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Listeria monocytogenes is a bacterial pathogen capable of causing severe infections but also thriving outside the host. To respond to different stress conditions, L. monocytogenes mainly utilizes the general stress response regulon, which largely is controlled by the alternative sigma factor Sigma B (SigB). In addition, SigB is important for virulence gene expression and infectivity. Upon encountering stress, a large multicomponent protein complex known as the stressosome becomes activated, ultimately leading to SigB activation. RsbX is a protein needed to reset a "stressed"stressosome and prevent unnecessary SigB activation in nonstressed conditions. Consequently, absence of RsbX leads to constitutive activation of SigB even without prevailing stress stimulus. To further examine the involvement of SigB in the virulence of this pathogen, we investigated whether a strain with constitutively active SigB would be affected in virulence factor expression and/or infectivity in cultured cells and in a chicken embryo infection model. Our results suggest that increased SigB activity does not substantially alter virulence gene expression compared with the wild-type (WT) strain at transcript and protein levels. Bacteria lacking RsbX were taken up by phagocytic and nonphagocytic cells at a similar frequency to WT bacteria, both in stressed and nonstressed conditions. Finally, the absence of RsbX only marginally affected the ability of bacteria to infect chicken embryos. Our results suggest only a minor role of RsbX in controlling virulence factor expression and infectivity under these conditions.

The survival of microbial cells under changing environmental conditions requires an efficient reprogramming of transcription, often mediated by alternative sigma factors. The Gram-positive human pathogen Listeria monocytogenes senses and responds to environmental stress mainly through the alternative sigma factor σB (SigB), which controls expression of the general stress response regulon. SigB activation is achieved through a complex series of phosphorylation/dephosphorylation events culminating in the release of SigB from its anti-sigma factor RsbW. At the top of the signal transduction pathway lies a large multi-protein complex known as the stressosome that is believed to act as a sensory hub for stresses. Following signal detection, stressosome proteins become phosphorylated. Resetting of the stressosome is hypothesized to be exerted by a putative phosphatase, RsbX, which presumably removes phosphate groups from stressosome proteins post-stress.We addressed the role of the RsbX protein in modulating the activity of the stressosome and consequently regulating SigB activity in L. monocytogenes. We show that RsbX is required to reduce SigB activation/levels under non-stress conditions and that it is required for appropriate SigB mediated stress-adaptation. A strain lacking RsbX displayed impaired motility and biofilm formation, but also an increased survival at low pH. Our results could suggest that absence of RsbX alter the multi-protein composition of the stressosome without dramatically affecting its phosphorylation status. Overall the data show that RsbX plays a critical role in modulating the signal transduction pathway by blocking SigB activation under non-stressed conditions.

Listeria monocytogenes is a ubiquitous foodborne Gram-positive bacterium. Despite being mainly a soil bacterium, it can reach the food processing environment and contaminate food destined for human consumption, causing outbreaks. Because of its pathogenicity, it poses a danger for certain high-risk groups, including children, elderly, and immune-compromised people, as well as pregnant women, being capable of causing a life-threatening systemic infection known as listeriosis.

All bacteria require an efficient transcriptional response and its fine-tuned modulation in order to survive the different stresses it encounters. This is especially true for L. monocytogenes, which presents an impressive range of stress adaptions that allows it survival in certain extreme conditions such as low temperature, low pH and high osmolarity. The alternative Sigma factor B, SigB, is responsible for the expression of the general stress response of this bacterium and plays a key role in the survival and adaption to new environments. The activation of SigB requires an intricate system of partner switching mechanisms, involving anti-sigma and anti-anti-sigma factors, triggered by a number of phosphorylation and dephosphorylation events that culminates with SigB being available to interact with RNA polymerase and lead the transcription of the general stress response regulon. At the top of this signal transduction pathway lies a large multi-protein complex, known as the stressosome. It is formed by RsbR (and its paralogs), RsbS and RsbT and is believed to function as a sensory hub for environmental stimuli. After signal detection, the stressosome proteins are phosphorylated and the complex goes through conformational changes that will ultimately allow for SigB activation. The reset of the stressosome to its pre-stress conformation, is hypothesized to be exerted by a putative phosphatase, RsbX, which most likely dephosphorylates the stressosome proteins post-stress.

The role of RsbX in modulating the activity and conformation of the stressosome as well as in subsequent regulation of SigB activity was investigated. RsbX was shown to be required for maintaining SigB levels and activity low in non-stressed conditions as well as for proper SigB mediated stress adaptation. A ΔrsbX mutant strain was shown to have a very slight growth defect, but it also exhibited impaired motility, reduced biofilm formation, as well as a more acid resistant phenotype. The absence of RsbX was shown to alter the composition of the stressosome without drastically affecting its phosphorylation pattern. In general, RsbX was shown to play a crucial role in modulating the signal transduction pathways by preventing SigB activation under non-stressed conditions.

