Genomic, Genetic, and Functional Interrogation of Mycobacterium tuberculosis Outbreak Strains

In the past 200 years, tuberculosis (TB) has caused more deaths than any other infectious disease and currently infects more people than it has at any other time in human history. Mycobacterium tuberculosis (Mtb), the etiological agent of TB, is an obligate human pathogen that has evolved through the millennia to become the archetypal human-adapted pathogen. This work focuses on the evolutionary framework by which Mtb emerged as a specialized human pathogen, and applying this perspective to outbreak strains and the strategies Mtb deploys to manipulate its host environment.

There are seven major lineages that define the human-adapted species of Mtb. Beijing lineage strains, also known as Lineage 2 strains, have emerged as important drivers of global Mtb burden due to the elevated rates of drug-resistance, rapid disease progression, and increased transmission characteristics they display. Beijing lineage strains are endemic to East Asia, but have recently expanded globally. In Chapter 2, we investigate circulating Beijing strains in Guatemala, a country with limited data regarding the molecular epidemiology of Mtb and few reported cases of disease caused by Beijing strains. We report the first whole genome sequencing of Central American Beijing-lineage strains of Mtb. We find that multiple Beijing-lineage strains, derived from independent founding events, are currently circulating in Guatemala, but overall still represent a relatively small proportion of disease burden. Finally, we identify a specific Beijing-lineage outbreak centered on a poor neighborhood in Guatemala City.

Pairing whole genome sequencing with outbreak strains displaying unusual disease phenotypes provides compelling insight into the genetic changes underlying the corresponding disease presentation. Mtb most commonly causes lung disease, but can also disseminate to other tissue sites. We identified an outbreak strain of Mtb that presented clinically with unusually high rates of extrapulmonary and bone disease in seemingly immunocompetent individuals. We find that the outbreak was caused by an ancient strain of Mtb that, like other ancestral strains and animal-adapted mycobacterial pathogens, carries a full-length version of the Type VII secreted effector EsxM. Here we show that EsxM is required for the full disseminative properties of mycobacterium-infected macrophages and is sufficient to directly enhance macrophage motility. Moreover, we find that EsxM has been inactivated in all modern strains of Mtb, suggesting a potential selective advantage to limiting dissemination as Mtb adopted and adapted to its modern pulmonary niche.

Pathogenic mycobacteria have long been known to utilize an array of effectors to manipulate their host environment. Mycobacterial infection initiates the assembly of granulomas, which are discrete host structures composed of tightly associated immune cell aggregates. Macrophages within the granuloma have been described as "epithelioid" due to the morphological transformation they undergo as they interdigitate with their neighboring cells. The epithelial transformation macrophages undergo is central to the formation of the granuloma, yet this transformation has not been well characterized at the molecular level during mycobacterial infection. Our laboratory has utilized the zebrafish to dissect the epithelial transformation of macrophages during mycobacterial infection. In Chapter 4, I outline the transcriptional reprogramming that occurs in granuloma macrophages that underlies this transformation. We find that granulomas exhibit distinct transcriptional profiles from macrophages, and, surprisingly, that immune cells deploy developmental programs to construct the granuloma.