Structure-function relationships in stressosome complexes from Listeria monocytogenes and Bacillus subtilis

Abstract

Listeria monocytogenes is a foodborne bacterial pathogen that can resist and overcome extreme environmental conditions, such as the extremes of temperature, salinity and pH that are encountered during food processing. Stress resistance is regulated by a supramolecular protein complex called the stressosome, which detects and integrates environmental stress signals that induce a partner-switching and phosphorylation cascade leading to the activation of an alternative RNA polymerase sigma factor, σB, which controls a regulon of ~200 genes involved in the general stress response. The stressosome comprises three main proteins, RsbR (which has four paralogues), RsbS and RsbT. The N-terminal domains of RsbR proteins have been proposed to act as stress sensors and they project from the core of the stressosome as ‘turrets’. However, the mechanism by which signals are perceived and transmitted is still unknown. Structural studies of the stressosome’s sensory domains resulted in the successful determination of the crystal structures of N-RsbR1, N-RsbR2, and N-RsbR3. Ligand binding pockets were identified in N-RsbR3 that yield insight into signal perception and transduction mechanisms. The interaction of the Prli42 miniprotein with N-RsbR proteins was also assessed and shown not to occur at biologically-relevant concentrations. Common ligands and drug-like fragments were screened against binding to the putative ligand binding pocket and candidate interacting molecules were identified. Native stressosomes pulled-down from B. subtilis cell lysates by affinity purification contained all four RsbR paralogues, along with RsbS and RsbT. Initial electron microscopy of the purified native stressosomes were consistent with the formation of a highly symmetric structure. Purification and EM studies of stressosome variants revealed that stressosomes can be formed by any of the RsbR paralogues. Stressosome variants were analysed by cryo-EM single particle analysis, which showed that the RsbR-RsbS complex displays similar features to the known stressosome complex of B. subtilis, albeit with markedly different stoichiometries.