Towards a better understanding of the physiological role and mechanism of the bacterial Hsp90 chaperone

Chaperone proteins are essential in all living cells to ensure protein homeostasis. Hsp90 is a major ATP-dependent chaperone highly conserved from bacteria to eukaryotes. Bacterial Hsp90 has recently been shown to be essential in stress conditions in some bacteria including our lab bacterial model Shewanella oneidensis, and to participate in the virulence of pathogenic bacteria. The goal of my PhD project is to uncover the physiological role of bacterial Hsp90 by identifying its substrates (called clients), and to understand the functional interactions between Hsp90 and other chaperones and proteases.
I first focused on the interplay between Hsp90 and the chaperone DnaK. Indeed, it was known that in vitro, bacterial Hsp90 directly interacts and collaborates with the Hsp70 chaperone DnaK to reactivate model substrate proteins. However, it was still unknown whether this collaboration is relevant in vivo with physiological substrates. To answer this point, I used site-directed mutagenesis on Hsp90 to impair DnaK binding, thereby uncoupling the activities of the two chaperones. Using two bacterial models, S. oneidensis and E. coli, we demonstrated the importance of the cooperation in vivo for physiological activities of Hsp90. We therefore demonstrate the essentiality of the direct collaboration between Hsp90 and DnaK in vivo to support client folding. In another part of my work, we also used quantitative proteomics approaches to identify new clients of Hsp90. We now have a first list of potential Hsp90 clients that are under study. Identifying the clients of Hsp90 will provide insights into the physiological role and mechanism of action of the chaperone in bacteria.