Identification of Mycobacterium tuberculosis genes that mediate inhibition of the host cell IFN-beta signaling

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Abstract It is estimated that about one third of the world’s population is latently infected with Mycobacterium tuberculosis (Mtb). The 10 million annual new cases of tuberculosis resulting in about one million deaths further underline the global public health impact of this pathogen. Mtb has developed a multitude of pathways to evade the host immune response. For the most part, the molecular mechanisms of these host pathogen interactions are only beginning to be understood. The premise of the current proposal is supported by a series of novel and unpublished results which demonstrate that Mtb inhibits IFN-a/b-receptor (IFNAR)-mediated cell signaling which results in improved intracellular growth of the bacteria. These findings thus describe a novel immunoevasion pathway engaged by Mtb. The central hypothesis of this application is that genetic screens will be able to identify the genes in Mtb important for inhibition of the IFNAR-mediated cell signaling. In Aim 1.1 we propose to execute a “gain-of-function” genetic screen using M. smegmatis clones transfected with a Mtb-DNA cosmid library. This approach has been successfully executed for two other screens by the PI’s lab. Next, in Aim 1.2 we describe how the relevant Mtb gene out of the approximately 40 genes in one of the isolated cosmids will be identified. Overall, we anticipate that the successful completion of the current project will generate at least one Mtb deletion mutant deficient in inhibition of IFNAR-mediated cell signaling and potentially the knowledge of several other Mtb regions that contain additional genes. These findings will be the basis for a competitive R01 application which will propose to: 1) complete the identification of all of the Mtb genes involved in inhibition of IFN-β-mediated cell signaling, 2) to use the mutants for detailed analysis of the molecular mechanisms of the inhibition and 3) to characterize the importance of inhibiting IFNAR-mediated cell signaling for in vivo virulence of Mtb.