基于CRISPR/Cas9构建伪结核棒状杆菌cp40敲除株及其生物学特性与致病性研究

Biological characteristics and pathogenicity of Corynebacterium pseudotuberculosis cp40-deleted strains constructed by CRISPR/Cas9

摘要:

【目的】建立更高效的伪结核棒状杆菌(Corynebacteriumpseudotuberculosis, Cp)靶基因敲除方法，敲除丝氨酸蛋白酶编码基因(cp40)，并评价其在Cp感染致病中的作用。【方法】以pECXK99E为载体，Cp宣汉株(XH02) cp40为靶基因，设计含CRISPR/Cas9的间隔序列(spacer)、导向RNA (guide RNA, gRNA)和cp40上下游同源臂的表达质粒(pEC-cp40gRNA-HDarm)，转入含pCas9gRNA-ccdB的Cp感受态细胞，构建CRISPR/Cas9基因编辑系统以敲除cp40基因。通过比较cp40敲除株和野生株的菌落形态及生长曲线、体外对J774A.1巨噬细胞活力及白细胞介素(interleukin, IL)-1β分泌，以及体内感染小鼠致死率及脏器载菌水平，研究cp40与该病原感染致病之间的关系。【结果】应用所建立的双质粒CRISPR/Cas9编辑系统成功获得XH02的cp40敲除株(XH02Δcp40)。与XH02相比，XH02Δcp40在菌落形态及生长曲线上无明显差异，但XH02Δcp40感染J774A.1巨噬细胞的乳酸脱氢酶(lactate dehydrogenase, LDH)释放(P=0.06)、碘化丙锭(propidium iodide, PI)染色比例均降低(P<0.01)，体内感染小鼠致死率下降50%，并且被感染小鼠肝脏和肾脏中Cp含量显著降低(P<0.001)。【结论】本研究所建立的CRISPR/Cas9编辑系统可较高效敲除Cp基因，证实cp40是该病原感染致病相关基因，为后续基于该基因研究Cp感染致病机制奠定基础。

Abstract:

[Objective] To establish a more efficient knockout method for the serine protease coding gene (cp40) of Corynebacterium pseudotuberculosis and evaluate the role of this gene in the pathogenicity of C. pseudotuberculosis. [Methods] A vector pEC-cp40gRNA-HDarm, with guide RNA, upstream and downstream sequences flanking cp40 of C. pseudotuberculosis Xuanhan strain (XH02), and spacer, was constructed from pECXK99E. The recombinant vector pEC-cp40gRNA-HDarm was transferred into C. pseudotuberculosis competent cells carrying pCas9gRNA-ccdB to form the CRISPR/Cas9 gene editing system for the deletion of cp40. The roles of cp40 in the pathogenicity of C. pseudotuberculosis were evaluated by comparison of the colony morphology and growth curves between the cp40-deleted (Δcp40) strain and wild type (WT) strain, the viability and interleukin (IL)-1β secretion of J774A.1 macrophages infected with Δcp40 and WT in vitro, and the mortality and organ bacterial loads in mice infected with Δcp40 and WT in vivo. [Results] We successfully constructed the cp40-deleted strain XH02Δcp40 by using the established dual-plasmid CRISPR/Cas9 editing system. Compared with WT (XH02), XH02Δcp40 showed no obvious difference in the colony morphology or growth curve. However, the J774A.1 cells infected with XH02Δcp40 showed decreased lactate dehydrogenase (LDH) release (P=0.06) and propidium iodide (PI) staining ratio (P<0.01) compared with those infected with XH02. The mortality of XH02Δcp40-infected mice reduced by 50% and the bacterial loads in the liver and kidney of XH02Δcp40-infected mice significantly reduced compared with those of XH02-infected mice (P<0.001). [Conclusion] The CRISPR/Cas9 gene editing system established in this study can effectively delete cp40 of C. pseudotuberculosis. The results confirm that cp40 is a virulence-related gene, providing a foundation for subsequent research on the infection of C. pseudotuberculosis based on this gene.