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Signal transduction pathways enable organisms to monitor their external environment and adjust gene regulation to appropriately modify their cellular processes. Second messenger nucleotides including cyclic adenosine monophosphate (c-AMP), cyclic guanosine monophosphate (c-GMP), cyclic di-guanosine monophosphate (c-di-GMP), and cyclic di-adenosine monophosphate (c-di-AMP) play key roles in many signal transduction pathways used by prokaryotes and/or eukaryotes. Among the various second messenger nucleotides molecules, c-di-AMP was discovered recently and has since been shown to be involved in cell growth, survival, and regulation of virulence, primarily within Gram-positive bacteria. The cellular level of c-di-AMP is maintained by a family of c-di-AMP synthesizing enzymes, diadenylate cyclases (DACs), and degradation enzymes, phosphodiesterases (PDEs). Genetic manipulation of DACs and PDEs have demonstrated that alteration of c-di-AMP levels impacts both growth and virulence of microorganisms. Unlike other second messenger molecules, c-di-AMP is essential for growth in several bacterial species as many basic cellular functions are regulated by c-di-AMP including cell wall maintenance, potassium ion homeostasis, DNA damage repair, etc. c-di-AMP follows a typical second messenger signaling pathway, beginning with binding to receptor molecules to subsequent regulation of downstream cellular processes. While c-di-AMP binds to specific proteins that regulate pathways in bacterial cells, c-di-AMP also binds to regulatory RNA molecules that control potassium ion channel expression in Bacillus subtilis. c-di-AMP signaling also occurs in eukaryotes, as bacterially produced c-di-AMP stimulates host immune responses during infection through binding of innate immune surveillance proteins. Due to its existence in diverse microorganisms, its involvement in crucial cellular activities, and its stimulating activity in host immune responses, c-di-AMP signaling pathway has become an attractive antimicrobial drug target and therefore has been the focus of intensive study in several important pathogens.

The bacterium Bacillus subtilis produces the DNA integrity scanning protein (DisA), a checkpoint protein that delays sporulation in response to DNA damage. DisA scans the chromosome and pauses at sites of DNA lesions. Structural analysis showed that DisA synthesizes the small molecule cyclic diadenosine monophosphate (c‐di‐AMP). Here, we demonstrate that the intracellular concentration of c‐di‐AMP rises markedly at the onset of sporulation in a DisA‐dependent manner. Furthermore, exposing sporulating cells to DNA‐damaging agents leads to a global decrease in the level of this molecule. This drop was associated with stalled DisA complexes that halt c‐di‐AMP production and with increased levels of the c‐di‐AMP‐degrading enzyme YybT. Reduced c‐di‐AMP levels cause a delay in sporulation that can be reversed by external supplementation of the molecule. Thus, c‐di‐AMP acts as a secondary messenger, coupling DNA integrity with progression of sporulation. This study shows that the bacterial DNA damage checkpoint protein, DisA, produces the small secondary messenger c‐di‐AMP to report DNA integrity prior to sporulation.

Antibiotic-producing Streptomyces use the diadenylate cyclase DisA to synthesize the nucleotide second messenger c-di-AMP, but the mechanism for terminating c-di-AMP signaling and the proteins that bind the molecule to effect signal transduction are unknown. Here, we identify the AtaC protein as a c-di-AMP-specific phosphodiesterase that is also conserved in pathogens such as Streptococcus pneumoniae and Mycobacterium tuberculosis. AtaC is monomeric in solution and binds Mn2+ to specifically hydrolyze c-di-AMP to AMP via the intermediate 5′-pApA. As an effector of c-di-AMP signaling, we characterize the RCK_C domain protein CpeA. c-di-AMP promotes interaction between CpeA and the predicted cation/proton antiporter, CpeB, linking c-di-AMP signaling to ion homeostasis in Actinobacteria. Hydrolysis of c-di-AMP is critical for normal growth and differentiation in Streptomyces, connecting ionic stress to development. Thus, we present the discovery of two components of c-di-AMP signaling in bacteria and show that precise control of this second messenger is essential for ion balance and coordinated development in Streptomyces.

