Phosphorylation State Dictates Bacterial Stressosome Assembly and Function

Area of Science:

• Bacterial pathogenesis
• Molecular microbiology
• Structural biology

Background:

• Bacteria utilize stressosomes, large protein complexes, to sense and respond to environmental stressors.
• The stressosome regulates the general stress response pathway, essential for bacterial survival and virulence.
• Understanding stressosome regulation is key to developing novel antimicrobial strategies.

Purpose of the Study:

• To elucidate the atomic structure of Listeria monocytogenes stressosomes.
• To determine the role of protein phosphorylation in stressosome assembly, activation, and function.
• To investigate the link between stressosome regulation, bacterial adaptation, and pathogenesis.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to determine stressosome structures.
• Site-directed mutagenesis to create phosphomimetic and phosphodeficient mutants.
• Functional assays including oxidative stress resistance and host cell virulence models.

Main Results:

• Atomic structures of five Listeria monocytogenes stressosomes were solved, revealing inactive and activated states.
• Phosphorylation at specific RsbR (T175, T209) and RsbS (S56) residues dictates stressosome assembly, stoichiometry, and activation dynamics.
• Phosphorylation at T175 primes activation, S56 triggers RsbT release, and T209 fine-tunes the response intensity.
• Mutants mimicking phosphorylation (T209E, S56D) enhanced stress resistance but reduced virulence.
• Mutants lacking phosphorylation (T175A, S56A) were stress-sensitive but retained virulence.

Conclusions:

• Phosphorylation acts as a critical regulatory switch controlling bacterial stressosome structure and function.
• Structural dynamics of the stressosome directly impact bacterial adaptation and virulence.
• Targeting stressosome phosphorylation offers a promising avenue for developing new antimicrobials against bacterial pathogens.