c-di-GMP-mediated signal transduction in the opportunistic pathogen Burkholderia cenocepacia

在它们的自然环境中，以及在宿主内部，细菌可以被组织成生物膜，这与增强对环境压力的抵抗力有关，如干燥、辐射、波动的pH条件。生长减少和由DNA、蛋白质和胞外多糖组成的细胞外基质的产生也导致对抗菌治疗的抵抗力增强。在包括伯克霍尔德氏菌在内的许多细菌中，生物膜的形成、运动和毒力都受到第二信使c-di-GMP(c-di-GMP)的调控，但对它的调控在该属中知之甚少。在过去的二十年里，囊性纤维症(CF)患者等免疫功能受损的患者已成为一种机会性病原体。鞭毛虫的定植和生物被膜的形成往往会导致肺功能的下降和病死率的增加。对大多数抗生素的抗药性以及胞外多糖对宿主防御机制的干扰，使伯克霍尔德氏菌感染的成功治疗复杂化。C-di-GMP由二鸟苷环化酶(DGC，具有保守的GGDEF结构域)合成，并被磷酸二酯酶(PDE，具有保守的EAL-或HD-Gyp结构域)降解。为本申请的目的而进行的比较BLAST分析表明，双毛虫H111基因组编码含有25个GGDEF/EAL/HD-GYP结构域的蛋白质，但到目前为止只有两个蛋白质被进一步描述，RpfR和CDpA。在这项研究中，我将表征GGDEF/EAL/HD-Gyp结构域包含的蛋白质，涉及它们在生物被膜形成和毒力形成中的产出。由于c-di-GMP代谢在转录、翻译和翻译后水平上影响多个细胞过程，分析基因敲除突变将有助于将单个GGDEF/EAL/HD-Gyp结构域蛋白与特定的表型输出联系起来，并识别影响同一靶点的DGC-PDE对。此外，我将研究c-di-GMP在体内和体外的结合和转换，并测试DGC、PDE和效应分子之间的蛋白质-蛋白质相互作用，以了解单个c-di-GMP模块的调控过程。C-di-GMP调节酶的多样性为我们提供了许多靶点，通过细胞内信号转导来控制生物膜的形成和毒力，本项目将拓宽对c-di-GMP信号转导模块及其对细菌生命周期的调控作用的知识。破译c-di-GMP代谢的本质及其在生物被膜形成、毒力因子合成和致病性的调节级联反应中的影响，将为控制和治疗CF患者、动物和植物中的伯克霍尔德氏菌感染开辟新的视角。

In their natural environment, as well as inside the host, bacteria can be organized in biofilms, which are linked with enhanced resistance towards environmental stresses such as desiccation, radiation, fluctuating pH conditions. Reduced growth and the production of an extracellular matrix consisting of DNA, proteins and exopolysaccharides also leads to enhanced resistance towards antibacterial treatment. Biofilm formation, motility and virulence are regulated by the ubiquitous second messenger cyclic-di-GMP (c-di-GMP) in numerous bacteria including Burkholderia, but its regulation is poorly understood in this genus. Over the last two decades, B. cenocepacia has emerged as an opportunistic pathogen for immunocompromised individuals such as patients with cystic fibrosis (CF). B. cenocepacia colonization and biofilm formation often leads to a decline in lung function and increased mortality of CF patients. Resistance to most antibiotics and interfering of exopolysaccharides with host defence mechanisms complicate successful treatment of Burkholderia infections. c-di-GMP is synthesized by diguanylate cyclases (DGCs with conserved GGDEF-domains) and degraded by phosphodiesterases (PDEs with conserved EAL- or HD-GYP domain). Comparative BLAST analysis performed for the purpose of the present application revealed that the B. cenocepacia H111 genome encodes for 25 GGDEF/EAL/HD-GYP domain containing proteins, but hitherto only two of them, RpfR and CdpA, were described further. In this study, I will characterize GGDEF/EAL/HD-GYP domain containing proteins concerning their output in biofilm formation and virulence. As c-di-GMP metabolism affects multiple cellular processes on the transcriptional, translational and post-translational level, analysing gene knockout mutants will help to link single GGDEF/EAL/HD-GYP domain proteins to specific phenotypic outputs and to identify DGC-PDE-pairs affecting the same target. Furthermore, I will study binding and turnover of c-di-GMP in vivo and in vitro and test for protein-protein-interactions between DGCs, PDEs and effector molecules to understand regulatory processes in single c-di-GMP modules. The multiplicity of c-di-GMP regulating enzymes provides us with numerous targets to control biofilm formation and virulence via intracellular signalling and this project will broaden the knowledge of c-di-GMP signalling modules and their regulatory effect on the bacterial life cycle. Deciphering the nature of c-di-GMP metabolism and its influence in regulatory cascades of biofilm formation, synthesis of virulence factors and pathogenicity will open new perspectives on the control and treatment of Burkholderia infections in CF patients, animals and plants.