ORCID

https://orcid.org/0009-0008-8915-5733

Date of Award

Summer 2024

Language

English

Embargo Period

8-8-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Program

Biomedical Sciences

First Advisor

Anil Ojha

Committee Members

Kathleen McDonough, Rajendra Agrawal, Janice Pata, Gabriele Fuchs, Richard Cole

Keywords

Microbiology, Tuberculosis, Persisters, Ribosome Hibernation, Infectious Diseases, Public Health

Subject Categories

Bacterial Infections and Mycoses | Bacteriology | Biology | Genetics | Microbiology | Molecular Genetics | Pathogenic Microbiology | Public Health | Respiratory Tract Diseases

Abstract

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, is a prescient global health threat and the leading cause of death by an infectious agent. Treatment of Mtb infection involves administering 2-4 antibiotics for a minimum of 6 months. This extended drug regimen is required to target a subpopulation of persistent bacilli. As their name implies, these bacteria can “persist” by acquiring nonhereditary and transient antibiotic tolerance. Although the precise mechanism is unknown, it’s often attributed to slowing cellular growth and metabolic processes. During zinc deprivation, bacteria can remodel their ribosomes by replacing their zinc-binding paralogues containing the CXXC motif (C+) with ribosomal protein paralogues that lack this motif and do not bind to zinc (C-). The C- paralogues in mycobacteria are located within an operon regulated by the zinc uptake regulator, Zur. During zinc starvation, the mycobacterial protein Y, Mpy binds to the decoding center of the 30S subunit of the C- ribosome, stabilizing 70S inactive and hibernating monosomes. Mpy binding to the ribosome is facilitated by the zinc-binding and regulated protein, Mpy recruitment factor, Mrf. Mrf is co-transcribed with the C- proteins but, in its zinc-bound form, is recognized and degraded by the caseinolytic protease (Clp) system in growth-permissive zinc concentrations. In its zinc-free form, Mrf binds to the 30S subunit to help recruit Mpy to the ribosome. Because Mpy is constitutively expressed, we used Mrf to create a novel fluorescent reporter to visualize zinc-starved mycobacteria with physiological conditions promoting ribosome hibernation in vitro and in vivo. Using this surrogate reporter system, we demonstrated that the subpopulation of cells with Mpy-dependent ribosome hibernation contributed to ~50-60% of the overall mycobacterial population in vitro. Cells expressing the reporter exhibited Mpy-dependent metabolic slowdown and growth arrest.

Furthermore, we determined that Mpy-dependent ribosome hibernation contributes to nascent polypeptide translation attenuation and helps to prevent oxidative stress. In a high-burden, C3HeB/FeJ mouse model of chronic infection, we observed ~2-3% of the bacterial population expressing our fluorescent reporter. Upon treatment with isoniazid, the percentage of bacteria fluorescing increased from 2% to ~40% in an Mpy-dependent manner. Using both the Mtb susceptible mouse strain, C3HeB/FeJ, and the Mtb resistant mouse strain, C57BL/6, we demonstrated that extensive neutrophil infiltration likely causes zinc-limited extracellular niches for which these bacilli reside. Through transcriptomics, we identified the zinc importer, ZIP2, and the metal chelator, S100A8/A9, as likely contributing factors depleting zinc from the extracellular environment, creating zinc-starved Mtb with Mpy-dependent hibernating ribosomes. These findings contribute to developing a new paradigm in which a subpopulation of non-replicating persisters of Mtb emerges in vivo through zinc chelation by extensive neutrophil infiltration during high-burden chronic infections. Our studies provide a novel means to study these bacteria by utilizing a fluorescent reporter system and microscopic visualization.

License

This work is licensed under the University at Albany Standard Author Agreement.

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