Pathsense Scientific Publications

PATHSENSE Scientific Publications

Impact of osmotic stress on the phosphorylation and subcellular location of Listeria monocytogenes stressosome proteins. Dessaux C, Guerreiro DN, Pucciarelli MG, O’Byrne CP, García-Del Portillo F. Sci Rep. 2020;10(1):20837. Published 2020 Nov 30.

doi: 10.1038/s41598-020-77738-z

Abstract: Listeria monocytogenes responds to environmental stress using a supra-macromolecular complex, the stressosome, to activate the stress sigma factor SigB. The stressosome structure, inferred from in vitro-assembled complexes, consists of the core proteins RsbR (here renamed RsbR1) and RsbS and, the kinase RsbT. The active complex is proposed to be tethered to the membrane and to support RsbR1/RsbS phosphorylation by RsbT and the subsequent release of RsbT following signal perception. Here, we show in actively-growing cells that L. monocytogenes RsbR1 and RsbS localize mostly in the cytosol in a fully phosphorylated state regardless of osmotic stress. RsbT however distributes between cytosolic and membrane-associated pools. The kinase activity of RsbT on RsbR1/RsbS and its requirement for maximal SigB activation in response to osmotic stress were demonstrated in vivo. Cytosolic RsbR1 interacts with RsbT, while this interaction diminishes at the membrane when RsbR1 paralogues (RsbR2, RsbR3 and RsbL) are present. Altogether, the data support a model in which phosphorylated RsbR1/RsbS may sustain basal SigB activity in unstressed cells, probably assuring a rapid increase in such activity in response to stress. Our findings also suggest that in vivo the active RsbR1-RsbS-RsbT complex forms only transiently and that membrane-associated RsbR1 paralogues could modulate its assembly.

Tentative model depicting the dynamics of interaction among the stressosome proteins analysed in this study. (A) Model proposed for stress response based on crystallographic data obtained in vitro with purified proteins in L. monocytogenes16; (B) Model integrating the observations obtained in vivo from L. monocytogenes about the distribution and interaction of distinct stressosome proteins. Note the putative negative role assigned to RsbR1 paralogues to prevent the formation of a stable RsbR1-RsbS-RsbT complex, which could be only transiently formed upon stress. The association of RsbT with the membrane is depicted as a direct interaction, although it could be indirect since it lacks defined hydrophobic domains. The model also highlights the presence of a large pools of cytosolic RsbR1, RsbS and RsbT, with the possibility of functional stressosomes in this location capable of activating SigB. See text for details.

rpoB mutations conferring rifampicin- resistance affect growth, stress response and motility in Vibrio vulnificus. Laura Cutugno, Jennifer Mc Cafferty, Jan Pané-Farré,Conor O’Byrne and Aoife Boyd. Microbiology 2020; 166:1160–1170

Abstract: Rifampicin is a broad-spectrum antibiotic that binds to the bacterial RNA polymerase (RNAP), compromising DNA transcription. Rifampicin resistance is common in several microorganisms and it is typically caused by point mutations in the gene encoding the β subunit of RNA polymerase, rpoB. Different rpoB mutations are responsible for various levels of rifampicin resistance and for a range of secondary effects. rpoB mutations conferring rifampicin resistance have been shown to be responsible for severe effects on transcription, cell fitness, bacterial stress response and virulence. Such effects have never been investigated in the marine pathogen Vibrio vulnificus , even though rifampicin-resistant strains of V. vulnificus have been isolated previously. Moreover, spontaneous rifampicin-resistant strains of V. vulnificus have an important role in conjugation and mutagenesis protocols, with poor consideration of the effects of rpoB mutations. In this work, effects on growth, stress response and virulence of V. vulnificus were investigated using a set of nine spontaneous rifampicin-resistant derivatives of V. vulnificus CMCP6. Three different mutations (Q513K, S522L and H526Y) were identified with varying incidence rates. These three mutant types each showed high resistance to rifampicin [minimal inhibitory concentration (MIC) >800 µg ml−1], but different secondary effects. The strains carrying the mutation H526Y had a growth advantage in rich medium but had severely reduced salt stress tolerance in the presence of high NaCl concentrations as well as a significant reduction in ethanol stress resistance. Strains possessing the S522L mutation had reduced growth rate and overall biomass accumulation in rich medium. Furthermore, investigation of virulence characteristics demonstrated that all the rifampicin-resistant strains showed compromised motility when compared with the wild-type, but no major effects on exoenzyme production were observed. These findings reveal a wide range of secondary effects of rpoB mutations and indicate that rifampicin resistance is not an appropriate selectable marker for studies that aim to investigate phenotypic behaviour in this organism.

