Dual RNAseq Characterization of Mycobacterium Tuberculosis Infection

Abstract

Tuberculosis (TB) disease causes 1.6 million deaths annually, making it the leading infectious killer in the world. The causative agent of TB, Mycobacterium tuberculosis (Mtb), must replicate in macrophages to cause disease within the host. Simultaneous profiling of host and pathogen transcription, dual RNAseq, can identify gene networks that mediate disease outcome and reveal new therapeutic targets. However, dual RNAseq profiling of host and pathogen during bacterial infections is limited by detection of lowly abundant bacterial mRNA within infected host cells. We develop an efficient, accurate, and reproducible method, Hybrid Capture (HC) to enrich for bacterial mRNA using transcriptome-specific probes. HC enables simultaneous analysis of host and pathogen transcriptional programs from a single sample. This method is broadly adaptable to the study of a range of bacterial pathogens and, more generally, to low-abundance species in mixed populations. We use HC to perform dual RNAseq profiling of bacterial and host transcription during Mtb infection. In chapter 3 we characterize the transcriptional dynamics of Mtb growth within the host, identifying iron limitation as a host-imposed stressor felt by intracellular bacteria. We then use this dataset to combine our transcriptional profiling of intracellular bacteria with phenotypic characterization of Mtb transcription factor (TF) mutants to characterize transcription regulatory networks important for Mtb growth in macrophages. In chapter 3, we identify the bacterial transcription factor, Rv2989, as a negative regulator of leuCD, which encodes an essential enzyme in leucine biosynthesis. We show that overexpression of leuCD attenuates Mtb intracellular growth suggesting that regulation of this enzyme is important for Mtb pathogenesis. In chapter 4 we focus on a second bacterial transcription factor, Rv0135c. We show that ∆Rv0135c up-regulates redox genes during infection, yet these regulatory targets do not appear to be important for Mtb infection. Combined, our studies suggest that these transcription factors function to constrain the expression of their regulatory targets during Mtb intracellular growth. The work presented in this dissertation expands our understanding of Mtb requirements for growth inside the host and provides a powerful tool for future studies of Mtb biology.