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Key Points Cyclic di-GMP (c-di-GMP) is a global bacterial second messenger that controls a wide range of cellular processes that contribute to surface adaptation, biofilm formation, cell cycle progression and virulence. Cellular levels of c-di-GMP are controlled by enzyme classes that have antagonistic activities, diguanylate cyclases that synthesize c-di-GMP and phosphodiesterases that degrade c-di-GMP. c-di-GMP controls cellular processes at the transcriptional, translational and post-translational level, and through an increasing number of c-di-GMP-binding proteins and riboswitches. The binding of c-di-GMP to specific proteins can influence their activity, stability, interaction with other proteins or their subcellular localization. c-di-GMP and other bacterial cyclic dinucleotides (CDNs) stimulate the mammalian innate immune response by binding to the stimulator of interferon genes (STING) receptor, thereby converging with the cyclic GMP–AMP synthase (cGAS)–cyclic GMP–AMP (cGAMP) cytosolic DNA-surveillance pathway. Cyclic dinucleotides are highly versatile signalling molecules that control important biological processes in bacteria, including motility, virulence, biofilm formation and cell cycle progression. In this Review, Jenal and colleagues discuss the molecular principles of cyclic di-GMP (c-di-GMP) synthesis and degradation, and the cellular functions that are exerted by c-di-GMP-binding effectors and their diverse targets. Cyclic dinucleotides (CDNs) are highly versatile signalling molecules that control various important biological processes in bacteria. The best-studied example is cyclic di-GMP (c-di-GMP). Known since the late 1980s, it is now recognized as a near-ubiquitous second messenger that coordinates diverse aspects of bacterial growth and behaviour, including motility, virulence, biofilm formation and cell cycle progression. In this Review, we discuss important new insights that have been gained into the molecular principles of c-di-GMP synthesis and degradation, which are mediated by diguanylate cyclases and c-di-GMP-specific phosphodiesterases, respectively, and the cellular functions that are exerted by c-di-GMP-binding effectors and their diverse targets. Finally, we provide a short overview of the signalling versatility of other CDNs, including c-di-AMP and cGMP–AMP (cGAMP).

Stimulator of interferon genes (STING) is a receptor in human cells that senses foreign cyclic dinucleotides that are released during bacterial infection and in endogenous cyclic GMP–AMP signalling during viral infection and anti-tumour immunity 1 – 5 . STING shares no structural homology with other known signalling proteins 6 – 9 , which has limited attempts at functional analysis and prevented explanation of the origin of cyclic dinucleotide signalling in mammalian innate immunity. Here we reveal functional STING homologues encoded within prokaryotic defence islands, as well as a conserved mechanism of signal activation. Crystal structures of bacterial STING define a minimal homodimeric scaffold that selectively responds to cyclic di-GMP synthesized by a neighbouring cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzyme. Bacterial STING domains couple the recognition of cyclic dinucleotides with the formation of protein filaments to drive oligomerization of TIR effector domains and rapid NAD + cleavage. We reconstruct the evolutionary events that followed the acquisition of STING into metazoan innate immunity, and determine the structure of a full-length TIR–STING fusion from the Pacific oyster Crassostrea gigas . Comparative structural analysis demonstrates how metazoan-specific additions to the core STING scaffold enabled a switch from direct effector function to regulation of antiviral transcription. Together, our results explain the mechanism of STING-dependent signalling and reveal the conservation of a functional cGAS–STING pathway in prokaryotic defence against bacteriophages. Structures of prokaryotic homologues of STING permit the reconstruction of the evolutionary trajectory of its incorporation into metazoan innate immunity, and reveal a role for the conserved cGAS–STING pathway in prokaryotic defence against bacteriophages.

The formation of microbial biofilms enables single planktonic cells to assume a multicellular mode of growth. During dispersion, the final step of the biofilm life cycle, single cells egress from the biofilm to resume a planktonic lifestyle. As the planktonic state is considered to be more vulnerable to antimicrobial agents and immune responses, dispersion is being considered a promising avenue for biofilm control. In this Review, we discuss conditions that lead to dispersion and the mechanisms by which native and environmental cues contribute to dispersion. We also explore recent findings on the role of matrix degradation in the dispersion process, and the distinct phenotype of dispersed cells. Last, we discuss the translational and therapeutic potential of dispersing bacteria during infection.In this Review, Rumbaugh and Sauer discuss the environmental cues and microorganism-derived signals that lead to the biofilm dispersal response, recent findings of matrix-degrading enzymes required for cells to liberate themselves from the biofilm matrix, novel insight into the mechanisms and regulation of dispersal, and the implications of these insights for biofilm control efforts.

