Intracellular lipopolysaccharide from Gram-negative bacteria including Escherichia coli, Salmonella typhimurium, Shigella flexneri, and Burkholderia thailandensis activates mouse caspase-11, causing pyroptotic cell death, interleukin-1β processing, and lethal septic shock. How caspase-11 executes these downstream signalling events is largely unknown. Here we show that gasdermin D is essential for caspase-11-dependent pyroptosis and interleukin-1β maturation. A forward genetic screen with ethyl-N-nitrosourea-mutagenized mice links Gsdmd to the intracellular lipopolysaccharide response. Macrophages from Gsdmd(-/-) mice generated by gene targeting also exhibit defective pyroptosis and interleukin-1β secretion induced by cytoplasmic lipopolysaccharide or Gram-negative bacteria. In addition, Gsdmd(-/-) mice are protected from a lethal dose of lipopolysaccharide. Mechanistically, caspase-11 cleaves gasdermin D, and the resulting amino-terminal fragment promotes both pyroptosis and NLRP3-dependent activation of caspase-1 in a cell-intrinsic manner. Our data identify gasdermin D as a critical target of caspase-11 and a key mediator of the host response against Gram-negative bacteria.

Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling

SUMMARY Twenty-five years have passed since the discovery of cyclic dimeric (3′→5′) GMP (cyclic di-GMP or c-di-GMP). From the relative obscurity of an allosteric activator of a bacterial cellulose synthase, c-di-GMP has emerged as one of the most common and important bacterial second messengers. Cyclic di-GMP has been shown to regulate biofilm formation, motility, virulence, the cell cycle, differentiation, and other processes. Most c-di-GMP-dependent signaling pathways control the ability of bacteria to interact with abiotic surfaces or with other bacterial and eukaryotic cells. Cyclic di-GMP plays key roles in lifestyle changes of many bacteria, including transition from the motile to the sessile state, which aids in the establishment of multicellular biofilm communities, and from the virulent state in acute infections to the less virulent but more resilient state characteristic of chronic infectious diseases. From a practical standpoint, modulating c-di-GMP signaling pathways in bacteria could represent a new way of controlling formation and dispersal of biofilms in medical and industrial settings. Cyclic di-GMP participates in interkingdom signaling. It is recognized by mammalian immune systems as a uniquely bacterial molecule and therefore is considered a promising vaccine adjuvant. The purpose of this review is not to overview the whole body of data in the burgeoning field of c-di-GMP-dependent signaling. Instead, we provide a historic perspective on the development of the field, emphasize common trends, and illustrate them with the best available examples. We also identify unresolved questions and highlight new directions in c-di-GMP research that will give us a deeper understanding of this truly universal bacterial second messenger.

Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger

On the stage of bacterial signal transduction and regulation, bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) has long played the part of Sleeping Beauty. c-di-GMP was first described in 1987, but only recently was it recognized that the enzymes that 'make and break' it are not only ubiquitous in the bacterial world, but are found in many species in huge numbers. As a key player in the decision between the motile planktonic and sedentary biofilm-associated bacterial 'lifestyles', c-di-GMP binds to an unprecedented range of effector components and controls diverse targets, including transcription, the activities of enzymes and larger cellular structures. This Review focuses on emerging principles of c-di-GMP signalling using selected systems in different bacteria as examples.

Principles of c-di-GMP signalling in bacteria.

The innate immune system detects infection by using germline-encoded receptors that are specific for conserved microbial molecules. The recognition of microbial ligands leads to the production of cytokines, such as type I interferons (IFNs), that are essential for successful pathogen elimination. Cytosolic detection of pathogen-derived DNA is one major mechanism of inducing IFN production, and this process requires signalling through TANK binding kinase 1 (TBK1) and its downstream transcription factor, IFN-regulatory factor 3 (IRF3). In addition, a transmembrane protein called STING (stimulator of IFN genes; also known as MITA, ERIS, MPYS and TMEM173) functions as an essential signalling adaptor, linking the cytosolic detection of DNA to the TBK1-IRF3 signalling axis. Recently, unique nucleic acids called cyclic dinucleotides, which function as conserved signalling molecules in bacteria, have also been shown to induce a STING-dependent type I IFN response. However, a mammalian sensor of cyclic dinucleotides has not been identified. Here we report evidence that STING itself is an innate immune sensor of cyclic dinucleotides. We demonstrate that STING binds directly to radiolabelled cyclic diguanylate monophosphate (c-di-GMP), and we show that unlabelled cyclic dinucleotides, but not other nucleotides or nucleic acids, compete with c-di-GMP for binding to STING. Furthermore, we identify mutations in STING that selectively affect the response to cyclic dinucleotides without affecting the response to DNA. Thus, STING seems to function as a direct sensor of cyclic dinucleotides, in addition to its established role as a signalling adaptor in the IFN response to cytosolic DNA. Cyclic dinucleotides have shown promise as novel vaccine adjuvants and immunotherapeutics, and our results provide insight into the mechanism by which cyclic dinucleotides are sensed by the innate immune system.

