Nucleotides are indispensable components of all living cells, as they make up DNA and RNA, and serve as important energy sources. Nucleotides also have key roles in signaling in eukaryotic, bacterial and archaeal cells. In bacteria, signaling nucleotides such as cyclic AMP and guanosine tetra- or pentaphosphate ((p)ppGpp) have been classically linked to carbon metabolism and the stringent response, which is caused by nutrient limitation. However, it has become clear that signaling nucleotides contribute to the regulation of multiple different pathways; for example, in addition to its involvement in central carbon metabolism, cAMP is also involved in the regulation of both biofilm formation and virulence gene expression in many pathogenic bacteria. One of the latest signaling nucleotides to be identified is cyclic di-AMP (c-di-AMP), which is the second cyclic dinucleotide shown to be produced by bacteria, after cyclic di-GMP (c-di-GMP). It has been suggested that c-di-AMP and c-di-GMP regulate very different processes.
c-di-AMP is produced from two molecules of ATP by diadenylyl cyclase (DAC) enzymes and is degraded to pApA by phosphodiesterase (PDE) enzymes. The dinucleotide was initially discovered during a structural study on Thermotoga maritima. DNA integrity scanning protein (DisA), which is a homologue of Bacillus subtilis DisA (formerly known as YacK), a bacterial DNA damage checkpoint protein that can delay sporulation in the event of DNA damage. The first report of c-di-AMP production by bacterial cells came in 2010, when the dinucleotide was identified as a molecule secreted into the cytosol of host cells by the intracellular bacterial pathogen Listeria monocytogenes. Since then, c-di-AMP has been detected in cellular extracts from Streptococcus pyogenes, B. subtilis, Chlamydia rachomatis and Staphylococcus aureus, and a DisA-type c-di-AMP-synthesizing enzyme from Mycobacterium tuberculdosis has been characterized biochemically.
Although most of the mechanistic details still await molecular characterization, the regulation of cellular pathways by c-di-AMP presumably follows the same general principles as for the other signaling nucleotides. Environmental changes are sensed either directly or indirectly by the nucleotide-synthesizing or nucleotide-degrading enzymes, leading to a change in the cellular nucleotide concentration. At high concentrations, c-di-AMP is expected to bind to a specific set of receptor or target proteins and allosterically alter their function or the function of downstream effector proteins, thus controlling specific cellular pathways. Although many details of the c-di-AMP signaling network remain to be discovered, this nucleotide has been linked to the regulation of fatty acid synthesis in Mycobacterium sminegatis, to the growth of S. aureus in low-potassium conditions, to the sensing of DNA integrity in B. subtilis and to cell wall homeostasis in multiple species.
The M. tuberculosis genome encodes a di-adenylate cyclase enzyme (disA, also called dacA; encoded by gene Rv3586 (also called MT3692) in the H37Rv genome or MT3692 in the CDC1551 genome) that synthesizes c-di-AMP from ATP or ADP4. Orthologs of disA exist in all mycobacterial genomes with the exception of M. leprae. However, the role of c-di-AMP in M. tuberculosis physiology and mechanism of its interaction with the host immune system is poorly understood. However, the existing model for M. tuberculosis infection is that extracellular mycobacterial DNA is the only ligand for CSP activation within macrophages, which leads to increased autophagy and bacterial clearance in an ESX-1 secretion system-dependent manner, excluding any role for bacterial CDNs in CSP activation.
The mammalian innate immune system is composed of receptors that collectively serve as a pathogen sensor to monitor the extracellular, vacuolar, and cytosolic cellular compartments. Recognition of microbes within these distinct compartments leads to cellular responses that are commensurate with the microbial threat. Although both pathogenic and nonpathogenic microbes interact with extracellular and vacuolar compartments, infectious disease agents often mediate their pathogenesis by directly entering the cytosol or through delivery of virulence factors into the host cell cytosolic compartment. Thus, the innate immune system may distinguish between pathogenic and nonpathogenic microbes by monitoring the cytosol.
Several distinct pathways of innate immunity are present in the host cell cytosol. One, termed the cytosolic surveillance pathway (CSP), detects bacterial, viral, and protozoan pathogens, leading to the activation of interferon regulatory factor 3 (IRF3) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), resulting in the induction of interferon-β (IFN-β) and co-regulated genes. Some ligands that activate this pathway are known, for example, viral and bacterial nucleic acids. However, the ligands and host receptors that lead to IFN-β production after exposure to nonviral microbes-including L. monocytogenes, M. tuberculoss, F. tularensis, L. pneumophila, B. abortis, and T. cruzi—remain unknown. The mechanisms and role of c-di-AMP signaling in Mycobacterium tuberculosis infection must be identified and treatments that prevent, alleviate, or cure tuberculosis must be developed.
Bacille Calmette Guerin (BCG) is the most widely used vaccination in the world. BCG is made of a live, weakened strain of Mycobacterium bovis, (a cousin of Mycobacterium tuberculosis, the TB bacteria). It was developed in the 1930's and it remains the only vaccination available against tuberculosis today. Despite its protection against active TB in children, BCG has failed to protect adults against TB infection and active disease development, especially in developing countries where the disease is endemic. Some of key reasons for failure of BCG is low immunogenicity and its inability to induce maturation of DC efficiently. Among various strategies that have been employed so far to improve the protective potential of BCG involve construction of rBCG, which could confer similar or higher protection along with induction of a better immunological memory than BCG. Most of the methodologies used to achieve greater immunogenicity involve (i) over-expression of promising immuno-dominant antigens either singularly or as fusion with other immuno-dominant antigens, (ii) over-expression and reintroduction of antigens lost during the attenuation process or (iii) over-expression of mammalian cytokines in BCG such as IL-2, IL-12, IL-15, and GM-CSF. New methods of tuberculosis vaccination are needed to prevent the spread of disease.
In addition, more than 60,000 new cases of bladder cancer are diagnosed each year in the United States accounting for approximately 13,000 deaths. BCG-based therapy is currently the most effective intravesical therapy for nonmuscle invasive bladder cancer (NMIBC) and it represents the only agent known to reduce the progression of invasive bladder cancer into muscle. It is widely accepted that an intact immune system is a prerequisite to a successful therapy. BCG-induced antitumor effects depend on a sequence of events involving a complex interplay of soluble and cellular immune mediators and a cross-talk between innate and adaptive immunity. Limitations of BCG therapy include recurrence of the disease after initiation of BCG therapy. Consequently, new BCG strains enhancing the prevention or cure, and minimizing the recurrence rate, of cancer in patients must be identified.