Antibiotic-resistant microbes are once again a major threat to human health worldwide. Drugs once crucial in fighting the spread of deadly pathogens have become ineffective. The development of novel antimicrobials to solve this crisis will require detailed knowledge of the underlying mechanisms of antibiotic resistance, including the structural basis of resistance caused by mutations in antibiotic targets. The most important antibiotic target is the ribosome, the universal site of protein synthesis, and resistanc mutations have been identified in numerous species. Through a strong collaborative effort, this proposal capitalizes on the current state of genetics and X-ray crystallography of the ribosome from Thermus thermophilus to address fundamental aspects of antibiotic resistance.
The aims of this proposal are: (1) determine the structural basis for phenotypic interaction among multiple antibiotic-resistance mutations that can potentially arise after sequential exposure to multiple antibiotic challenges; (2) monitor the evolution of secondary mutations that compensate for the fitness cost of deleterious antibiotic-resistance mutations.
These aims are designed to give fundamental insights into mechanisms of antibiotic resistance, and findings will be applicable to the rational design of drugs directed against antibiotic-resistant pathogens. Achieving these aims will involve an innovative fusion of genetics and X-ray crystallography to determine the structures of mutant ribosomes. The bacterium T. thermophilus is especially suited as a model system due to its amenability to genetic manipulation and the suitability of its ribosomes for crystallization. Our proposal rests on a strong foundation of: (1) extensive experience in both genetics and X-ray crystallography, including a proven track record of the skills needed to achieve the proposed aims; (2) extensive preliminary results, including the structure determination of a number of antibiotic-resistant ribosomes; (3) an extensive working knowledge of the protein synthesis field and antibiotic-resistance, as indicated by a long track record of publications; (4) an excellent collaborative relationship between the two PIs. The accomplishment of the goals of this proposal will provide fundamental insights into the mechanism of antibiotic resistance, which will have predictive power regarding resistance mutations not yet identified. They will also create a valuable framework for the rational design of novel antimicrobials.

Public Health Relevance

A comprehensive understanding of the mechanisms of antibiotic resistance is essential for the development of novel antimicrobial agents by rational drug design. More than half of all antibiotics target the ribosome, the universal site of protein synthesis. We will combine genetics, experimental evolution and X-ray crystallography to determine the precise structural basis for antibiotic resistance conferred by mutations in the ribosome.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM094157-07
Application #
9266791
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Sledjeski, Darren D
Project Start
2010-09-15
Project End
2020-04-30
Budget Start
2017-05-01
Budget End
2018-04-30
Support Year
7
Fiscal Year
2017
Total Cost
Indirect Cost
Name
Brown University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
001785542
City
Providence
State
RI
Country
United States
Zip Code
02912
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Kamath, Divya; Gregory, Steven T; O'Connor, Michael (2017) The Loop 2 Region of Ribosomal Protein uS5 Influences Spectinomycin Sensitivity, Translational Fidelity, and Ribosome Biogenesis. Antimicrob Agents Chemother 61:
VanNice, John; Gregory, Steven T; Kamath, Divya et al. (2016) Alterations in ribosomal protein L19 that decrease the fidelity of translation. Biochimie 128-129:122-6
Carr, Jennifer F; Danziger, Michael E; Huang, Athena L et al. (2015) Engineering the genome of Thermus thermophilus using a counterselectable marker. J Bacteriol 197:1135-44
Carr, Jennifer F; Lee, Hannah J; Jaspers, Joshua B et al. (2015) Phenotypic Suppression of Streptomycin Resistance by Mutations in Multiple Components of the Translation Apparatus. J Bacteriol 197:2981-8
Carr, Jennifer F; Gregory, Steven T; Dahlberg, Albert E (2015) Transposon mutagenesis of the extremely thermophilic bacterium Thermus thermophilus HB27. Extremophiles 19:221-8
Demirci, Hasan; Murphy 4th, Frank V; Murphy, Eileen L et al. (2014) Structural analysis of base substitutions in Thermus thermophilus 16S rRNA conferring streptomycin resistance. Antimicrob Agents Chemother 58:4308-17
Gregory, Steven T; Connetti, Jacqueline L; Carr, Jennifer F et al. (2014) Phenotypic interactions among mutations in a Thermus thermophilus 16S rRNA gene detected with genetic selections and experimental evolution. J Bacteriol 196:3776-83
Demirci, Hasan; Murphy 4th, Frank; Murphy, Eileen et al. (2013) A structural basis for streptomycin-induced misreading of the genetic code. Nat Commun 4:1355
Demirci, Hasan; Wang, Leyi; Murphy 4th, Frank V et al. (2013) The central role of protein S12 in organizing the structure of the decoding site of the ribosome. RNA 19:1791-801

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