Regulation of the ppGpp-dependent virulence gene programmes of S. Typhimurium

肠沙门氏菌是一种在哺乳动物宿主细胞内入侵、生存和繁殖的致病细菌，可导致从胃肠炎到伤寒等一系列疾病。沙门氏菌的感染和持续过程需要毒力基因，这些毒力基因通常聚集在致病岛上。沙门氏菌致病岛1和2(SPI1和SPI2)是最大的，分别含有摄取细菌和在宿主细胞内生长所需的基因。参与宿主入侵和定植的毒力基因转录网络(包括SPI1和SPI2)被定义为沙门氏菌胞外(STEX)和胞内(STIN)毒力基因表达程序。沙门氏菌必须对它在感染期间遇到的新环境做出快速反应。这就需要迅速改变毒力基因的表达。引起基因表达快速变化的一种细胞分子被称为鸟苷四磷酸‘ppGpp’。两种酶，relA和Spot，在细胞内合成ppGpp，对饥饿等环境线索做出反应。PpGpp通过与RNA聚合酶(RNAP)结合来发挥作用，RNAP是负责基因表达的酶。PpGpp与RNAP的结合导致RNAP重新分配到允许细菌在不同环境中生存的基因。我们以前的研究表明，ppGpp是沙门氏菌在宿主细胞内感染和生存所必需的，因此它介导了导致STEX和STIN程序表达的环境信号。通过使用转录转录方法，我们证明了在沙门氏菌中，由肠道低氧环境触发的SPI1基因的表达需要ppGpp。我们还发现，由限制宿主细胞内的无机营养或酸度触发的SPI2表达也需要ppGpp。然而，SPI1和SPI2的表达调控是非常复杂的，人们对其了解还很少。彻底了解环境控制的毒力基因表达程序是了解沙门氏菌感染的关键。我们将使用一种不能产生ppGpp(携带relA和Spot基因的缺失)的沙门氏菌突变株来鉴定控制SPI1和SPI2毒力基因表达的细胞蛋白。我们将通过首先确定ppGpp是否将RNAP特异性地重新分配给毒力基因的启动子来做到这一点。这将为解释ppGpp如何控制毒力基因表达提供机械论的见解。然而，RNAP的活性也受许多其他间接或直接作用于RNAP的细胞调节因子的控制，包括调节RNAP特异性的辅助蛋白。这些调控因子在缺少ppGpp的情况下不起作用，因为没有足够的RNAP被重新分配给毒力基因启动子。与ppGpp一起控制RNAP活性的辅助蛋白之一被称为DksA。我们已经知道Dks A控制着沙门氏菌的毒力。我们将检验这样一种假设，即DksA与ppGpp共同作用，控制沙门氏菌中所有毒力基因的表达，或者控制属于SPI1或SPI2的基因的子集。我们将使用relA点缺失菌株来鉴定与SPI1和SPI2表达相关的其他调控蛋白。我们已经设计了一种使用该菌株的实验，并表明它可以用于识别SPI1表达的调节因子。我们将使用该分析，结合RelA斑点缺失菌株与野生型菌株的转录转录比较来寻找SPI1和SPI2的潜在调节蛋白。然后，我们将制造突变的沙门氏菌菌株，这些菌株已经被删除，用于这些潜在的调节者。这些潜在的SPI1和/或SPI2调节因子在沙门氏菌在巨噬细胞和人类上皮细胞中的入侵和存活中的作用将被确定。

Salmonella enterica is one of several pathogenic bacteria that invade, survive and proliferate within mammalian host cells causing diseases ranging from gastroenteritis to typhoid. The course of infection and persistence of Salmonella requires virulence genes that are often clustered together within pathogenicity islands. Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) are the largest, and contain genes required for the uptake of bacteria and growth within host cells, respectively. The virulence gene transcriptional (TR) networks involved in successful host invasion and colonisation (including SPI1 and SPI2 respectively) have been defined as the Salmonella extracellular (STEX) and the Salmonella intracellular (STIN) virulence gene expression programmes. Salmonella must respond quickly to the new environments it encounters during infection. This necessitates swift changes in the expression of virulence genes. One of the cellular molecules that causes rapid changes in gene expression is called guanosine tetraphosphate 'ppGpp'. Two enzymes, RelA and SpoT, synthesise ppGpp inside the cell in response to environmental cues such as starvation. ppGpp acts by binding to RNA polymerase (RNAP), the enzyme responsible for gene expression. The binding of ppGpp to RNAP causes the re-allocation of RNAP to genes which allow the bacteria to survive in different environments. Our previous research has shown that ppGpp is required for infection and survival of Salmonella within host cells and therefore mediates the environmental signals that result in expression of the STEX and STIN programmes. By using a transcriptomic approach we showed that expression of SPI1 genes in Salmonella, triggered by the low-oxygen environment of the gut, requires ppGpp. We also showed that SPI2 expression, triggered by limiting inorganic nutrients or acidity encountered by Salmonella inside host cells, also requires ppGpp. However, the control of expression of SPI1 and SPI2 is very complex and only partially understood. A thorough knowledge of the environmentally controlled virulence gene expression programmes is the key to understanding Salmonella infection. We will use a mutant strain of Salmonella unable to make ppGpp (carrying deletions of the relA & spoT genes), to identify cellular proteins that control the expression of SPI1 and SPI2 virulence genes. We will do this by firstly determining whether ppGpp specifically re-allocates RNAP to the promoters of virulence genes. This will provide mechanistic insight to explain how ppGpp controls virulence gene expression. However, RNAP activity is also controlled by many other cellular regulators that act on it indirectly or directly, including accessory proteins which modulate RNAP specificity. These regulators do not function in the absence of ppGpp since insufficient RNAP is re-allocated to virulence gene promoters. One of the accessory proteins that acts with ppGpp to control RNAP activity is called DksA. We already know that DksA controls Salmonella virulence. We will test the hypothesis that DksA acts with ppGpp to control either all of virulence gene expression in Salmonella or a subset of genes belonging to SPI1 or SPI2. We will use the relA spoT deletion strain to identify other regulatory proteins involved in SPI1 and SPI2 expression. We have designed an assay using this strain and shown that it can be used to identify regulators of SPI1 expression. We will use the assay, together with a transcriptomic comparisons of the relA spoT deletion strain to the wild-type strain to search for potential regulatory proteins of SPI1 and SPI2. We will then make mutant Salmonella strains that have been deleted for these potential regulators. The role of these potential regulators of SPI1 and/or SPI2 in the invasion and survival of Salmonella in macrophages and human epithelial cells will be determined.