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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

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Abstract

Objective

To investigate the antibacterial mechanism of high-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

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-32ac(+)- 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

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

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.

摘 要

目 的

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

从分子水平上探讨了HMGN2 对大肠埃希菌的抗 菌机制。

方 法

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

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

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。

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References

  • Bolintineanu, D., Hazrati, E., Davis, H.T., et al., 2010. Antimicrobial mechanism of pore-forming protegrin peptides: 100 pores to kill E. coli. Peptides, 31(1):1–8. http://dx.doi.org/10.1016/j.peptides.2009.11.010

    Article  CAS  PubMed  Google Scholar 

  • Bratton, D.L., Henson, P.M., 2011. Neutrophil clearance: when the party is over, clean-up begins. Trends Immunol., 32(8):350–357. http://dx.doi.org/10.1016/j.it.2011.04.009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Brogden, K.A., 2005. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol., 3(3):238–250. http://dx.doi.org/10.1038/nrmicro1098

    CAS  Google Scholar 

  • Cao, Y., Wu, G., Fan, B., et al., 2011. High mobility group nucleosomal binding domain 2 protein protects bladder epithelial cells from Klebsiella pneumoniae invasion. Biol. Pharm. bull., 34(7):1065–1071. http://dx.doi.org/10.1248/bpb.34.1065

    Article  CAS  PubMed  Google Scholar 

  • Chen, H., Wang, B., Gao, D., et al., 2013. Broad-spectrum antibacterial activity of carbon nanotubes to human gut bacteria. Small, 9(16):2735–2746. http://dx.doi.org/10.1002/smll.201202792

    Article  CAS  PubMed  Google Scholar 

  • Costerton, J.W., Stewart, P.S., Greenberg, E., 1999. Bacterial biofilms: a common cause of persistent infections. Science, 284(5418):1318–1322. http://dx.doi.org/10.1126/science.284.5418.1318

    Article  CAS  PubMed  Google Scholar 

  • Degryse, B., Resnati, M., Rabbani, S.A., et al., 1999. Srcdependence and pertussis-toxin sensitivity of urokinase receptor-dependent chemotaxis and cytoskeleton reorganization in rat smooth muscle cells. Blood, 94(2): 649–662.

    CAS  PubMed  Google Scholar 

  • Deng, L.X., Wu, G.X., Cao, Y., et al., 2011. The chromosomal protein HMGN2 mediates lipopolysaccharide-induced expreßsion of ß-defensins in A549 cells. FEBS J., 278(12): 2152–2166. http://dx.doi.org/10.1111/j.1742-4658.2011.08132.x

    Article  CAS  PubMed  Google Scholar 

  • Deng, L.X., Wu, G.X., Cao, Y., et al., 2012. The chromosomal protein HMGN2 mediates the LPS-induced expreßsion of ß-defensins in mice. Inflammation, 35(2):456–473. http://dx.doi.org/10.1007/s10753-011-9335-3

    Article  CAS  PubMed  Google Scholar 

  • Feng, Y., Huang, N., Wu, Q., et al., 2005. HMGN2: a novel antimicrobial effector molecule of human mononuclear leukocytes? J. Leukoc. Biol., 78(5):1136–1141. http://dx.doi.org/10.1189/jlb.0505280

    Article  CAS  PubMed  Google Scholar 

  • Feng, Y., He, F., Zhang, P., et al., 2009. Inhibitory effect of HMGN2 protein on human hepatitis B virus expression and replication in the HepG2.2.15 cell line. Antivir. Res., 81(3):277–282. http://dx.doi.org/10.1016/j.antiviral.2008.12.011

    Article  CAS  PubMed  Google Scholar 

  • Furusawa, T., Cherukuri, S., 2010. Developmental function of HMGN proteins. BBA-Gene Regul. Mech., 1799(1):69–73.

