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

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.