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（英文）

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

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(tH MGN2)。诱导重组表达质粒PET-32a-c(+)-HMGN2表达重组蛋白HMGN2(rH MGN2)。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16.25μg/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。

Objective： To investigate the antibacterial mechanism of high-mobility group nucleosomal-binding domain 2 （HMGN2） on Escherichia coil 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 HPl100 reversed-phase high-performance liquid chromatography （RP-HPLC）. Recombinant human HMGN2 （rHMGN2） was generated in E. coil 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 migra- tion 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. coil K12. HMGN2 could also bind the bacterial plasmid and genomic DNA in a dose-dependent manner. The antibiofilm effect of HMGN2 on E. coil 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. Con- clusions： As an antimicrobial peptide （AMP）, HMGN2 possessed a good capacity for antibacterial and antibiofilm activities on E. coil 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. coil.