    CAS  Google Scholar 

  • Hawkey, P.M., Jones, A.M., 2009. The changing epidemiology of resistance. J. Antimicorob. Chemoth., 64(Suppl. 1): i3–i10. http://dx.doi.org/10.1093/jac/dkp256

    Article  CAS  Google Scholar 

  • Høiby, N., Bjarnsholt, T., Givskov, M., et al., 2010. Antibiotic resistance of bacterial biofilms. Int. J. Antimicrob. Ag., 35(4):322–332. http://dx.doi.org/10.1016/j.ijantimicag.2009.12.011

    Article  Google Scholar 

  • Lai, Y., Gallo, R.L., 2009. AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense. Trends Immunol., 30(3):131–141. http://dx.doi.org/10.1016/j.it.2008.12.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lehrer, R.I., Rosenman, M., Harwig, S.S., et al., 1991. Ultrasensitive assays for endogenous antimicrobial polypeptides. J. Immunol. Methods, 137(2):167–173. http://dx.doi.org/10.1016/0022-1759(91)90021-7

    Article  CAS  PubMed  Google Scholar 

  • Liu, Y., Knapp, K.M., Yang, L., et al., 2013. High in vitro antimicrobial activity of ß-peptoid-peptide hybrid oligomers against planktonic and biofilm cultures of Staphylococcus epidermidis. Int. J. Antimicrob. Ag., 41(1):20–27. http://dx.doi.org/10.1016/j.ijantimicag.2012.09.014

    Article  Google Scholar 

  • Mitra, A., Palaniyandi, S., Herren, C.D., et al., 2013. Pleiotropic roles of uvrY on biofilm formation, motility and virulence in uropathogenic Escherichia coli CFT073. PLoS ONE, 8(2):e55492. http://dx.doi.org/10.1371/journal.pone.0055492

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • O'Toole, G., Kaplan, H.B., Kolter, R., 2000. Biofilm formation as microbial development. Annu. Rev. Microbiol., 54(1): 49–79. http://dx.doi.org/10.1146/annurev.micro.54.1.49

    Article  PubMed  Google Scholar 

  • Park, B., Fikrig, S., Smithwick, E., 1968. Infection and nitroblue-tetrazolium reduction by neutrophils: a diagnostic aid. Lancet Oncol., 292(7567):532–534. http://dx.doi.org/10.1016/S0140-6736(68)92406-9

    Article  Google Scholar 

  • Park, C.B., Kim, H.S., Kim, S.C., 1998. Mechanism of action of the antimicrobial peptide buforin II: buforin II kills microorganisms by penetrating the cell membrane and inhibiting cellular functions. Biochem. Bioph. Res. Commun., 244(1):253–257. http://dx.doi.org/10.1006/bbrc.1998.8159

    Article  CAS  Google Scholar 

  • Reddy, K., Yedery, R., Aranha, C., 2004. Antimicrobial peptides: premises and promises. Int. J. Antimicrob. Ag., 24(6):536–547. http://dx.doi.org/10.1016/j.ijantimicag.2004.09.005

    Article  CAS  Google Scholar 

  • Reeves, R., 2010. Nuclear functions of the HMG proteins. BBA-Gene Regul. Mech., 1799(1):3–14.

    CAS  Google Scholar 

  • Rovere-Querini, P., Capobianco, A., Scaffidi, P., et al., 2004. HMGB1 is an endogenous immune adjuvant released by necrotic cells. EMBO Rep., 5(8):825–830. http://dx.doi.org/10.1038/sj.embor.7400205

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sarda-Mantel, L., Saleh-Mghir, A., Welling, M., et al., 2007. Evaluation of 99mTc-UBI 29-41 scintigraphy for specific detection of experimental Staphylococcus aureus prosthetic joint infections. Eur. J. Nucl. Med. Mol. Imaging, 34(8): 1302–1309. http://dx.doi.org/10.1007/s00259-007-0368-7

    Article  PubMed  Google Scholar 

  • Steinstraesser, L., Kraneburg, U., Jacobsen, F., et al., 2011. Host defense peptides and their antimicrobialimmunomodulatory duality. Immunobiology, 216(3):322–333. http://dx.doi.org/10.1016/j.imbio.2010.07.003

