Capturing Antibiotic Resistant Ribosomes
Abstract
The universally conserved nucleotide (A2058) of 23S rRNA in all bacterial ribosomes, when methylated (m6A2058),causes cross-resistance to multiple families of therapeutically important antibiotics. The abundance and essentiality of ribosomes make them an attractive target for the detection of resistant pathogens based on the unique m6A2058 signature. The goal of this study is to develop immunoreagents and synthetic binders that can be used as a rapid diagnostic tool for antibiotic resistant bacteria. They can also be used as a capturing tool to isolate homogenous populations of m6A2058-ribosomes for structural and biochemical determination, a critical step to delineate the molecular mechanisms of new ribosome-targeting antibiotics. In this 18-month project, our success in the first phase (first 12 months, Aims 1-2) has offered a strong proof-of-principle to further improve the immunoreagents that specifically recognize the resistant ribosomes bearing them6A2058 modification. However, extending the oligonucleotide helper did not improve the specificity and binding of the antibody-oligo conjugate, possibly because the additional antisense regions are inaccessible to the A2058 nucleotide inside the fully assemble ribosomes. We generated anti-m6A2058 antibody and successfully used it for immunoprecipitation to perform selective ribosome profiling. In the final 6 months, we performed an in vitro phage display screen (Aim 3) to identify several synthetic peptide binders of m6A2058 rRNA, one of these peptides was labeled with fluorescent dye, allowing in situ discrimination of m6A2058 rRNA from unmethylated rRNA.
Document Details
- Document Type
- Technical Report
- Publication Date
- May 18, 2020
- Accession Number
- AD1115137
Entities
People
- Mee-Ngan F Yap
Organizations
- Northwestern University