Student Theses and Dissertations
Date of Award
2023
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Jeremy M. Rock
Keywords
Mycobacterium tuberculosis, CRISPR-Cas, functional genomics, antibiotic targets, gene vulnerability, bacterial fitness
Abstract
Essential bacterial genes orchestrate core biological processes and represent the targets of nearly all antibacterial drugs. Traditional genetic approaches such as knockout studies and transposon mutagenesis have cataloged hundreds of essential bacterial genes but provide no basis on which to identify the most attractive targets for antibiotics. In contrast to the binary definition of gene essentiality, the gene vulnerability is an expression that relates the magnitude of gene inhibition with the resulting impact on bacterial fitness. Partial gene inhibition results in stronger fitness costs for vulnerable genes than invulnerable genes. Understanding vulnerability is critical for prioritizing targets for drug development and in understanding the growth rate limiting steps in bacterial physiology. To quantify gene vulnerability, we developed a CRISPR interference-based functional genomics method to systematically titrate the expression of nearly all essential genes and monitor fitness outcomes in the global pathogen, Mycobacterium tuberculosis (Mtb). Additionally we created an approach to study differential vulnerability in clinical Mtb strains and under host-relevant growth conditions. We find that different biological pathways, while all essential for Mtb viability, vary widely in their vulnerability. We define highly vulnerable steps in Mtb central dogma processes, protein secretion and cell wall synthesis, including targets currently unexplored in the drug discovery pipeline. Likewise, we identify invulnerable processes that can withstand substantial inhibition with little impact on bacterial fitness, including targets of failed efforts in drug discovery. Comparison of vulnerability in Mtb and the model bacterium M. smegmatis resulted in substantial overlap in vulnerability predictions but with notable exceptions, suggesting considerable but incomplete conservation of expression-fitness relationships within mycobacteria. Bioinformatic analysis of 10,000 Mtb clinical isolates showed that vulnerable Mtb genes are under higher purifying selection than invulnerable genes, indicating that these gene classes are under distinct evolutionary pressures. Evolutionary analysis further identified that vulnerable genes are more likely to be conserved and essential across >2 billion years of evolutionary separation in bacteria. Differential vulnerability analyses between the reference H37Rv Mtb strain and a hypervirulent Mtb isolate HN878 as well as between standard culture conditions and different iron and carbon source conditions, revealed clinically relevant differences in vulnerability. Lastly, we find that differential genetic vulnerability can predict differential susceptibility to chemical inhibitors. This work provides a quantitative redefinition of essential bacterial processes and serves as a roadmap for developing more effective drugs against Mtb and other bacterial pathogens.
DOI
10.48496/a71v-gt15
License and Reuse Information
This work is licensed under a Creative Commons Attribution-NonCommercial-Share Alike 4.0 International License.
Recommended Citation
Bosch, Barbara, "Genome-Scale Quantification of Target Vulnerability in Mycobacterium Tuberculosis" (2023). Student Theses and Dissertations. 834.
https://doi.org/10.48496/a71v-gt15
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy