| 1. |
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| 2. |
- de Oliveira, Ana Henriques, et al.
(författare)
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Listeria monocytogenes requires the RsbX protein to prevent SigB-activation under non-stressed conditions
- 2022
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Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 204:1
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- de Oliveira, Ana H., 1995-, et al.
(författare)
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The virulence and infectivity of Listeria monocytogenes are not substantially altered by elevated SigB activity
- 2023
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Ingår i: Infection and Immunity. - : American Society for Microbiology. - 0019-9567 .- 1098-5522. ; 91:6
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 4. |
- Gripenland, Jonas, et al.
(författare)
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RNAs : regulators of bacterial virulence
- 2010
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Ingår i: Nature Reviews Microbiology. - : Springer Science and Business Media LLC. - 1740-1526 .- 1740-1534. ; 8:12, s. 857-866
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Tidskriftsartikel (refereegranskat)abstract
- RNA-based pathways that regulate protein expression are much more widespread than previously thought. Regulatory RNAs, including 5' and 3' untranslated regions next to the coding sequence, cis-acting antisense RNAs and trans-acting small non-coding RNAs, are effective regulatory molecules that can influence protein expression and function in response to external cues such as temperature, pH and levels of metabolites. This Review discusses the mechanisms by which these regulatory RNAs, together with accessory proteins such as RNases, control the fate of mRNAs and proteins and how this regulation influences virulence in pathogenic bacteria.
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| 5. |
- Guerreiro, Duarte N., et al.
(författare)
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Acid stress signals are integrated into the σb-dependent general stress response pathway via the stressosome in the food-borne pathogen Listeria monocytogenes
- 2022
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Ingår i: PLoS Pathogens. - : Public Library of Science. - 1553-7366 .- 1553-7374. ; 18:3
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Tidskriftsartikel (refereegranskat)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 (rsbTN49A). 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.
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| 6. |
- Guerreiro, Duarte N., et al.
(författare)
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Mild Stress Conditions during Laboratory Culture Promote the Proliferation of Mutations That Negatively Affect Sigma B Activity in Listeria monocytogenes
- 2020
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Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 202:9
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Loh, Edmund, 1981-, et al.
(författare)
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A trans-acting riboswitch controls expression of the virulence regulator PrfA in Listeria monocytogenes
- 2009
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Ingår i: Cell. - : Elsevier BV. - 0092-8674 .- 1097-4172. ; 139:4, s. 770-779
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Tidskriftsartikel (refereegranskat)abstract
- Riboswitches are RNA elements acting in cis, controlling expression of their downstream genes through a metabolite-induced alteration of their secondary structure. Here, we demonstrate that two S-adenosylmethionine (SAM) riboswitches, SreA and SreB, can also function in trans and act as noncoding RNAs in Listeria monocytogenes. SreA and SreB control expression of the virulence regulator PrfA by binding to the 5´-untranslated region of its mRNA. Absence of the SAM riboswitches SreA and SreB increases the level of PrfA and virulence gene expression in L. monocytogenes. Thus, the impact of the SAM riboswitches on PrfA expression highlights a link between bacterial virulence and nutrient availability. Together, our results uncover an unexpected role for riboswitches and a distinct class of regulatory noncoding RNAs in bacteria.
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| 8. |
- O'Donoghue, Beth, et al.
(författare)
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Blue-Light Inhibition of Listeria monocytogenes Growth Is Mediated by Reactive Oxygen Species and Is Influenced by sigma(B) and the Blue-Light Sensor Lmo0799
- 2016
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Ingår i: Applied and Environmental Microbiology. - 0099-2240 .- 1098-5336. ; 82:13, s. 4017-4027
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Tidskriftsartikel (refereegranskat)abstract
- Listeria monocytogenes senses blue light via the flavin mononucleotide-containing sensory protein Lmo0799, leading to activation of the general stress response sigma factor SigB (sigma(B)). In this study, we investigated the physiological response of this foodborne pathogen to blue light. We show that blue light (460 to 470 nm) doses of 1.5 to 2 mW cm(-2) cause inhibition of growth on agar-based and liquid culture media. The inhibitory effects are dependent on cell density, with reduced effects evident when high cell numbers are present. The addition of 20 mM dimethylthiourea, a scavenger of reactive oxygen species, or catalase to the medium reverses the inhibitory effects of blue light, suggesting that growth inhibition is mediated by the formation of reactive oxygen species. A mutant strain lacking sigma(B) (Delta sigB) was found to be less inhibited by blue light than the wild type, likely indicating the energetic cost of deploying the general stress response. When a lethal dose of light (8 mW cm(-2)) was applied to cells, the Delta sigB mutant displayed a marked increase in sensitivity to light compared to the wild type. To investigate the role of the blue-light sensor Lmo0799, mutants were constructed that either had a deletion of the gene (Delta lmo0799) or alteration in a conserved cysteine residue at position 56, which is predicted to play a pivotal role in the photocycle of the protein (lmo0799 C56A). Both mutants displayed phenotypes similar to the Delta sigB mutant in the presence of blue light, providing genetic evidence that residue 56 is critical for light sensing in L. monocytogenes. Taken together, these results demonstrate that L. monocytogenes is inhibited by blue light in a manner that depends on reactive oxygen species, and they demonstrate clear light-dependent phenotypes associated with sigma(B) and the blue-light sensor Lmo0799.
