Many bacterial pathogens including Staphylococcus, Streptococcus, Pseudomonas, Yersiniae, Listeria and Mycobacterial
species employ key secondary messengers including c-di-GMP or c-di-AMP that play essential modulatory roles in bacteria4, 7
. Although several substrates and effectors have been identified for these cyclic dinucleotide monophosphate species within the bacterial cell, our understanding of these bacteria-specific secondary messengers on the innate immune response within the mammalian host cell is just beginning to emerge. c-di-GMP and c-di-AMP activate the host type I IFN response in a manner dependent on the STING adaptor10, 11
and our findings indicate that the DNA sensor helicase DDX41 functions as a direct PRR for these cyclic dinucleotides in both murine and human cells. Our results showed that unlabeled c-di-GMP or c-di-AMP could disrupt the DDX41-c-di-GMP interaction. We found that GMP and AMP, but not the bulkier GTP or ATP molecules, could also competitively disrupt the DDX41-c-di-GMP complex. Although two molecules of GMP or AMP are structurally similar to c-di-GMP or c-di-AMP, respectively, they are not known to function as PAMPs in mammalian cells9
. It will therefore be of further interest to determine how these species play a modulatory role in the type I IFN response. Our competition experiments additionally revealed that B-DNA could disrupt the DDX41-c-di-GMP complex. Indeed DDX41 has been shown to function as a sensor for B-DNA as well17
. The mechanism by which DDX41 binds B-DNA, as well as cyclic dinucleotides, as revealed by co-crystallization studies and point mutation analysis will be the subject of future investigation.
Our results additionally indicated that c-di-GMP and c-di-AMP mediated activation of innate signaling and type I IFN induction were similarly defective between cells in which DDX41 or STING had been knocked down , suggesting DDX41 and STING share a common signaling pathway. STING-deficient cells displayed a very modest defect in NF-κB activation in response to c-di-GMP or c-di-AMP. The reason for this phenomenon is not entirely clear10
, however it may be possible that there is redundancy or compensation in signaling to NF-κB. Another DNA sensor, IFI16 (also known as p204) was also shown to facilitate some viral DNA triggered signaling via the STING adaptor21, 22
. Although a role for IFI16 cannot be ruled out in the c-di-GMP and c-di-AMP signaling pathway, it is however unlikely that IFI16 functions as a primary sensor for these molecules since its’ basal expression is low and is rather induced in a type I IFN-dependent manner. DDX41 expression on the other hand, is greater at the basal state and is not modulated by type I IFNs17
Our data suggests that DDX41 serves as the PRR for c-di-GMP and c-di-AMP, which upon receptor binding signals to TBK1-IRF3 via the STING adaptor. Lending further credibility as a scaffolding molecule, STING was recently shown to bridge TBK1-IRF3 interactions for optimal signaling23
. Nevertheless, consistent with a published report20
, we also found that c-di-GMP associated with STING, however, with lower affinity than DDX41. Although the physiological relevance of this interaction requires further investigation, our data shows that c-di-GMP interaction with STING is significantly enhanced in the presence of DDX41 in cells. The solved structure of the C-terminal domain of STING in complex with c-di-GMP revealed that one molecule of c-di-GMP binds one dimer of STING24-28
. We propose that c-di-GMP detection and binding to DDX41 promotes enhanced DDX41-STING interactions leading to an increase in binding affinity of STING toward c-di-GMP, ultimately driving downstream signaling events. Thus, STING may function as a secondary receptor or co-factor in the cyclic dinucleotide signaling pathway.
The significance of type I IFN induction in the context of anti-bacterial innate immunity is currently unclear and somewhat controversial, particularly due to conflicting reports on whether type I IFN either functions to support or inhibit bacterial growth29-32
. It will therefore be of interest to further study how different bacteria and host cells use secondary messengers and DDX41 as virulence factors and innate immune receptors, respectively, in their battle of infection and immunity33-35
. As such, cyclic dinucleotide species and DDX41 represent new targets such that modulation of their interaction during certain bacterial infections can alter the host immune response in a manner to suppress bacterial replication and spread.