ncRNA伴侣分子Hfq对单核细胞增生李斯特菌胞内寄生及致病力的调控作用及机制研究

Listeria monocytogenes (LM) is one of important zoonotic pathogens，which poses a serious threat to livestock production and hygiene and safety of animal food. As an intracellular bacterium, LM can achieve the purpose of long-term intracellular parasitism, proliferation and spread in various ways to regulate of expression of virulence genes. It has been proved that expression and regulation of virulence genes in LM are closely related to its intracellular survival and pathogenicity. Recent studies found that there were non-coding RNA (ncRNA) which play an important role except regulatory factor PrfA and σB in the LM. These ncRNA can regulate expression of virulence genes in LM, which were mediated by the chaperone Hfq. However, molecular mechanisms about Hfp regulation in LM virulence gene expression remain unclear to date. The project is to delete Hfp gene in LM virulent strain, constructing LM △Hfp gene mutant, comparing differences of intracellular survival phenotype,virulence gene expression and pathogenicity among △Hfp mutant, complementary strain and wild strain, analyzing the virulence genes and proteins regulate by the chaperone Hfq at transcriptional and translational levels, which is intended to reveal the mechanism of intracellular survival and pathogenicity regulated by chaperone Hfq at the cellular and molecular levels. The clarification of regulation mechanism of intracellular survival and pathogenicity in LM may provide scientific basis for development of attenuated LM live vaccine vector and screening of new targets for drug.

单核细胞增生李斯特菌（LM）是一种重要的人兽共患病原菌，严重威胁畜牧业生产和公共卫生安全。作为胞内寄生菌，LM依赖各种途径调控毒力基因的表达来实现胞内寄生、繁殖和细胞间的扩散。近期研究发现，除了蛋白类的毒力调控因子PrfA和σB之外，LM中还存在着具有调控作用的非编码RNA（ncRNA），这些ncRNA在伴侣分子Hfq 的介导下可对LM许多基因的表达产生调控作用。然而，目前有关伴侣分子Hfp对LM胞内寄生和致病力的调控作用及分子机制仍不清楚。本项目拟利用基于Red系统的基因缺失技术对LM 野毒株Hfp基因缺失突变，通过细菌同源重组技术构建LM △Hfp 突变株和互补株，开展△Hfp突变株、互补株和野毒株胞内寄生相关表型和致病性差异研究，分析Hfq调控的毒力基因和蛋白，旨在从分子水平揭示Hfq对LM胞内寄生和致病力的调控作用及机制，为减毒LM活疫苗载体的研发和药物新靶点的筛选提供科学依据。

单核细胞增生李斯特菌（Listeria monocytogenes, LM）是一种能引起人和动物李斯特菌病的人兽共患病原菌，也是李斯特菌属中致病力最强的细菌。LM既能在专业吞噬细胞也可在非专业吞噬细胞内繁殖，其胞内寄生和致病的分子机制一直是研究的热点。作为ncRNA伴侣分子，hfq可通过ncRNA对LM毒力基因的表达发挥调控作用，从而影响LM的胞内寄生和致病性。然而，有关hfq对LM胞内寄生及致病力的调控作用及机制尚不清楚。项目组以LM-SB5强毒株为研究对象，通过SOE-PCR基因缺失和同源重组技术构建了LM-△hfq基因缺失突变株和LM-△hfq+hfq互补株，并对突变株、互补株的胞内寄生相关表型和毒力基因的表达调控开展研究，分析了hfq对LM菌体蛋白表达的影响。通过人工感染小鼠试验进行LM-△hfq突变株、互补株和LM-SB5强毒株的LD50、脏器载菌量的测定及病理组织学分析，结果发现LM-△hfq 在小鼠上的LD50升高了6.067倍，毒力显著降低；肝、脾载菌量显著下降；对肝、脾、肾的病理损伤减弱，LM-△hfq较LM-SB5溶血能力下降了2倍，生物被膜生成能力显著降低。通过细菌粘附侵袭试验、细胞凋亡诱导试验、胞内存活试验证实，与LM-SB5强毒株相比，LM-△hfq 对细胞粘附侵袭能力显著下降，毒力显著降低，胞内存活能力也明显减弱。荧光定量RT-PCR检测结果表明，LM-△hfq 突变株在感染小鼠的肝脏和脾脏细胞内inlA、inlB和inlC毒力基因的转录水平显著上调，而prfA、SigmaB、VirB和VirS毒力调控相关基因的转录水平显著下调。蛋白双向电泳分析结果发现，与LM-SB5强毒株相比，LM-△hfq 突变株中有21个蛋白表达丰度上调，8个蛋白表达丰度下调。对部分差异表达蛋白的MALDI-TOF质谱鉴定和功能预测分析，发现这些蛋白分别归类为热休克相关蛋白、内化素蛋白和转录调控因子三类蛋白家族。研究结果表明，ncRNA伴侣分子hfq基因缺失后对部分热休克蛋白、内化素蛋白和转录调控因子蛋白的表达具有调控作用，提示hfq可能通过某些与其互作ncRNA介导的蛋白基因表达调控作用来维持LM的胞内寄生和致病性。本项目完成了任务书中的所有研究内容，达到了预期目标，为LM减毒活疫苗载体的研发和药物新靶点的筛选提供了科学依据。