Background: Increasingly, bacterial RNAs are recognized as potent inducers of type I interferons. In M. tuberculosis, high levels of IFN-β are associated with active forms of the disease and promote host pathology and bacterial pathogenesis. While mycobacterial RNAs have been shown to be released from bacteria in a SecA2-dependent manner and gain access to the host cytosol in an ESX-I dependent manner, little is known about the mechanisms regulating this pathogenesis mechanisms or whether it is conserved across other pathogenic mycobacteria.Methods: To identify genes/proteins integral to SecA2-dependent release of M. tuberculosis RNA, we developed a primary transposon library screen for low IFN-β inducing mutants in a closely related mycobacterial species, M. marinum (Chapter 2). As SecA2-dependent activation of Rig-I/MAVS has not been characterized in M. marinum, we examined the conservation of this critical virulence mechanism by generating ΔsecA2 knockout and complementation strains. These strains were tested for their ability to induce IFN-β from host cells following infection (Chapter 3). Finally, we applied existing computational tools to characterize six previously identified RNAs secreted by M. tuberculosis and trialled an RNA labeling and pulldown approach to identify RNA binding proteins (Chapter 4). Results: Our results indicate the proposed primary screening technique can identify low IFN-β inducing transposon mutants. However, our M. marinum ΔsecA2 strain showed intracellular growth rates and secreted levels of IFN-β equal to or higher than wild type. Comparative analysis of M. tuberculosis secreted RNAs identified shared motifs and heterogenous RNA secondary structures. Dot blots and biotin-RNA pulldown trials showed the incorporation of biotin in synthesized mce1B but non-specificity between labelled and unlabelled RNA following pull down.Conclusions: Together, our results show that while the proposed IFN-β screen can identify low IFN-β inducing mutants, M. marinum is not an appropriate model under the conditions tested. This screen could potentially be applied to an M. tuberculosis transposon library following validation. In silico analysis of secreted M. tuberculosis RNAs in addition to RNA synthesis and pulldown experiments suggests these tools could provide valuable information on shared structural attributes and RNA binding proteins involved in the SecA2-dependent activation of host Rig-I/MAVS signaling.