Project Summary 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 that gain-of-function (GOF) genetic screens provide a currently underappreciate approach towards identification of Mtb virulence factors. In the specific aims 1 and 2 we propose to generate and characterize a barcoded, arrayed cosmid library 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 genes inhibiting host cell death and for Mtb genes that mediate increased virulence in an in vivo GOF screen in immunodeficient SCID mice. Overall, our work will provide a novel resource to the research community that may have a major impact in the discovery of virulence genes of Mtb.