System Dynamics of the Salmonella Virulence Regulatory Network

描述（由申请人提供）：细胞依靠复杂的调节网络来感知和响应环境线索。调控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被理解，而是作为多种蛋白、mRNA和DMA之间复杂的生化相互作用网络的结果而出现的。我们的长期目标是发展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，将细菌效应蛋白注射到宿主细胞质中。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、衣原体和博德氏菌。其中，负责入侵哺乳动物细胞的沙门氏菌TTSS （SPI-1）具有最明确的结构和调控特征，是本研究的重点。在初步实验中，我们观察到：1。SPI-1基因的表达有一定的时间顺序。结构基因和效应基因独立控制；效应器的表达存在滞后性；基因表达的随机成分受到差异控制。基于这些实验，我们假设这种动态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。第二种是双稳态开关，在针结构完成后，效应器被不可逆地激活。本提案旨在结合实验、理论和工程来定量表征这些电路。目的1：表征SPI-1调控通路中的两个遗传回路。第一个是负责整合许多环境输入，并致力于表达TTSS。第二种形成双稳态开关，使效应表达在输入刺激被移除后持续存在。目标2：通过添加人工反馈回路来设计网络动态。为了确定调节相互作用的拓扑结构如何编码网络动力学，将使用人工反馈回路对网络进行遗传干扰。这将使我们深入了解复杂的动态是如何演变的。

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.