Through transcriptional profiling, we were able to identify a gene expression signature of Mycobacterium tuberculosis persister cells that survive four days of treatment with isoniazid, a lytic first-line antitubercular drug. This signature was then used to develop reporter constructs in which the regulatory regions of genes that are induced in persister cells are fused to the coding sequence of fluorescent protein genes. Initially, these constructs were delivered by mycobacteriophage, which allowed us to detect cells that are likely to be persisters in sputum from tuberculosis patients. Time-lapse imaging of M. tuberculosis cells infected with the reporter-bearing mycobacteriophage indicated that cells expressing high levels of the persister-induced reporter construct had enhanced survival during isoniazid treatment. We have employed phage-delivered reporter constructs to investigate several aspects of persister cell biology in vitro and will now extend our studies to the behavior of these cells during experimental infection. Currently, we know that persister cells are present during infection based on the biphasic response of bacterial burden to antimicrobial drug treatment, but we are in the dark about the niches occupied by persister cells over the course of infection and treatment, particularly in relation to the well-organized structure of the characteristic lesion of human tuberculosis, the necrotic granuloma. To address these unresolved questions, we are using a recently reported iNOS knockout mouse model of tuberculosis that has been shown to develop human-like pathology and to more accurately reproduce the response to drug treatment that has been observed in human patients. We are infecting these mice with strains bearing persister reporter constructs, and characterizing the occurrence of persister cells during the stages of infection and antitubercular drug treatment by advanced imaging techniques, with particular interest in where persister cells are found in relation to the structural and cellular features of the granuloma. The knowledge gained from these studies will be invaluable in the development of improved therapies that can clear persister cells from the niches where they survive, thus shortening treatment.