鼠伤寒沙门菌VI型分泌系统效应因子Rhs生物学特性分析

Biological characterization of Rhs effectors of type VI secretion system of Salmonella typhimurium

摘要:

【背景】细菌的VI型分泌系统作为杀死真核捕食者或原核竞争对手的“武器”，其杀伤作用是通过释放有毒物质即效应因子来实现。尽管已发现一些效应因子，但大多数效应因子的功能仍然未知。【目的】研究rhs基因编码的效应因子Rhs对鼠伤寒沙门菌生物学特性的影响。【方法】利用Red同源重组的方法构建鼠伤寒沙门菌rhs基因缺失株及相应的基因回补株。通过试验分析比较亲本株与缺失株、回补株在生化特性、生物被膜形成、耐药性、细菌间竞争、抗血清补体杀菌能力、组织载菌量及感染小鼠后炎症因子IL-18、IL-1β释放量上的差异。【结果】效应因子Rhs不影响鼠伤寒沙门菌的生化代谢、生物被膜形成、耐药性及抗血清补体杀菌能力。细菌种间竞争试验中，基因缺失株CVCC541Δrhs1、CVCC541Δrhs2和CVCC541Δrhs1-2的竞争指数(competition index，CI)值分别为0.85、0.77和0.87，毒力均被轻度致弱。体内组织载菌量试验中，CVCC541Δrhs1、CVCC541Δrhs2和CVCC541Δrhs1-2基因缺失株在小鼠肝脏和脾脏中的细菌数均较亲本株明显下降(P<0.05)；机体分泌IL-1β、IL-18的能力与鼠伤寒沙门菌效应因子Rhs具有相关性(P<0.05)。【结论】鼠伤寒沙门菌Rhs效应因子能在细菌间的竞争及细菌在宿主器官内的定殖上发挥作用，同时参与调控宿主体内炎性因子IL-1β和IL-18的分泌。

Abstract:

[Background] The type VI secretion system of bacteria acts as a weapon to kill eukaryotic predators or prokaryotic competitors by releasing toxins, known as effectors. Although some effectors have been identified, the functions of most effectors remain unknown. [Objective] To study the role of the effector Rhs encoded by rhs gene in Salmonella typhimurium. [Methods] The rhs-deleted mutants of Salmonella typhimuriumCVCC541 were constructed via Red recombination, including single gene deletion strains CVCC541Δrhs1 and CVCC541Δrhs2 and the double genes deletion strain CVCC541Δrhs1-2. Meanwhile, the corresponding gene complemented strains C-Δrhs1, C-Δrhs2 and C-Δrhs1-2 were constructed. The biochemical characteristics, biofilm formation, antibiotic resistance, competition between bacteria, bactericidal ability of complement in antiserum, dynamic distribution in vivo and the amounts of IL-18 and IL-1β released by host after infection were compared among the wild-type strain, gene-deleted strains and gene-complemented strains. [Results] Rhs did not affect the metabolism, biofilm formation, antibiotic resistance or complement bactericidal ability of Salmonella typhimurium. The competition index (CI) of CVCC541Δrhs1, CVCC541Δrhs2 and CVCC541Δrhs1-2 was 0.85, 0.77 and 0.87, respectively, which indicated slightly weakened virulence. Compared with the wild-type strain, CVCC541Δrhs1, CVCC541Δrhs2 and CVCC541Δrhs1-2 showed decreased number in the liver and spleen of mice (P<0.05). The host secretion of IL-1β and IL-18 was correlated with the Rhs of Salmonella typhimurium(P<0.05). [Conclusion] The Rhs effectors of Salmonella typhimuriumplay a role in the competition between bacteria, the colonization of bacteria in host organs, and the regulation of the secretion of inflammatory cytokines IL-1β and IL-18 in the host.