Cyclic dinucleotides (CDNs) are agonists of stimulator of IFN genes (STING) and have potential as vaccine adjuvants. However, cyclic di-GMP (cdGMP) injected s.c. shows minimal uptake into lymphatics/draining lymph nodes (dLNs) and instead is rapidly distributed to the bloodstream, leading to systemic inflammation. Here, we encapsulated cdGMP within PEGylated lipid nanoparticles (NP-cdGMP) to redirect this adjuvant to dLNs. Compared with unformulated CDNs, encapsulation blocked systemic dissemination and markedly enhanced dLN accumulation in murine models. Delivery of NP-cdGMP increased CD8+ T cell responses primed by peptide vaccines and enhanced therapeutic antitumor immunity. A combination of a poorly immunogenic liposomal HIV gp41 peptide antigen and NP-cdGMP robustly induced type I IFN in dLNs, induced a greater expansion of vaccine-specific CD4+ T cells, and greatly increased germinal center B cell differentiation in dLNs compared with a combination of liposomal HIV gp41 and soluble CDN. Further, NP-cdGMP promoted durable antibody titers that were substantially higher than those promoted by the well-studied TLR agonist monophosphoryl lipid A and comparable to a much larger dose of unformulated cdGMP, without the systemic toxicity of the latter. These results demonstrate that nanoparticulate delivery safely targets CDNs to the dLNs and enhances the efficacy of this adjuvant. Moreover, this approach can be broadly applied to other small-molecule immunomodulators of interest for vaccines and immunotherapy.

Inherited retinal degeneration (RD) is a devastating and currently untreatable neurodegenerative condition that leads to loss of photoreceptor cells and blindness. The vast genetic heterogeneity of RD, the lack of “druggable” targets, and the access-limiting blood–retinal barrier (BRB) present major hurdles toward effective therapy development. Here, we address these challenges (i) by targeting cGMP (cyclic guanosine- 3′,5′-monophosphate) signaling, a disease driver common to different types of RD, and (ii) by combining inhibitory cGMP analogs with a nanosized liposomal drug delivery system designed to facilitate transport across the BRB. Based on a screen of several cGMP analogs we identified an inhibitory cGMP analog that interferes with activation of photoreceptor cell death pathways. Moreover, we found liposomal encapsulation of the analog to achieve efficient drug targeting to the neuroretina. This pharmacological treatment markedly preserved in vivo retinal function and counteracted photoreceptor degeneration in three different in vivo RD models. Taken together, we show that a defined class of compounds for RD treatment in combination with an innovative drug delivery method may enable a single type of treatment to address genetically divergent RD-type diseases.

Using methodology developed herein, it is found that reactive persulfides and polysulfides are formed endogenously from both small molecule species and proteins in high amounts in mammalian cells and tissues. These reactive sulfur species were biosynthesized by two major sulfurtransferases: cystathionine β-synthase and cystathionine γ-lyase. Quantitation of these species indicates that high concentrations of glutathione persulfide (perhydropersulfide >100 μM) and other cysteine persulfide and polysulfide derivatives in peptides/proteins were endogenously produced and maintained in the plasma, cells, and tissues of mammals (rodent and human). It is expected that persulfides are especially nucleophilic and reducing. This view was found to be the case, because they quickly react with H ₂O ₂ and a recently described biologically generated electrophile 8-nitroguanosine 3′,5′-cyclic monophosphate. These results indicate that persulfides are potentially important signaling/effector species, and because H ₂S can be generated from persulfide degradation, much of the reported biological activity associated with H ₂S may actually be that of persulfides. That is, H ₂S may act primarily as a marker for the biologically active of persulfide species.

Abstract Pseudomonas aeruginosa is a versatile opportunistic pathogen capable of infecting a broad range of hosts, in addition to thriving in a broad range of environmental conditions outside of hosts. With this versatility comes the need to tightly regulate its genome to optimise its gene expression and behaviour to the prevailing conditions. Two-component systems (TCSs) comprising sensor kinases and response regulators play a major role in this regulation. This minireview discusses the growing number of TCSs that have been implicated in the virulence of P. aeruginosa, with a special focus on the emerging theme of multikinase networks, which are networks comprising multiple sensor kinases working together, sensing and integrating multiple signals to decide upon the best response. The networks covered in depth regulate processes such as the switch between acute and chronic virulence (GacS network), the Cup fimbriae (Roc network and Rcs/Pvr network), the aminoarabinose modification of lipopolysaccharide (a network involving the PhoQP and PmrBA TCSs), twitching motility and virulence (a network formed from the Chp chemosensory pathway and the FimS/AlgR TCS), and biofilm formation (Wsp chemosensory pathway). In addition, we highlight the important interfaces between these systems and secondary messenger signals such as cAMP and c-di-GMP. Two-component systems controlling the virulence of the opportunistic pathogen, Pseudomonas aeruginosa.

