鼠伤寒沙门氏菌Ⅵ型分泌系统相关基因敲除及其功能特性

Knockout and functional characteristics of CVCC541 VI secretion system related genes in Salmonella typhimurium

摘要:

Ⅵ型分泌系统（T6SS）是大多数革兰氏阴性细菌中都存在的一种重要的分泌系统，能介导细菌与细菌之间以及细菌和宿主细胞之间的相互作用，溶血素共调节蛋白（Hcp）和缬氨酸甘氨酸重复蛋白G（VgrG）是组成T6SS穿刺装置的重要组分。但鼠伤寒沙门氏菌Ⅵ型分泌系统的Hcp与VgrG在该菌入侵宿主细胞及抗吞噬过程中发挥的作用尚不十分清楚。[目的] 本研究旨在利用基因敲除技术构建的鼠伤寒沙门氏菌hcp及vgrg基因缺失株体外接种真核上皮细胞和巨噬细胞，并以其亲本株作为对照，以研究Hcp及VgrG在该菌粘附、侵入上皮细胞及抗吞噬过程中所发挥的作用。[方法] 通过优化Red同源重组系统操作过程中各个条件，建立一套快速敲除鼠伤寒沙门氏菌Ⅵ型分泌系统相关基因的操作系统，成功构建鼠伤寒沙门氏菌CVCC541的hcp及vgrg单基因缺失株、双基因缺失株及三基因缺失株，并用Hela细胞接种试验和菌落计数试验，评估不同菌株的粘附和侵袭能力；用小鼠巨噬细胞RAW 264.7接种试验，评估不同菌株的抗吞噬能力。[结果] 与亲本株CVCC541粘附侵袭Hela细胞相比，基因缺失株CVCC541Δvgrg、CVCC541Δhcp2Δvgrg和CVCC541Δhcp1Δhcp2Δhcp3的粘附率分别为17.17%±2.1%、14.73%±2.5%和82%±3.7%；CVCC541Δvgrg、CVCC541Δhcp2Δvgrg和CVCC541Δhcp1Δhcp2Δhcp3的侵袭率分别为7.05%±1.05%、6.21%±1.35%和87%±3.25%；与亲本株CVCC541在小鼠巨噬细胞RAW 264.7中的存活相比，基因缺失株CVCC541Δvgrg、CVCC541Δhcp2Δvgrg和CVCC541Δhcp1Δhcp2Δhcp3的存活率分别为15.67%±2.9%、14.47%±1.87%和56.12%±3.48%。[结论] 鼠伤寒沙门氏菌Ⅵ型分泌系统VgrG和Hcp对该菌入侵细胞和抗吞噬方面具有重要作用，该研究为鼠伤寒沙门氏菌通过六型分泌系统与宿主细胞相互作用的机制研究奠定了基础。

Abstract:

The type VI secretion system (T6SS) was an important secretory system found in most Gram-negative bacteria. It mediates the interaction between bacteria and bacteria and between bacteria and host cells. Haemolysin co-regulatory protein (Hcp) and valine glycine repeat protein G (VgrG) were important components of the T6SS puncturing device. However, the role of Hcp and VgrG of the Salmonella typhimurium type VI secretion system in the process of invading host cells and anti-phagocytosis is not completely clear.[Objective] The aim of this study was to inoculate eukaryotic epithelial cells and macrophages in vitro using the hcp and vgrg gene deletion strains constructed by gene knockout technology, and to use their parental strains as a control group, to study the role of Hcp and VgrG genes in the process of adhesioning and invading epithelial cell and anti-phagocytosis.[Methods] We optimized the conditions in the operation of Red homologous recombination system, and established an operating system that rapidly knockout genes related to the type VI secretion system of Salmonella typhimurium, and successfully constructed CVCC541 single deletion strain, double deletion strain, and triple deletion strain, and Hela cell inoculation test and colony count test were used to evaluate the adhesion and invasion ability of different strains; the macrophage RAW 264.7 inoculation test was used to evaluate the anti-phagocytic ability of different strains.[Results] Compared with the CVCC541 strain, the adhesion rates of CVCC541Δvgrg, CVCC541Δhcp2Δvgrg, and CVCC541Δhcp1Δhcp2Δhc3 were 16.17%±2.1%, 14.73%±2.5% and 82%±3.7%, respectively; the invasive rates of CVCC541Δvgrg, CVCC541Δhcp2Δvgrg, and CVCC541Δhcp1Δhcp2Δhcp3 were 7.05%±1.05%, 6.21%±1.35% and 87%±3.25%, respectively. The survival rates of CVCC541Δvgrg, CVCC541Δhcp2Δvgrg and CVCC541Δhcp1Δhcp2Δhcp3 were 15.67%±2.9%, 14.47%±1.87% and 56.12%±3.48%, respectively.[Conclusion] Salmonella typhimurium type VI secretion system VgrG and Hcp played an important role in the process invasioning host cells and anti-phagocytosis. This work created the conditions for the research mechanism of interaction between Salmonella typhimurium and host cells through type VI secretion system secretory system.