Date of Award
Doctor of Philosophy (PhD)
Tuberculosis (TB) is the leading cause of death from any single infectious disease, killing over 1.5 million people each year. Treatment of TB is extraordinarily difficult, requiring at least 6 months of combination antibiotic therapy to cure patients. Even with this prolonged treatment, 5 to 10% of patients are not fully cured and experience disease relapse. Mycobacterium tuberculosis (Mtb), the causative agent of TB, is intrinsically resistant to most antibiotics and can acquire an additional level of antibiotic resistance through specific chromosomal mutations. A better understanding of intrinsic and acquired drug resistance in Mtb is essential to develop faster treatments and better drug resistance diagnostics. We sought to understand the genetic basis drug resistance in Mtb using a novel CRISPR interference (CRISPRi)-based chemical genetics platform. Our lab developed a CRISPRi library containing 100,000 unique knockdown strains and treated this library with a panel of some of the most important antitubercular drugs. The fitness of each mutant in the library was assessed by next generation sequencing. Across over thirty different drug treatment conditions, we identified hundreds of chemical-genetic hits. We were then able to leverage this data set to gain novel biological insights into the mechanisms of intrinsic drug resistance in Mtb and identify genetic strategies to disarm these mechanisms to potentiate the activity of existing antibiotics. These data provided a nuanced picture of how the mycobacterial cell envelope serves as a selective barrier to antibiotic penetration. We found that antibiotic activity can be potentiated via selective inhibition of cell envelope synthetic enzymes as well as regulatory proteins. These data also led to the identification of a novel ribosomal protection protein, which we have termed OcrA, that confers resistance against ribosome inhibitors such as linezolid and chloramphenicol. This may allow for the rational engineering of linezolid analogs to avoid the activity of OcrA. The CRISPRi chemical-genetic screen data was also used to uncover several novel mechanisms of acquired drug resistance amongst clinical Mtb isolates. Among the strongest hits identified from these chemical-genetic screens was ettA, a ribosome-associated ATPase that regulates translation initiation. CRISPRi mutants for ettA were resistant to a diverse set of drugs including streptomycin, levofloxacin, and ethambutol. Mining a whole genome sequence database of over 40,000 clinical Mtb isolates we identified several ettA mutations which phenocopy the CRISPRi mutants and confer multidrug resistance. Of particular interest, strains harboring an EttA ATP binding site mutation (Gly41Glu) were found to be concentrated in South America, especially in Perú, the location of a widespread multidrug-resistant TB outbreak. Molecular epidemiology suggests that this mutation likely arose early in the TB chemotherapy era. By conferring low-level resistance to multiple antibiotics, it likely served as a stepping-stone to the evolution of higher order drug resistance mutations. A current collaborative project is aimed at investigating the role of this ettA mutation in patient treatment outcomes in Perú.
Poulton, Nicholas, "A Chemical-Genetic Map of Drug Resistance in Mycobacterium Tuberculosis" (2023). Student Theses and Dissertations. 738.
Available for download on Sunday, April 27, 2025