ABSTRACT Cyclic dimeric adenosine 3′,5′-monophosphate (c-di-AMP) is an emerging second messenger in bacteria and archaea that is synthesized from two molecules of ATP by diadenylate cyclases and degraded to pApA or two AMP molecules by c-di-AMP-specific phosphodiesterases. Through binding to specific protein- and riboswitch-type receptors, c-di-AMP regulates a wide variety of prokaryotic physiological functions, including maintaining the osmotic pressure, balancing central metabolism, monitoring DNA damage and controlling biofilm formation and sporulation. It mediates bacterial adaptation to a variety of environmental parameters and can also induce an immune response in host animal cells. In this review, we discuss the phylogenetic distribution of c-di-AMP-related enzymes and receptors and provide some insights into the various aspects of c-di-AMP signaling pathways based on more than a decade of research. We emphasize the key role of c-di-AMP in maintaining bacterial osmotic balance, especially in Gram-positive bacteria. In addition, we discuss the future direction and trends of c-di-AMP regulatory network, such as the likely existence of potential c-di-AMP transporter(s), the possibility of crosstalk between c-di-AMP signaling with other regulatory systems, and the effects of c-di-AMP compartmentalization. This review aims to cover the broad spectrum of research on the regulatory functions of c-di-AMP and c-di-AMP signaling pathways. This review describes the latest outlook on c-di-AMP signaling pathways that are involved in its homeostasis, reception, various physiological functions, and emphasizing on its role in regulating bacterial osmotic balance, as well as several issues to be explored in the near future.

IntroductionSubunit vaccines represent a safer alternative to live attenuated formulations. However, they often require potent adjuvants and delivery systems to elicit robust immunity, particularly against highly contagious diseases such as Classical Swine Fever (CSF).MethodsIn this study, we investigated the immunogenicity and protective efficacy of a novel mucosal subunit vaccine comprising the chimeric E2-CD154 protein, co-administered with the mucosal adjuvant c-di-AMP, in domestic pigs. Optimal dosing and immunization schedules for sublingual immunization were determined, followed by a challenge experiment using a highly virulent CSF virus (CSFV) strain.ResultsOur results showed that sublingual co-administration of E2-CD154 and the STING agonist c-di-AMP conferred robust clinical protection, effectively prevented viral replication, and restricted the dissemination of infectious virus. This combination induced strong systemic IgG and IgA responses and neutralizing antibodies against multiple CSFV strains, achieving outcomes comparable with the commercial Porvac® vaccine, administered intramuscularly. Importantly, virus isolation from tonsils confirmed the absence of infectious virus in pigs immunized with E2-CD154 and c-di-AMP, unlike those receiving E2-CD154 or the adjuvant alone. Moreover, immunized animals exhibited minimal IFN-α serum levels post-challenge, indicating reduced innate activation and viral replication.DiscussionThese findings provide evidence, in a large mammalian host such as the pig, that c-di-AMP functions as an adjuvant for a recombinant E2-CD154 protein delivered sublingually, enhancing immune responses consistent with protection against viral replication. Together, these results offer insights into the development of non-replicating, DIVA-compatible platforms against CSFV and support the rational design of next-generation subunit vaccines targeting viral pathogens relevant to both veterinary and human medicine.

