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

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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.