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

在假结核耶尔森菌中，重要的毒力相关特征受到转录调控因子RoVa的控制，该转录调控因子使用内置的温度传感器来控制其活性。当宿主进入和离开时，在25℃到37℃之间的温度漂移会引起RoVa二聚体的可逆构象变化，从而减弱RoVa与DNA的结合能力，并使调节蛋白更容易被Lon蛋白酶降解。RoVa的合成受到正负反馈回路的严格自动调节，除了温度控制外，它还受到生长阶段和培养基营养成分的调节。利用延时荧光显微镜和流式细胞仪对RoVa-GFP融合基因在不同温度下多代表达的分析表明，假结核杆菌在一定的温度范围内出现了两个不同的亚群(开和关)，以响应RoVa的双峰行为。我们阐明了温度可调双稳开关的分子机制，并建立了确定性和随机模型，这些模型允许我们解释和预测温度和控制RoVa合成的调控元件的变化对表型开关的影响。在小鼠感染模型中对以RoVa的双峰行为为特征的突变株的分析进一步表明，RoVa的表型异质性有利于致病和感染期间的适应。然而，RoVa的双稳态行为影响了哪些生物学功能，哪些功能支持更好的适应能力和/或提高细菌的致病性，仍是未知的，将是我们未来研究的一部分。为了解决这些问题，我们将：(I)分析感染宿主组织中不同亚群(On和Off)的时空分布，以了解RoVa在感染期间在哪里、何时和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，并探讨它们对细菌适合性和致病机理的意义；(Iii)研究营养物质(代谢物)在感染期间RoVa双稳定性产生中的作用，并确定它们对持久性和细菌致病性的重要性。关于代谢物对RoVa双稳态表型的影响的知识将被整合到数学模型中，并用于模型指导的系统调节，以验证和优化模型。这将提供关于细菌群落中RoVa表达和非表达细胞在感染过程中的存在、性质和作用的有价值的信息，以及系统的双稳态如何促进对快速变化的适应，例如当它们遇到不同的宿主内外环境时。

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.