This study unveils the intricate functional association between cyclic di-3’,5’-adenylic acid (c-di-AMP) signaling, cellular bioenergetics, and the regulation of lipopolysaccharide (LPS) profile in Porphyromonas gingivalis, a Gram-negative obligate anaerobe considered as a keystone pathogen involved in the pathogenesis of chronic periodontitis. Previous research has identified variations in P. gingivalis LPS profile as a major virulence factor, yet the underlying mechanism of its modulation has remained elusive. We employed a comprehensive methodological approach, combining two mutants exhibiting varying levels of c-di-AMP compared to the wild type, alongside an optimized analytical methodology that combines conventional mass spectrometry techniques with a novel approach known as FLATn. We demonstrate that c-di-AMP acts as a metabolic nexus, connecting bioenergetic status to nuanced shifts in fatty acid and glycosyl profiles within P. gingivalis LPS. Notably, the predicted regulator gene cdaR, serving as a potent regulator of c-di-AMP synthesis, was found essential for producing N-acetylgalactosamine and an unidentified glycolipid class associated with the LPS profile. The multifaceted roles of c-di-AMP in bacterial physiology are underscored, emphasizing its significance in orchestrating adaptive responses to stimuli. Furthermore, our findings illuminate the significance of LPS variations and c-di-AMP signaling in determining the biological activities and immunostimulatory potential of P. gingivalis LPS, promoting a pathoadaptive strategy. The study expands the understanding of c-di-AMP pathways in Gram-negative species, laying a foundation for future investigations into the mechanisms governing variations in LPS structure at the molecular level and their implications for host-pathogen interactions.

The nucleotide cyclic di-3′,5′- adenosine monophosphate (c-di-AMP) was recently identified as an essential and widespread second messenger in bacterial signaling. Among c-di-AMP–producing bacteria, altered nucleotide levels result in several physiological defects and attenuated virulence. Thus, a detailed molecular understanding of c-di-AMP metabolism is of both fundamental and practical interest. Currently, c-di-AMP degradation is recognized solely among DHH-DHHA1 domain-containing phosphodiesterases. Using chemical proteomics, we identified the Listeria monocytogenes protein PgpH as a molecular target of c-di-AMP. Biochemical and structural studies revealed that the PgpH His-Asp (HD) domain bound c-di-AMP with high affinity and specifically hydrolyzed this nucleotide to 5′-pApA. PgpH hydrolysis activity was inhibited by ppGpp, indicating a cross-talk between c-di-AMP signaling and the stringent response. Genetic analyses supported coordinated regulation of c-di-AMP levels in and out of the host. Intriguingly, a L. monocytogenes mutant that lacks c-di-AMP phosphodiesterases exhibited elevated c-di-AMP levels, hyperinduced a host type-I IFN response, and was significantly attenuated for infection. Furthermore, PgpH homologs, which belong to the 7TMR-HD family, are widespread among hundreds of c-di-AMP synthesizing microorganisms. Thus, PgpH represents a broadly conserved class of c-di-AMP phosphodiesterase with possibly other physiological functions in this crucial signaling network. Significance The small nucleotide cyclic di-3′,5′-adenosine monophosphate (c-di-AMP) recently emerged as a ubiquitous signaling molecule among bacteria, with essential roles in both bacterial physiology and host–pathogen interactions. Bacterial mutants with abnormal c-di-AMP levels exhibit growth and virulence defects, reflecting the importance of regulating c-di-AMP synthesis and degradation for normal signal transduction and adaptation to changing environments. Previously documented phosphodiesterases hydrolyze c-di-AMP via the DHH-DHHA1 domain, but they are not present in all c-di-AMP synthesizing species. We identified a previously unrecognized class of His-Asp -domain phosphodiesterases that are widespread across several taxonomic groups. Furthermore, for the bacterial pathogen Listeria monocytogenes , phosphodiesterase mutants exhibit enhanced host inflammation, growth defects inside host cells, and significantly attenuated virulence in a murine model of infection.

Small molecule can be utilized to restore the effectiveness of existing major classes of antibiotics against antibiotic‐resistant bacteria. In this study, it is demonstrated that celastrol, a natural compound, can modify the bacterial cell wall and subsequently render bacteria more suceptible to β‐lactam antibiotics. It is shown that celastrol leads to incomplete cell wall crosslinking by modulating levels of c‐di‐AMP, a secondary messenger, in methicillin‐resistant Staphylococcus aureus (MRSA). This mechanism enables celastrol to act as a potentiator, effectively rendering MRSA susceptible to a range of penicillins and cephalosporins. Restoration of in vivo susceptibility of MRSA to methicillin is also demonstrated using a sepsis animal model by co‐administering methicillin along with celastrol at a much lower amount than that of methicillin. The results suggest a novel approach for developing potentiators for major classes of antibiotics by exploring molecules that re‐program metabolic pathways to reverse β‐lactam‐resistant strains to susceptible strains. This study provides insight into the mechanism of celastrol as a potentiator of β‐lactams against methicillin‐resistant Staphylococcus aureus (MRSA). Celastrol reduces the cellular c‐di‐AMP level by activating GdpP and increases muropeptides with shorter glycine branches that cannot be processed by PBP2a of MRSA, making MRSA susceptible to β‐lactams. The findings suggest that modulation of c‐di‐AMP can be a therapeutic target for enhancing β‐lactams.

