Project SummaryIt is estimated that about one third of the world?s population is latently infected withMycobacterium tuberculosis (Mtb). The 10 million annual new cases of tuberculosis resulting inabout one million deaths further underline the global public health impact of this pathogen. Mtbhas 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 beunderstood. The premise of the current proposal is that gain-of-function (GOF) genetic screensprovide a currently underappreciate approach towards identification of Mtb virulence factors. Inthe specific aims 1 and 2 we propose to generate and characterize a barcoded, arrayed cosmidlibrary of Mtb regions of about 50kbp expressed in the host mycobacterial strain M. kansasii.Specific aim 3 will test this newly generated resource in ex vivo GOF screens for genesinhibiting host cell death and for Mtb genes that mediate increased virulence in an in vivo GOFscreen in immunodeficient SCID mice. Overall, our work will provide a novel resource to theresearch community that may have a major impact in the discovery of virulence genes of Mtb.