Chemistry and Chemical Biology ETDs

Publication Date

Winter 11-15-2016

Abstract

The ribosome is the quintessential antibacterial drug target, with many structurally and mechanistically distinct classes of antibacterial agents acting by inhibiting ribosome function. Detecting and quantifying ribosome inhibition by small molecules and investigating their binding modes and mechanisms of action are critical to antibacterial drug discovery and development efforts. To develop a ribosome inhibition assay that is operationally simple, yet provides direct information on the drug target and the mechanism of action, we have developed engineered E. coli strains harboring an orthogonal ribosome controlled green fluorescent protein reporter that produce fluorescent signal when the O-ribosome is inhibited. As a proof of concept, we demonstrate that these strains act as sensitive and quantitative detectors of ribosome inhibition by a set of 12 structurally diverse 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics, which target the A-site in helix 44 of the 16S rRNA. To prove the generality and promote the application of the system, we extended this system for detecting ribosome inhibition by a variety of structurally and mechanistically distinct drug classes targeting both small and large subunits of the ribosome. We have engineered E. coli strains capable of detecting O-ribosome inhibition by other drugs targeting the 16S rRNA including the non-2-DOS aminoglycosides streptomycin and kasugamycin, and the aminocyclitol spectinomycin. Through integration of the Ribo-T tethered ribosome system with our system, we can also detect ribosome inhibition by the 23S rRNA inhibitors erythromycin, lincomycin and linezolid. These results suggest the generalizability of our strategy. We then applied the system to screen a set of spectinomycin analogs with unknown activity to demonstrate potential application. Three spectinomycin analogs were shown to possess higher or similar anti-ribosome activity compared to the parent compound.

We have also modified our system to enable rapid directed evolution of rRNA variants with specific properties from large rRNA libraries to study the impact of rRNA sequence variation on ribosome activity and drug resistance. By replacing the fluorescence reporter gene with the chloramphenicol acetyltransferase (CAT) gene, we are able to select for functional rRNA mutants and quantify their catalytic activities. A variety of drug-resistant and drug-dependent rRNA mutants, as well as hyper- and hypo-active mutants were found. We also performed directed evolution to select ribosome mutants capable of supporting cell growth, yet confer resistance to aminoglycoside antibiotics. Only two mutants (A1408G and G1491U) were identified from this experiment; and both mutants have broad spectrum aminoglycoside resistance. Our results demonstrate that the O-ribosome controlled reporter system is capable of efficiently identifying new rRNA mutants with unique properties. We suggest that our directed evolution approach has the potential to provide new insights into mechanisms of ribosome inhibition and evolution of antibiotic resistance.

Language

English

Keywords

Antibiotics, Ribosome, Ribosome inhibition, Synthetic biology

Document Type

Dissertation

Degree Name

Chemistry

Level of Degree

Doctoral

Department Name

Department of Chemistry and Chemical Biology

First Committee Member (Chair)

Charles E. Melançon III

Second Committee Member

Debra Dunaway-Mariano

Third Committee Member

Jeremy S. Edwards

Fourth Committee Member

Fu-Sen Liang

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