Underlying principles of bistability in the expression of the pivotal virulence regulator RovA in Yersinia pseudotuberculosis and role for virulence

假结核耶尔森菌关键毒力调节因子 RovA 表达双稳定性的基本原理及其毒力作用

• 批准号: 218303910
• 负责人: Professorin Dr. Petra Dersch
• 金额: --
• 依托单位: Institut für Infektiologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/218303910?language=en
• 关键词: Underlying; principles; bistability; expression; pivotal

In Yersinia pseudotuberculosis, important virulence-related traits are under the control of the transcriptional regulator RovA, which uses a built-in thermosensor to control its activity. Thermal shifts between 25°C and 37°C, encountered upon host entry and exit, induce reversible conformational changes in the RovA dimer that attenuate DNA-binding capacity of RovA and renders the regulatory protein more susceptible to degradation by the Lon protease. The synthesis of RovA is strictly autoregulated by a positive and a negative feedback loop, and besides thermal control, it is regulated in response to growth phase and nutrient composition of the medium. Expression analysis of rovA-gfp fusions at different temperatures over multiple generations using time-lapse fluorescence microscopy and flow cytometry revealed that two distinct subpopulations (ON and OFF) of Y. pseudotuberculosis emerge in a certain temperature range in response to a bimodal behavior of RovA. We elucidated the molecular mechanisms underlying the temperature-tunable bistable switch and developed deterministic and stochastic models, which allow us to explain and predict the influence of temperature and alterations of regulatory elements controlling RovA synthesis on phenotypic switching. The analysis of mutant strains that are characterized by a modified bimodal behavior of RovA in a mouse infection model further indicated that phenotypic heterogeneity of RovA is advantageous for pathogenesis and fitness during infection. However, which biological functions are influenced by the bistable behavior of RovA and which of them support a better fitness and/or improve pathogenicity of the bacteria are still unknown and will be part of our future study. To address these questions we will: (i) analyze the spatio-temporal distribution of the distinct subpopulation (ON and OFF) in infected host tissues to gain insight where, when and to what extent rovA is heterogenously expressed during the infection(ii) unravel the biological functions which are differently expressed in the RovA ON and OFF subpopulation in vitro and during the infection, and address their significance for bacterial fitness and pathogenesis(iii) investigate the role of nutrients (metabolites) for the generation of RovA bistability during the infection and determine their importance for persistence and bacterial pathogenicity. Gained knowledge about the influence of metabolites on the bistable phenotype of rovA will be incooperated into the mathematical models and used for model-guided modulation of the system to validate and optimize the models.This will provide valuable information about the existence, properties and role of the RovA-expressing and non-expressing cells within bacterial communities during the infection, and about how bistability of the system promote adaptation to rapid changes, e.g. when they encounter a different environment inside or outside their hosts.

在假结核耶尔森菌中，重要的毒力相关性状受转录调节因子RovA的控制，它使用内置的热传感器来控制其活性。在宿主进入和退出时，在25°C和37°C之间的温度变化会引起RovA二聚体的可逆构象变化，从而减弱RovA的dna结合能力，并使调节蛋白更容易被Lon蛋白酶降解。RovA的合成受正负反馈回路的严格自动调节，除热控制外，还受培养基生长阶段和营养成分的调节。利用延时荧光显微镜和流式细胞术对RovA -gfp融合物在不同温度下多代的表达分析显示，在一定温度范围内，假结核杆菌出现了两个不同的亚群（ON和OFF），以响应RovA的双峰行为。我们阐明了温度可调双稳态开关的分子机制，并建立了确定性和随机模型，使我们能够解释和预测温度和控制RovA合成的调节元件的改变对表型开关的影响。在小鼠感染模型中对具有RovA修饰双峰行为的突变株的分析进一步表明，RovA的表型异质性有利于其发病和感染期间的适应度。然而，哪些生物学功能受到RovA双稳态行为的影响，哪些功能支持更好的适应度和/或提高细菌的致病性仍然未知，这将是我们未来研究的一部分。为了解决这些问题，我们将：(i)分析受感染宿主组织中不同亚群（ON和OFF）的时空分布，以了解在感染期间rovA在何处、何时以及在多大程度上异质表达；（ii）揭示在体外和感染期间rovA ON和OFF亚群中不同表达的生物学功能；（iii）研究营养物质（代谢物）在感染过程中对RovA双稳定性的产生所起的作用，并确定其对持久性和细菌致病性的重要性。所获得的关于代谢物对rovA双稳态表型影响的知识将被纳入数学模型，并用于模型导向的系统调制，以验证和优化模型。这将提供有价值的信息，了解感染期间细菌群落中表达和不表达rova的细胞的存在、性质和作用，以及系统的双稳态如何促进对快速变化的适应，例如当它们遇到宿主内外不同的环境时。