Conservation and similarity of bacterial and eukaryotic innate immunity.

Pathogens are ubiquitous and a constant threat to their hosts, which has led to the evolution of sophisticated immune systems in bacteria, archaea and eukaryotes. Bacterial immune systems encode an astoundingly large array of antiviral (antiphage) systems, and recent investigations have identified unexpected similarities between the immune systems of bacteria and animals. In this Review, we discuss advances in our understanding of the bacterial innate immune system and highlight the components, strategies and pathogen restriction mechanisms conserved between bacteria and eukaryotes. We summarize evidence for the hypothesis that components of the human immune system originated in bacteria, where they first evolved to defend against phages. Further, we discuss shared mechanisms that pathogens use to overcome host immune pathways and unexpected similarities between bacterial immune systems and interbacterial antagonism. Understanding the shared evolutionary path of immune components across domains of life and the successful strategies that organisms have arrived at to restrict their pathogens will enable future development of therapeutics that activate the human immune system for the precise treatment of disease.

Bacterial antiviral defense pathways encode eukaryotic-like ubiquitination systems.

Ubiquitination and related pathways play crucial roles in protein homeostasis, signaling, and innate immunity1-3. In these pathways, an enzymatic cascade of E1, E2, and E3 proteins conjugates ubiquitin or a ubiquitin-like protein (Ubl) to target-protein lysine residues4. Bacteria encode ancient relatives of E1 and Ubl proteins involved in sulfur metabolism5,6 but these proteins do not mediate Ubl-target conjugation, leaving open the question of whether bacteria can perform ubiquitination-like protein conjugation. Here, we demonstrate that a bacterial antiviral immune system encodes a complete ubiquitination pathway. Two structures of a bacterial E1:E2:Ubl complex reveal striking architectural parallels with canonical eukaryotic ubiquitination machinery. The bacterial E1 encodes an N-terminal inactive adenylation domain (IAD) and a C-terminal active adenylation domain (AAD) with a mobile α-helical insertion containing the catalytic cysteine (CYS domain). One structure reveals a pre-reaction state with the bacterial Ubl C-terminus positioned for adenylation, and the E1 CYS domain poised nearby for thioester formation. A second structure mimics an E1-to-E2 transthioesterification state, with the E1 CYS domain rotated outward and its catalytic cysteine adjacent to the bound E2. We show that a deubiquitinase (DUB) in the same pathway pre-processes the bacterial Ubl, exposing its C-terminal glycine for adenylation. Finally, we show that the bacterial E1 and E2 collaborate to conjugate Ubl to target-protein lysine residues. Together, these data reveal that bacteria possess bona fide ubiquitination systems with strong mechanistic and architectural parallels to canonical eukaryotic ubiquitination pathways, suggesting that these pathways arose first in bacteria.

Bacterial cGAS-like enzymes produce 2',3'-cGAMP to activate an ion channel that restricts phage replication.

The mammalian innate immune system uses cyclic GMP-AMP synthase (cGAS) to synthesize the cyclic dinucleotide 2',3'-cGAMP during antiviral and antitumor immune responses. 2',3'-cGAMP is a nucleotide second messenger that initiates inflammatory signaling by binding to and activating the stimulator of interferon genes (STING) receptor. Bacteria also encode cGAS/DncV-like nucleotidyltransferases (CD-NTases) that produce nucleotide second messengers to initiate antiviral (antiphage) signaling. Bacterial CD-NTases produce a wide range of cyclic oligonucleotides but have not been documented to produce 2',3'-cGAMP. Here we discovered bacterial CD-NTases that produce 2',3'-cGAMP to restrict phage replication. Bacterial 2',3'-cGAMP binds to CD-NTase associated protein 14 (Cap14), a transmembrane protein of unknown function. Using electrophysiology, we show that Cap14 is a chloride-selective ion channel that is activated by 2',3'-cGAMP binding. Cap14 adopts a modular architecture, with an N-terminal transmembrane domain and a C-terminal nucleotide-binding SAVED domain. Domain-swapping experiments demonstrated the Cap14 transmembrane region could be substituted with a nuclease, thereby generating a biosensor that is selective for 2',3'-cGAMP. This study reveals that 2',3'-cGAMP signaling extends beyond metazoa to bacteria. Further, our findings suggest that transmembrane proteins of unknown function in bacterial immune pathways may broadly function as nucleotide-gated ion channels.

