Phosphorylation State Dictates Bacterial Stressosome Assembly and Function

Bacterial pathogens rely on their ability to sense and respond to environmental stressors to survive and maintain virulence. The stressosome, a 1.8-megadalton nanomachine, serves as a critical sensor and regulator of the general stress response. It is composed of multiple copies of three proteins RsbR, RsbS, and the kinase RsbT which together orchestrate activation of downstream stress adaptation pathways. Using cryo-electron microscopy, we solved the atomic structure of five Listeria monocytogenes stressosomes, capturing structural mimics of the transition between inactive and activated states using phosphomimetic and phosphodeficient mutants. Our findings reveal that phosphorylation at specific residues T175 and T209 on RsbR, and S56 on RsbS dictates stressosome assembly, stoichiometry, and activation. Specifically, phosphorylation at T175 primes the stressosome for activation, while S56 phosphorylation destabilizes the core, triggering the release of RsbT to propagate the stress response. In contrast, phosphorylation at T209 modulates stressosome composition and appears to fine-tune the intensity of the stress response. Functional analyses reveal that phosphomimetic mutants (T209E, S56D) resist oxidative stress but lose virulence in host cell model, while phosphodeficient mutants (T175A, S56A) are stress-sensitive but retain virulence. These findings establish phosphorylation as a central regulatory switch linking structural dynamics to bacterial adaptation and pathogenesis, highlighting potential targets for antimicrobial intervention.