Phenotypic heterogeneity generated by histidine kinases based signaling networks

越来越多的研究表明，表型异质性是细菌种群的一个共同特征。本项目侧重于基于组氨酸激酶的信号网络的比较分析，以揭示产生表型异质性的设计原则。在第一个资助期，研究了哈维氏弧菌中基于多输入组氨酸激酶的群体感应级联与表型异质性的相关性。我们的结果清楚地表明，这种由三个平行的双功能组氨酸激酶和一个sRNA调节的开关组成的信号级联具有导致表型异质性的固有特性。具体地说，群体感应组氨酸激酶活性与磷酸酶活性的比值(受三种自身诱导物的不同影响)和主调控子LuxR的拷贝数对表型异质性的程度至关重要。最后，将哈维氏弧菌暴露在人工高浓度的所有自体诱导剂中，使其转变为同质种群，产生的生物膜比异质种群少。在第二个资助期，我们将重点研究在大肠杆菌中的多输入组氨酸激酶营养传感网络。这个网络由两个组氨酸激酶和两个转运蛋白组成。它感知几种细胞内外代谢物的水平，通过蛋白质-蛋白质相互作用传递这些信息，并诱导表型异质性。这种营养感知网络也存在于肠杆菌科的其他成员中，包括许多病原体，如克雷伯氏菌、克隆氏菌、柠檬酸杆菌。所有可用的数据都强烈表明，大肠杆菌种群的个体通过这个网络感知可用的碳源，并制定更好的对冲策略，以适应并在营养过渡期间生存。这些更好的对冲策略可能会导致不同的细胞命运，如生长和分裂细胞、生物膜形成细胞或宿存细胞。我们将通过定量分析和数学建模来比较群体感应和营养感应网络与表型异质性的相关性，重点关注稳健性、信号转导的分子机制和sRNA开关的作用。最后，我们将分析营养可获得性和新陈代谢对大肠杆菌个体细胞命运的影响。此外，我们还将探讨营养感知和表型异质性在致尿路感染大肠杆菌寄主定植过程中的生物学意义。我们的目标是通过特定的饮食来控制这些大肠杆菌种群，使其变得均匀，因此更容易获得免疫反应或抗生素治疗。

More and more studies reveal that phenotypic heterogeneity is a common feature in bacterial populations. This project focuses on the comparative analysis of histidine kinases based signaling networks to unravel design principles that generate phenotypic heterogeneity. The multi-input histidine kinases based quorum sensing cascade in Vibrio harveyi was studied in correlation with phenotypic heterogeneity in the first funding period. Our results clearly indicate that this signaling cascade with three parallel bifunctional histidine kinases and a sRNA-regulated switch has the inherent property to cause phenotypic heterogeneity. Specifically, the ratio between kinase to phosphatase activities of the quorum sensing histidine kinases, which is differentially influenced by the three autoinducers, and the copy number of the master regulator LuxR were found to be crucial for the degree of phenotypic heterogeneity. Finally, exposure of V. harveyi to an artificial high concentration of all autoinducers caused the switch into a homogeneous population, which produced less biofilm than a heterogeneous one. We will focus on the multi-input histidine kinases nutrient sensing network in Escherichia coli in the second funding period. This network is composed of two histidine kinases and two transporters. It senses the level of several intra- and extracellular metabolites, transduces this information by protein-protein interactions and induces phenotypic heterogeneity. This nutrient sensing network is also found in other members of the Enterobacteriaceae including many pathogens, e.g., Klebsiella, Cronobacter, Citrobacter. All available data strongly suggest that the individuals of the E. coli population sense the available carbon sources through this network and develop bet-hedging strategies to adapt and survive during nutrient transitions. These bet-hedging strategies could result in different cellular fates, such as growing and dividing cells, biofilm-forming cells, or persister cells. We will compare the quorum sensing and the nutrient sensing networks in correlation with phenotypic heterogeneity by quantitative analysis and mathematical modeling with a specific focus on robustness, the molecular mechanism of signal transduction and the role of sRNA switches. Finally, we will analyze the impact of nutrient availability and metabolism on individual cellular fates of E. coli. In addition, we will investigate the biological significance of nutrient sensing and phenotypic heterogeneity during host colonization of uropathogenic E. coli. We aim to manipulate these E. coli populations by specific diets to become homogeneously and therefore, easier accessible to the immune response or antibiotic treatments.