How Molecular Chaperones Promote Pathogen Survival During Starvation

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PROJECT SUMMARY All life forms synthesize and maintain proteins to carry out fundamental cell processes. Protein synthesis and maintenance require tremendous energy and resources, including Mg2+, the most abundant divalent cation in living cells. Microbial pathogens often face nutrient limitation inside mammalian macrophages and must restore protein homeostasis to persist in host tissues. I propose to determine how the facultative intracellular pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) uses molecular chaperones to control protein homeostasis, thereby enabling survival during Mg2+ starvation. I discovered two novel functions for DnaK, the highly conserved molecular chaperone that functions with cochaperones in folding proteins under nutrient-replete conditions. First, I established that, surprisingly, DnaK represses protein synthesis by binding ribosomes in a cochaperone-independent manner when S. Typhimurium experiences low Mg2+, thereby helping conserve energy and resources. And second, I determined that DnaK antagonizes the canonical ribosome-associated chaperone Trigger Factor, assuming its role in cotranslational folding of nascent polypeptides also during low Mg2+. I will now elucidate the mechanism by which DnaK takes over cotranslational polypeptide folding from Trigger Factor during Mg2+ starvation; and identify the physiological benefits of this novel DnaK function. I will also examine how Mg2+ starvation changes the balance between the canonical DnaK/DnaJ/GrpE and GroEL/GroES chaperone systems that act on existing proteins because protein homeostasis involves not only synthesis of new proteins but also maintenance of existing proteins. By altering the activities of these two chaperone systems, S. Typhimurium is hypothesized to maintain certain proteins soluble and active, and other proteins insoluble and inactive. The starvation-induced transition to a slow growth state renders bacteria phenotypically resistant to antibacterial agents, hindering the cure of bacterial infections. The molecular and physiological results from this research will reveal novel control of chaperone-mediated adaptations in human pathogens. Moreover, the universal nature of Mg2+ dependence, chaperones, and protein homeostasis makes this study widely applicable to diverse organisms across all domains of life.