Bacterial NLR-related proteins protect against phage.

Bacteria use a wide range of immune pathways to counter phage infection. A subset of these genes shares homology with components of eukaryotic immune systems, suggesting that eukaryotes horizontally acquired certain innate immune genes from bacteria. Here, we show that proteins containing a NACHT module, the central feature of the animal nucleotide-binding domain and leucine-rich repeat containing gene family (NLRs), are found in bacteria and defend against phages. NACHT proteins are widespread in bacteria, provide immunity against both DNA and RNA phages, and display the characteristic C-terminal sensor, central NACHT, and N-terminal effector modules. Some bacterial NACHT proteins have domain architectures similar to the human NLRs that are critical components of inflammasomes. Human disease-associated NLR mutations that cause stimulus-independent activation of the inflammasome also activate bacterial NACHT proteins, supporting a shared signaling mechanism. This work establishes that NACHT module-containing proteins are ancient mediators of innate immunity across the tree of life.

An E1-E2 fusion protein primes antiviral immune signalling in bacteria.

In all organisms, innate immune pathways sense infection and rapidly activate potent immune responses while avoiding inappropriate activation (autoimmunity). In humans, the innate immune receptor cyclic GMP-AMP synthase (cGAS) detects viral infection to produce the nucleotide second messenger cyclic GMP-AMP (cGAMP), which initiates stimulator of interferon genes (STING)-dependent antiviral signalling1. Bacteria encode evolutionary predecessors of cGAS called cGAS/DncV-like nucleotidyltransferases2 (CD-NTases), which detect bacteriophage infection and produce diverse nucleotide second messengers3. How bacterial CD-NTase activation is controlled remains unknown. Here we show that CD-NTase-associated protein 2 (Cap2) primes bacterial CD-NTases for activation through a ubiquitin transferase-like mechanism. A cryo-electron microscopy structure of the Cap2-CD-NTase complex reveals Cap2 as an all-in-one ubiquitin transferase-like protein, with distinct domains resembling eukaryotic E1 and E2 proteins. The structure captures a reactive-intermediate state with the CD-NTase C terminus positioned in the Cap2 E1 active site and conjugated to AMP. Cap2 conjugates the CD-NTase C terminus to a target molecule that primes the CD-NTase for increased cGAMP production. We further demonstrate that a specific endopeptidase, Cap3, balances Cap2 activity by cleaving CD-NTase-target conjugates. Our data demonstrate that bacteria control immune signalling using an ancient, minimized ubiquitin transferase-like system and provide insight into the evolution of the E1 and E2 machinery across domains of life.

Molecular basis of CD-NTase nucleotide selection in CBASS anti-phage defense.

cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes are signaling proteins that initiate antiviral immunity in animal cells and cyclic-oligonucleotide-based anti-phage signaling system (CBASS) phage defense in bacteria. Upon phage recognition, bacterial CD-NTases catalyze synthesis of cyclic-oligonucleotide signals, which activate downstream effectors and execute cell death. How CD-NTases control nucleotide selection to specifically induce defense remains poorly defined. Here, we combine structural and nucleotide-analog interference-mapping approaches to identify molecular rules controlling CD-NTase specificity. Structures of the cyclic trinucleotide synthase Enterobacter cloacae CdnD reveal coordinating nucleotide interactions and a possible role for inverted nucleobase positioning during product synthesis. We demonstrate that correct nucleotide selection in the CD-NTase donor pocket results in the formation of a thermostable-protein-nucleotide complex, and we extend our analysis to establish specific patterns governing selectivity for each of the major bacterial CD-NTase clades A-H. Our results explain CD-NTase specificity and enable predictions of nucleotide second-messenger signals within diverse antiviral systems.

