鸭疫里默氏杆菌SOS反应调控机制研究

The bacterial SOS response principally targets at addressing DNA damage and closely interrelates with multi-drug resistance. The SOS response can be induced by some antibiotics in some bacteria, but which remains elusive so far in Riemerella anatipestifer (RA). Our preliminary studies indicate that the quinolone antibiotics can induce the SOS stress response in RA. The purpose of this study is to probe the regulation mechanism of SOS response in RA based on the preliminary work. Firstly, the key genes regulating RA SOS response will be identified by SOS response defects strains derived from mutant library. Secondly, the functions of the genes are identified by means of construction of the gene mutants and complementary strains. Furthermore, in order to screen the differential genes, the cDNAs, derived from the RNA extracting from the strains (field strains, gene mutants and complementary strains), are hybridized with DNA microarray of RA-GD strain. Based on the above studies, the key genes and pathways regulating RA SOS response are clarified. This work will provide a theoretical basis for further elucidating the mechanisms of RA SOS response and multiple-antibiotic resistance mediated by SOS response in bacteria.

SOS反应是细菌应对DNA损伤的重要保护机制，又与其耐药性的发生和传播密切相关。一些抗生素可以诱导细菌发生SOS反应，但关于鸭疫里默氏杆菌（RA）的SOS反应还未见报道。本研究团队证实喹诺酮类抗生素可以诱导RA发生SOS反应，这为进一步探索RA SOS反应的调控机制奠定了基础。本研究通过构建RA突变体库，筛选SOS反应缺陷株，鉴定参与RA SOS反应的关键基因；构建相关基因缺失突变株和对应的互补株，验证基因的功能。制备RA菌株基因组表达谱芯片，用喹诺酮类抗生素诱导RA野生株、基因缺失突变株及其互补株发生SOS反应后，提取它们的mRNA，制备荧光标记的cDNA，分别与RA菌株基因组表达谱芯片杂交，筛选差异表达基因并验证。通过以上研究，明确参与RA SOS反应的关键基因及其功能，探索相关基因参与RA SOS反应的信号通路，为阐明RA SOS反应调控机制及其介导的细菌耐药机制提供科学依据。

细菌DNA损伤诱导反应（SOS反应），是普遍存在的细菌应对DNA损伤的重要保护机制，目前在鸭疫里默氏杆菌中还未见相关报道。本研究以含转座子Tn4001mod的pISM2062质粒成功构建了RA-GD菌株的突变体文库，得到2556株突变株。共筛选到8株SOS反应缺陷株，鉴定出4个与RA-GD菌株发生SOS反应相关的基因，包括RIA_0042、RIA_0163、RIA_0641和RIA_1469（RecA）。用紫外线辐照菌株后，用qRT-PCR方法检测上述基因的表达量，发现RecA基因的转录水平上升5.2倍，RIA_0042和RIA_0641基因的转录水平上升1.6和2.3倍，RIA_0163基因的转录水平无显著差异，据此推测RecA基因在RA-GD株发生SOS反应中发挥重要作用，因此对该基因的生物学功能进行验证。构建了RecA基因缺失株RA-GD△RecA和回复株cRA-GD△RecA，检测了亲本株、缺失株和回复株的生长能力、毒力以及在紫外线辐照条件下基因组DNA的损伤程度。结果表明，RecA蛋白缺失后不影响菌株的生长，但降低了菌株的毒力；经紫外线辐照处理后，亲本株RecA基因的转录水平显著上升，但蛋白表达水平无显著差异；与亲本株相比，缺失株基因组DNA的损伤程度显著升高。上述结果证实，RecA蛋白在鸭疫里默氏杆菌的SOS反应中发挥正向调控作用。另外，还对鸭疫里默氏杆菌中外排泵介导的耐药机制进行了拓展研究，鉴定了RND外排泵RaeE-RaeF-RopN和ABC外排泵蛋白RanB；还证实该菌可以穿越雏鸭血脑屏障，导致脑膜炎。