RsbX and stress response in Listeria monocytogenes

Sammanfattning: 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.