Rifampicin cluster positions and sequence in rpoB gene and RpoB protein. Figure adapted from [2]. The map and sequence of V. vulnificus rpoB gene and RNA polymerase β subunit (RpoB) and position of the rifampicin clusters I, II, III (black segments) are shown, together with mutations found in the nine rifampicin-resistant strains isolated in this work. Nucleotide mutations and corresponding amino acid changes are shown respectively at the top and bottom of the figure. Mutations and strains are grouped by colour, the H526Y mutation in red, the Q513K mutation in blue and the S522L mutation in green. Asterisks above the single amino acids indicate residues involved in direct binding to rifampicin.

Flick of a switch: regulatory mechanisms allowing Listeria monocytogenes to transition from a saprophyte to a killer. Teresa Tiensuu, Duarte N. Guerreiro, Ana H. Oliveira, Conor O’Byrne and Jörgen Johansson. Microbiology 2019;165:819–833

Abstract: 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 σB and PrfA regulatory systems used by L. monocytogenes to sense the changing environ-ment 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.

Schematic overview of Listeria monocytogenes colonization of the intestine by pathways being controlled by σB and/or PrfA. The human microbiota is depicted on top, lying over the microvilli-containing epithelial cells. Below are shown the events, which are controlled by regulators (σB or PrfA) that in turn regulate expression of important factors. A time-line of events during L. monocytogenes passage of the intestinal barrier is shown at the bottom.

Comparative Review of the Responses of Listeria monocytogenes and Escherichia coli to Low pH Stress

Arcari T, Feger ML, Guerreiro DN, Wu J, O’Byrne CP. Genes (Basel). 2020 Nov 11; 11(11):1330.

DOI: 10.3390/genes11111330

Abstract: Acidity is one of the principal physicochemical factors that influence the behavior of microorganisms in any environment, and their response to it often determines their ability to grow and survive. Preventing the growth and survival of pathogenic bacteria or, conversely, promoting the growth of bacteria that are useful (in biotechnology and food production, for example), might be improved considerably by a deeper understanding of the protective responses that these microorganisms deploy in the face of acid stress. In this review, we survey the molecular mechanisms used by two unrelated bacterial species in their response to low pH stress. We chose to focus on two well-studied bacteria, Escherichia coli (phylum Proteobacteria) and Listeria monocytogenes (phylum Firmicutes), that have both evolved to be able to survive in the mammalian gastrointestinal tract. We review the mechanisms that these species use to maintain a functional intracellular pH as well as the protective mechanisms that they deploy to prevent acid damage to macromolecules in the cells. We discuss the mechanisms used to sense acid in the environment and the regulatory processes that are activated when acid is encountered. We also highlight the specific challenges presented by organic acids. Common themes emerge from this comparison as well as unique strategies that each species uses to cope with acid stress. We highlight some of the important research questions that still need to be addressed in this fascinating field.

Schematic representation of the sensory, protective, and regulatory mechanisms triggered by environmental low pH conditions in E. coli (A) and L. monocytogenes (B)…..

The σB-dependent regulatory sRNA Rli47 represses isoleucine biosynthesis in Listeria monocytogenes through a direct interaction with the ilvA transcript. Catarina M. Marinho, Patrícia T. Dos Santos, Birgitte H. Kallipolitis, Jörgen Johansson, Dmitriy Ignatov, Duarte N. Guerreiro, Pascal Piveteau, Conor P. O’Byrne. RNA Biology2019, Vol. 16, No. 10, 1424-1437

Abstract: The facultative intracellular pathogen Listeria monocytogenes can persist and grow in a diverse range of environmental conditions, both outside and within its mammalian host. The alternative sigma factor Sigma B (σB) plays an important role in this adaptability and is critical for the transition into the host. While some of the functions of the σB regulon in facilitating this transition are understood the role of σB-dependent small regulatory RNAs (sRNAs) remain poorly characterized. In this study, we focused on elucidating the function of Rli47, a σB-dependent sRNA that is highly induced in the intestine and in macrophages. Using a combination of in silico and in vivo approaches, a binding interaction was predicted with 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 a deletion mutant lacking the rli47 gene. The Δrli47 mutant displayed a shorter growth lag in isoleucine-depleted growth media relative to the wild-type, and a similar phenotype was also observed in a mutant lacking σB. The impact of the Δrli47 on the global transcription profile of the cell was investigated using RNA-seq, and a significant role for Rli47 in modulating amino acid metabolism was uncovered. Taken together, the data point to a model where Rli47 is responsible for specifically repressing isoleucine biosynthesis as a way to restrict growth under harsh conditions, potentially contributing to the survival of L. monocytogenes in niches both outside and within the mammalian host.

Proposed model of the regulatory effect of Rli47 on isoleucine biosynthesis in L. monocytogenes. Transcription of rli47 is under σB control and when it is expressed it interacts directly with mRNA from the ilv-leu operon at the predicted Shine-Dalgarno sequence upstream from the ilvA start codon. The Rli47-ilv interaction blocks the first step of isoleucine biosynthesis by preventing the translation of ilvA to produce threonine deaminase (TD), and also by affecting the stability of the ilv-leu transcript. σB negatively influences the activity of TD through an Rli47-independent route which remains to be identified. CodY represses transcription of the ilv-leu operon in a manner that depends on the availability of isoleucine (ile) through an interaction with two binding sites, shown in green [10,12]. When σB is active Rli47 influences the CodY regulon through an effect on the cytoplasmic pool of isoleucine.

The σB-Mediated General Stress Response of Listeria monocytogenes: Life and Death Decision Making in a Pathogen. Guerreiro DN, Arcari T, O’Byrne CP. Front Microbiol. 2020; 11:1505. Published 2020 Jul 7.

doi: 10.3389/fmicb.2020.01505

Abstract: Sensing and responding to environmental cues is critical for the adaptability and success of the food-borne bacterial pathogen Listeria monocytogenes. A supramolecular multi-protein complex known as the stressosome, which acts as a stress sensing hub, is responsible for orchestrating the activation of a signal transduction pathway resulting in the activation of σB, the sigma factor that controls the general stress response (GSR). When σB is released from the anti-sigma factor RsbW, a rapid up-regulation of the large σB regulon, comprised of ≥ 300 genes, ensures that cells respond appropriately to the new environmental conditions. A diversity of stresses including low pH, high osmolarity, and blue light are known to be sensed by the stressosome, resulting in a generalized increase in stress resistance. Appropriate activation of the stressosome and deployment of σB are critical to fitness as there is a trade-off between growth and stress protection when the GSR is deployed. We review the recent developments in this field and describe an up-to-date model of how this sensory organelle might integrate environmental signals to produce an appropriate activation of the GSR. Some of the outstanding questions and challenges in this fascinating field are also discussed.

Schematic representation of the alterations in resource allocation that occur during the GSR. Cell growth largely depends on the housekeeping sigma factor σA in the absence nutrient limitations or stressful conditions. Under these conditions (no stress), most of the transcriptional machinery is dedicated to the transcription of housekeeping genes that preceded by σA promoters. In the absence of stress, σB is sequestered by the anti-sigma factor RsbW. At the onset of stress σB is released from its anti-sigma factor RsbW, resulting in competition between σB and σA and the displacement of σA from a proportion of the RNA polymerase pool. It is possible that the interaction of σB with RNA polymerase is specifically regulated as has been described in other species. Consequently, genes under σA control that are associated with growth functions are downregulated and σB dependent genes (the GSR regulon) are upregulated. The energy resources needed to maintain the general stress response reduces the availability of ATP for growth and reproduction. σB may specifically regulate growth rate to allow for improved maintenance and repair, thereby increasing the likelihood of survival.

Genomic Differences between Listeria monocytogenes EGDe Isolates Reveal Crucial Roles for SigB and Wall Rhamnosylation in Biofilm Formation. Hsu CY, Cairns L, Hobley L, Abbott J, O’Byrne C, Stanley-Wall NR.  J Bacteriol. 2020 Mar 11; 202(7):e00692-19.