    Article  CAS  PubMed  Google Scholar 

  • Stewart, P.S., Costerton, J.W., 2001. Antibiotic resistance of bacteria in biofilms. Lancet, 358(9276):135–138. http://dx.doi.org/10.1016/S0140-6736(01)05321-1

    Article  CAS  PubMed  Google Scholar 

  • Tunc, O., Thompson, J., Tremellen, K., 2010. Development of the NBT assay as a marker of sperm oxidative stress. Int. J. Androl., 33(1):13–21. http://dx.doi.org/10.1111/j.1365-2605.2008.00941.x

    Article  CAS  PubMed  Google Scholar 

  • Wang, K., Yan, J., Dang, W., et al., 2014. Dual antifungal properties of cationic antimicrobial peptides polybia-MPI: membrane integrity disruption and inhibition of biofilm formation. Peptides, 56:22–29. http://dx.doi.org/10.1016/j.peptides.2014.03.005

    Article  CAS  PubMed  Google Scholar 

  • Wang, L., Rao, C., Gao, K., et al., 2013. Development of a reference standard of Escherichia coli DNA for residual DNA determination in China. PLoS ONE, 8(9):e74166. http://dx.doi.org/10.1371/journal.pone.0074166

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wiegand, I., Hilpert, K., Hancock, R.E., 2008. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc., 3(2):163–175. http://dx.doi.org/10.1038/nprot.2007.521

    Article  CAS  PubMed  Google Scholar 

  • Wu, G., Cao, Y., Fan, B., et al., 2011. High-mobility group protein N2 (HMGN2) inhibited the internalization of Klebsiella pneumoniae into cultured bladder epithelial cells. Acta Bioch. Bioph. Sin., 43(9):680–687. http://dx.doi.org/10.1093/abbs/gmr064

    Article  CAS  Google Scholar 

  • Xie, Y., Fleming, E., Chen, J.L., et al., 2011. Effect of proline position on the antimicrobial mechanism of buforin II. Peptides, 32(4):677–682. http://dx.doi.org/10.1016/j.peptides.2011.01.010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yang, D., Postnikov, Y.V., Li, Y., et al., 2012. High-mobility group nucleosome-binding protein 1 acts as an alarmin and is critical for lipopolysaccharide-induced immune responses. J. Exp. Med., 209(1):157–171. http://dx.doi.org/10.1084/jem.20101354

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zanetti, M., 2004. Cathelicidins, multifunctional peptides of the innate immunity. J. Leukocyte Biol., 75(1):39–48. http://dx.doi.org/10.1189/jlb.0403147

    Article  PubMed  Google Scholar 

  • Zhang, L., Wang, Y.W., Lu, Z.Q., 2015. Midgut immune responses induced by bacterial infection in the silkworm, Bombyx mori. J. Zhejiang Univ.-Sci. B (Biomed. & Biotechnol.), 16(10):875–882. http://dx.doi.org/10.1631/jzus.B1500060

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Research Unit of Infection and Immunity, Sichuan University, Chengdu, 610041, China

    Heng Li, Xiao-fei Shen & Ning Huang

  2. Department of Pathophysiology, West China College of Basic and Forensic Medicine, Sichuan University, Chengdu, 610041, China

    Heng Li, Xiao-fei Shen, Xin-e Zhou, Yan-e Shi, Lu-xia Deng, Yi Ma, Xiao-ying Wang, Jing-yu Li & Ning Huang

  3. Sichuan University “985 Project Science and Technology Innovation Platform for Novel Drug Development”, Sichuan University, Chengdu, 610041, China

    Ning Huang

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  1. Heng Li
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Correspondence to Jing-yu Li or Ning Huang.

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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

ORCID: Heng LI, http://orcid.org/0000-0002-1293-7744

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Li, H., Shen, Xf., Zhou, Xe. et al. Antibacterial mechanism of high-mobility group nucleosomal-binding domain 2 on the Gram-negative bacteria Escherichia coli . J. Zhejiang Univ. Sci. B 18, 410–420 (2017). https://doi.org/10.1631/jzus.B1600139

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