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STING is a direct innate immune sensor of cyclic di-GMP

The lipid A moiety of lipopolysaccharide forms the outer monolayer of the outer membrane of most gram-negative bacteria. Escherichia coli lipid A is synthesized on the cytoplasmic surface of the inner membrane by a conserved pathway of nine constitutive enzymes. Following attachment of the core oligosaccharide, nascent core-lipid A is flipped to the outer surface of the inner membrane by the ABC transporter MsbA, where the O-antigen polymer is attached. Diverse covalent modifications of the lipid A moiety may occur during its transit from the outer surface of the inner membrane to the outer membrane. Lipid A modification enzymes are reporters for lipopolysaccharide trafficking within the bacterial envelope. Modification systems are variable and often regulated by environmental conditions. Although not required for growth, the modification enzymes modulate virulence of some gram-negative pathogens. Heterologous expression of lipid A modification enzymes may enable the development of new vaccines.

Lipid a modification systems in gram-negative bacteria

Cyclic diguanylate (c-di-GMP) is a bacterial second messenger of growing recognition involved in the regulation of a number of complex physiological processes. This review describes the biosynthesis and hydrolysis of c-di-GMP and several mechanisms of regulation of c-di-GMP metabolism. The contribution of c-di-GMP to regulating biofilm formation and motility, processes that affect pathogenesis of many bacteria, is described, as is c-di-GMP regulation of virulence gene expression. Finally, ways in which c-di-GMP may mediate these regulatory effects are proposed.

Roles of Cyclic Diguanylate in the Regulation of Bacterial Pathogenesis

https://www.jbc.org/content/280/39/33324.full.pdf

The EAL Domain Protein VieA Is a Cyclic Diguanylate Phosphodiesterase

The second messenger cyclic diguanylate (c-di-GMP) controls the transition between motile and sessile growth in eubacteria, but little is known about the proteins that sense its concentration. Bioinformatics analyses suggested that PilZ domains bind c-di-GMP and allosterically modulate effector pathways. We have determined a 1.9 A crystal structure of c-di-GMP bound to VCA0042/PlzD, a PilZ domain-containing protein from Vibrio cholerae. Either this protein or another specific PilZ domain-containing protein is required for V. cholerae to efficiently infect mice. VCA0042/PlzD comprises a C-terminal PilZ domain plus an N-terminal domain with a similar β-barrel fold. C-di-GMP contacts seven of the nine strongly conserved residues in the PilZ domain, including three in a seven-residue long N-terminal loop that undergoes a conformational switch as it wraps around c-di-GMP. This switch brings the PilZ domain into close apposition with the N-terminal domain, forming a new allosteric interaction surface that spans these domains and the c-di-GMP at their interface. The very small size of the N-terminal conformational switch is likely to explain the facile evolutionary diversification of the PilZ domain.

/pdf/the-structural-basis-of-cyclic-diguanylate-signal-3whxne44gt.pdf

Rita Tamayo

Papers

Roles of Cyclic Diguanylate in the Regulation of Bacterial Pathogenesis

The EAL Domain Protein VieA Is a Cyclic Diguanylate Phosphodiesterase

The structural basis of cyclic diguanylate signal transduction by PilZ domains

PilZ domain proteins bind cyclic diguanylate and regulate diverse processes in Vibrio cholerae.

Genes induced late in infection increase fitness of Vibrio cholerae after release into the environment