Sigma 因子对沙门氏菌生物被膜形成和毒力基因的调控作用

Biofilm formation of Salmonella plays an important role in resistance to stresses and its pathogenicity, and RpoS is a critical sigma factor for regulation of biofilm formation in Salmonella. However, some Salmonella strains can form biofilm by a RpoS-independent pathway. In order to identify new sigma factor and elucidate its regulation pathway for biofilm formation and virulence genes, the following experiments were performed. First, the representative RpoS-dependent and RpoS-independent Salmonella strains were selected by screening of Salmonella isolates. Second, sigma factor related to biofilm formation in a RpoS-independent strain was identified by insertion of transposon, determination of expression array, RNA sequencing and construction of mutants with deletion of sigma factor gene. Third, the regulation pathway of sigma factor in biofilm formation was investigated by determination of expression activity of reporter genes and RNA sequencing, and critical sequences of promoters were further determined by mutagenesis of promoters. Fourth, the pathogenesis of mutants with deletion of sigma factor gene was explored by comparison of wild-type strain and mutant strain in vitro and in vivo. Finally, virulence genes regulated by sigma factor were identified by comparison of genes expression in both wild-type strain and mutant strain. The identification of new sigma factors and their downstream genes related to both biofilm formation and virulence in Salmonella will provide useful target genes for medicine design and vaccine development.

沙门氏菌生物被膜在抵抗不利条件和致病过程中起着重要作用，虽然RpoS是调控沙门氏菌生物被膜形成的关键Sigma（σ）因子,但是一些沙门氏菌为RpoS非依赖型生物被膜形成株。为了鉴定新的σ因子，阐明它所依赖的生物被膜形成调控途径，本研究拟筛选出沙门氏菌RpoS依赖和非依赖型生物被膜形成代表株；通过转座子插入、表达芯片测定、RNA转录组测序和缺失株构建的方法确定RpoS非依赖型生物被膜形成株中参与生物被膜形成的σ因子；通过报告基因表达活性测定和RNA转录组测序，确定σ因子调控生物被膜形成的基因通路；通过启动子定点突变，鉴定σ因子识别的关键序列；比较野生株和σ因子缺失株在体外和体内的致病性差异；比较野生株和σ因子缺失株的基因表达差异，确定σ因子调控的毒力基因。通过鉴定出新的σ因子及其调控同时与生物被膜形成和毒力相关的基因，为药物设计和疫苗创制提供靶标。

沙门菌中的Sigma（σ）因子 RpoS可结合RNA聚合酶的核心酶，识别下游基因启动子，调控生物被膜形成，但是部分沙门菌生物被膜形成并不依赖RpoS。为了鉴定新的σ因子，阐明它所依赖的生物被膜形成调控途径及对毒力的影响，首先从鸡白痢沙门菌和鼠伤寒沙门菌筛选出生物被膜形成rpoS基因依赖株和非依赖株各一对，通过沙门菌生物被膜形成状态下σ因子表达的测定，确定rpoS基因非依赖株的生物被膜形成主要是通过rpoE基因来调控的。通过同源重组技术构建沙门菌rpoE基因缺失株，rpoE基因缺失后均不能形成生物被膜，不表达csgA基因，无卷曲菌毛条带，表明RpoE在rpoS基因非依赖株中主要通过影响CsgA表达调节生物被膜的形成。rpoE基因对不同血清型沙门菌的致病性影响不一致，在鸡白痢沙门菌中缺失rpoE基因可导致毒力上升，但在鼠伤寒沙门菌中缺失rpoE基因可导致毒力下降；进一步研究表明rpoE基因可通过调节III型分泌系统及其效应蛋白分泌来影响其毒力。其次，通过比较rpoS基因依赖株与非依赖株的rpoS基因序列发现不同菌株间存在L193P的突变，193P的RpoS结合RNA聚合酶和下游启动子序列的能力更强，表明rpoS基因的193P是影响鸡白痢沙门菌rpoS基因依赖株生物被膜形成的关键氨基酸。再次，利用RNA-seq分析鸡白痢沙门菌rpoS基因非依赖株生物被膜状态和浮游状态转录组信息，发现含有GGDEF结构域的2428蛋白和σ因子RpoN为负调控蛋白，基因缺失后可显著增加生物被膜的形成。最后，通过构建鸡白痢沙门菌和鼠伤寒沙门菌 csgA、bcsA基因缺失株，测定其生物被膜形成能力发现，这两个基因的缺失均可以减少生物被膜形成；同时csgA基因的缺失导致沙门菌缺失株对1日龄SPF鸡的毒力下降。综上所述，我们发现了在rpoS基因非依赖株中新的生物被膜调控基因rpoE，证实了rpoS基因的193P是影响鸡白痢沙门菌rpoS基因依赖株生物被膜形成的关键氨基酸，揭示了其影响生物被膜形成和毒力的机制。本研究为沙门菌生物被膜形成的调节机制提供了新观点，为药物设计和疫苗创制提供了新靶标。