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| 9. |
- Tiensuu, Teresa, 1970-
(författare)
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Cell fate specification by Ras-mediated cell signalling in C. elegans
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Induction of vulval fates in the C. elegans hermaphrodite is mediated by a conserved RTK/Ras/MAP kinase signalling pathway, in which the core components can be placed into a linear genetic and biochemical pathway. However, the events that occur downstream of this pathway are not yet well understood. This thesis describes studies on three genes, lin-1, lin-25 and sur-2 that function genetically downstream of the RTK/Ras/MAP kinase pathway in vulva induction. lin-1 encodes an ETS protein that appears to be a direct target of the RTK/Ras/MAP kinase pathway during the induction of vulval fates. To understand more in detail how Ras signalling in C. elegans affects cell fate specification we have analysed the effects of lin-1 mutations on various Ras-mediated cell fate specification events. Our results show that lin-1, besides its function in vulval induction, functions in most other Ras-mediated cell fate specification events in C. elegans, and that lin-1 appears to have a negative function in a majority of these events. Two other genes, lin-25 and sur-2, also function genetically downstream of the RTK/Ras/MAP kinase pathway during induction of vulval fates. Previously, two different models have been proposed for the function of these genes (I) that they function together with a gene in the homeotic cluster to specify the identity of the vulval precursor cells or (II) that they constitute components of the RTK/Ras/MAP kinase signalling pathway. To help clarify the role of lin-25 and sur-2, we have caried out studies of the effects of lin-25 and sur-2 mutations on other cells in the worm in which the RTK/Ras/MAP kinase pathway functions. The results exclude the possibility that lin-25 and sur-2 solely function in vulva induction and suggest that the two genes are intimately involved in Ras-mediated signalling. In addition we show that the major focus for lin-25 during vulval induction is in the vulva precursor cells themselves. Furthermore, results presented here suggest that LIN-25 and SUR-2 function together in the same process in the cell. We show here by both genetic and immunological experiments that LIN-25 is associated with Mediator in C. elegans, a multiprotein complex required for transcriptional regulation. Taken together, these results suggest that lin-25 and sur-2 function in regulating transcription of genes in response to Ras signalling.
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| 10. |
- Tiensuu, Teresa, et al.
(författare)
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Cycles of light and dark co-ordinate reversible colony differentiation in Listeria monocytogenes
- 2013
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 87:4, s. 909-924
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Tidskriftsartikel (refereegranskat)abstract
- Recently, several light receptors have been identified in non-phototrophic bacteria, but their physiological roles still remain rather elusive. Here we show that colonies of the saprophytic bacterium Listeria monocytogenes undergo synchronized multicellular behaviour on agar plates, in response to oscillating light/dark conditions, giving rise to alternating ring formation (opaque and translucent rings). On agar plates, bacteria from opaque rings survive increased levels of reactive oxygen species (ROS), as well as repeated cycles of light and dark, better than bacteria from translucent rings. The ring formation is strictly dependent on a blue-light receptor, Lmo0799, acting through the stress-sigma factor, sigma B. A transposon screening identified 48 mutants unable to form rings at alternating light conditions, with several of them showing a decreased sigma B activity/level. However, some of the tested mutants displayed a varied sigma B activity depending on which of the two stress conditions tested (light or H2O2 exposure). Intriguingly, the transcriptional regulator PrfA and the virulence factor ActA were shown to be required for ring formation by a mechanism involving activation of sigma B. All in all, this suggests a distinct pathway for Lmo0799 that converge into a common signalling pathway for sigma B activation. Our results show that night and day cycles co-ordinate a reversible differentiation of a L.monocytogenes colony at room temperature, by a process synchronized by a blue-light receptor and sigma B.
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