# Alternative Ribosomes & Antibiotic Tolerance in Mycobacteria

> **NIH NIH R01** · WADSWORTH CENTER · 2024 · $454,818

## Abstract

Summary
Changes in abundance of zinc, an essential nutrient but toxic at high concentrations, necessitates
adaptive mechanisms in mycobacteria. Under zinc-rich conditions, mycobacteria synthesize
ribosomes with a set of ribosomal (r-) proteins containing the zinc-binding CXXC (C+) motif, while
zinc starvation induces paralogs of these proteins called C- (for the lack of CXXC motif) as
replacements in the 70S ribosome. Evidence of C+ to C- ribosome remodeling is reported in Mtb from
chronic lung infections in mice and humans. Clinical relevance of ribosome remodeling is supported
by greater antibiotic resistance in the C- than C+ ribosomes, highlighting the challenges in using
ribosome targeting drugs in TB therapy. Previously, we solved the structures of C+ and C- ribosomes
to decipher the mechanism of their differential antibiotic resistance. We discovered the C- ribosome
as the preferred target of mycobacterial protein Y (Mpy). Mpy is a member of highly conserved family
of bacterial protein Y, which binds to the decoding region of the ribosome, thereby hibernating non-
translating ribosomes as 70S monosomes in growth arrested cells. The structure identified two
distinct drug resistance mechanisms associated with the C- ribosome. Aminoglycoside resistance is
largely associated with the preferential binding of Mpy to the decoding center of the C- ribosome,
which directly impacts the streptomycin and kanamycin binding pockets within the C- ribosome.
Unlike aminoglycosides, tolerance to spectinamides is a direct consequence of the structural changes
in the 30S ribosomal subunit due to C+ to C- substitution of the r-protein, S14. During our investigation
of ribosome hibernation as a specific response to zinc starvation, we discovered a zinc-sensing Mpy-
recruitment factor (Mrf), that ensures Mpy recruitment to C- ribosome. The mechanism by which Mpy
is recruited to C- ribosomes, resulting in ribosome hibernation, remains unknown. In this proposal,
we will specifically focus on: a) deciphering the mechanism of Mpy recruitment to the ribosome, and
b) identifying the mechanistic basis for Mpy-dependent and -independent drug resistance in
mycobacteria expressing C- ribosomes. The findings will expand our understanding of antibiotic
tolerance associated with ribosome remodeling and hibernation in zinc-starved mycobacteria. Given
that lung environment of Mtb in humans is likely zinc-limiting, our findings will inform and improve the
therapeutic strategies for TB, possibly leading to a shorter treatment regimen.

## Key facts

- **NIH application ID:** 10932987
- **Project number:** 5R01AI132422-07
- **Recipient organization:** WADSWORTH CENTER
- **Principal Investigator:** RAJENDRA K AGRAWAL
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $454,818
- **Award type:** 5
- **Project period:** 2017-06-01 → 2028-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10932987

## Citation

> US National Institutes of Health, RePORTER application 10932987, Alternative Ribosomes & Antibiotic Tolerance in Mycobacteria (5R01AI132422-07). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10932987. Licensed CC0.

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