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[Objective] To establish a more efficient knockout method for the serine protease coding gene (cp40) ofCorynebacteriumpseudotuberculosis and evaluate the role of this gene in the pathogenicity ofC.pseudotuberculosis. [Methods] A vector pEC-cp40gRNA-HDarm, with guide RNA, upstream and downstream sequences flankingcp40 ofC.pseudotuberculosis Xuanhan strain (XH02), and spacer, was constructed from pECXK99E. The recombinant vector pEC-cp40gRNA-HDarm was transferred intoC.pseudotuberculosis competent cells carrying pCas9gRNA-ccdB to form the CRISPR/Cas9 gene editing system for the deletion ofcp40. The roles ofcp40 in the pathogenicity ofC.pseudotuberculosis were evaluated by comparison of the colony morphology and growth curves between thecp40-deleted (Δcp40) strain and wild type (WT) strain, the viability and interleukin (IL)-1β secretion of J774A.1 macrophages infected with Δcp40 and WTinvitro, and the mortality and organ bacterial loads in mice infected with Δcp40 and WTinvivo. [Results] We successfully constructed thecp40-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 deletecp40 ofC.pseudotuberculosis. The results confirm thatcp40 is a virulence-related gene, providing a foundation for subsequent research on the infection ofC.pseudotuberculosis based on this gene.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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