ABCC1/MRP1 exports cGAMP and modulates cGAS-dependent immunity

The DNA sensor cyclic GMP-AMP synthase (cGAS) is important for antiviral and anti-tumor immunity. cGAS generates cyclic GMP-AMP (cGAMP), a diffusible cyclic dinucleotide that activates the antiviral response through the adapter protein Stimulator of Interferon Genes (STING). cGAMP is negatively charged and cannot passively cross cell membranes, but recent advances have established a role for extracellular cGAMP as an “immunotransmitter” that can be imported into cells. However, the mechanism by which cGAMP exits cells remains unknown. Here, we identify ABCC1/MRP1 as an ATP-dependent cGAMP exporter that influences STING signaling and type I interferon production. We demonstrate that ABCC1 deficiency exacerbates cGAS-dependent autoimmunity in the Trex1-/- mouse model of Aicardi-Goutières syndrome. These studies identify ABCC1-mediated cGAMP export as a key regulatory mechanism of the cGAS-STING pathway.

STING mediates immune responses in the closest living relatives of animals

Animals have evolved unique repertoires of innate immune genes and pathways that provide their first line of defense against pathogens. To reconstruct the ancestry of animal innate immunity, we have developed the choanoflagellate Monosiga brevicollis, one of the closest living relatives of animals, as a model for studying mechanisms underlying pathogen recognition and immune response. We found that M. brevicollis is killed by exposure to Pseudomonas aeruginosa bacteria. Moreover, M. brevicollis expresses STING, which, in animals, activates innate immune pathways in response to cyclic dinucleotides during pathogen sensing. M. brevicollis STING increases the susceptibility of M. brevicollis to P. aeruginosa-induced cell death and is required for responding to the cyclic dinucleotide 2'3' cGAMP. Furthermore, similar to animals, autophagic signaling in M. brevicollis is induced by 2'3' cGAMP in a STING-dependent manner. This study provides evidence for a pre-animal role for STING in antibacterial immunity and establishes M. brevicollis as a model system for the study of immune responses.

NrnA is a linear dinucleotide phosphodiesterase with limited function in cyclic dinucleotide metabolism in Listeria monocytogenes

Listeria monocytogenes produces both c-di-AMP and c-di-GMP to mediate many important cellular processes, but the levels of both nucleotides must be regulated. C-di-AMP accumulation attenuates virulence and diminishes stress response, and c-di-GMP accumulation impairs bacterial motility. An important regulatory mechanism to maintain c-di-AMP and c-di-GMP homeostasis is to hydrolyze them to the linear dinucleotides pApA and pGpG, respectively, but the fates of these hydrolytic products have not been examined in L. monocytogenes . We found that NrnA, a stand-alone DHH-DHHA1 phosphodiesterase, has a broad substrate range, but with a strong preference for linear dinucleotides over cyclic dinucleotides. Although NrnA exhibited detectable cyclic dinucleotide hydrolytic activities in vitro, NrnA had negligible effects on their levels in the bacterial cell, even in the absence of the c-di-AMP phosphodiesterases PdeA and PgpH. The Δ nrnA mutant had a mammalian cell infection defect that was fully restored by E. coli Orn. Together, our data indicate that L. monocytogenes NrnA is functionally orthologous to Orn, and its preferred physiological substrates are most likely linear dinucleotides. Furthermore, our findings revealed that, unlike some other c-di-AMP and c-di-GMP-producing bacteria, L. monocytogenes does not employ their hydrolytic products to regulate their phosphodiesterases, at least at the pApA and pGpG levels in the Δ nrnA mutant. Finally, the Δ nrnA infection defect was overcome by constitutive activation of PrfA, the master virulence regulator, suggesting that accumulated linear dinucleotides might inhibit the expression, stability, or function of PrfA-regulated virulence factors. IMPORTANCE Listeria monocytogenes produces both c-di-AMP and c-di-GMP, and encodes specific phosphodiesterases that degrade them into pApA and pGpG, respectively, but the metabolism of these products has not been characterized in this bacterium. We found that L. monocytogenes NrnA degrades a broad range of nucleotides. Among the tested cyclic and linear substrates, it exhibits a strong biochemical and physiological preference the linear dinucleotides pApA, pGpG, and pApG. Unlike in some other bacteria, these oligoribonucleotides do not appear to interfere with cyclic dinucleotide hydrolysis. The absence of NrnA is well tolerated by L. monocytogenes in broth cultures but impairs its ability to infect mammalian cells. These findings indicate a separation of cyclic dinucleotide signaling and oligoribonucleotide metabolism in L. monocytogenes .