Date of Award
Spring 2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Microbiology
First Advisor
Groisman, Eduardo
Abstract
All organisms require metal ions to help carry out biological processes. Mg2+ is the most abundant divalent cation in all living cells, where it neutralizes negative charges on DNA and RNA, acts as a cofactor for enzymatic reactions, stabilizes macromolecular complexes, and fulfills numerous other essential roles. Due to the critical nature of Mg2+ in all cellular compartments, cells have developed intricate mechanisms of sensing and responding to changes in intracellular and extracellular Mg2+. Inside the cell, Mg2+ concentration in the cytoplasm impacts the synthesis, maintenance, and degradation of proteins, which perform the vast majority of biochemical functions. First, because both ribosomes and the nucleotide triphosphates ATP and GTP that power them exist in Mg2+-bound forms in cells, Mg2+ is required for the structure and function of protein synthesis machinery. Second, Mg2+ availability regulates intracellular ATP amounts, thereby affecting ATP-dependent protein chaperoning and solubilization. Finally, Mg2+ controls ATP-dependent proteolysis both globally and of specific proteins. Cells facing cytoplasmic Mg2+ starvation must thus enact major adaptations to protect protein homeostasis and survive during low Mg2+. In every domain of life, molecular chaperones play a key role in maintaining a functional proteome by aiding protein folding, protecting proteins from aggregation, targeting proteins for degradation, and facilitating assembly and disassembly of protein complexes. Chaperones are abundant and active under all investigated growth conditions and are upregulated in response to many stresses, including heat, acid, and oxidative damage. Here, our research establishes that molecular chaperones adopt fundamentally different biochemical and physiological functions during Mg2+ starvation, thereby promoting bacterial survival during infection-relevant conditions. In Chapter 1, we discuss the cellular role of Mg2+ and the mechanisms that diverse microbes use to sense and respond to cytoplasmic Mg2+ limitation, which many organisms face in their natural lifestyles. The facultative intracellular pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium), which infects a broad range of animal hosts and causes gastrointestinal disease, resides in a low Mg2+ environment when it infiltrates immune cells. Bacteria capable of overcoming Mg2+ starvation survive in host tissues, trigger host cell death, and spread to other organs. Therefore, Mg2+ availability connects protein homeostasis with virulence. In Chapter 2, we uncover how S. Typhimurium repurposes a molecular chaperone in response to low Mg2+. We determine that the widely conserved heat shock protein 70-kDa (Hsp70) chaperone DnaK associates with ribosomes and represses protein synthesis. Increased ribosome binding by DnaK is accompanied by a decrease in ribosome binding by Trigger Factor, the canonical ribosome-associated chaperone during nutrient abundance. We identify the region of DnaK required for interaction with ribosomes and reducing protein synthesis. By coordinating protein synthesis with protein folding capacity, DnaK protects protein homeostasis and promotes bacterial survival against Mg2+ starvation. How does DnaK reduce protein synthesis? In Chapter 3, we determine that DnaK slows translation elongation. The slower speed of translation elongation enhances activity of proteins undergoing synthesis, likely by providing polypeptides more time to fold. Slowed translation directly furthers survival against low Mg2+ because a DnaK-independent reduction in translation elongation speed fully restored survival to a mutant of dnaK unable to reduce protein synthesis. We engineer a variant of DnaK capable of reducing protein synthesis but not increasing protein specific activity, which leads to accumulation of insoluble protein aggregates, thereby compromising proteostasis. Our results indicate that S. Typhimurium preserves protein activity while making fewer proteins during low Mg2+ by coupling slowed translation to protein folding. During nutrient abundant conditions, expression of DnaK is coordinated with that of its cochaperones because DnaK cooperates with cochaperones to post-translationally fold proteins. However, during Mg2+ limitation, DnaK represses protein synthesis independent from the cochaperones it typically cooperates with, providing the first example of an Hsp70 chaperone functioning separately from cochaperones. In Chapter 4, we determine that S. Typhimurium selectively upregulates expression of DnaK but not cochaperones during low Mg2+ and when inside mammalian macrophages. Differential expression of dnaK and cochaperone-encoding genes is controlled by the master regulator of Mg2+ homeostasis and virulence in S. Typhimurium. While DnaK is required for survival within macrophages and macrophage killing, cochaperones are dispensable. Moreover, cochaperone-independent expression of DnaK is critical for both intramacrophage survival and host cell killing. These results reveal how differential gene control promotes virulence and establishes an independent role of DnaK in pathogenesis. Our findings provide a genetic, biochemical, and physiological basis for the central role of chaperone-mediated adaptations in microbial survival against infection-relevant stresses. The broad and high degree of conservation of the chaperones studied in this work, as well as the universal need for Mg2+ in all cells, makes it likely for our findings to extend to other species. As chaperones are essential for bacterial proliferation, virulence, and persistence, pathways identified in this study present ideal targets to reduce disease burden. Furthermore, these findings can be harnessed to improve human health: many diseases stem from dysregulated protein homeostasis, and, as established here, chaperones have the potent ability to reestablish protein homeostasis even during unfavorable conditions.
Recommended Citation
Chan, Carissa, "How Molecular Chaperones Promote Pathogen Survival" (2024). Yale Graduate School of Arts and Sciences Dissertations. 1283.
https://elischolar.library.yale.edu/gsas_dissertations/1283
