Regulation and evolution of the σB stress sensing pathway in Listeria monocytogenes: impact on stress tolerance, fitness and virulence
Neves Guerreiro, Duarte João
Neves Guerreiro, Duarte João
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Publication Date
2022-06-15
Type
Thesis
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Abstract
The Gram-positive bacterium Listeria monocytogenes is the aetiological agent of listeriosis, a high death rate infection in susceptible groups, caused by the ingestion of contaminated food. L. monocytogenes is a highly adaptable bacterium capable of withstanding a wide range of environmental assaults. This feature is partially attributed to the upregulation of the General Stress Response (GSR) regulon comprising approximately 300 genes regulated by the alternative sigma factor B (σB). Under stressful conditions, the stressosome, a multi-protein complex and a putative sensory organelle in L. monocytogenes, was hypothesized to activate the downstream signal cascade that ultimately liberates σ B from its anti-sigma factor and upregulates the GSR. In this study, the role of the stressosome as a stress sensor and its impact on the activation of σ B, growth, survival, and virulence of L. monocytogenes was assessed. Mild stresses activate σ B and enhance L. monocytogenes resistance towards extreme stresses in exchange for growth rate. Herein, it was shown that growth temperatures of 42°C, which are routinely used in the laboratory to construct mutant strains, promote the emergence and proliferation of σ B lossof-function alleles. In contrast, lower temperatures of 30°C promote the emergence of Agr loss-of-function alleles instead of the σ B operon. In both cases, these mutations inactivated the respective systems and conferred a competitive advantage at the respective temperatures. Additionally, the analysis of all publicly available whole genome sequences of L. monocytogenes strains allowed the identification of a high rate of premature stop codons at both sigB and agr operons. Overall, the results obtained suggest that both σ B and Agr are subjected to a negative selective pressure in certain natural habitats as well as in laboratory conditions. The stressosome components, RsbR1 and its paralogues, RsbS and RsbT, were genetically manipulated and their sensory response towards mild acidic stress (pH 5.0) was assessed through the expression of highly σ B-dependent genes, such as lmo2230 and lmo0596. It was determined that the phosphorylation of RsbS S56 by the action of the kinase RsbT was essential for the transduction of the acid stress signal and increase of stress resistance at extreme acidic conditions (pH 2.5). Moreover, RsbR1 T209 and its putative interaction with RsbR1 T241 are essential for the suppression of the signal propagation within the stressosome core and consequently for controlling the intensity of the σ B activity. Furthermore, the stressosome controls the transcription of the internalins inlA and inlB and modulates the internalization of L. monocytogenes on epithelial cells. The sensory properties of the stressosome under low pH are also essential for the upregulation of homeostatic mechanisms that contribute to the acid tolerance of L. monocytogenes, such as the glutamate decarboxylase, arginine, and agmatine deiminase systems. Overall, the activation of σ B often results in a trade-off between stress resistance and growth rate under mild stresses, culminating in the emergence of alleles that impair the normal activation of σ B. Furthermore, the stressosome and the downstream signal cascade are not only responsible for activating σ B, but also for modulating its intensity under mild acidic pH. The stressosome is essential for the perception of environmental shifts and respond by quickly enhancing its stress resistance L. monocytogenes against imminent environmental assaults.
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NUI Galway