ABSTRACT - PROJECT 4 Perhaps the greatest barrier to improving tuberculosis (TB) treatment outcomes is the six months of multi- drug therapy that is required to reliably cure a patient with active disease. Lengthy therapy is both costly and leads to poor adherence. This project aims to understand the bacterial factors responsible for lengthy TB treatment, TB treatment failure, and relapse. Mycobacterium tuberculosis (Mtb), the causative agent of TB has developed an exquisite ability to adapt to its environment. Drug treatment can lead to the development of “phenotypically drug-resistant” (i.e. drug tolerant) subpopulations that persist for long periods, as well as atypical very-low level drug resistant mutants (i.e. elevated sub-breakpoint MIC strains) that can eventually relapse despite months of seemingly effective drug treatment. Our overriding hypothesis is that the length of time required to treat TB and the adverse TB outcomes that can occur even with adequate therapy are strongly linked to phenotypic drug resistance and low-level genetic drug resistance mechanisms that can be present in Mtb subpopulation pre-treatment, or that arise during treatment. This project will apply novel tools we have developed, including One-cell Doubling Evaluation of Living Arrays of Mycobacterium (ODELAM), a robotic Transwell Tolerance and Resistance (TTR) system, a complete library of transcription factor inducible (TFI) strains, and constitutively tolerant glpK mutant Mtb strains to investigate the mechanisms that underlie these bacterial states, as well as the heterogeneous expression of tolerance and low level resistance in different Mtb sub-populations. Expanding from well-defined laboratory strains to a diverse collection of well characterized clinical Mtb strains associated with either cure or relapse, we will develop a mechanistic understanding of these still poorly characterized bacterial phenotypes and their role in treatment outcome. These discoveries will lead to important opportunities for developing targeted TB treatments that increase therapeutic success while shortening treatment times. This work will be conducted in three related aims: Aim 1. Define the links between heterogeneity and phenotypic drug tolerance in Mtb. Aim 2. Determine the role of Mtb phase variation on population heterogeneity, drug tolerance and emergent drug resistance. Aim 3. Assess role(s) of genes, networks, and population heterogeneity in clinical isolates with defined treatment-related phenotypes.