Mycobacterium tuberculosis SecA2-dependent activation of host Rig-I/MAVs signaling is not conserved in Mycobacterium marinum

Retinoic acid inducible gene I (Rig-I) is a cytosolic pattern recognition receptor canonically described for its important role in sensing viral RNAs. Increasingly, bacterially-derived RNA from intracellular bacteria such as Mycobacterium tuberculosis, have been shown to activate the same host Rig-I/Mitochondrial antiviral sensing protein (MAVS) signaling pathway to drive a type-I interferon response that contributes to bacterial pathogenesis in vivo. In M. tuberculosis, this response is mediated by the protein secretion system SecA2, but little is known about whether this process is conserved in other pathogenic mycobacteria or the mechanism by which these nucleic acids gain access to the host cytoplasm. Because the M. tuberculosis and M. marinum SecA2 protein secretion systems share a high degree of genetic and functional conservation, we hypothesized that Rig-I/MAVS activation and subsequent induction of IFN-beta secretion by host macrophages will also be conserved between these two mycobacterial species. To test this, we generated a Delta secA2 M. marinum strain along with complementation strains expressing either the M. marinum or M. tuberculosis secA2 genes. Our results suggest that the Delta secA2 strain has a growth defect in vitro but not in host macrophages. These intracellular growth curves also suggested that the calculation applied to estimate the number of bacteria added to macrophage monolayers in infection assays underestimates bacterial inputs for the Delta secA2 strain. Therefore, to better examine secreted IFN-beta levels when bacterial infection levels are equal across strains we plated bacterial CFUs at 2hpi alongside our ELISA based infections. This enabled us to normalize secreted levels of IFN-beta to a standard number of bacteria. Applying this approach to both WT and MAVS-/- bone marrow derived macrophages we observed equal or higher levels of secreted IFN-beta from macrophages infected with the Delta secA2 M. marinum strain as compared to WT. Together our findings suggest that activation of host Rig-I/MAVS cytosolic sensors and subsequent induction of IFN-beta response in a SecA2-dependent manner is not conserved in M. marinum under the conditions tested.