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

Abstract

The general stress response (GSR) in Listeria monocytogenes plays a critical role in the survival of this pathogen in the host gastrointestinal tract. The GSR is regulated by the alternative sigma factor B (σB), whose role in protection against acid stress is well established. Here, we investigated the involvement of the stressosome, a sensory hub, in transducing low pH signals to induce the GSR. Mild acid shock (15 min at pH 5.0) activated σB and conferred protection against a subsequent lethal pH challenge. A mutant strain where the stressosome subunit RsbR1 was solely present retained the ability to induce σB activity at pH 5.0. The role of stressosome phosphorylation in signal transduction was investigated by mutating the putative phosphorylation sites in the core stressosome proteins RsbR1 (rsbR1-T175A, -T209A, -T241A) and RsbS (rsbS-S56A), or the stressosome kinase RsbT (rsbT-N49A). The rsbS S56A and rsbT N49A mutations abolished the response to low pH. The rsbR1-T209A and rsbR1-T241A mutants displayed constitutive σB activity. Mild acid shock upregulates invasion genes inlAB and stimulates epithelial cell invasion, effects that were abolished in mutants with an inactive or overactive stressosome. Overall, the results show that the stressosome is required for acid-induced activation of σB in L. monocytogenes. Furthermore, they show that RsbR1 can function independently of its paralogues and signal transduction requires RsbT-mediated phosphorylation of RsbS on S56 and RsbR1 on T209 but not T175. These insights shed light on the mechanisms of signal transduction that activate the GSR in L. monocytogenes in response to acidic environments, and highlight the role this sensory process in the early stages of the infectious cycle.

The dynamics and function of stressosome proteins in Listeria monocytogenes

“…The D L falls in the range of characterized DNA/RNA-binding proteins 45,46 , ribosomes and ribosome-binding proteins 36,37,40,47 , which suggests that the diffusion coefficient reflects the mobility of RsbR1 present in supramolecular complexes with RsbS (and probably RsbT) (RsbR1-RsbS-RsbT); the full stressosome complex has a molecular mass (1.8 MDa) comparable to that of ribosomes 22 . We also determined the mobility of RsbR1 under mild acid stress conditions (pH 5.0) as the protein has been reported to be involved in acid stress sensing in L. monocytogenes 35 . Mild acid stress triggers enhanced expression of listerial virulence genes expression and invasiveness in epithelial cells 48,49 .…”

“…Since RsbR1 senses acid stress independently of the presence of paralogous RsbRs 35 , we tested whether or not the diffusion of RsbR1 was affected by subjecting cells to a mild acidic condition at pH 5.0 (Fig. 3f).…”

The stressosome is required to transduce low pH signals leading to increased transcription of the amino acid-based acid tolerance mechanisms in Listeria monocytogenes

“…The foodborne pathogen Listeria monocytogenes , the aetiological agent of listeriosis, is a robust bacterium capable of surviving in harsh environments including the extremely low pH of the human stomach [ 1, 2 ]. L. monocytogenes senses acidification of the environment through a multi-protein complex designated as the stressosome, composed of putative sensory proteins RsbR1 and its four paralogues, the scaffold protein RsbS and the serine–threonine kinase RsbT, which is responsible for the phosphorylation of RsbR1 and RsbS [ 3, 4 ]. Under stressful conditions, the stressosome activates a signal cascade that ultimately releases the alternative sigma factor B (σ B ) from an anti-sigma factor, culminating in the upregulation of approximately 300 genes that comprise the general stress response (GSR) regulon.…”

“…σ B probably contributes to this response since it is activated at the same low pH values that trigger the ATR, although some researchers have suggested that it is not the main regulator controlling the response [ 16 ]. In a recent study, we demonstrated the pivotal role of the stressosome in the sensing of low pH and the subsequent activation of σ B [ 3 ]. Pre-treating mid-log phase cultures at pH 5 for 15 min increased the transcription of highly σ B -dependent genes lmo2230 and lmo0596 , and enhanced L. monocytogenes acid tolerance in a stressosome-dependent manner.…”

“…Pre-treating mid-log phase cultures at pH 5 for 15 min increased the transcription of highly σ B -dependent genes lmo2230 and lmo0596 , and enhanced L. monocytogenes acid tolerance in a stressosome-dependent manner. The genes lmo2230 and lmo0596 encode a putative arsenate reductase and a transmembrane protein with unknown function, respectively [ 3 ]. It is currently unknown whether the stressosome is required for upregulation of the amino acid-based acid resistance mechanisms in response to acidification of the medium, although σ B is known to play a role in regulating some elements of this system, including the glutamate decarboxylase (GAD) system [ 17 ] and the arginine deiminase (ADI) system [ 18 ] ( Fig.…”