A Global Analysis of the Adaptations Required for Sterol Catabolism in Mycobacterium Tuberculosis: A Dissertation

Abstract

Systems biology approaches have allowed for comprehensive understanding of complicated biological processes. Here, we’ve developed a global phenotypic profiling method by improving upon transposon mutagenesis methods for identifying genes required for bacterial growth in various conditions. By using the massively parallel power of Illumina sequencing, we precisely redefined the genes required for the growth of Mycobacterium Tuberculosis (Mtb) in vitro. This adapted technique provided more informative data with both increased dynamic range and resolution. As a result, we quantitatively assessed the fitness of individual mutants, as well as identified sub-genic essentiality. Mtb is well adapted to its nutrient-limiting intracellular niche. One important and novel adaptation is its ability to consume cholesterol for both energy and carbon. A combination of this genome-wide phenotypic analysis and global metabolite profiling was used to define the dedicated cholesterol catabolic pathway, as well as important transcriptional and metabolic adaptations required for the consumption of this carbon source. We identified the methylcitrate cycle (MCC) and an unexpected gluconeogenic route as essential pathways. Furthermore, we found that the cholesterol-dependent transcriptional induction of these metabolic enzymes was also essential for growth on this substrate, a function mediated by the Rv1129c regulatory protein. Using a combination of genetic and chemical methods to inhibit these pathways, we show that cholesterol represents a significant source of carbon during intracellular growth in macrophages. Finally, we have begun to define the mechanism by which lipids, such as cholesterol, are imported into the cell by investigating the assembly of the ABC-like lipid transporter, Mce1. The subunits of this system are localized to the cell wall and data is provided to support a novel mechanism for Mce-dependent import of lipids, such as cholesterol. In sum, this global analysis of host cholesterol utilization during infection provides insight into each step of this complicated process; import into the bacterial cell, the degradation of the molecule into primary metabolites, and the transformation of these metabolites into carbon and energy.