氨基糖苷类抗生素诱导大肠杆菌发生适应性耐药的小RNA调控机制研究

Adaptive resistance is one of the reasons that lead to the treatment failure of clinical infections caused by gram-negative bacteria. Currently, the mechanisms of adaptive resistance to aminoglycosides of gram-negative bacteria remain unclear. Previous studies show that small RNAs (sRNAs) modulate the target genes and enhance the bacterial fitness to the environmental stress. We hypothesize that adaptive resistance is also related with the regulation of sRNA on its target gene, which causes the reduced drug accumulation in bacteria. In this study, we will screen the Escherichia coli isolates which are susceptible to aminoglycosides. Deep sequencing technique will be used to analyse the level of sRNAs in Escherichia coli with or without adaptive resistance to aminoglycosides. Northern blot and real-time PCR are also used to detect the level of sRNAs in regulating of outer membrane proteins. Small RNA knockout mutant and complement strain are constructed to investigate the function of sRNA in adaptive resistance mechanisms. This study will help to reveal the molecular mechanisms involved in adaptive resistance of Escherichia coli and provide the theoretical basis for new drugs in future that may be used in the infections caused by bacteria with adaptive resistance.

适应性耐药可导致临床革兰阴性菌感染治疗失败。目前氨基糖苷类抗生素诱导革兰阴性菌产生适应性耐药的具体机制仍不明确。已有研究发现，细菌可通过小RNA(sRNA)调控其靶基因，以适应不同的应激环境。我们推测细菌获得适应性耐药与sRNA通过调控其下游靶基因的表达水平，使菌体内蓄积药物浓度降低有关。在本课题中，我们拟分离对氨基糖苷类药物敏感的大肠杆菌临床分离株，采用深度测序技术，筛选氨基糖苷类抗生素诱导大肠杆菌发生适应性耐药的差异sRNA；并利用northern blot 和real-time PCR等分子生物学技术比较已知的大肠杆菌膜蛋白相关sRNA的表达水平。通过构建适应性耐药相关sRNA敲除株和回复株，研究sRNA在大肠杆菌发生适应性耐药过程中发挥的作用。本课题的研究成果，有助于揭示大肠杆菌产生适应性耐药的分子机制，并为今后寻找防治相关耐药菌感染的药物新靶点做出理论探索。

适应性耐药普遍存在多种革兰阴性菌中，可引起临床抗感染治疗的失败。本项目拟采用深度测序技术，筛选获得与大肠杆菌对氨基糖苷类药物发生庆大霉素适应性耐药的sRNA，通过构建适应性耐药相关sRNA敲除株和回复株，研究sRNA在大肠杆菌发生适应性耐药过程中发挥的作用。本项目在执行过程中, 对亚抑制浓度的庆大霉素诱导适应性耐药的特点进行了研究。但经转录组测序发现，经庆大霉素诱导后大肠杆菌的上调的sRNA仅有45个，下调的仅有1个，且全部是功能未知的sRNA，增大了筛选工作的难度，因此，我们在继续筛选sRNA工作的同时，对与适应性耐药相关的功能未知基因进行了筛选，成功找到一个与适应性耐药发生有关的膜蛋白编码基因gene972。转录组测序还发现亚抑制浓度的庆大霉素可影响多数大肠杆菌运动相关基因的表达水平，作为本课题的延伸，我们进一步发现亚抑制浓度的庆大霉素处理后，大肠杆菌的swarm运动能力降低，该机制与庆大霉素抑制琥珀酸脱氢酶的表达，导致延胡索酸产生减少有关。本项目明确了一个新的与大肠杆菌适应性耐药发生有关的基因，有助于开发防止适应性耐药发生的新技术。另外，本研究中亚抑制浓度庆大霉素对大肠杆菌运动性的抑制作用提示其用于大肠杆菌的感染仍然具备其自身优势。