Phosphodiesterases (PDEs), enzymes that degrade 3',5'-cyclic nucleotides, are being pursued as therapeutic targets for several diseases, including those affecting the nervous system, the cardiovascular system, fertility, immunity, cancer and metabolism. Clinical development programmes have focused exclusively on catalytic inhibition, which continues to be a strong focus of ongoing drug discovery efforts. However, emerging evidence supports novel strategies to therapeutically target PDE function, including enhancing catalytic activity, normalizing altered compartmentalization and modulating post-translational modifications, as well as the potential use of PDEs as disease biomarkers. Importantly, a more refined appreciation of the intramolecular mechanisms regulating PDE function and trafficking is emerging, making these pioneering drug discovery efforts tractable.

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Therapeutic targeting of 3',5'-cyclic nucleotide phosphodiesterases: inhibition and beyond.

A Bioinformatic Analysis of Integrative Mobile Genetic Elements Highlights Their Role in Bacterial Adaptation

Bacterial signal transduction systems are responsible for sensing environmental cues and adjusting the cellular behaviour and/or metabolism in response to these cues. They also monitor the intracellular conditions and the status of the cell envelope and the cytoplasmic membrane and trigger various stress responses to counteract adverse changes. This surveillance involves several classes of sensor proteins: histidine kinases; chemoreceptors; membrane components of the sugar phosphotransferase system; adenylate, diadenylate and diguanylate cyclases and certain cAMP, c-di-AMP and c-di-GMP phosphodiesterases; extracytoplasmic function sigma factors and Ser/Thr/Tyr protein kinases and phosphoprotein phosphatases. We have compiled a detailed listing of sensor proteins that are encoded in the genomes of Escherichia coli, Bacillus subtilis and 10 widespread pathogens: Chlamydia trachomatis, Haemophilus influenzae, Helicobacter pylori, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Porphyromonas gingivalis, Rickettsia typhi, Streptococcus pyogenes and Treponema pallidum, and checked what, if anything, is known about their functions. This listing shows significant gaps in the understanding of which environmental and intracellular cues are perceived by these bacteria and which cellular responses are triggered by the changes in the respective parameters. A better understanding of bacterial preferences may suggest new ways to modulate the expression of virulence factors and therefore decrease the reliance on antibiotics to fight infection.

What bacteria want

Microbial biofilms are the cause of persistent infections associated with various medical implants and distinct body sites such as the urinary tract, lungs, and wounds. Compared with their free living counterparts, bacteria in biofilms display a highly increased resistance to immune system activities and antibiotic treatment. Therefore, biofilm infections are difficult or impossible to treat with our current armory of antibiotics. The challenges associated with biofilm infections have urged researchers to pursue a better understanding of the molecular mechanisms that are involved in the formation and dispersal of biofilms, and this has led to the identification of several steps that could be targeted in order to eradicate these challenging infections. Here we describe mechanisms that are involved in the regulation of biofilm development in Pseudomonas aeruginosa, Escherichia coli, and Acinetobacter baumannii, and provide examples of chemical compounds that have been developed to specifically inhibit these processes. These compounds include (i) pilicides and curlicides which inhibit the initial steps of biofilm formation by E. coli; (ii) compounds that interfere with c-di-GMP signaling in P. aeruginosa and E. coli; and (iii) compounds that inhibit quorum-sensing in P. aeruginosa and A. baumannii. In cases where compound series have a defined molecular target, we focus on elucidating structure activity relationship (SAR) trends within the particular compound series.

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Small Molecule Anti-biofilm Agents Developed on the Basis of Mechanistic Understanding of Biofilm Formation.

High-specificity local and global c-di-GMP signaling.

The bacterial second messenger bis-(3'-5')-cyclic diguanosine monophosphate (c-di-GMP) ubiquitously promotes bacterial biofilm formation. Intracellular pools of c-di-GMP seem to be dynamically negotiated by diguanylate cyclases (DGCs, with GGDEF domains) and specific phosphodiesterases (PDEs, with EAL or HD-GYP domains). Most bacterial species possess multiple DGCs and PDEs, often with surprisingly distinct and specific output functions. One explanation for such specificity is "local" c-di-GMP signaling, which is believed to involve direct interactions between specific DGC/PDE pairs and c-di-GMP-binding effector/target systems. Here we present a systematic analysis of direct protein interactions among all 29 GGDEF/EAL domain proteins of Escherichia coli Since the effects of interactions depend on coexpression and stoichiometries, cellular levels of all GGDEF/EAL domain proteins were also quantified and found to vary dynamically along the growth cycle. Instead of detecting specific pairs of interacting DGCs and PDEs, we discovered a tightly interconnected protein network of a specific subset or "supermodule" of DGCs and PDEs with a coregulated core of five hyperconnected hub proteins. These include the DGC/PDE proteins representing the c-di-GMP switch that turns on biofilm matrix production in E. coli Mutants lacking these core hub proteins show drastic biofilm-related phenotypes but no changes in cellular c-di-GMP levels. Overall, our results provide the basis for a novel model of local c-di-GMP signaling in which a single strongly expressed master PDE, PdeH, dynamically eradicates global effects of several DGCs by strongly draining the global c-di-GMP pool and thereby restricting these DGCs to serving as local c-di-GMP sources that activate specific colocalized effector/target systems.IMPORTANCE c-di-GMP signaling in bacteria is believed to occur via changes in cellular c-di-GMP levels controlled by antagonistic and potentially interacting pairs of diguanylate cyclases (DGCs) and c-di-GMP phosphodiesterases (PDEs). Our systematic analysis of protein-protein interaction patterns of all 29 GGDEF/EAL domain proteins of E. coli, together with our measurements of cellular c-di-GMP levels, challenges both aspects of this current concept. Knocking out distinct DGCs and PDEs has drastic effects on E. coli biofilm formation without changing the cellular c-di-GMP level. In addition, rather than generally coming in interacting DGC/PDE pairs, a subset of DGCs and PDEs operates as central interaction hubs in a larger "supermodule," with other DGCs and PDEs behaving as "lonely players" without contacts to other c-di-GMP-related enzymes. On the basis of these data, we propose a novel concept of "local" c-di-GMP signaling in bacteria with multiple enzymes that make or break the second messenger c-di-GMP.

