Characterizing Functional Genetic Diversity in Mycobacterium tuberculosis

Abstract

The bacterium Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), which is the deadliest infectious disease in the world. Mtb is genetically conserved compared to most other human pathogens, so the field has traditionally considered Mtb genetically and thus phenotypically monomorphic. Therefore, the functional consequences of Mtb genetic diversity are underexplored, especially mutations acquired early in the evolutionary history of Mtb. Since antiquity, Mtb has evolved to adapt to the host environment in which it infects. This evolution is still occurring as Mtb currently infects nearly one third of the human population. Both ancient and contemporary host adaption results in genetic diversity that could have important implications for how we understand TB pathogenesis, patient clinical outcomes, and the efficacy of the TB antibiotic regimen.

This dissertation opens with Chapter One, where we examine the progress and potential of harnessing what we know about Mtb genetic variation to improve antitubercular therapy. In Chapter Two, we describe a pipeline we developed that allowed us to complete high-throughput in vitro phenotyping and genotyping of a large cohort of Mtb clinical strains. By leveraging this methodology, we uncover a diverse Mtb phenotypic landscape in response to host-relevant metabolic and antibiotic stress, the genetic determinants of which we describe. We find that a subset of these ancestral and contemporary mutations that are associated with altered metabolic and antibiotic stress fitness are also associated with patient cavitary disease, treatment failure, and transmission. As a result, we make a case for the contribution of Mtb phenogenomic variation in shaping TB clinical outcomes. In Chapter Three, we apply the same pipeline to identify monogenic variants within the Mtb clinical strain cohort that are predictive of bacterial fitness during macrophage and mouse infection. In Chapter 4, we describe the stepwise evolution of lldD2, a gene that encodes the Mtb lactate dehydrogenase LldD2. Lactate is an important feature of the host intracellular environment in which Mtb replicates, and we show that both ancestral and modern mutations in lldD2 improve the ability of Mtb to utilize lactate as a carbon source. Further, we demonstrate that lldD2 mutations modulate antibiotic sensitivity and virulence pathways. Lastly, in Chapter 5 we summarize the implications of this body of work where we defined the functional impacts of mutations that have been historically overlooked. We also discuss future directions, address questions posed by our results, and define ways in which our finding can be translated to improve TB diagnostics and treatment. In all, the goal of our work is to provide evidence that signifies the importance of dissecting the impact of functional Mtb genetic diversity. Ultimately, this will enrich our understanding of the evolution of pathogenesis and the subsequent effects on TB disease and bacterial drug sensitivity.