Leveraging a transcription regulatory network to understand Salmonella invasion of host epithelial cells

摘要 非伤寒沙门氏菌菌株，包括鼠伤寒沙门氏菌(STM)，是一种新的致病因素 儿童中的侵袭性疾病和免疫功能受损。虽然疫苗开发工作仍在进行中， STM多药耐药菌株的出现表明有必要寻求替代策略来保护 高危人群不受感染。STM先侵袭肠粘膜，然后全身传播。 侵袭需要通过3型分泌系统将特定的效应蛋白注入宿主细胞 (T3SS)。侵袭相关T3SS及其分泌的效应蛋白的大部分结构成分 编码在称为沙门氏菌致病岛1(SPI-1)的基因组区域。SPI-1基因的调控 代表了病原菌如何响应环境信号以诱导表达的模型系统 毒力基因。SPI-1基因转录的主要调控因子是DNA结合转录因子HILD， 其本身在SPI-1中编码。其中5个由Hild激活的基因编码调节子：HilC、RTSA、InvF、SprB 和Hila，我们推测它们参与了SPI-1基因表达的时间调控。然而，几乎没有 已知不同的调控因子如何在感染过程中对靶基因的表达时间做出贡献。 我们全面绘制了Hild、HilC、RTSA、InvF、SprB和Hila的监管目标，定义了 入侵的“超级规则”。值得注意的是，我们发现的大多数&gt；100直接监管互动 都是新奇的。通过分析已发表的数据，我们在体外鉴定了12个侵袭超调节基因的成员 表达谱与已知入侵基因的表达谱相关联。我们把这些称为“入侵--共同调控” 基因“(ICGS)。另一个小组进行的一项大规模转座子突变研究表明，大多数或 所有ICGS都是有效感染多种动物模型所必需的。我们将剖析ICGS的功能 在感染的不同阶段，使用体外和体内感染模型，我们将确定表达 侵袭超调节基因在液体生长和感染过程中的激活和失活 在体外培养上皮细胞，从而确定调控因子和表达时机之间的关系。这项工作 这里提出的将是确定具有强烈联系的未表征基因的作用的第一步 在入侵过程中。我们还希望表明侵袭性超调节基因的表达时机是 由相关的转录因子决定，这将是我们的 了解调控网络如何促进细菌发病。

SUMMARY Non-typhoidal Salmonella enterica strains, including serovar Typhimurium (STm), are an emerging cause of invasive disease among children and the immunocompromised. While vaccine development efforts are ongoing, the emergence of multidrug resistant strains of STm affirms the need to seek alternative strategies to protect high-risk individuals from infection. STm invades the intestinal mucosa before disseminating systemically. Invasion requires injection of specific effector proteins into host cells through a Type Three Secretion System (T3SS). Most of the structural components of the invasion-associated T3SS and its secreted effector proteins are encoded in a genomic region known as Salmonella Pathogenicity Island 1 (SPI-1). Regulation of SPI-1 genes represents a model system for how pathogenic bacteria respond to environmental signals to induce expression of virulence genes. The master regulator of SPI-1 gene transcription is a DNA-binding transcription factor, HilD, which is itself encoded within SPI-1. Five of the HilD-activated genes encode regulators, HilC, RtsA, InvF, SprB and HilA, which we postulate are involved in temporal regulation of SPI-1 gene expression. However, very little is known about how the different regulators contribute to the timing of expression of target genes during infection. We comprehensively mapped the regulatory targets of HilD, HilC, RtsA, InvF, SprB and HilA, defining the invasion “super-regulon”. Remarkably, the large majority of the >100 direct regulatory interactions we identified were novel. By analyzing published data, we identified 12 members of the invasion super-regulon whose in vitro expression profiles correlate with those of known invasion genes. We refer to these as “Invasion-Co-Regulated Genes” (ICGs). A large-scale transposon mutagenesis study performed by another group suggests that most or all of the ICGs are required for efficient infection of multiple animal models. We will dissect the function of ICGs at different stages of infection using in vitro and in vivo infection models, and we will determine the expression profiles of invasion super-regulon genes upon activation and inactivation in liquid growth and during infection of epithelial cells in vitro, thereby defining the relationship between regulator and expression timing. The work proposed here will represent the first step in establishing the role of uncharacterized genes that have strong ties to the invasion process. We also expect to show that expression timing for invasion super-regulon genes is determined by the associated transcription factors, which would represent an important advance in our understanding of how regulatory networks contribute to bacterial pathogenesis.