System Dynamics of the Salmonella Virulence Regulatory Network

描述（由申请人提供）：细胞依赖于复杂的调节网络来感知和响应环境线索。控制细胞反应的调控网络的动力学不能在单个调控蛋白的水平上理解，而是作为多种蛋白质、mRNA和DMA之间的复杂的生化相互作用网络的结果而出现。我们的长期目标是开发计算和实验方法来解剖和分析调控网络。关于人类健康，最重要的原核调节网络之一是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菌的毒力至关重要，包括沙门氏菌、假单胞菌、大肠杆菌。大肠杆菌、志贺氏菌、耶尔森氏菌、衣原体和博德特氏菌。其中，负责入侵哺乳动物细胞的沙门氏菌TTSS（SPI-1）具有最充分表征的结构和调控，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.效应子的表达存在滞后; 4.基因表达的随机成分受到差异控制。基于这些实验，我们假设动力学是由通路中的两个遗传回路决定的。第一个类似于提交SPI-1表达式的多信号积分器。第二个是一个不可逆开关，在针结构完成后，效应器被不可逆地激活。该提案旨在使用实验，理论和工程的组合来定量表征这些电路。目的1：描述SPI-1调控通路中的两个遗传回路。第一个部门负责整合许多环境投入，并致力于表达TTSS。第二个形成了一个信号开关，使效应表达持续输入刺激后，被删除。目标2：通过添加人工反馈回路来设计网络动态。为了确定调控相互作用的拓扑结构如何编码网络动态，将使用人工反馈回路来遗传地扰动网络。这将提供对复杂动态如何演变的洞察。

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.