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线索。的动态 控制细胞反应的调节网络不能在个体调节水平上理解 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而出现的 蛋白质、mRNA 和 DMA。我们的长期目标是开发计算和实验方法 剖析和分析监管网络。就人类健康而言，最重要的因素之一 原核调节网络是 III 型分泌系统 (TTSS) 的基础。 TTSS 的作用就像 将细菌效应蛋白注射到宿主细胞质中的分子注射器。 TTSS 对于毒力至关重要 对于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) 具有最明确的结构和监管，也是本提案的重点。 在初步实验中，我们观察到： 1. SPI-1的表达存在时间顺序 基因，2.结构和效应基因的独立控制，3.存在滞后 效应子的表达，以及4.基因表达的随机成分受到差异性控制。 基于这些实验，我们假设动力学是由两个遗传电路决定的 途径。第一个就像一个致力于 SPI-1 表达式的多信号积分器。第二个是一个 双稳态开关，在针结构完成后，效应器不可逆地激活。这个 该提案旨在结合实验、理论和工程来定量表征 这些电路。 目标 1：表征 SPI-1 调控途径中的两个遗传回路。第一个负责 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，在输入刺激移除后导致效应器表达持续存在。 目标 2：通过添加人工反馈循环来设计网络动态。确定拓扑结构 监管相互作用编码网络动态，人工反馈循环将用于遗传 扰乱网络。这将提供对复杂动态如何演变的深入了解。

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.