Although circumstantial evidence supports enhanced Toll-like receptor 7 (TLR7) signalling as a mechanism of human systemic autoimmune disease 1 – 7 , evidence of lupus-causing TLR7 gene variants is lacking. Here we describe human systemic lupus erythematosus caused by a TLR7 gain-of-function variant. TLR7 is a sensor of viral RNA 8 , 9 and binds to guanosine 10 – 12 . We identified a de novo, previously undescribed missense TLR7 Y264H variant in a child with severe lupus and additional variants in other patients with lupus. The TLR7 Y264H variant selectively increased sensing of guanosine and 2',3'-cGMP 10 – 12 , and was sufficient to cause lupus when introduced into mice. We show that enhanced TLR7 signalling drives aberrant survival of B cell receptor (BCR)-activated B cells, and in a cell-intrinsic manner, accumulation of CD11c + age-associated B cells and germinal centre B cells. Follicular and extrafollicular helper T cells were also increased but these phenotypes were cell-extrinsic. Deficiency of MyD88 (an adaptor protein downstream of TLR7) rescued autoimmunity, aberrant B cell survival, and all cellular and serological phenotypes. Despite prominent spontaneous germinal-centre formation in Tlr7 Y264H mice, autoimmunity was not ameliorated by germinal-centre deficiency, suggesting an extrafollicular origin of pathogenic B cells. We establish the importance of TLR7 and guanosine-containing self-ligands for human lupus pathogenesis, which paves the way for therapeutic TLR7 or MyD88 inhibition. The missense TLR7 Y264H gain-of-function genetic variation causes systemic lupus erythematosus in humans and mice.

Biofilms are communities of sessile microbes that are phenotypically distinct from their genetically identical, free-swimming counterparts. Biofilms initiate when bacteria attach to a solid surface. Attachment triggers intracellular signaling to change gene expression from the planktonic to the biofilm phenotype. For Pseudomonas aeruginosa, it has long been known that intracellular levels of the signal cyclic-di-GMP increase upon surface adhesion and that this is required to begin biofilm development. However, what cue is sensed to notify bacteria that they are attached to the surface has not been known. Here, we show that mechanical shear acts as a cue for surface adhesion and activates cyclic-di-GMP signaling. The magnitude of the shear force, and thereby the corresponding activation of cyclic-di-GMP signaling, can be adjusted both by varying the strength of the adhesion that binds bacteria to the surface and by varying the rate of fluid flow over surface-bound bacteria. We show that the envelope protein PilY1 and functional type IV pili are required mechanosensory elements. An analytic model that accounts for the feedback between mechanosensors, cyclic-di-GMP signaling, and production of adhesive polysaccharides describes our data well.

Nucleotide second messengers, such as cAMP and c-di-GMP, regulate many physiological processes in bacteria, including biofilm formation. There is evidence of cross-talk between pathways mediated by c-di-GMP and those mediated by the cAMP receptor protein (CRP), but the mechanisms are often unclear. Here, we show that cAMP-CRP modulates biofilm maintenance in Shewanella putrefaciens not only via its known effects on gene transcription, but also through direct interaction with a putative c-di-GMP effector on the inner membrane, BpfD. Binding of cAMP-CRP to BpfD enhances the known interaction of BpfD with protease BpfG, which prevents proteolytic processing and release of a cell surface-associated adhesin, BpfA, thus contributing to biofilm maintenance. Our results provide evidence of cross-talk between cAMP and c-di-GMP pathways through direct interaction of their effectors, and indicate that cAMP-CRP can play regulatory roles at the post-translational level. Nucleotide second messengers, such as cAMP and c-di-GMP, regulate many physiological processes in bacteria, including biofilm formation. Here, the authors provide evidence of cross-talk between cAMP and c-di-GMP pathways through direct interaction of their effectors, showing that the cAMP receptor protein (CRP) can play regulatory roles at the post-translational level.