沙门氏菌毒力岛SPI-1、2和3的缺失诱导其显著的形态和代谢变化以及保护性免疫潜力

doi:10.1016/j.jia.2023.11.009

摘要/Abstract

摘要：

为了研究沙门菌三种主要毒力岛SPIs（SPI-1、2和3）对其生理学、致病性和免疫原性的影响，本研究通过构建改进的λ-Red同源重组系统，以肠炎沙门菌SM6野毒株为亲本制备了三种主要SPIs（SPI-1、2和3）的单缺失（SM6ΔSPI1, SM6ΔSPI2, SM6ΔSPI3）和三缺失突变体（SM6ΔSPI1&2&3）；对突变体的生长和稳定性进行检测；采用扫描电镜观察菌体形态；利用Biolog微生物鉴定系统检测碳源代谢情况；检测突变体对Caco-2细胞的粘附和侵袭能力；以1×10⁷ CFU剂量腹腔注射7日龄来航鸡于注射后第2、4、8天(dpi)检测突变体在肝脏和脾脏内的定殖情况；另外，按1×10⁷ CFU剂量将各突变体分别于7、21和35日龄腹腔免疫后在首免2、3、4、5、6周（wpi）收集血清检测特异性IgG水平，并于42日龄腹腔注射1×10⁹ CFU剂量的SM6野毒株，于攻毒后第7和14天检测血液中细胞因子水平，同时收集脾细胞检测CD3⁺CD4⁺和CD3⁺CD8⁺比率，对免疫和攻毒后鸡只的临床表现和组织病理损伤进行打分，对各突变体的免疫效力进行评价。结果表明，单缺失突变体的表面表现出更粗糙的质地，并似乎被一层透明的胶体包裹，而与亲本菌株相比，三缺失突变体的形态保持不变。SPI突变体的碳代谢谱发生了深刻的变化，在95个碳源中的30个碳源（主要是碳水化合物）中观察到了显著且具有统计学意义的变化（30个碳来源中的17个）。此外，与亲本菌株相比，四种突变体对Caco-2细胞的粘附能力显著降低。此外，突变体SM6ΔSPI1和SM6ΔSPI1&2&3的入侵能力显著降低，而SM6ΔSPI3和SM6△SPI2的入侵能力不同程度地增强。重要的是，四个突变体中没有一个在雏鸡中引起任何临床症状。然而，它们确实在脾脏和肝脏短暂定植。值得注意的是，SM6ΔSPI1&2&3突变体在感染后8天内从脾脏和肝脏中迅速清除，并且在器官中没有观察到显著的病理变化。此外，当受到攻击时，突变体免疫组的抗体水平显著增加，CD3⁺CD4⁺和CD3+CD8⁺亚群发生变化，SM6ΔSPI1&2&3免疫鸡血清中的IL-4和IFN-γ细胞因子水平超过其他组。总之，四个SPI突变体的成功构建为进一步探索SPI的致病（包括代谢）机制和开发安全有效的减毒沙门氏菌活疫苗或载体奠定了基础。

Abstract:

The Salmonella pathogenicity islands (SPIs) play crucial roles in the progression of Salmonella infection. In this study, we constructed an improved λ Red homologous recombination system to prepare single and triple deletion mutants of 3 prominent SPIs (SPI-1, 2, and 3), aiming at the impact of deletion on morphology, carbon source metabolism, adhesion and invasion capacity, in vivo colonization, and immune efficacy in chicks. Our examination revealed that the surface of the single deletion mutants (SM6ΔSPI1, ΔSPI2, and ΔSPI3) exhibited a more rugged texture and appeared to be enveloped in a layer of transparent colloid, whereas the morphology of the triple deletion mutant (SM6ΔSPI1&2&3) remained unaltered when compared to the parent strain. The carbon metabolic spectrum of the SPI mutants underwent profound alterations, with a notable and statistically significant modification observed in 30 out of 95 carbon sources, primarily carbohydrates (17 out of 30). Furthermore, the adhesion capacity of the 4 mutants to Caco-2 cells was significantly reduced when compared to that of the parent strain. Moreover, the invasion capacity of mutants SM6ΔSPI1 and SM6ΔSPI1&2&3 exhibited a substantial decrease, while it was enhanced to varying degrees for SM6ΔSPI3 and SM6ΔSPI2. Importantly, none of the 4 mutants induced any clinical symptoms in the chicks. However, they did transiently colonize the spleen and liver. Notably, the SM6ΔSPI1&2&3 mutant was rapidly cleared from both the spleen and liver within 8 days post-infection and no notable pathological changes were observed in the organs. Additionally, when challenged, the mutants immunized groups displayed a significant increase in antibody levels and alterations in the CD3⁺CD4⁺ and CD3⁺CD8⁺ subpopulations, and the levels of IL-4 and IFN-γ cytokines in the SM6ΔSPI1&2&3 immunized chicken serum surpassed those of other groups. In summary, the successful construction of the 4 SPI mutants lays the groundwork for further exploration into the pathogenic (including metabolic) mechanisms of SPIs and the development of safe and effective live attenuated Salmonella vaccines or carriers.

Key words: Salmonella Pathogenicity Islands (SPIs) , morphology , carbon source metabolism , pathogenicity , immunogenicity , live attenuated vaccine

Gaosong Liu, Xuelian Lü, Qiufeng Tian, Wanjiang Zhang, Fei Yi, Yueling Zhang, Shenye Yu. 沙门氏菌毒力岛SPI-1、2和3的缺失诱导其显著的形态和代谢变化以及保护性免疫潜力[J]. Journal of Integrative Agriculture, 2025, 24(1): 272-289.

Gaosong Liu, Xuelian Lü, Qiufeng Tian, Wanjiang Zhang, Fei Yi, Yueling Zhang, Shenye Yu. Deletion of Salmonella pathogenicity islands SPI-1, 2 and 3 induces substantial morphological and metabolic alternation and protective immune potential[J]. Journal of Integrative Agriculture, 2025, 24(1): 272-289.

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