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국외단행본

Elucidation of an Essential Genetic Pathway Under Antibiotic Selection in Mycobacterium tuberculosis [electronic resource].

Harvard University. Medical Sciences.

[S.l.] : Harvard University. 2024 Ann Arbor : ProQuest Dissertations & Theses , 2024

1 online resource(153 p.)

Source: Dissertations Abstracts International, Volume: 85-12, Section: B.
Advisor: Fortune, Sarah M.

Thesis (Ph.D.)--Harvard University, 2024.

Tuberculosis remains the world's deadliest infectious disease caused by a single agent. Although tuberculosis is curable, treatment success is limited by our narrow understanding of genetic factors allowing its causative agent, Mycobacterium tuberculosis (Mtb), to evade antibiotic clearance. Large-scale sequencing of clinical Mtb populations revealed ongoing selection on genetic variants that could confer fitness advantages in the presence of drug pressure. This unbiased approach allowed identification of genes with no previous link to drug resistance, including two essential genes dnaA and resR. Although initially investigated independently, DnaA and ResR share a common binding site at the Rv0010c-Rv0011c intergenic region (IGR) and this IGR itself is one of the highly mutated non-coding regions on Mtb genome. Clinical IGR variants overlap with DnaA and ResR binding sites and phenocopy dnaA and resR variants, revealing a genetic pathway under selection. Yet this genetic pathway and the function of Rv0010c-Rv0011c IGR remains uncharacterized.Isogenic variants in the Rv0010c-Rv0011c IGR phenocopy dnaA and resR variants, showing similar increases in cell length, antibiotic resilience, and low-level isoniazid resistance. We found that DnaA and ResR bind at neighboring sites in the most conserved regions of this IGR, which paradoxically are where more recent clinical mutations accumulate. Knockout of the entire the Rv0010c-Rv0011c operon, including its 155bp IGR, resulted in shorter cells with increased sensitivity to isoniazid. This defect can only be complemented with the entire operon, though this complementation does not require translation of the two coding genes. Complementation with the intact operon carrying clinically relevant IGR variants recapitulates isogenic variant phenotypes. Meanwhile, complementation with the intact operon carrying DnaA or ResR binding site deletions failed, highlighting the requirement of protein binding in its downstream function.To understand the functional consequence of protein binding, we used biochemical approaches and found that DnaA and ResR bind cooperatively at this IGR. Clinical IGR variants increase the binding affinity of two proteins and binding site deletions reduce their affinity. Using transcriptomics, we identified genes that are differentially expressed in strains with clinical IGR variants versus strains with binding site deletions to pinpoint transcriptional changes correlating with divergent phenotypes in these strains. These genes included whiB2 and its regulon of division related genes. Notably, the promoter of whiB2 is a known direct target of ResR and is also highly mutated in clinical Mtb populations. We propose a model where clinical variations sequester ResR through its interaction with DnaA at the Rv0010c-Rv0011c IGR. This sequestration reduces ResR's ability to activate division related genes and alter division dynamics, resulting in morphology and drug phenotypes.Together, the data in this dissertation provide functional insight into an essential and previously uncharacterized genetic pathway under selection in clinical Mtb populations. We propose that clinical mutations in this pathway alter dynamics of cell cycle events and contribute to changes in Mtb morphology and drug response. Understanding non-canonical drug determinants is critical to elucidate other mechanisms Mtb use to evade antibiotics killing and we hope to inspire future studies on intergenic regions and unknown genetic pathways to better understand Mtb biology and improve treatment design.

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