Extracellular cyclic dinucleotides induce polarized responses in barrier epithelial cells by adenosine signaling.

Cyclic dinucleotides (CDNs) are secondary messengers used by prokaryotic and eukaryotic cells. In mammalian cells, cytosolic CDNs bind STING (stimulator of IFN gene), resulting in the production of type I IFN. Extracellular CDNs can enter the cytosol through several pathways but how CDNs work from outside eukaryotic cells remains poorly understood. Here, we elucidate a mechanism of action on intestinal epithelial cells for extracellular CDNs. We found that CDNs containing adenosine induced a robust CFTR-mediated chloride secretory response together with cAMP-mediated inhibition of Poly I:C-stimulated IFNß expression. Signal transduction was strictly polarized to the serosal side of the epithelium, dependent on the extracellular and sequential hydrolysis of CDNs to adenosine by the ectonucleosidases ENPP1 and CD73, and occurred via activation of A2B adenosine receptors. These studies highlight a pathway by which microbial and host produced extracellular CDNs can regulate the innate immune response of barrier epithelial cells lining mucosal surfaces.

The Linguistics of Bacterial Conflict Systems Reveal Ancient Origins of Eukaryotic Innate Immunity.

The arms race between bacteria and their competitors has produced an astounding variety of conflict systems that are shared via horizontal gene transfer across bacterial populations. In this issue of the Journal of Bacteriology, Burroughs and Aravind investigate how these biological conflict systems have been mixed and matched into new configurations, often with novel protein domains (A. M. Burroughs and L. Aravind, J Bacteriol 202:e00365-20, 2020, https://doi.org/10.1128/JB.00365-20). The authors additionally characterize the evolutionary history of genes in eukaryotes that appear to have been acquired from these prokaryotic defense systems.

(p)ppGpp and c-di-AMP Homeostasis Is Controlled by CbpB in Listeria monocytogenes.

The facultative intracellular pathogen Listeria monocytogenes, like many related Firmicutes, uses the nucleotide second messenger cyclic di-AMP (c-di-AMP) to adapt to changes in nutrient availability, osmotic stress, and the presence of cell wall-acting antibiotics. In rich medium, c-di-AMP is essential; however, mutations in cbpB, the gene encoding c-di-AMP binding protein B, suppress essentiality. In this study, we identified that the reason for cbpB-dependent essentiality is through induction of the stringent response by RelA. RelA is a bifunctional RelA/SpoT homolog (RSH) that modulates levels of (p)ppGpp, a secondary messenger that orchestrates the stringent response through multiple allosteric interactions. We performed a forward genetic suppressor screen on bacteria lacking c-di-AMP to identify genomic mutations that rescued growth while cbpB was constitutively expressed and identified mutations in the synthetase domain of RelA. The synthetase domain of RelA was also identified as an interacting partner of CbpB in a yeast-2-hybrid screen. Biochemical analyses confirmed that free CbpB activates RelA while c-di-AMP inhibits its activation. We solved the crystal structure of CbpB bound and unbound to c-di-AMP and provide insight into the region important for c-di-AMP binding and RelA activation. The results of this study show that CbpB completes a homeostatic regulatory circuit between c-di-AMP and (p)ppGpp in Listeria monocytogenesIMPORTANCE Bacteria must efficiently maintain homeostasis of essential molecules to survive in the environment. We found that the levels of c-di-AMP and (p)ppGpp, two nucleotide second messengers that are highly conserved throughout the microbial world, coexist in a homeostatic loop in the facultative intracellular pathogen Listeria monocytogenes Here, we found that cyclic di-AMP binding protein B (CbpB) acts as a c-di-AMP sensor that promotes the synthesis of (p)ppGpp by binding to RelA when c-di-AMP levels are low. Addition of c-di-AMP prevented RelA activation by binding and sequestering CbpB. Previous studies showed that (p)ppGpp binds and inhibits c-di-AMP phosphodiesterases, resulting in an increase in c-di-AMP. This pathway is controlled via direct enzymatic regulation and indicates an additional mechanism of ribosome-independent stringent activation.

