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.