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

总结 非伤寒性肠道沙门氏菌菌株，包括鼠伤寒血清型（STm），是引起肠伤寒的新原因。 儿童的侵袭性疾病和免疫功能低下。虽然疫苗开发工作正在进行中， STm多药耐药菌株的出现证实了寻求替代策略以保护 高危人群感染。STm在全身传播之前侵入肠粘膜。 入侵需要通过第三型分泌系统将特定的效应蛋白注入宿主细胞 （T3SS）。侵袭相关的T3 SS及其分泌的效应蛋白的大部分结构组分 在称为沙门氏菌致病岛1（SPI-1）的基因组区域中编码。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基因转录的主要调节因子是DNA结合转录因子HilD， 其本身编码在SPI-1中。HilD激活的五个基因编码调节因子：HilC、RtsA、InvF、SprB 和HilA，我们推测它们参与SPI-1基因表达的时间调节。然而， 关于不同的调节因子如何在感染过程中对靶基因表达的时机做出贡献， 我们全面绘制了HilD、HilC、RtsA、InvF、SprB和HilA的调控靶点， 入侵“超级调节子”。值得注意的是，在我们确定的100多个直接监管相互作用中， 是新奇的。通过分析已发表的数据，我们鉴定了12个侵袭超调节子成员，其在体外 表达谱与已知的侵袭基因的表达谱相关。我们称之为“入侵共同管制 基因”（ICG）。另一个小组进行的大规模转座子诱变研究表明，大多数或 所有ICG都是有效感染多种动物模型所必需的。我们将剖析ICG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 在液体生长中和感染期间激活和失活的入侵超调节子基因的概况 上皮细胞在体外，从而确定调节和表达时机之间的关系。工作 这里提出的将代表建立具有强联系的未表征基因的作用的第一步， 入侵的过程。我们还希望表明入侵超调节子基因的表达时机是 这是由相关的转录因子决定的，这将代表我们在研究中的一个重要进展。 了解调控网络如何促进细菌发病机制。

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.