Final Report Summary - SECMESSBIOFILM (Cyclic-di-GMP: New Concepts in Second Messenger Signaling and Bacterial Biofilm Formation)
Biofilms are large multicellular aggregates of bacteria that causes many medical (e.g. in chronic diseases) and technical problems. Biofilms form intricate three-dimensional structures, in which bacteria are embedded in an extracellular matrix of adhesins, amyloid-forming proteins (e.g. curli fibres) and exopolysaccharides (e.g. cellulose) that generates resistance against antibiotics and host immune defense. Using macrocolony biofilms of Escherichia coli – a commensal inhabitant of the human intestine that also occurs in important pathogenic variants – as a model system, the overall objective of the project was to clarify the genetic control network and its integration with environmentally responsive signaling pathways that determine physiological differentiation in a biofilm and its three-dimensional architecture and function.
Biofilm formation is promoted by the ubiquitous intracellular signaling molecule cyclic diguanylate (c-di-GMP). In most bacterial species c-di-GMP is produced by multiple diguanylate cyclases (DGCs, with GGDEF domains) and degraded by multiple phosphodiesterases (PDEs, with EAL or HD-GYP domains). The project therefore aimed at elucidating the regulation, interactions and molecular functions of the GGDEF/EAL domain proteins of E. coli (29 in E. coli K-12, six additional ones in distinct pathogenic E. coli), to identify and characterize major c-di-GMP-binding effectors and their targets, and to clarify the roles of all these components during the molecular events that generate a biofilm.
Using a combination of genetic, genomic, biochemical and cell/tissue biology approaches, the project demonstrated and visualized the existence of a highly complex 3D matrix architecture of biofilms controlled by c-di-GMP signaling – thereby also introducing the spatial dimension into a complex regulatory network. At the molecular level, it provided the first mechanistic paradigms for 'locally' acting c-di-GMP-driven switch modules. These operate by combining the enzymatic activities of DGCs and PDEs with highly specific direct protein-protein interactions that can have regulatory or scaffolding functions. One of these paradigms is the c-di-GMP-specific PDE PdeR, a 'trigger enzyme', whose direct inhibition of partner proteins – which activate the expression of the biofilm regulator CsgD – is modulated by its binding and degradation of its substrate (c-di-GMP), making PdeR also the first active PDE that serves as a c-di-GMP effector. Additional 'peripheral' DGCs and PDEs can conditionally either dock onto and thereby directly modulate this central switch module or contribute by modulating just the cellular c-di-GMP level that is then sensed by PdeR. The second paradigm for 'local signaling' is provided by DgcC and PdeK, which directly interact with cellulose synthase and thereby control the local c-di-GMP level in close proximity to this c-di-GMP-activated enzyme complex. Furthermore, it was demonstrated that (i) the DGC DgcM also acts as a direct co-activator in a novel mechanism of transcriptional activation, (ii) CSS domain-containing PDEs show a completely novel redox sensory input mechanism into c-di-GMP signaling, (iii) c-di-GMP signaling is tightly interlinked with regulation by small RNAs and (iv) c-di-GMP signaling and biofilm-related features are connected to virulence of certain pathogenic E. coli (in particular, the 2011 O104:H4 outbreak strain).
In conclusion, the project revealed how multi-modular global and local c-di-GMP signaling – interlinked with global stress responses and RNA-based regulatory circuits – controls the elaborate microarchitecture and macroscopic morphogenesis as well as the physiological functions of a bacterial biofilm. It also provided a fundamentally novel view of biofilm formation by showing that biofilm formation is not the result of a developmental process – leading to a physiological 'biofilm state' – as was previously widely believed. Finally, this new knowledge on biofilm biogenesis and the regulation and mode of action of the ubiquitously biofilm-promoting c-di-GMP opens new and general perspectives for developing anti-biofilm drugs.
Biofilm formation is promoted by the ubiquitous intracellular signaling molecule cyclic diguanylate (c-di-GMP). In most bacterial species c-di-GMP is produced by multiple diguanylate cyclases (DGCs, with GGDEF domains) and degraded by multiple phosphodiesterases (PDEs, with EAL or HD-GYP domains). The project therefore aimed at elucidating the regulation, interactions and molecular functions of the GGDEF/EAL domain proteins of E. coli (29 in E. coli K-12, six additional ones in distinct pathogenic E. coli), to identify and characterize major c-di-GMP-binding effectors and their targets, and to clarify the roles of all these components during the molecular events that generate a biofilm.
Using a combination of genetic, genomic, biochemical and cell/tissue biology approaches, the project demonstrated and visualized the existence of a highly complex 3D matrix architecture of biofilms controlled by c-di-GMP signaling – thereby also introducing the spatial dimension into a complex regulatory network. At the molecular level, it provided the first mechanistic paradigms for 'locally' acting c-di-GMP-driven switch modules. These operate by combining the enzymatic activities of DGCs and PDEs with highly specific direct protein-protein interactions that can have regulatory or scaffolding functions. One of these paradigms is the c-di-GMP-specific PDE PdeR, a 'trigger enzyme', whose direct inhibition of partner proteins – which activate the expression of the biofilm regulator CsgD – is modulated by its binding and degradation of its substrate (c-di-GMP), making PdeR also the first active PDE that serves as a c-di-GMP effector. Additional 'peripheral' DGCs and PDEs can conditionally either dock onto and thereby directly modulate this central switch module or contribute by modulating just the cellular c-di-GMP level that is then sensed by PdeR. The second paradigm for 'local signaling' is provided by DgcC and PdeK, which directly interact with cellulose synthase and thereby control the local c-di-GMP level in close proximity to this c-di-GMP-activated enzyme complex. Furthermore, it was demonstrated that (i) the DGC DgcM also acts as a direct co-activator in a novel mechanism of transcriptional activation, (ii) CSS domain-containing PDEs show a completely novel redox sensory input mechanism into c-di-GMP signaling, (iii) c-di-GMP signaling is tightly interlinked with regulation by small RNAs and (iv) c-di-GMP signaling and biofilm-related features are connected to virulence of certain pathogenic E. coli (in particular, the 2011 O104:H4 outbreak strain).
In conclusion, the project revealed how multi-modular global and local c-di-GMP signaling – interlinked with global stress responses and RNA-based regulatory circuits – controls the elaborate microarchitecture and macroscopic morphogenesis as well as the physiological functions of a bacterial biofilm. It also provided a fundamentally novel view of biofilm formation by showing that biofilm formation is not the result of a developmental process – leading to a physiological 'biofilm state' – as was previously widely believed. Finally, this new knowledge on biofilm biogenesis and the regulation and mode of action of the ubiquitously biofilm-promoting c-di-GMP opens new and general perspectives for developing anti-biofilm drugs.