Chemogenomic investigation of Burkholderia cenocepacia K56-2 reveals the targets of antibiotics and potentiators
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Date
2022-12-05
Authors
Hogan, Andrew
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Abstract
To counter the rise in resistance, new antibiotics are desperately needed. Part of characterizing these compounds involves identifying the target, mechanism of action, and determinants of resistance and susceptibility. Chemogenomic assays coupled to next-generation sequencing (NGS) can rapidly probe genome-wide contributions to antibiotic action. Tools such as transposons have enabled genome-wide studies of species in the Burkholderia cepacia complex, a group of Gram-negative opportunistic pathogens noted for high levels of intrinsic antibiotic resistance. Thus, the goal of my thesis was to validate transposon-based methodologies for target and mechanism of action identification of antibiotics in B. cenocepacia.
I started my investigation with an uncharacterized compound called C109, which displayed broad-spectrum bactericidal activity. To identify the target of C109, I used a transposon with outward-facing rhamnose-inducible promoters, allowing tunable knockdown of mutants in essential genes. By NGS-based tracking of a pool of mutants in essential genes, I identified that C109 targeted FtsZ, a highly-conserved essential cell division protein. In vitro, C109 inhibited the GTPase and polymerization of FtsZ, which are critical for its function.
To improve on our set of genetic tools to manipulate essential genes, I adapted robust CRISPR-interference (CRISPRi) technology for Burkholderia. The single guide RNA cassette controlling gene targeting can be easily replaced, enabling rapid mutant creation.
Lastly, I constructed a randomly-barcoded transposon mutant library to probe genome-wide fitness determinants in the presence of a large panel of cell envelope-targeting antibiotics. I validated over a hundred new functional annotations for genes not previously associated with antibiotic activity in B. cenocepacia. I made new connections between β-lactam susceptibility and disruptions in undecaprenyl phosphate metabolic pathways, likely due to a weakened peptidoglycan matrix. Dissecting the effect of antibiotic combinations revealed that the PenB carbapenemase was the primary target of avibactam, paving the way for more effective antibiotic combinations active against Burkholderia. Overall, my thesis demonstrates how chemogenomics can be used to determine antibiotic targets, and mechanisms of action and resistance. This work has implications for antibiotic development, target prioritization, and using rational combinations to extend the utility of our current clinical arsenal.
See also Hogan, Andrew. (2022) Table S5 for Hogan et al. 2019. [Dataset]. UM Dataverse; https://doi.org/10.34990/FK2/ZS89DI
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Keywords
Transposon mutagenesis, Antibiotic combinations, Beta-lactam, Undecaprenyl phosphate, Avibactam, Beta-lactamase