electrostatic interaction (Brogden, 2005 2005 ). Then, the AMPs insert into the hydrophobic membrane core, and eventually form transmembrane pores that cause an unrestricted, lethal flux of intracellular ions and other molecules from the cytoplasm (Bolintineanu et al., 2010 2010 ). Therefore, the release of intracellular components including DNA, RNA, and proteins serves as a useful indicator of bacterial membrane integrity (Chen et al., 2013 2013 ). We observed a dose-dependent manner for DNA, RNA, or protein release (Fig. 3 3 ) when treated with HMGN2 by testing the absorbance at 260 and 280 nm. Therefore, we can suggest that HMGN2 damaged the cell membrane integrity of the bacteria. HMGN2 is a cationic and amphipathic molecule (Feng et al., 2005 2005 ), which may target the bacterial membrane through binding to the negatively charged membrane components of E. coli (such as phosphate groups from LPS). Then the α-helix of HMGN2 inserts into the membrane and induces the membrane pore formation so that the intracellular contents can be released into the extracellular medium, eventually resulting in the death of bacteria. Furthermore, a result from our laboratory showed that HMGN2 inhibited the synthesis of capsular polysaccharide in Gram-negative bacteria K . pneumoniae (data not shown). When approaching the bacterial surface, AMPs traverse capsular polysaccharides in order to act on the bacterial membrane. The inhibitory potential of HMGN2 on capsular polysaccharides may contribute to the fact that HMGN2 more easily accesses the cytoplasmic membrane, and acts on the bacterial lipid bilayers. It is also possible that some AMPs target other different intracellular components, and membrane disruption may not be the only cause of bacterial cell death (Brogden, 2005 2005 ). Once translocate into cells, AMPs alter cytoplasmic membrane septum formation, inhibit cell-wall/nucleic acid/protein synthesis, or suppress enzymatic activity (Brogden, 2005 2005 ; Sarda-Mantel et al., 2007 2007 ). For example, a histone-derived AMP named buforin II, which first crosses the cytoplasmic membrane, and then binds to nucleic acids, eventually causes bacterial cell death (Xie et al., 2011 2011 ). Similar to histone, HMGN2 functions as a nuclear factor that can bind to nucleosomes, thereby regulating chromosome architecture and gene transcription in the nucleus (Furusawa and Cherukuri, 2010 2010 ; Reeves, 2010 2010 ). Upon release into the extracellular medium, we explored whether HMGN2 exerts similar effects on bacteria to those of the histone-derived AMPs. We found that HMGN2 could bind genomic DNA of E. coli K12 and alter the electrophoretic mobility of DNA in 1% agarose gels in a dose-dependent manner (Fig. 4 4 ). This result also suggested that the antibacterial activity of HMGN2 on E. coli might be associated with its ability to bind the DNA. HMGN2 also can bind to plasmid DNA. Plasmids are responsible for the capture of different antibiotic resistance genes, and for inducing the multidrug resistance phenotype (Hawkey and Jones, 2009 2009 ). The plasmid DNA binding capacity might imbue HMGN2 with the potential to reduce antibiotic resistance. We assume that the ability of HMGN2 to bind the genomic and plasmid DNAs might disrupt gene transcription and translation, and thereby affect growth and reproduction of bacteria. However, proper experiments on the transcription and translational level should be conducted to explain the antibiotic resistance of HMGN2. Generally, bacteria adhere to damaged tissue by formation of a biofilm consisting of a polysaccharide, protein, and DNA (Stewart and Costerton, 2001 2001 ), leading to persistent and chronic infections (Costerton et al., 1999 1999 ) and inherent resistance to antibiotic chemotherapy as well as phagocytosis and other components of the human body’s immune defense system (Høiby et al., 2010 2010 ). As is well known, the process of a biofilm consists of five stages: initial attachment, irreversible attachment, microcolony and early biofilm formation, biofilm maturation, and final dispersion (O'Toole et al., 2000 2000 ). The crystal violet staining and scanning electron microscopy showed that the treatment of HMGN2 in the E. coli K12 strain reduced the early (3 d) and mature (6 d) biofilm formation (Figs. 5a and 5c 5a and 5c – 5f 5f ). Simultaneously, HMGN2 also could damage the early-and maturely-formed biofilm of E. coli (Figs. 5b and 5g 5b and 5g – 5j 5j ). Additionally, HMGN2-treated E. coli with impaired biofilms might be more easily recognized by the host immune system or be killed by antibiotics. Therefore, the suppression of biofilm formation by HMGN2 is also conducive to the prevention and treatment of E. coli -induced chronic infections. Recently, a growing body of work suggests that AMPs also act as gene-encoded natural antibiotics (Brogden, 2005 2005 ). Some AMPs such as defensins and cathelicidins activate a number of responses in host innate immune cells by inducing chemokines and cytokines release, promoting leukocytes recruitment, and activating antigen presenting cells (Steinstraesser et al., 2011 2011 ). Previous studies indicated that some endogenous mediators that are not traditionally considered as AMPs, such as HMG superfamily protein HMGB1 and HMGN1, act as alarmins, and are also shown to exhibit chemoattracting and activating effects on dendritic cells (Rovere-Querini et al., 2004 2004 ; Yang et al., 2012 2012 ). Apart from the dendritic cells, neutrophils are other key innate immune cells in the human immune system. They can be rapidly recruited to the site of infection in response to chemokines, and trigger microbicidal mechanisms such as AMP release and rapid reactive oxygen species (ROS) production (Bratton and Henson, 2011 2011 ). The NBT reduction assay has been used to test ROS production and neutrophil activation (Tunc et al., 2010 2010 ) and the results showed that 100 μg/ml HMGN2 could not induce the activation of human neutrophils (Fig. 6a 6a ). On the other hand, Transwell chamber assay suggested that HMGN2 did not exhibit a chemoattracting effect on neutrophils in vitro (Figs. 6b and 6c 6b and 6c ). Yang et al. ( 2012 2012 ) reported that HMGN2 did not exhibit the capacity to induce phenotypic maturation of human monocyte­derived dendritic cells, which also confirms our results. Hence, HMGN2 does not possess an alarmin-like effect and does not exhibit the ability to modulate the host immune function. Although HMGN2 has limited homology (about 50%) with HMGN1 and is only 10 amino acids shorter than HMGN1 (Furusawa and Cherukuri, 2010 2010 ), it is unclear why HMGN2 does not possess an alarmin-like effect on innate immune cells. 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Midgut immune responses induced by bacterial infection in the silkworm, Bombyx mori Compliance with ethics guidelines Heng LI, Xiao-fei SHEN, Xin-e ZHOU, Yan-e SHI, Lu-xia DENG, Yi MA, Xiao-ying WANG, Jing-yu LI, and Ning HUANG declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1600139 Project supported by the National Natural Science Foundation of China (Nos. 30470763, 81470931, and 31401188), the China Medical Board of New York (No. 98-861), and the Youth Foundation of Sichuan University (No. 2014SCU11042), China 2016-03-29 2017-04-19 2016-06-23 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2017 2017 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 05 18