Bacterial Hsp90 chaperone: comprehensive identification of clients to reveal Hsp90 functional roles – Bact90ome

In every living organism, protein homeostasis (also called proteostasis) is ensured by a class of proteins called chaperones. One of them, Hsp90, is a very abundant ATP-dependent protein conserved from bacteria to human. In eukaryotes, Hsp90 participates to the folding and activation of hundreds of substrate proteins (called clients) including oncoproteins in cancer cells. In bacteria, despite a growing number of studies indicating its importance in bacterial physiology and virulence, very few Hsp90 clients are known. To fill this gap and better understand the role of Hsp90 in bacteria, it becomes of tremendous importance to comprehensively identify its clients.
The goal of this project is to identify the Hsp90 client proteins in bacteria in order to define the physiological role of Hsp90, its involvement in virulence, and its mechanism of action. We will focus on two bacterial models: the environmental bacterium Shewanella oneidensis that is an exquisite model for Hsp90 chaperone study, and pathogenic Escherichia coli strains in which we have shown that Hsp90 plays a major role for virulence. This project will be divided in three main axes:
(i) Using a combination of global complementary approaches from a genetic selection to proteomics approaches, we will obtain a comprehensive list of Hsp90 clients in S. oneidensis. Analyzing these clients will allow to determine in which physiological pathways Hsp90 is involved.
(ii) We will determine the mechanism of action of Hsp90 using the physiological clients identified. We will use in vitro protein refolding assays to dissect how Hsp90 participates in the folding of its clients together with other chaperones like the DnaK chaperone system. We will also study the interplay between Hsp90 and some proteases that finely control the proteome. Finally, we propose that Hsp90 is involved in protein complex assembly. This hypothesis will be evaluated on a protein complex involved in bacterial chemotaxis by combining microscopy and biochemistry approaches.
(iii) We will define how Hsp90 participates in the virulence of pathogenic E. coli strains. To do that, we will identify the Hsp90 clients involved in toxin and siderophore biosynthesis whose amount is dramatically reduced in the absence of Hsp90. Then, based on the already known eukaryotic Hsp90 inhibitors, we will identify molecules that block E. coli virulence, in animal and cultured epithelial cell infection models.
Therefore, we will demonstrate that Hsp90 is a key actor that controls proteostasis in bacteria. The project will allow a global understanding of bacterial Hsp90 and could lead to new antivirulence strategies to combat multi-resistant bacteria. This project involves three partners with complementary expertise in molecular microbiology, pathogenicity, and proteomics.