海分枝杆菌中sRNA5.85的功能及调控机制探索

Exploration of the Function and Regulatory Mechanism of sRNA5.85 in Mycobacterium Marinum

目的:鉴定海分枝杆菌(Mycobacterium marinum)中s RNA5.85的存在,探究s RNA5.85对分枝杆菌的生理影响及调控机制。方法:Northern-blot验证分枝杆菌中s RNA5.85的存在后,利用pMV261质粒,构建s RNA5.85过表达重组海分枝杆菌(5.85-OE株),观察重组菌株在体外生长、滑动能力及菌落形态变化。通过人源巨噬细胞感染模型,评估重组菌株的入胞及胞内增殖能力,LDH检测感染后的细胞活力。利用斑马鱼感染模型,验证s RNA5.85对细菌毒力的影响。结合targetRNA2与IntaRNA预测工具,预测s RNA5.85的潜在靶标及互作位点,并使用qRT-PCR验证。结果:海分枝杆菌基因组中存在s RNA5.85。过表达s RNA5.85不改变重组菌株的体外生长、滑动能力和菌落形态;5.85-OE株感染巨噬细胞48 h后,不影响THP-1的细胞活力,细菌胞内增殖能力与野生型无显著差异;s RNA5.85过表达会提高海分枝杆菌长期感染过程中,在斑马鱼体内的载菌量。targetRNA2预测的4个潜在靶基因MMAR_3476、MMAR_4052、MMAR_3482、MMAR_2920的转录水平随s RNA5.85过表达而显著上调,MMAR_3476及MMAR_4052共享同样的与s RNA5.85的互作位点。结论:s RNA5.85可通过调控低氧响应相关基因的表达,提高长期感染过程中对斑马鱼的毒力。本研究为致病性分枝杆菌中s RNA的挖掘和功能解析提供了重要线索。

Objective:To identify the presence of sRNA5.85 and investigate its physiological effects and regulatory mechanisms in Mycobacterium marinum(M.marinum).Methods:The presence of sRNA5.85 was validated through Northern blot analysis in M.mar-inum.Following this,the sRNA5.85 overexpression recombinant strain(referred to as the 5.85-OE strain)was constructed using the pMV261 plasmid.And M.marinum wild type strain with empty pMV261 plasmid was used as control strain(referred to as the WT-EV strain).In vitro experiments were conducted to observe the growth kinetics,sliding motility,and colony morphology of the 5.85-OE strain.To examine its behavior within human macrophages THP-1,an infection model was employed to assess the entry and intracellular proliferation of the 5.85-OE strain,with cell viability post-infection determined using LDH assays.Furthermore,a zebrafish infection model was utilized to validate the impact of sRNA5.85 on bacterial virulence.Prediction of potential target genes and interaction sites of sRNA5.85 was carried out using targetRNA2 and IntaRNA tools,with subsequent validation performed using qRT-PCR.Results:The sRNA5.85 was found to exist in the M.marinum.Overexpression of sRNA5.85 did not alter the in vitro growth,sliding ability,or colony morphology of the recombinant strain.After infecting macrophages with the 5.85-OE strain for 48 hours,THP-1 cell viability was not affected,and bacterial intracellular proliferation ability was not significantly different from the wild type strain.Overexpression of sRNA5.85 increased the bacterial load in zebrafish during long-term infection.The transcription levels of four potential target genes(MMAR_3476,MMAR_4052,MMAR_3482,MMAR_2920)predicted by targetRNA2 were significantly upregulated with sRNA5.85 overexpression.MMAR_3476 and MMAR_4052 shared the same interaction site with sRNA5.85.Conclusions:The findings reveal that sRNA5.85 enhances virulence towards zebrafish during extended infection periods by modulating the expression of low-oxygen response-related genes.This study offers crucial insights into understanding and unraveling the role of sRNA in pathogenic mycobacteria.