Exploring the SecA2-Dependent Induction of Host Type I Interferon Response by Pathogenic Mycobacteria

<p> <b>Background</b>: Increasingly, bacterial RNAs are recognized as potent inducers of type I interferons. In <i>M. tuberculosis</i>, 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.</p><p><b>Methods</b>: To identify genes/proteins integral to SecA2-dependent release of <i>M. tuberculosis</i> RNA, we developed a primary transposon library screen for low IFN-β inducing mutants in a closely related mycobacterial species, <i>M. marinum </i>(Chapter 2). As SecA2-dependent activation of Rig-I/MAVS has not been characterized in <i>M. marinum</i>,<i> </i>we examined the conservation of this critical virulence mechanism by generating Δ<i>secA2</i> 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 <i>M. tuberculosis</i> and trialled an RNA labeling and pulldown approach to identify RNA binding proteins (Chapter 4). </p><p><b>Results</b>: Our results indicate the proposed primary screening technique can identify low IFN-β inducing transposon mutants. However, our <i>M. marinum</i> Δ<i>secA2</i> strain showed intracellular growth rates and secreted levels of IFN-β equal to or higher than wild type. Comparative analysis of <i>M. tuberculosis</i> secreted RNAs identified shared motifs and heterogenous RNA secondary structures. Dot blots and biotin-RNA pulldown trials showed the incorporation of biotin in synthesized <i>mce1B</i> but non-specificity between labelled and unlabelled RNA following pull down.</p><p><b>Conclusions</b>: Together, our results show that while the proposed IFN-β screen can identify low IFN-β inducing mutants, <i>M. marinum</i> is not an appropriate model under the conditions tested. This screen could potentially be applied to an <i>M. tuberculosis</i> transposon library following validation. <i>In silico </i>analysis of secreted <i>M. tuberculosis</i> 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.</p>