CBASS Immunity Uses CARF-Related Effectors to Sense 3'-5'- and 2'-5'-Linked Cyclic Oligonucleotide Signals and Protect Bacteria from Phage Infection.

cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes are immune sensors that synthesize nucleotide second messengers and initiate antiviral responses in bacterial and animal cells. Here, we discover Enterobacter cloacae CD-NTase-associated protein 4 (Cap4) as a founding member of a diverse family of >2,000 bacterial receptors that respond to CD-NTase signals. Structures of Cap4 reveal a promiscuous DNA endonuclease domain activated through ligand-induced oligomerization. Oligonucleotide recognition occurs through an appended SAVED domain that is an unexpected fusion of two CRISPR-associated Rossman fold (CARF) subunits co-opted from type III CRISPR immunity. Like a lock and key, SAVED effectors exquisitely discriminate 2'-5'- and 3'-5'-linked bacterial cyclic oligonucleotide signals and enable specific recognition of at least 180 potential nucleotide second messenger species. Our results reveal SAVED CARF family proteins as major nucleotide second messenger receptors in CBASS and CRISPR immune defense and extend the importance of linkage specificity beyond mammalian cGAS-STING signaling.

Structure and Mechanism of a Cyclic Trinucleotide-Activated Bacterial Endonuclease Mediating Bacteriophage Immunity.

Bacteria possess an array of defenses against foreign invaders, including a broadly distributed bacteriophage defense system termed CBASS (cyclic oligonucleotide-based anti-phage signaling system). In CBASS systems, a cGAS/DncV-like nucleotidyltransferase synthesizes cyclic di- or tri-nucleotide second messengers in response to infection, and these molecules activate diverse effectors to mediate bacteriophage immunity via abortive infection. Here, we show that the CBASS effector NucC is related to restriction enzymes but uniquely assembles into a homotrimer. Binding of NucC trimers to a cyclic tri-adenylate second messenger promotes assembly of a NucC homohexamer competent for non-specific double-strand DNA cleavage. In infected cells, NucC activation leads to complete destruction of the bacterial chromosome, causing cell death prior to completion of phage replication. In addition to CBASS systems, we identify NucC homologs in over 30 type III CRISPR/Cas systems, where they likely function as accessory nucleases activated by cyclic oligoadenylate second messengers synthesized by these systems' effector complexes.

Analysis of human cGAS activity and structure.

Cyclic GMP-AMP synthase (cGAS) is an innate immune system enzyme responsible for recognition of double-stranded DNA aberrantly localized in the cell cytosol. cGAS binds DNA and is activated to catalyze production of the nucleotide second messenger 2'-5'/3'-5' cyclic GMP-AMP (2'3' cGAMP). In spite of a major role for cGAS in the cellular immune response, a complete understanding of cGAS biology has been limited by a lack of genetic tools to rapidly screen cGAS activity, instability of human cGAS-DNA interactions in vitro, and a previous absence of structural information for the human cGAS-DNA complex. Here we detail procedures to map the molecular determinants of cGAS activation and describe methods developed to prepare human cGAS-DNA crystals for structural analysis. Together with earlier systems established to study mammalian homologs of cGAS, these innovations provide a foundation to understand and therapeutically target human cGAS biology.

Bacterial cGAS-like enzymes synthesize diverse nucleotide signals.

Cyclic dinucleotides (CDNs) have central roles in bacterial homeostasis and virulence by acting as nucleotide second messengers. Bacterial CDNs also elicit immune responses during infection when they are detected by pattern-recognition receptors in animal cells. Here we perform a systematic biochemical screen for bacterial signalling nucleotides and discover a large family of cGAS/DncV-like nucleotidyltransferases (CD-NTases) that use both purine and pyrimidine nucleotides to synthesize a diverse range of CDNs. A series of crystal structures establish CD-NTases as a structurally conserved family and reveal key contacts in the enzyme active-site lid that direct purine or pyrimidine selection. CD-NTase products are not restricted to CDNs and also include an unexpected class of cyclic trinucleotide compounds. Biochemical and cellular analyses of CD-NTase signalling nucleotides demonstrate that these cyclic di- and trinucleotides activate distinct host receptors and thus may modulate the interaction of both pathogens and commensal microbiota with their animal and plant hosts.

