基于SOS调控通路牛荚膜A型多杀性巴氏杆菌耐药抑制剂靶标的筛选

Respiratory disease caused by Pasteurella multocida (Pm) serotype A is becoming increasingly serious, especially the emergence of highly resistant strains has caused huge economic losses to animal husbandry. To explore the Pm drug resistancemechanismand screen drug resistance inhibitor target has become the current research spot. Our research team has monitored the resistance of clinical Pm for many yearsand found that the bacteria can easily produce drug resistance adaptation to commonly used clinical drugs, and confirmed that significant changeshad happened to the SOS response pathway of Pm. The related genes in the response pathway have potential application value as drug resistance inhibitor targets. In this study, genomic and transcriptome sequencing techniques were used to analyze the genetic environment of SOS-response pathway in Pm resistant and susceptible strains, and screen differentially expressed genes that were validated by real-time quantitative RT-PCR and ELISA; Red recombination techniquewas used to construct differentially expressed gene deleted strains and complementary strains. Furthermore, to perform drug sensitivity and biological activity tests on gene deletedand complementary strains to find the target genes that are critical to Pm drugre sistance. Our study will lay the foundation of the screening of drug-resistant inhibitors of Pm.

牛荚膜A型多杀性巴氏杆菌引发的牛呼吸系统疾病日趋严重，特别是其高度耐药株的出现给畜牧业造成了巨额经济损失，探究该菌耐药适应性机制，筛选其耐药抑制剂靶标已成为当前研究重点。本项目组连续多年对我国临床牛荚膜A型多杀性巴氏杆菌进行耐药性监测，发现该菌极易对临床常用药物产生耐药适应性，且证实该耐药菌SOS应答调控网络发生了显著变化，应答通路中相关基因有成为耐药抑制剂靶标的潜在应用价值。本研究拟采用基因组及转录组测序技术对该菌耐药株、敏感株SOS应答调控网络的基因环境进行分析，筛选出差异表达基因，并采用实时荧光定量RT-PCR、ELISA方法对差异表达基因进行验证；采用red重组技术构建差异表达基因缺失株和互补株，进一步对缺失株、互补株进行药物敏感性及生物活性检测，筛选得到对牛荚膜A型多杀性巴氏杆菌耐药适应性起关键作用的靶基因。上述研究将为牛荚膜A型多杀性巴氏杆菌耐药抑制剂的筛选奠定基础。

兽医临床主要病原菌对氟喹诺酮类抗生素产生耐药性的问题已日益严重，其新型耐药抑制靶标的筛选及新型耐药抑制剂的研发迫在眉睫。为此，项目组以对氟喹诺酮类抗生素呈不同耐药水平的牛荚膜A型多杀性巴氏杆菌为模式菌，对其耐药表型、耐药基因型进行确定，采用转录组（RNAseq）、蛋白质组 (iTRAQ)对不同耐药表型、不同耐药基因型菌株SOS应答调控网络的基因环境进行分析，筛选出差异表达基因，进一步构建了差异表达基因缺失株、互补株，对缺失株相关生物学活性进行了检测。研究结果显示，本项目共筛选获得了5株对氟喹诺酮类抗生素具有不同耐药表型、不同耐药基因型的菌株；基于RNAseq、iTRAQ和qRT-PCR，共筛选获得了3个显著差异表达基因，分别为recO、recA、rucA；成功构建了敏感株P3、耐药株P32recO基因缺失株、互补株；对缺失基因的功能进行了初步确证，结果证明recO基因的缺失可在一定程度上抑制SOS反应，同时通过体外耐药性诱导证明其可一定程度上延缓牛荚膜A型Pm氟喹诺酮类抗生素耐药菌的产生，降低耐受性，该基因具有作为氟喹诺酮类抗生素耐药抑制剂靶标的潜力。该项目共筛选获得具有氟喹诺酮类药物SOS反应耐药抑制剂的潜在药物靶标1个，发表国内外重要期刊收录文章5篇，培养硕士研究生2名。本项目为兽医临床致病菌氟喹诺酮类药物耐药抑制剂靶标的发现提供了一个新的策略，也为氟喹诺酮类抗生素耐药抑制剂的研发奠定了坚实的基础。