Environments with high concentrations of salt or other solutes impose an osmotic stress on cells, ultimately limiting viability by dehydration of the cytosol. A very common cellular response to high osmolarity is to immediately import high levels of potassium ion (K + ), which helps prevent dehydration and allows time for the import or synthesis of biocompatible solutes that allow a resumption of growth. Osmotic stress is a significant physical challenge for free-living cells. Cells from all three domains of life maintain viability during osmotic stress by tightly regulating the major cellular osmolyte potassium (K + ) and by import or synthesis of compatible solutes. It has been widely established that in response to high salt stress, many bacteria transiently accumulate high levels of K + , leading to bacteriostasis, with growth resuming only when compatible solutes accumulate and K + levels are restored to biocompatible levels. Using Bacillus subtilis as a model system, we provide evidence that K + fluxes perturb Mg 2+ homeostasis: import of K + upon osmotic upshift is correlated with Mg 2+ efflux, and Mg 2+ reimport is critical for adaptation. The transient growth inhibition resulting from hyperosmotic stress is coincident with loss of Mg 2+ and a decrease in protein translation. Conversely, the reimport of Mg 2+ is a limiting factor during resumption of growth. Furthermore, we show the essential signaling dinucleotide cyclic di-AMP fluctuates dynamically in coordination with Mg 2+ and K + levels, consistent with the proposal that cyclic di-AMP orchestrates the cellular response to osmotic stress. IMPORTANCE Environments with high concentrations of salt or other solutes impose an osmotic stress on cells, ultimately limiting viability by dehydration of the cytosol. A very common cellular response to high osmolarity is to immediately import high levels of potassium ion (K + ), which helps prevent dehydration and allows time for the import or synthesis of biocompatible solutes that allow a resumption of growth. Here, using Bacillus subtilis as a model, we demonstrate that concomitant with K + import there is a large reduction in intracellular magnesium (Mg 2+ ) mediated by specific efflux pumps. Further, it is the reimport of Mg 2+ that is rate-limiting for the resumption of growth. These coordinated fluxes of K + and Mg 2+ are orchestrated by cyclic-di-AMP, an essential second messenger in Firmicutes . These findings amend the conventional model for osmoadaptation and reveal that Mg 2+ limitation is the proximal cause of the bacteriostasis that precedes resumption of growth.

Antibiotic resistance and biofilm-forming capacity contribute to the success of Staphylococcus aureus as a human pathogen in both healthcare and community settings. These virulence factors do not function independently of each other and the biofilm phenotype expressed by clinical isolates of S. aureus is influenced by acquisition of the methicillin resistance gene mecA. Methicillin-sensitive S. aureus (MSSA) strains commonly produce an icaADBC operon-encoded polysaccharide intercellular adhesin (PIA)-dependent biofilm. In contrast, the release of extracellular DNA (eDNA) and cell surface expression of a number of sortase-anchored proteins, and the major autolysin have been implicated in the biofilm phenotype of methicillin-resistant S. aureus (MRSA) isolates. Expression of high level methicillin resistance in a laboratory MSSA strain resulted in (i) repression of PIA-mediated biofilm production, (ii) down-regulation of the accessory gene regulator (Agr) system, and (iii) attenuation of virulence in murine sepsis and device infection models. Here we review the mechanisms of MSSA and MRSA biofilm production and the relationships between antibiotic resistance, biofilm and virulence gene regulation in S. aureus.