禽滑液囊支原体SOS反应相关基因的鉴定及功能研究

Mycoplasma synoviae is an important avian pathogen and causes respiratory infection and synovitis in poultry. Antibiotic resistance development has been linked to the bacterial SOS response (SOSr), which is an important DNA repair mechanism in bacteria. However, whether the Mycoplasma synoviae is involved in antibiotic-induced SOSr remain unclear. We had identified SOSr in Mycoplasma synoviae isolate K6695 induced by enrofloxacin and tetracycline using qRT-PCR and western-blot in previous studies. To further investigate the correlation mechanism of SOSr and modulation of antibiotic resistance in Mycoplasma synoviae, the methods including construction of random transposon mutagenesis libraries, the SOSr phenotypic screening, and identification of insertion sites will be used. The key SOSr genes involved in Mycoplasma synoviae will be identified through comparison of deletion mutants and complementary mutants in vitro and vivo. Taken together, this study will be contribute to understanding the SOSr-mediated antibiotic resistance mechanisms of Mycoplasma synoviae and providing theoretical basis for discovery of drug targets and development of new drugs.

禽滑液囊支原体（Mycoplasma synoviae）是引起家禽呼吸道疾病、传染性滑膜炎的重要病原之一。SOS应答（SOS response，SOSr）作为细菌重要的DNA修复机制，在细菌抗药性的形成中起着重要作用，但有关禽滑液囊支原体的SOSr还未见相关报道。本项目前期利用qRT-PCR和Western-blot技术，在恩诺沙星和四环素选择压力下，筛选到可发生SOSr的禽滑液囊支原体K6695株。基于此，为进一步探究SOSr调控禽滑液囊支原体耐药产生的分子机制，本项目通过转座子诱变构建K6695株突变体库，筛选SOSr缺陷株并分析参与禽滑液囊支原体SOSr的关键基因，构建相关基因的缺失突变株和互补株，结合体内和体外实验，验证参与禽滑液囊支原体SOSr的相关基因功能。该研究为进一步揭示禽滑液囊支原体SOSr在其抗药性产生的分子机制，以及药物靶标的筛选和新药物开发提供理论依据和思路。

禽滑液囊支原体（Mycoplasma synoviae，MS）是引起家禽呼吸道疾病、传染性滑膜炎的重要病原之一。SOS应答（SOS response，SOSr）作为细菌重要的DNA修复机制，在细菌抗药性的形成中起着重要作用。本项目按计划执行，通过流行病学调查、致病力及药敏试验筛选出抗大环内酯类及喹诺酮类药物的耐药强毒株CHN-BZJ2-2015、耐药弱毒株CHN-JNB19-2016和敏感疫苗株MS-H；以这三株菌株为试验对象，通过差异蛋白质组学研究及RT-PCR验证，明确了参与耐药强毒株的SOSr关键基因，分别为uvrD及recX；进一步实验验证表明，uvrD和recX基因在转录水平和蛋白水平表达一致，其中uvrD的表达升高增加了CHN-BZJ2-2015株的耐药，而recX在CHN-BZJ2-2015株中表达降低，证明它在SOSr中起负调控，有稳定基因组的作用；本研究还鉴定出一些和MS多重耐药、粘附及入侵相关的基因，如tktA-1，tpiA、phdB、fusA、dppD及pstA，为下一步研究MS耐药及感染机制提供理论依据。另外，我们用强毒株CHN-WF224-2016制备的灭活抗原与MontanideTM ISA 71 VG佐剂配伍制备的疫苗不仅可以激活机体的体液免疫反应还可以激活细胞免疫反应，可提供很好的免疫保护力，具有良好的前景，可弥补国内空白。