Stress-dependent activation of the <i>Listeria monocytogenes</i> virulence program ensures bacterial resilience during infection

<i>Listeria monocytogenes (Lm</i>) is a Gram-positive, facultative intracellular pathogen that uses both a housekeeping (P1) and stress-activated (Sigma B-dependent) promoter (P2) to express the master virulence regulator PrfA. The Sigma B regulon contains over 300 genes known to respond to different stressors. However, the role of Sigma B in the regulation of <i>prfA</i> during the infection remains uncertain. To define pathways that lead to Sigma B-dependent <i>prfA</i> activation, we performed a genetic screen in L2 fibroblasts using ΔP1 <i>Lm</i> that only has the Sigma B-dependent promoter directly upstream of <i>prfA</i>. The screen identified transposon insertions in a large bacterial sensory organelle known as the stressosome. The absence of functional stressosome components resulted in heterogeneity within bacterial populations, with some bacteria behaving like wild type, while other members of the population exhibited defects in either vacuolar escape and/or cell-to-cell spread. We show that the heterogeneity of the stressosome mutants cannot be rescued by constitutive activation of PrfA. These data defined the importance of the stressosome in controlling bacterial homogeneity and characterized the function of the stressosome in robust virulence activation during infection. ΔP1 <i>Lm</i> model provides new opportunities to identify host-specific signals necessary for stressosome-dependent signaling during <i>Listeria</i> pathogenesis.IMPORTANCEMicrobial pathogens must adapt to varying levels of stress to survive. This study uncovered a link between stress sensing and activation of the virulence program in a facultative intracellular pathogen, <i>Listeria monocytogenes</i>. We show that host-imposed stress is sensed by the signaling machinery known as the stressosome to ensure robust and resilient virulence responses <i>in vivo</i>. Stressosome-dependent activation of the master virulence regulator PrfA was necessary to maintain <i>L. monocytogenes</i> homogeneity within the bacteria population during the transition between early and late stages of intracellular infection. This work also provides a model to further characterize how specific stress stimuli affect bacterial survival within the host<i>,</i> which is critical for our understanding of bacterial pathogenesis.