System Dynamics of the Salmonella Virulence Regulatory Network

描述(由申请人提供)：细胞依赖复杂的调控网络来感知和响应环境提示。调控细胞反应的调控网络的动态不能在单个调控蛋白的水平上理解，而是作为多个蛋白质、mRNA和DMA之间复杂的生化相互作用网络的结果。我们的长期目标是开发计算和实验方法来剖析和分析监管网络。在人类健康方面，最重要的原核调控网络之一是III型分泌系统(TTSS)。TTSS就像一个分子注射器，将细菌效应蛋白注入宿主细胞质。TTSS对许多革兰氏阴性病原体的毒力至关重要，包括沙门氏菌、假单胞菌、大肠杆菌、志贺氏菌、耶尔森氏菌、衣原体和波尔德氏菌。其中，负责入侵哺乳动物细胞的沙门氏菌TTSS(SPI-1)具有最典型的结构和调控，也是本提案的重点。在初步实验中，我们观察到：1.SPI-1基因的表达存在时间顺序；2.结构基因和效应基因的独立控制；3.效应基因的表达存在滞后性；4.基因表达的随机成分是差异控制的。在这些实验的基础上，我们假设这种动力学是由该途径中的两个遗传回路决定的。第一个像一个致力于SPI-1表达的多信号积分器。第二种是双稳态开关，在针结构完成后，效应器被不可逆转地激活。这项建议寻求使用实验、理论和工程相结合的方法来定量描述这些电路。目的1：研究SPI-1调控通路中的两个遗传回路。第一个责任是整合许多环境投入，并致力于TTSS的表达。第二个形成一个双稳态开关，使效应器的表达在输入刺激被移除后保持不变。目标2：通过添加人工反馈环来设计网络动力学。为了确定调控相互作用的拓扑结构如何编码网络动态，将使用人工反馈环路从基因上扰乱网络。这将提供对复杂动力学如何演变的洞察。

DESCRIPTION (provided by applicant): Cells rely on complex regulatory networks to sense and respond to environmental cues. The dynamics of the regulatory network governing cellular responses cannot be understood at the level of individual regulatory proteins, but rather emerges as a result of a complex web of biochemical interactions between multiple proteins, mRNA, and DMA. Our long-term objective to develop computational and experimental methods to dissect and analyze regulatory networks. With respect to human health, one of the most important prokaryotic regulatory networks underlies the type III secretion system (TTSS). The TTSS acts like a molecular syringe to inject bacterial effector proteins into the host cytosol. The TTSS is critical for virulence for many gram-negative pathogens, including Salmonella, Pseudomonas, E. coli, Shigella, Yersinia, Chlamydia, and Bordetella. Of these, the Salmonella TTSS responsible for invading mammalian cells (SPI-1) has the most well-characterized structure and regulation and is the focus of this proposal. In preliminary experiments, we have observed: 1. there is a temporal order in the expression of SPI-1 genes, 2. there is independent control of structural and effector genes, 3. there is hysteresis in the expression of effectors, and 4. the stochastic component of gene expression is differentially controlled. Based on these experiments, we hypothesize that the dynamics are dictated by two genetic circuits in the pathway. The first acts like a multi-signal integrator that commits to SPI-1 expression. The second is a bistable switch, where effectors are irreversibly activated after the needle structure is completed. This proposal seeks to use a combination of experiments, theory, and engineering to quantitatively characterize these circuits. Aim 1: Characterize two genetic circuits in the SPI-1 regulatory pathway. The first is responsible for integrating many environmental inputs and committing to the expression of the TTSS. The second forms a bistable switch that causes effector expression to persist after the input stimulus is removed. Aim 2: Engineer the network dynamics by adding artificial feedback loops. To determine how the topology of regulatory interactions encodes network dynamics, artificial feedback loops will be used to genetically perturb the network. This will provide insight into how complex dynamics evolve.