Novel Mechanisms of Protein Homeostasis by the Molecular Chaperone DnaK

PROJECT SUMMARY Proteins carry out almost all functions in all living cells. At the same time, cells must coordinate protein synthesis with folding capacity to avoid misfolding and the accumulation of highly toxic protein aggregates. Critically, chaperones enable most proteins to adopt conformations required for essential biological activities. In bacteria, the chaperone trigger factor (TF), which is as abundant as ribosomes, had long been thought responsible for cotranslational protein folding when nutrients are plentiful. However, we have now identified cytoplasmic Mg2+ starvation as an infection-relevant stress condition in which the facultative intracellular pathogen Salmonella enterica serovar Typhimurium requires Hsp70 chaperone DnaK instead of TF. Surprisingly, DnaK directly decreases protein synthesis, thus conferring survival against cytoplasmic Mg2+ starvation. This is the first example of DnaK performing protein homeostasis activities independently of its J-domain cochaperones and nucleotide exchange factor. We now propose to combine genetic, biochemical, and biophysical approaches to determine how DnaK decreases protein synthesis, in course solving the structure of DnaK-bound ribosomes harvested during cytoplasmic Mg2+ starvation. We will use selective ribosome profiling to identify nascent polypeptides associated with DnaK versus TF during cytoplasmic Mg2+ starvation and abundance. We will also interrogate DnaK binding to the ribosome as necessary for bacterial resistance to proline-rich antimicrobial peptides that attack the translation machinery and for survival against cytoplasmic Mg2+ starvation and inside murine macrophages. The proposed experiments will provide fundamental tools and knowledge to solve how slower protein synthesis enables bacterial survival during infection. Moreover, results will apply broadly to diverse microbes as this physiologically important chaperone is widely distributed, shares high amino acid sequence identity across species, and is required for virulence by multiple pathogens.