Activation of the Listeria monocytogenes Stressosome in the Intracellular Eukaryotic Environment.

Listeria monocytogenes is a ubiquitous environmental bacterium and intracellular pathogen that responds to stress using predominantly the alternative sigma factor SigB. Stress is sensed by a multiprotein complex, the stressosome, extensively studied in bacteria grown in nutrient media. Following signal perception, the stressosome triggers a phosphorylation cascade that releases SigB from its anti-sigma factor. Whether the stressosome is activated during the intracellular infection is unknown. Here, we analyzed the subcellular distribution of stressosome proteins in L. monocytogenes located inside epithelial cells following their immunodetection in membrane and cytosolic fractions prepared from intracellular bacteria. Unlike bacteria in laboratory media, intracellular bacteria have a large proportion of the core stressosome protein RsbR1 associated with the membrane. However, another core protein, RsbS, is undetectable. Despite the absence of RsbS, a SigB-dependent reporter revealed that SigB activity increases gradually from early (1 h) to late (6 h) postinfection times. We also found that RsbR1 paralogues attenuate the intensity of the SigB response and that the miniprotein Prli42, reported to tether the stressosome to the membrane in response to oxidative stress, plays no role in associating RsbR1 to the membrane of intracellular bacteria. Altogether, these data indicate that, once inside host cells, the L. monocytogenes stressosome may adopt a unique configuration to sense stress and to activate SigB in the intracellular eukaryotic niche.