Structure of the Human cGAS-DNA Complex Reveals Enhanced Control of Immune Surveillance.

Cyclic GMP-AMP synthase (cGAS) recognition of cytosolic DNA is critical for immune responses to pathogen replication, cellular stress, and cancer. Existing structures of the mouse cGAS-DNA complex provide a model for enzyme activation but do not explain why human cGAS exhibits severely reduced levels of cyclic GMP-AMP (cGAMP) synthesis compared to other mammals. Here, we discover that enhanced DNA-length specificity restrains human cGAS activation. Using reconstitution of cGAMP signaling in bacteria, we mapped the determinant of human cGAS regulation to two amino acid substitutions in the DNA-binding surface. Human-specific substitutions are necessary and sufficient to direct preferential detection of long DNA. Crystal structures reveal why removal of human substitutions relaxes DNA-length specificity and explain how human-specific DNA interactions favor cGAS oligomerization. These results define how DNA-sensing in humans adapted for enhanced specificity and provide a model of the active human cGAS-DNA complex to enable structure-guided design of cGAS therapeutics.

Listeria monocytogenes triggers noncanonical autophagy upon phagocytosis, but avoids subsequent growth-restricting xenophagy.

Xenophagy is a selective macroautophagic process that protects the host cytosol by entrapping and delivering microbes to a degradative compartment. Both noncanonical autophagic pathways and xenophagy are activated by microbes during infection, but the relative importance and function of these distinct processes are not clear. In this study, we used bacterial and host mutants to dissect the contribution of autophagic processes responsible for bacterial growth restriction of Listeria monocytogenesL. monocytogenes is a facultative intracellular pathogen that escapes from phagosomes, grows in the host cytosol, and avoids autophagy by expressing three determinants of pathogenesis: two secreted phospholipases C (PLCs; PlcA and PlcB) and a surface protein (ActA). We found that shortly after phagocytosis, wild-type (WT) L. monocytogenes escaped from a noncanonical autophagic process that targets damaged vacuoles. During this process, the autophagy marker LC3 localized to single-membrane phagosomes independently of the ULK complex, which is required for initiation of macroautophagy. However, growth restriction of bacteria lacking PlcA, PlcB, and ActA required FIP200 and TBK1, both involved in the engulfment of microbes by xenophagy. Time-lapse video microscopy revealed that deposition of LC3 on L. monocytogenes-containing vacuoles via noncanonical autophagy had no apparent role in restricting bacterial growth and that, upon access to the host cytosol, WT L. monocytogenes utilized PLCs and ActA to avoid subsequent xenophagy. In conclusion, although noncanonical autophagy targets phagosomes, xenophagy was required to restrict the growth of L. monocytogenes, an intracellular pathogen that damages the entry vacuole.

Activation of the Listeria monocytogenes Virulence Program by a Reducing Environment.

