Mycobacterium tuberculosisSecA2-dependent activation of host Rig-I/MAVs signaling is not conserved inMycobacterium marinum

Abstract

ABSTRACTRetinoic 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 asMycobacterium 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 pathogenesisin vivo. InM. 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 theM. tuberculosisandM. marinumSecA2 protein secretion systems share a high degree of genetic and functional conservation, we hypothesized that Rig-I/MAVS activation and subsequent induction of IFN-β secretion by host macrophages will also be conserved between these two mycobacterial species. To test this, we generated a ΔsecA2 M. marinumstrain along with complementation strains expressing either theM. marinumorM. tuberculosis secA2genes. Our results suggest that the ΔsecA2strain has a growth defectin vitrobut 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 ΔsecA2strain. Therefore, to better examine secreted IFN-β 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-β 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-β from macrophages infected with the ΔsecA2 M. marinumstrain as compared to WT. Together our findings suggest that activation of host Rig-I/MAVS cytosolic sensors and subsequent induction of IFN-β response in a SecA2-dependent manner is not conserved inM. marinumunder the conditions tested.