Type I IFN bioassay and luciferase reporter assay. Cell-culture supernatants from stimulated cells were overlayed on top of ISRE-L929 IFN reporter cells (Jiang et al., 2005) and incubated for 4-6 h (96-well plate). The reporter cells were lysed in Passive Lysis Buffer (Promega) for 5 min at room temperature, mixed with firefly luciferin substrate (Biosynth), and measured on a luminometer (LmaxII 384 ; MDS Analytical Technologies). Levels of type I IFN were calculated from a standard curve using recombinant mouse IFN- (R&D Systems). secretion system of F. tularensis, the Dot/Icm system of L. pneumophila, or the MdrM multidrug efflux pump of L. monocytogenes. It is tempting to speculate that a small, bacterially derived molecule such as c-di-GMP could be transported or leak through these secretion systems. It has not yet been possible to test this idea directly because all of these bacterial species encode numerous c-di-GMP synthases, and a strain lacking all c-di-GMP synthesis has not been reported. In any case, interpretation of these experiments would be complicated by the fact that c-di-GMP plays important regulatory roles in bacterial physiology and pathogenesis. Nevertheless, our results with synthetic purified c-di-GMP suggest that in addition to DNA, c-di-GMP is a candidate for a conserved molecule unique to bacteria that is responsible for triggering transcription of type I IFN genes. In light of a recent report that bacteria appear to be able to synthesize c-di-AMP (Witte et al., 2008) , it is interesting to consider whether additional nucleic acids produced specifically by bacteria might also trigger host immunosurveillance pathways. Non-nucleic acid small molecules, such as the drug DMXAA, also appear to be able to stimulate the TBK1-IRF3 axis (Roberts et al., 2007) . Indeed, there may be multiple redundant pathways for cytosolic sensing of bacteria. Collectively, the available evidence suggests that host cells may sense a wider array of bacterial ligands than was previously appreciated.