Upon entry into the host cell cytosol, the facultative intracellular pathogen Listeria monocytogenes coordinates the expression of numerous essential virulence factors by allosteric binding of glutathione (GSH) to the Crp-Fnr family transcriptional regulator PrfA. Here, we report that robust virulence gene expression can be recapitulated by growing bacteria in a synthetic medium containing GSH or other chemical reducing agents. Bacteria grown under these conditions were 45-fold more virulent in an acute murine infection model and conferred greater immunity to a subsequent lethal challenge than bacteria grown in conventional media. During cultivation in vitro, PrfA activation was completely dependent on the intracellular levels of GSH, as a glutathione synthase mutant (ΔgshF) was activated by exogenous GSH but not reducing agents. PrfA activation was repressed in a synthetic medium supplemented with oligopeptides, but the repression was relieved by stimulation of the stringent response. These data suggest that cytosolic L. monocytogenes interprets a combination of metabolic and redox cues as a signal to initiate robust virulence gene expression in vivoIMPORTANCE Intracellular pathogens are responsible for much of the worldwide morbidity and mortality from infectious diseases. These pathogens have evolved various strategies to proliferate within individual cells of the host and avoid the host immune response. Through cellular invasion or the use of specialized secretion machinery, all intracellular pathogens must access the host cell cytosol to establish their replicative niches. Determining how these pathogens sense and respond to the intracellular compartment to establish a successful infection is critical to our basic understanding of the pathogenesis of each organism and for the rational design of therapeutic interventions. Listeria monocytogenes is a model intracellular pathogen with robust in vitro and in vivo infection models. Studies of the host-sensing and downstream signaling mechanisms evolved by L. monocytogenes often describe themes of pathogenesis that are broadly applicable to less tractable pathogens. Here, we describe how bacteria use external redox states as a cue to activate virulence.

A Redox-Responsive Transcription Factor Is Critical for Pathogenesis and Aerobic Growth of Listeria monocytogenes.

Bacterial pathogens have evolved sophisticated mechanisms to sense and adapt to redox stress in nature and within the host. However, deciphering the redox environment encountered by intracellular pathogens in the mammalian cytosol is challenging, and that environment remains poorly understood. In this study, we assessed the contributions of the two redox-responsive, Spx-family transcriptional regulators to the virulence of Listeria monocytogenes, a Gram-positive facultative intracellular pathogen. Spx-family proteins are highly conserved in Firmicutes, and the L. monocytogenes genome contains two paralogues, spxA1 and spxA2 Here, we demonstrate that spxA1, but not spxA2, is required for the oxidative stress response and pathogenesis. SpxA1 function appeared to be conserved with the Bacillus subtilis homologue, and resistance to oxidative stress required the canonical CXXC redox-sensing motif. Remarkably, spxA1 was essential for aerobic growth, demonstrating that L. monocytogenes SpxA1 likely regulates a distinct set of genes. Although the ΔspxA1 mutant did not grow in the presence of oxygen in the laboratory, it was able to replicate in macrophages and colonize the spleens, but not the livers, of infected mice. These data suggest that the redox state of bacteria during infection differs significantly from that of bacteria growing in vitro Further, the host cell cytosol may resemble an anaerobic environment, with tissue-specific variations in redox stress and oxygen concentration.

c-di-AMP modulates Listeria monocytogenes central metabolism to regulate growth, antibiotic resistance and osmoregulation.

Cyclic diadenosine monophosphate (c-di-AMP) is a conserved nucleotide second messenger critical for bacterial growth and resistance to cell wall-active antibiotics. In Listeria monocytogenes, the sole diadenylate cyclase, DacA, is essential in rich, but not synthetic media and ΔdacA mutants are highly sensitive to the ß-lactam antibiotic cefuroxime. In this study, loss of function mutations in the oligopeptide importer (oppABCDF) and glycine betaine importer (gbuABC) allowed ΔdacA mutants to grow in rich medium. Since oligopeptides were sufficient to inhibit growth of the ΔdacA mutant we hypothesized that oligopeptides act as osmolytes, similar to glycine betaine, to disrupt intracellular osmotic pressure. Supplementation with salt stabilized the ΔdacA mutant in rich medium and restored cefuroxime resistance. Additional suppressor mutations in the acetyl-CoA binding site of pyruvate carboxylase (PycA) rescued cefuroxime resistance and resulted in a 100-fold increase in virulence of the ΔdacA mutant. PycA is inhibited by c-di-AMP and these mutations prompted us to examine the role of TCA cycle enzymes. Inactivation of citrate synthase, but not down-stream enzymes suppressed ΔdacA phenotypes. These data suggested that c-di-AMP modulates central metabolism at the pyruvate node to moderate citrate production and indeed, the ΔdacA mutant accumulated six times the concentration of citrate present in wild-type bacteria.