Strains that acquire sigB operon mutations have been shown to have a growth advantage under certain mild stress conditions recurrent in a laboratory set. These strains were shown to outcompete the wild-type strain when grown in these conditions, demonstrating how a deficient SigB activity poses an advantage to the cell. On the other hand, and the ΔrsbX mutant strain was shown to have a growth disadvantage, since it was outcompeted by the wild-type strain when co-cultured. The data highlights the significant cost stress protection presents to this pathogen, since deploying the general stress response is a burden on cellular resources, and in its absence the cell can redirect energy for growth. In contrast, in the presence of a lethal stress (low pH) the strains with impaired SigB activity showed a reduced survival and an overall increased sensitivity to the stress. Hence demonstrating that in a more stressful condition the high cost of the general stress response regulon is outweighed by the protection benefits it confers to the cell. The importance of RsbX, which prevents unnecessary SigB activation, is even more evident. RsbX is not only critical to shut down the general stress response post-stress and subsequent recovery of homeostasis, but it also keeps SigB activity to low levels in non-stressed conditions, avoiding unwarranted gene expression and contributing to important energy saving. 

SigB also plays an important role in the transition of L. monocytogenes from a saprophytic to a pathogenic lifestyle. Even though most of the virulence factors are under the control of PrfA, the master regulator of virulence, SigB is fundamental in the survival of the bacteria inside the host’s gastro-intestinal tract (e.g., stomach high acidity and bile salt release in the duodenum), as well as in the early stages of infection, such as internalization into not phagocytic cells. Because of the importance of SigB for virulence, we speculated if RsbX, by controlling activity of SigB, would also impact the virulence of the bacteria. The data showed somewhat contradicting results, but in general it suggests that even though the expression of the virulence genes responsible for the uptake of the bacteria are increased in a strain lacking RsbX compared with the wild-type strain, the effect on the general infectivity of this strain was either minimal or not existent at all. A reason for this could be the suggested growth defect caused by the absence of RsbX, which could also jeopardize the bacteria’s ability to efficiently grow within infected cells or organisms.

Overall, RsbX seems to play a crucial role for L. monocytogenes, since it is responsible to maintain a very important, but extremely costly, stress protection mechanism in an inactive mode in absence of stress. Its functions span from alteration of stressosome conformation and subsequent modulation of stress response, to homeostasis recovery, motility, biofilm formation, stress survival, and even to indirect impact in the bacteria’s infectivity. This shows the diversified, but impactful range of effects RsbX seems to have for the bacterial cell.

In Listeria monocytogenes, the full details of how stress signals are integrated into the σB regulatory pathway are not yet available. To help shed light on this question, we investigated a collection of transposon mutants that were predicted to have compromised activity of the alternative sigma factor B (σB). These mutants were tested for acid tolerance, a trait that is known to be under σB regulation, and they were found to display increased acid sensitivity, similar to a mutant lacking σB (ΔsigB). The transposon insertions were confirmed by whole-genome sequencing, but in each case, the strains were also found to carry a frameshift mutation in the sigB operon. The changes were predicted to result in premature stop codons, with negative consequences for σB activation, independently of the transposon location. Reduced σB activation in these mutants was confirmed. Growth measurements under conditions similar to those used during the construction of the transposon library revealed that the frameshifted sigB operon alleles conferred a growth advantage at higher temperatures, during late exponential phase. Mixed-culture experiments at 42°C demonstrated that the loss of σB activity allowed mutants to take over a population of parental bacteria. Together, our results suggest that mutations affecting σB activity can arise during laboratory culture because of the growth advantage conferred by these mutations under mild stress conditions. The data highlight the significant cost of stress protection in this foodborne pathogen and emphasize the need for whole-genome sequence analysis of newly constructed strains to confirm the expected genotype.

IMPORTANCE: In the present study, we investigated a collection of Listeria monocytogenes strains that all carried sigB operon mutations. The mutants all had reduced σB activity and were found to have a growth advantage under conditions of mild heat stress (42°C). In mixed cultures, these mutants outcompeted the wild type when mild heat stress was present but not at an optimal growth temperature. An analysis of 22,340 published L. monocytogenes genome sequences found a high rate of premature stop codons present in genes positively regulating σB activity. Together, these findings suggest that the occurrence of mutations that attenuate σB activity can be favored under conditions of mild stress, probably highlighting the burden on cellular resources that stems from deploying the general stress response.

In contrast to obligate intracellular pathogens that can remain in relatively stable host-associated environments, the soil-living bacterial pathogen Listeria monocytogenes has to sense and respond to physical and chemical cues in a variety of quite different niches. In particular, the bacterium has to survive the dramatic transition from its saprophytic existence to life within the host where nutritional stress, increased temperature, acidity, osmotic stress and the host defences present a new and challenging landscape. This review focuses on the sB and PrfA regulatory systems used by L. monocytogenes to sense the changing environment and implement survival mechanisms that help to overcome the disparate conditions within the host, but also to switch from a harmless saprophyte to an impressively effective pathogen.