Overlaps in the regulatory networks of Listeria monocytogenes in response to environmental cues
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The facultative intracellular pathogen Listeria monocytogenes is a highly adaptable organism widely distributed in the environment. The ingestion of food contaminated with L. monocytogenes by at-risk individuals can ultimately lead to listeriosis, one of the leading causes of foodborne fatalities in developed countries. Regulatory networks are crucial for the adaptation and survival of this pathogen. The accessory gene regulator (Agr) system has been shown to be involved in virulence, biofilm formation, and survival of L. monocytogenes, affecting the transcription of over 700 genes. The alternative sigma factor Sigma B (sB) controls the general stress response in L. monocytogenes, regulating the transcription of almost 300 genes in response to stress. Earlier studies have suggested a regulatory overlap between the AgrA and the σB regulons in L. monocytogenes. While the functions of some regulatory systems are well understood, the biological role of most small regulatory RNAs (sRNAs) remains poorly characterized. This thesis aimed to decipher the interconnections between the AgrA and σB regulons in the regulatory network of L. monocytogenes, including the possible involvement of sRNAs. Growth was observed in sterile soil and similar population dynamics were shown in the wild- type (WT) strain, ΔagrA and ΔsigB mutants. In biotic soil microcosms, viability of the WT strain declined steadily, underlining the challenging nature of live soil environments. The inactivation of the two systems simultaneously (∆agrA∆sigB) further affected survival. Transcriptional analysis confirmed the expected effects of the mutations on known Agr- and σB-dependent genes. The ability to colonise the rhizosphere was also significantly compromised in the double mutant. Data highlighted the important role that these global regulatory systems play in the natural ecology of this pathogen. A decrease in cell attachment was observed as a consequence of the Agr system inactivation. Using strains constructed to carry fluorescent reporters of either Agr or σB activity the spatiotemporal regulation of these systems was followed during biofilm formation. Deletion of the Agr system is beneficial for biofilm production under osmotic stress. Results also suggested a role for σB in biofilm formation that depended on the environmental conditions encountered. Delayed activation of agrA was shown in the ∆sigB background. Deletion of sigB resulted in no activation of Agr in biofilm produced under osmotic stress. Overall, both Agr and σB systems were found to contribute to L. monocytogenes biofilm formation. Using a combination of in silico and in vivo approaches, a binding interaction was predicted between the sB-dependent sRNA Rli47 and the Shine-Dalgarno region of the ilvA mRNA, which encodes threonine deaminase, an enzyme required for branched-chain amino acid biosynthesis. Both ilvA transcript levels and threonine deaminase activity were increased in the Drli47 mutant. The Drli47 mutant displayed a shorter growth lag in isoleucine-depleted media than the WT, and a similar phenotype was also observed in a mutant lacking sB. RNA- seq analysis uncovered a significant role for Rli47 in modulating amino acid metabolism. The data point to a model where Rli47 is responsible for specifically repressing isoleucine biosynthesis as a way to restrict growth under harsh conditions, contributing to the survival of L. monocytogenes in niches both outside and within the mammalian host. The diversity of global regulators as well as the crosstalk between the various regulatory systems in L. monocytogenes add complexity to its transcriptional networks under the most diverse environments. Understanding how individual stress signals are sensed and transcriptomic rearrangements achieved for a quick adaptation response might be a critical step in helping to develop strategies to prevent L. monocytogenes growth, survival, and dissemination.
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