内源性抗生素肽HMGN2抗细菌内化作用及机制初探

Preliminary study on the effect of endogenous antibiotic peptides HMGN2 on internalization of Klebsiella pneumoniae and corresponding mechanisms

目的探讨HMGN2抗细菌内化作用及其机制。方法利用生物学在线软件对人及常见医学模式生物HMGN2进行生物信息学分析;用免疫打点杂交技术检测临床标本HMGN2表达;设置以HMGN2预处理Klebsiella pneumoniae细菌后感染细胞组(简称共孵育组)和HMGN2预刺激细胞后再感染KP组(简称预刺激细胞组),平板菌落计数法检测两种状态下胞内活菌数;用荧光显微镜、流式细胞术观测各组细胞微丝形态和构成变化,用光镜观测HMGN2处理菌及未处理菌细胞骨架表型变化。结果不同属种生物HMGN2具有很高同源性,带阳离子电荷,具有α螺旋结构,分子量小于10kD;在炎性和非炎性标本中均可见HMGN2阳性信号,而在炎性标本中信号强度更大。HMGN2共孵育组和HMGN2预刺激细胞组胞内活细菌数与对照组相比较都明显减少,但共孵育组比预刺激细胞组胞内活菌数减少更为明显;荧光显微镜观察显示,两组微丝均有解聚成颗粒状向核周聚集现象,流式细胞术检测发两组细胞骨架微丝蛋白的平均荧光量减少,而油镜下HMGN2处理菌与未处理菌比较,其菌体长短径无明显的变化。结论 HMGN2属于内源性抗菌肽家族分子,可能通过促进细胞内微丝蛋白解聚成单体,减弱了KP内化入膀胱上皮细胞。

Objective To study the effect of HMGN2 on intracellular infection and investigate its possible mechanisms. Methods ProtParam tool and PHD secondary structure prediction were used to analyze antibacterial peptides HMGN2 in different species; Expressions of HMGN2 in clinical specimens were detected by immune dot hybridization assay. Define pre-treating KP with HMGN2 then incubating with T24 cells as co-incubated group, while, pre-stimulating T24 cells with HMGN2 then incubating with KP as pre-stimulated group. Viable organisms of both groups were shown by culture plates; Cytoskeleton changes were displayed using fluorescence microscope and flowcytometry. Results Bioinformation results indicated HMGN2 was highly conserved in eukaryotes, with positively charged residues and alpha helix structures, also their molecular weights were less than 10kD. HMGN2 signals can be detected in both inflammatory and non-inflammatory specimens were much stronger. The viable organisms of co-incubated group and pre-stimulated group were both less than control. And the number of viable organism of pre-stimulated group was lower than co-incubated group. Fluorescence microscope result showed microfilament depolymerized and clustered around nucleolus. Flow cytometer data revealed weaker fluorescence of actin of co- incubated group and pre-stimulated group. Compared with the control, bacteria morphology had no obvious change. Conclusion As an endogenous antibiotic peptide, HMGN2 could weaken KP bacillus internalization to urinary bladder epithelial cell by promoting F-actin depolymerization.