Summary Transmission remains the driving force behind the global drug resistant Tuberculosis (TB) epidemic. This occurs despite the observation that the acquisition of drug resistance has a fitness cost on the pathogen. Fitness costs have been associated with rpoB mutations which confer resistance to rifampicin. The physiological basis of the mutant rpoB induced fitness cost remains largely unknown with the exception that compensatory mutations have been found to ameliorate the fitness cost and have been associated with transmissibility. The current advancements in next generation RNA sequencing (RNA-seq) enables us to generate and compare the transcriptomic profiles of rpoB mutations with various levels of fitness. Using this technology, we aim to elucidate how different resistance-conferring rpoB mutations alter the function of RNA polymerase and thereby the transcriptome, how a transcriptome evolves with the addition of a compensatory rpoC mutation and whether the combination of these events alters fitness and the propensity of the isolate to not only acquire additional resistance but also to influence drug susceptibility to second-line drugs. Furthermore to elucidate how different fitness mutations influence their respective transcriptomes to ensure survive within the host environment as well as to determine the host gene expression response to the mutated M. tuberculosis (MTB). We propose to address these questions using the following three aims: 1) Determine how the combination of different rpoB mutations with or without a compensatory (rpoC) mutation influences the transcriptome of MTB, 2) Determine the in vivo transcriptome of MTB harbouring different fitness rpoB mutations with or without a rpoC mutation and 3) Determine whether poor treatment outcome of rifampicin-resistant MTB is related to rpoB mutations influencing the MIC of second-line anti-TB drugs in vivo. To achieve these aims we will select rpoB in vitro mutants with a clinically relevant genetic background which has been associated with TB outbreaks and a predisposition to develop multidrug resistance. Competition fitness assays will be used to select isolates harbouring rpoB mutations spanning the spectrum of in vitro growth fitness phenotypes. Mutations in rpoC will be engineered into the selected rpoB mutants, and RNA-seq will be used to determine the transcriptomic profiles. Macrophages will be infected to determine how stress changes the transcriptome of the bacteria and whether these mutants have an effect on the macrophage itself using dual RNA-seq. Genes governing and compensating for fitness together with regulatory genes (as seen in preliminary data) will be identified using a in silico modelling. Lastly, mutants will be exposed to second-line drug to determine whether they more rapidly acquire additional resistance and decreases susceptibility to second-line drugs thereby resulting in poor treatment outcome for MDR strains. Understanding how pathogenicity of fitness, drives the evolution of resistance acquisition and transmission of rifampicin resistance strains, thereby adapting patient management programs.