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-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: 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.

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]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.

[2]Bratton, D.L., Henson, P.M., 2011. Neutrophil clearance: when the party is over, clean-up begins. Trends Immunol., 32(8):350-357.

[3]Brogden, K.A., 2005. Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol., 3(3):238-250.

[4]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.

[5]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.

[6]Costerton, J.W., Stewart, P.S., Greenberg, E., 1999. Bacterial biofilms: a common cause of persistent infections. Science, 284(5418):1318-1322.

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

[8]Deng, L.X., Wu, G.X., Cao, Y., et al., 2011. The chromosomal protein HMGN2 mediates lipopolysaccharide-induced expression of β-defensins in A549 cells. FEBS J., 278(12): 2152-2166.

[9]Deng, L.X., Wu, G.X., Cao, Y., et al., 2012. The chromosomal protein HMGN2 mediates the LPS-induced expression of β-defensins in mice. Inflammation, 35(2):456-473.

[10]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.

[11]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.

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

[13]Hawkey, P.M., Jones, A.M., 2009. The changing epidemiology of resistance. J. Antimicorob. Chemoth., 64(Suppl. 1): i3-i10.

[14]Høiby, N., Bjarnsholt, T., Givskov, M., et al., 2010. Antibiotic resistance of bacterial biofilms. Int. J. Antimicrob. Ag., 35(4):322-332.

[15]Lai, Y., Gallo, R.L., 2009. AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense. Trends Immunol., 30(3):131-141.

[16]Lehrer, R.I., Rosenman, M., Harwig, S.S., et al., 1991. Ultrasensitive assays for endogenous antimicrobial polypeptides. J. Immunol. Methods, 137(2):167-173.

[17]Liu, Y., Knapp, K.M., Yang, L., et al., 2013. High in vitro antimicrobial activity of β-peptoid-peptide hybrid oligomers against planktonic and biofilm cultures of Staphylococcus epidermidis. Int. J. Antimicrob. Ag., 41(1):20-27.

[18]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.

[19]O'Toole, G., Kaplan, H.B., Kolter, R., 2000. Biofilm formation as microbial development. Annu. Rev. Microbiol., 54(1): 49-79.

[20]Park, B., Fikrig, S., Smithwick, E., 1968. Infection and nitroblue-tetrazolium reduction by neutrophils: a diagnostic aid. Lancet Oncol., 292(7567):532-534.

[21]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.

[22]Reddy, K., Yedery, R., Aranha, C., 2004. Antimicrobial peptides: premises and promises. Int. J. Antimicrob. Ag., 24(6):536-547.

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

[24]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.

[25]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.

[26]Steinstraesser, L., Kraneburg, U., Jacobsen, F., et al., 2011. Host defense peptides and their antimicrobial-immunomodulatory duality. Immunobiology, 216(3):322-333.

[27]Stewart, P.S., Costerton, J.W., 2001. Antibiotic resistance of bacteria in biofilms. Lancet, 358(9276):135-138.

[28]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.

[29]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.

[30]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.

[31]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.

[32]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.

[33]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.

[34]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.

[35]Zanetti, M., 2004. Cathelicidins, multifunctional peptides of the innate immunity. J. Leukocyte Biol., 75(1):39-48.

[36]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.