More than Enzymes That Make or Break Cyclic Di-GMP-Local Signaling in the Interactome of GGDEF/EAL Domain Proteins of Escherichia coli.

The nucleotide second messenger c-di-GMP nearly ubiquitously promotes bacterial biofilm formation, with enzymes that synthesize and degrade c-di-GMP being controlled by diverse N-terminal sensor domains. Here, we describe a novel class of widely occurring c-di-GMP phosphodiesterases (PDE) that feature a periplasmic "CSS domain" with two highly conserved cysteines that is flanked by two transmembrane regions (TM1 and TM2) and followed by a cytoplasmic EAL domain with PDE activity. Using PdeC, one of the five CSS domain PDEs of Escherichia coli K-12, we show that DsbA/DsbB-promoted disulfide bond formation in the CSS domain reduces PDE activity. By contrast, the free thiol form is enzymatically highly active, with the TM2 region promoting dimerization. Moreover, this form is processed by periplasmic proteases DegP and DegQ, yielding a highly active TM2 + EAL fragment that is slowly removed by further proteolysis. Similar redox control and proteolysis was also observed for a second CSS domain PDE, PdeB. At the physiological level, CSS domain PDEs modulate production and supracellular architecture of extracellular matrix polymers in the deeper layers of mature E. coli biofilms.

https://www.embopress.org/doi/pdf/10.15252/embj.201797825

Transmembrane redox control and proteolysis of PdeC, a novel type of c-di-GMP phosphodiesterase.

The ubiquitous second messenger c-di-GMP promotes bacterial biofilm formation by playing diverse roles in the underlying regulatory networks. This is reflected in the multiplicity of diguanylate cyclases (DGC) and phosphodiesterases (PDE) that synthesize and degrade c-di-GMP, respectively, in most bacterial species. One of the 12 DGCs of Escherichia coli, DgcE, serves as the top-level trigger for extracellular matrix production during macrocolony biofilm formation. Its multi-domain architecture–a N-terminal membrane-inserted MASE1 domain followed by three PAS, a GGDEF and a degenerate EAL domain–suggested complex signal integration and transmission through DgcE. Genetic dissection of DgcE revealed activating roles for the MASE1 domain and the dimerization-proficient PAS3 region, whereas the inhibitory EALdeg domain counteracts the formation of DgcE oligomers. The MASE1 domain is directly targeted by the GTPase RdcA (YjdA), a dimer or oligomer that together with its partner protein RdcB (YjcZ) activates DgcE, probably by aligning and promoting dimerization of the PAS3 and GGDEF domains. This activation and RdcA/DgcE interaction depend on GTP hydrolysis by RdcA, suggesting GTP as an inhibitor and the pronounced decrease of the cellular GTP pool during entry into stationary phase, which correlates with DgcE-dependent activation of matrix production, as a possible input signal sensed by RdcA. Furthermore, DgcE exhibits rapid, continuous and processive proteolytic turnover that also depends on the relatively disordered transmembrane MASE1 domain. Overall, our study reveals a novel GTP/c-di-GMP-connecting signaling pathway through the multi-domain DGC DgcE with a dual role for the previously uncharacterized MASE1 signaling domain.

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Genetic dissection of Escherichia coli's master diguanylate cyclase DgcE: Role of the N-terminal MASE1 domain and direct signal input from a GTPase partner system

Olga Sarenko

Papers

More than Enzymes That Make or Break Cyclic Di-GMP-Local Signaling in the Interactome of GGDEF/EAL Domain Proteins of Escherichia coli.

Transmembrane redox control and proteolysis of PdeC, a novel type of c-di-GMP phosphodiesterase.

Genetic dissection of Escherichia coli's master diguanylate cyclase DgcE: Role of the N-terminal MASE1 domain and direct signal input from a GTPase partner system