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Journal of Zhejiang University-SCIENCE B

, Volume 18, Issue 5, pp 410–420 | Cite as

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

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

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.

Key words

High-mobility group nucleosomal-binding domain 2 (HMGN2) Bioactivity Membrane permeability Biofilm Chemotactic activity 

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

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。

关键词

高迁移率族蛋白N2(HMGN2) 膜通透性 生物被膜 趋化作用 

CLC number

R392.11 

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Copyright information

© Zhejiang University and Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Heng Li
    • 1
    • 2
  • Xiao-fei Shen
    • 1
    • 2
  • Xin-e Zhou
    • 2
  • Yan-e Shi
    • 2
  • Lu-xia Deng
    • 2
  • Yi Ma
    • 2
  • Xiao-ying Wang
    • 2
  • Jing-yu Li
    • 2
  • Ning Huang
    • 1
    • 2
    • 3
  1. 1.Research Unit of Infection and ImmunitySichuan UniversityChengduChina
  2. 2.Department of Pathophysiology, West China College of Basic and Forensic MedicineSichuan UniversityChengduChina
  3. 3.Sichuan University “985 Project Science and Technology Innovation Platform for Novel Drug Development”Sichuan UniversityChengduChina

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