Activation of the Listeria monocytogenes Stressosome in the Intracellular Eukaryotic Environment

Abstract

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 analysed 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. Another core protein, RsbS, is however undetectable. Despite the absence of RsbS, a SigB-dependent reporter revealed that SigB activity increases gradually from early (1 h) to late (6 h) post-infection 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. IMPORTANCE The response to stress mediated by the alternative sigma factor SigB has been extensively characterized in Bacillus subtilis and Listeria monocytogenes. These bacteria sense stress using a supra-macromolecular complex, the stressosome, which triggers a cascade that releases SigB from its anti-sigma factor. Despite much structural data of the complex available and analyses performed in mutants lacking components of the stressosome or the signalling cascade, the integration of the stress signal and the dynamics of stressosome proteins following environmental changes remain poorly understood. Our study provides data at the protein level on essential stressosome components and SigB activity when L. monocytogenes, normally a saprophytic bacterium, adapts to an intracellular lifestyle. Our results support activation of the stressosome complex in intracellular bacteria. The apparent loss of the stressosome core protein RsbS in intracellular L. monocytogenes also challenges current models, favouring the idea of a unique stressosome architecture responding to intracellular host cues.

Listeria monocytogenes Requires the RsbX Protein To Prevent SigB Activation under Nonstressed Conditions

“…Stressosome-mediated activation of SigB requires a series of RsbT-mediated phosphorylation events of RsbS and RsbR1 ( 23 , 32 ). RsbR1 becomes phosphorylated at positions T175 and T209 through the kinase activity of RsbT ( 14 , 29 ). The phosphorylation pattern of RsbR1 can be monitored by using Phos-tag gels, which allow separation of different protein isoforms depending on their phosphorylation status.…”

“…Subcellular fractionation was performed as previously described ( 14 , 29 ), with some minor alterations. Cultures were grown overnight at 37°C, diluted 1:100 in 50 ml of BHI and grown at 37°C with agitation until an OD 600 of ∼0.4 was reached.…”

“…The details of the signal transduction pathway leading to SigB activation in L. monocytogenes , as well as the stressosome structure, are now becoming better understood ( 2 , 14 , 20 , 25 , 26 , 29 , 30 ). Having SigB constitutively active is, however, costly, making the bacterium less competitive, as has been observed for a strain lacking RsbX ( 30 ).…”

Acid stress signals are integrated into the σB-dependent general stress response pathway via the stressosome in the food-borne pathogen Listeria monocytogenes

“…From studies on the non-pathogenic bacterium B. subtilis we have learned that the stressosome is a dynamic protein complex whose activation depends on phosphorylation of residues in RsbRA and RsbS [50,53], although acid stress has not been investigated as a stressor in this organism. In L. monocytogenes, RsbR1 is phosphorylated on residues T175 [77] and T209, but the phosphorylation sites are not conserved in the RsbR1 paralogues (Fig 1B). In this study, we confirm the phosphorylation of RsbR1 T209 and show that loss of this phosphorylation leads to derepressed σ B activity under no-stress conditions and loss of responsiveness to acid.…”

Acid stress signals are integrated into the σB-dependent general stress response pathway via the stressosome in the food-borne pathogen Listeria monocytogenes

“…From studies on the non-pathogenic bacterium B. subtilis we have learned that the stressosome is a dynamic protein complex whose activation depends on phosphorylation of residues in RsbRA and RsbS (48, 51), although acid stress has not been investigated as a stressor in this organism. In L. monocytogenes, RsbR1 is phosphorylated on residues T175 (74) and T209, but the phosphorylation sites are not conserved in the RsbR1 paralogues (Fig 1B). In this study, we confirm the phosphorylation of RsbR1 T209 and show that loss of this phosphorylation leads to derepressed σ B activity under no-stress conditions and loss of responsiveness to acid.…”