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

在其自然环境中，以及在宿主体内，细菌可以组织成生物膜，这与对环境应力（如干燥，辐射，波动的pH条件）的增强抵抗力有关。减少的生长和由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抗性增强。生物膜的形成，运动性和毒力的调节普遍存在的第二信使环二GMP（c-di-GMP）在许多细菌，包括伯克霍尔德氏菌，但其调控知之甚少，在这个属。在过去的二十年里，B。新洋葱虫病已经成为免疫功能低下个体如囊性纤维化（CF）患者的机会致病菌。B。新洋葱细菌定殖和生物膜形成通常导致CF患者的肺功能下降和死亡率增加。对大多数抗生素的耐药性和胞外多糖对宿主防御机制的干扰使伯克霍尔德菌感染的成功治疗复杂化。c-di-GMP由二鸟苷酸环化酶（具有保守GGDEF结构域的DGC）合成 并被磷酸二酯酶（具有保守的EAL-或HD-GYP结构域的PDE）降解。为了本申请的目的而进行的比较BLAST分析揭示了B. Cenocepacia H111基因组编码25个GGDEF/EAL/HD-GYP结构域蛋白，但迄今为止只有RpfR和CdpA两个蛋白得到了进一步的描述。在这项研究中，我将描述GGDEF/EAL/HD-GYP结构域含有蛋白质的生物膜形成和毒力的输出。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，分析基因敲除突变体将有助于将单个GGDEF/EAL/HD-GYP结构域蛋白与特定表型输出联系起来，并鉴定影响相同靶标的DGC-PDE对。此外，我将研究c-di-GMP在体内和体外的结合和周转，并测试DGCs，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，通过细胞内信号传导来控制生物膜形成和毒力，该项目将拓宽c-di-GMP信号传导模块及其对细菌生命周期的调节作用的知识。破译c-di-GMP代谢的性质及其在生物膜形成、毒力因子合成和致病性的调节级联中的影响，将为CF患者、动物和植物中伯克霍尔德菌感染的控制和治疗开辟新的视角。

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.