System Dynamics of the Salmonella Virulence Regulatory Network

细胞依靠复杂的调控网络来感知和响应环境提示。这场运动的动向 调控细胞反应的调控网络不能在个体调控的层面上被理解 蛋白质，而是作为多种生物化学相互作用的复杂网络的结果 蛋白质、信使核糖核酸和DNA。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。关于人类健康，最重要的是 原核调控网络是III型分泌系统(TTSS)的基础。TTSS的作用就像一个 分子注射器，将细菌效应蛋白注入宿主胞浆。TTSS对毒力是至关重要的 对许多革兰氏阴性病原体，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，沙门氏菌TTSS负责入侵哺乳动物细胞(SPI-1) 拥有最具特色的结构和监管，是本提案的重点。 在初步实验中，我们观察到：1.spi-1的表达具有时序性。 基因，2，结构基因和效应基因独立控制，3，存在滞后效应。 效应器的表达；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，我们假设动力学是由两个遗传回路决定的 路径。第一个像一个致力于SPI-1表达的多信号积分器。第二个是一个 双稳态开关，其中效应器在针结构完成后被不可逆转地激活。这 该提案寻求使用实验、理论和工程的组合来定量描述 这些电路。 目的1：研究SPI-1调控通路中的两个遗传回路。第一个是负责 整合多个环境投入，致力于TTSS的表达。第二种形式构成一个 双稳态开关，使效应器的表达在输入刺激移除后保持不变。 目标2：通过添加人工反馈环来设计网络动力学。要确定拓扑结构如何 监管互动编码网络动态，人工反馈环路将用于遗传 扰乱网络。这将提供对复杂动力学如何演变的洞察。

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.