结核分枝杆菌sRNA Mpr5的功能验证

Functional verification of sRNA Mpr5 in Mycobacterium tuberculosis

摘要:

【目的】探究结核分枝杆菌(Mycobacterium tuberculosis,M.tb)中sRNA Mpr5对分枝杆菌的抗逆性及宿主细胞生理的影响。【方法】构建结核分枝杆菌sRNA Mpr5过表达的重组耻垢分枝杆菌(Mycolicibacterium smegmatis,M.smeg) M3-Atc，以转入空载质粒(pSI)的野生型耻垢分枝杆菌T1-Atc作为对照,观察细菌体外生长能力和菌落形态变化。通过非生物胁迫处理(低氧、饥饿、0.02%十二烷基硫酸钠)探究重组菌株的抗逆能力。用重组耻垢分枝杆菌侵染人非小细胞肺癌上皮细胞系A549，活菌涂板计算细菌的胞内增殖能力，利用免疫荧光染色观察感染后细胞的生理结构变化。【结果】sRNA Mpr5过表达菌株的体外生长与菌落形态均与野生型相似。抗逆性实验表明Mpr5过表达菌株(M3-Atc)的抗表面活性剂能力在4 h时显著提高(P<0.05)；在饥饿模型中M3-Atc早期(2–12 h)就表现出生长劣势(P<0.05)；低氧模型中M3-Atc菌株0–3 d均处于生长优势状态，增长均高于对照组(P<0.05)，3 d后增长幅度低于对照组。Mpr5过表达不影响菌株对上皮细胞侵染能力及细胞毒力，但降低了细菌早期胞内存活及增殖能力。【结论】sRNA Mpr5的过表达影响细菌对低氧、饥饿的环境应激，改变其对上皮细胞的感染能力，可能影响分枝杆菌的致病能力。

Abstract:

[Objective] To explore the effect of sRNA Mpr5 of Mycobacterium tuberculosis (M.tb) on stress resistance of Mycobacterium and host cell physiology. [Methods] The recombinant Mycolicibacterium smegmatis (M.smeg) strain (M3-Atc) overexpressing the sRNA Mpr5 from M.tb was developed. The wild-type M.smeg (T1-Atc) which was transformed with an empty plasmid (pSI) was used as control. The in vitro growth status and colony morphology of these two strains were observed. The resistance of the recombinant strain to hypoxia, starvation, and 0.02% sodium dodecyl sulfate was investigated. M3-Atc was used to infect the human non-small-cell lung cancer A549 cell line and the proliferation in the cells was detected with the spread plate method. Meanwhile, the physiological structure changes of the cells were observed based on immunofluorescence staining.[Results] The in vitro growth and colony morphology of M3-Atc were similar to those of the wild type. The anti-surfactant ability of M3-Atc was significantly improved at 4 h (P<0.05). In the case of starvation, colony number of M3-Atc was smaller than that of the wild type at the early stage (2-12 h) (P<0.05). In the instance of hypoxia, colony number of M3-Atc was larger than that of the wild type in 0-3 days (P<0.05), and the growth rate was lower than that of the wild type after 3 days. Mpr5 overexpression failed to affect the infection to epithelial cells and the cytotoxicity, but reduced the early intracellular survival rate and proliferation.[Conclusion] Overexpressing Mpr5 influences the response of Mycobacterium to hypoxia and starvation and changes its ability to infect epithelial cells, which may finally affect the pathogenicity.