结核分枝杆菌PE_PGRS15调控分枝杆菌细胞包膜结构与耐药性

Mycobacterium tuberculosis PE_PGRS15 modulates the envelope structure and stress resistance of mycobacteria

摘要:

【目的】研究结核分枝杆菌PE_PGRS15的功能。【方法】构建过表达PE_PGRS15蛋白的重组耻垢分枝菌酸杆形菌，通过细胞分级分离实验检测其细胞定位。通过涂布实验、扫描电镜和透射电镜观察细菌菌落形态、细菌表面形态及细胞包膜(cell envelope)结构。通过杀菌曲线法及微量肉汤稀释法检测重组菌对环境压力及抗生素的耐受性。通过染料摄取实验检测重组菌细胞壁通透性，并用气相色谱-质谱联用仪检测重组菌细胞壁脂肪酸谱。通过蛋白截短及融合实验分析PE_PGRS15蛋白结构域的功能。【结果】PE_PGRS15蛋白定位于重组菌细胞壁，其表达影响重组菌菌落形态和细胞包膜结构，增强重组菌对环境压力和抗生素的耐受。PE_PGRS15的表达导致重组菌细胞包膜脂肪酸含量增加，也降低了重组菌的细胞壁通透性。PE_PGRS15蛋白的PE结构域负责将该蛋白转运到细胞表面，而PGRS结构域介导重组菌对压力条件和抗生素的耐受。【结论】PE_PGRS15蛋白可能通过调控耻垢分枝菌酸杆形菌细胞包膜的结构进而影响细菌菌落形态、细胞壁通透性及耐药性，为解析PE/PPE家族蛋白的功能奠定了一定的基础。

关键词:PE_PGRS15;耻垢分枝菌酸杆形菌;压力耐受;细胞壁通透性;过表达

Abstract:

[Objective] To reveal the function of PE_PGRS15 from Mycobacterium tuberculosis. [Methods] A recombinant Mycolicibacterium smegmatis strain heterologously expressing PE_PGRS15 (MS-PE_PGRS15) was generated. The colony morphology, cell surface morphology, and envelope structure were observed by a plating method, a scanning electron microscope, and a transmission electron microscope, respectively. The localization of PE_PGRS15 was detected by the cell fractionation assay. The resistance of the recombinant strain to environmental stresses and antibiotics was measured by the killing curve method and micro-broth dilution method. The permeability and fatty acid profile of the cell wall of the recombinant strain were determined by dye uptake assay and gas chromatography-mass spectrometry, respectively. The functions of different domains of PE_PGRS15 were analyzed by protein truncation and fusion experiments. [Results] PE_PGRS15 was located on the cell envelope of MS-PE_PGRS15. MS-PE_PGRS15 showed altered colony morphology, envelope structure, and cell wall fatty acid profile, with noticeable increase in resistance to multiple environmental stresses and antibiotics. The dye uptake experiments with ethidium bromide and Nile red suggested that the cell wall of MS-PE_PGRS15 was more impermeable than that of the control strain. The PGRS domain of PE_PGRS15 affected mycobacterial cell wall permeability and stress resistance, while the PE domain was involved in the transport of the protein to the cell surface. [Conclusion] PE_PGRS15 was present in the cell wall fraction of MS-PE_PGRS15 and influenced cell wall permeability and colony morphology, ultimately enhancing the resistance of recombinant M. smegmatis to stresses.

Key words:PE_PGRS15;Mycolicibacterium smegmatis;stress resistance;cell wall permeability;overexpression