DOI: 10.1128/JB.00692-19

Abstract: Listeria monocytogenes is a Gram-positive firmicute that causes foodborne infections, in part due to its ability to use multiple strategies, including biofilm formation, to survive adverse growth conditions. As a potential way to screen for genes required for biofilm formation, we harnessed the ability of bacteria to accumulate mutations in the genome over time, diverging the properties of seemingly identical strains. By sequencing the genomes of four laboratory reference strains of the commonly used L. monocytogenes EGDe, we showed that each isolate contains single nucleotide polymorphisms (SNPs) compared with the reference genome. We discovered that two SNPs, contained in two independent genes within one of the isolates, impacted biofilm formation. Using bacterial genetics and phenotypic assays, we confirmed that rsbU and rmlA influence biofilm formation. RsbU is the upstream regulator of the alternative sigma factor SigB, and mutation of either rsbU or sigB increased biofilm formation. In contrast, deletion of rmlA, which encodes the first enzyme for TDP-l-rhamnose biosynthesis, resulted in a reduction in the amount of biofilm formed. Further analysis of biofilm formation in a strain that still produces TDP-l-rhamnose but which cannot decorate the wall teichoic acid with rhamnose (rmlT mutant) showed that it is the decorated wall teichoic acid that is required for adhesion of the cells to surfaces. Together, these data uncover novel routes by which biofilm formation by L. monocytogenes can be impacted. IMPORTANCE Biofilms are an important mode of growth in many settings. Here, we looked at small differences in the genomes of the bacterium Listeria monocytogenes isolate EGDe and used them to find out how biofilms form. This important fundamental information may help new treatments to be developed and also highlights the fact that isolates of the same identity often diverge.

RmlA and RsbU influence biofilm formation by L. monocytogenes EGDe. (A) The biomasses of WT1031, WT1030, WT1031 Δlmo0184 (LSW1024), WT1031 ΔrsbU (LSW1028), WT1031 ΔrmlA (LSW1040), and WT1031 ΔrmlA ΔrsbU (LSW1051) strains that were adherent to the substratum were quantified. The samples were incubated at 30°C for the time points indicated. The values presented for WT1031 and WT1030 are reproduced from Fig. 3. The means from ≥4 experiments are presented for the remaining strains. The error bars are the standard errors of the means. The data were analyzed by one-way ANOVA, comparing with WT1031. *, P ≤ 0.05; **, P ≤ 0.01. The biomasses adherent to the substratum were imaged using scanning electron microscopy for WT1031 (B), WT1030 (C), WT1031 Δlmo0184 (D), WT1031 ΔrsbU (E), WT1031 ΔrmlA (F), and WT1031 ΔrmlA ΔrsbU (G). The representative images shown were taken at the midpoint of the peg after 48 h of incubation.

Measurement of Protein Mobility in Listeria monocytogenes Reveals a Unique Tolerance to Osmotic Stress and Temperature Dependence of Diffusion. Tran Buu Minh Tran, Haritha Prabha, Aditya Iyer, Conor O’Byrne, Tjakko Abee, Bert Poolman.

Abstract: Protein mobility in the cytoplasm is essential for cellular functions, and slow diffusion may limit the rates of biochemical reactions in the living cell. Here, we determined the apparent lateral diffusion coefficient (DL) of GFP in Listeria monocytogenes as a function of osmotic stress, temperature, and media composition. We find that DL is much less affected by hyperosmotic stress in L. monocytogenes than under similar conditions in Lactococcus lactis and Escherichia coli. We find a temperature optimum for protein diffusion in L. monocytogenes at 30°C, which deviates from predicted trends from the generalized Stokes-Einstein equation under dilute conditions and suggests that the structure of the cytoplasm and macromolecular crowding vary as a function of temperature. The turgor pressure of L. monocytogenes is comparable to other Gram-positive bacteria like Bacillus subtilis and L. lactis but higher in a knockout strain lacking the stress-inducible sigma factor SigB. We discuss these findings in the context of how L. monocytogenes survives during environmental transmission and interaction with the human host.

Diffusion of anionic and cationic GFP in Listeria monocytogenes. (A) (top) Recovery of +25 GFP fluorescence, corresponding to DL of 0.81 ± 0.042 μm2/s. (bottom) The fluorescent intensity along the orange line is shown as a function of time for the experimental data (left) and the one-dimensional heat-equation simulation (middle), and the residuals of the data (right). (B) Histograms of diffusion coefficients of –8 GFP (top) and +25 GFP (bottom) of cells grown in BHI and CDM as well as structural models the surface-modified GFP variants; the colors display the surface charge. The models are based on the structure of super-folder GFP (PDBID: 2B3P), and the images were created using UCSF Chimera (Pettersen et al., 2004). Poisson-Boltzmann electrostatics calculations and evaluations were done by PDB2PQR and APBS packages (Baker et al., 2001; Dolinsky et al., 2004). (C) The diffusion coefficient of –8 and +25 GFP in L. monocytogenes, and –7 and +25 GFP in E. coli, L. lactis, and Hfx. volcanii; the latter have been taken from Schavemaker et al. (2017)

“This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 721456”

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