Increasing the cellular (pp)pGpp level is associated with activation of stress response genes in Staphylococcus aureus
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ABSTRACT:
ORGANISM(S): Staphylococcus aureus
PROVIDER: GSE145144 | GEO | 2020/09/01
REPOSITORIES: GEO
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Project description:The stringent response is initiated by rapid (p)ppGpp synthesis, which leads to a profound reprogramming of gene expression in most bacteria. The stringent phenotype seems to be species specific and may be mediated by fundamentally different molecular mechanisms. In Staphylococcus aureus, (p)ppGpp synthesis upon amino acid deprivation is achieved through the synthase domain of the bifunctional enzyme RSH (RelA/SpoT homolog). In several firmicutes, a direct link between stringent response and the CodY regulon was proposed. Wild-type strain HG001, rshSyn, codY and rshSyn, codY double mutants were analyzed by transcriptome analysis to delineate different consequences of RSH-dependent (p)ppGpp synthesis after induction of the stringent response by amino-acid deprivation. Under these conditions genes coding for major components of the protein synthesis machinery and nucleotide metabolism were down-regulated only in rsh positive strains. Genes which became activated upon (p)ppGpp induction are mostly regulated indirectly via de-repression of the GTP-responsive repressor CodY. Only seven genes, including those coding for the cytotoxic phenol-soluble modulins (PSMs), were found to be up-regulated via RSH independently of CodY. qtRT-PCR analyses of hallmark genes of the stringent response indicate that an RSH activating stringent condition is induced after uptake of S. aureus in human polymorphonuclear neutrophils (PMNs). The RSH activity in turn is crucial for intracellular expression of psms. Accordingly, rshSyn and rshSyn, codY mutants were less able to survive after phagocytosis similar to psm mutants. Intraphagosomal induction of psma1-4 and/or psmM-CM-^_1,2 could complement the survival of the rshSyn mutant. Thus, an active RSH synthase is required for intracellular psm expression which contributes to survival after phagocytosis. Microarray was used to evaluate alteration in the transcriptome of codY and rsh mutants and compared to the wild type
2013-04-05 | E-GEOD-45617 | biostudies-arrayexpress
Project description:The stringent response is initiated by rapid (p)ppGpp synthesis, which leads to a profound reprogramming of gene expression in most bacteria. The stringent phenotype seems to be species specific and may be mediated by fundamentally different molecular mechanisms. In Staphylococcus aureus, (p)ppGpp synthesis upon amino acid deprivation is achieved through the synthase domain of the bifunctional enzyme RSH (RelA/SpoT homolog). In several firmicutes, a direct link between stringent response and the CodY regulon was proposed. Wild-type strain HG001, rshSyn, codY and rshSyn, codY double mutants were analyzed by transcriptome analysis to delineate different consequences of RSH-dependent (p)ppGpp synthesis after induction of the stringent response by amino-acid deprivation. Under these conditions genes coding for major components of the protein synthesis machinery and nucleotide metabolism were down-regulated only in rsh positive strains. Genes which became activated upon (p)ppGpp induction are mostly regulated indirectly via de-repression of the GTP-responsive repressor CodY. Only seven genes, including those coding for the cytotoxic phenol-soluble modulins (PSMs), were found to be up-regulated via RSH independently of CodY. qtRT-PCR analyses of hallmark genes of the stringent response indicate that an RSH activating stringent condition is induced after uptake of S. aureus in human polymorphonuclear neutrophils (PMNs). The RSH activity in turn is crucial for intracellular expression of psms. Accordingly, rshSyn and rshSyn, codY mutants were less able to survive after phagocytosis similar to psm mutants. Intraphagosomal induction of psma1-4 and/or psmß1,2 could complement the survival of the rshSyn mutant. Thus, an active RSH synthase is required for intracellular psm expression which contributes to survival after phagocytosis.
2013-04-05 | GSE45617 | GEO
Project description:In the intracellular pathogen Brucella spp., the activation of the stringent response, a global regulatory network providing rapid adaptation to a variety of growth-affecting stress conditions such as nutrient deficiency, is essential for replication in the host. A single, bi-functional enzyme Rsh catalyzes synthesis and hydrolysis of the alarmone (p)ppGpp, responsible for differential gene expression under stringent conditions. cDNA microarray analysis allowed characterization of the transcriptional profiles of the B. suis 1330 wild-type and rsh mutant in a synthetic minimal medium, partially mimicking the nutrient-poor environment of the intramacrophagic vacuole. A total of 379 genes (11.6% of the genome) were differentially expressed in a rsh-dependent manner, of which 52% were up-regulated and 48% were down-regulated. The pleiotropic character of the response was confirmed, as the genes encoded factors belonging to various functional groups, comprising an important number of transcriptional regulators, cell envelope proteins, stress factors, transport systems, and energy metabolism proteins. Several virulence genes were under the positive control of (p)ppGpp. Methionine was the only amino acid whose biosynthesis was absolutely dependent on stringent response in B. suis. The study illustrated the complexity of the processes involved in adaptation to nutrient starvation, and contributed to a better understanding of the correlation between stringent response and Brucella virulence. Most interestingly, it clearly indicated (p)ppGpp-dependent cross-talk between at least three stress responses playing a central role in Brucella adaptation to the host: nutrient, oxidative, and low-oxygen stress. Two-condition experiment, wild-type against rsh mutant. Biological replicates: 4 wild-type, 4 rsh mutant, independently grown in minimal medium (under nutrient starvation condition) and harvested. One replicate per array. Please note that Channel 1 (Cy3) on each hybridization was a universal reference (Uref) used as a reference point to merge two (2 channel arrays) into a virtual 2 channel array with wild-type versus rsh mutant.
2013-07-08 | E-GEOD-44688 | biostudies-arrayexpress
Project description:Cells respond to stress and starvation by adjusting their growth rate and enacting stress defense programs. In eukaryotes this involves inactivation of TORC1, which in turn triggers downregulation of ribosome and protein synthesis genes and upregulation of stress response genes. Here we report that the highly conserved inositol pyrophosphate second messengers (including 1-PP-IP5, 5-PP-IP4, and 5-PP-IP5) are also critical regulators of cell growth and the general stress response, acting in parallel to the TORC1 pathway to control the activity of the class I HDAC Rpd3L. In fact, yeast cells that cannot synthesize any of the PP-IPs mount little to no transcriptional response in osmotic, heat, or oxidative stress. Furthermore, PP-IP dependent regulation of Rpd3L occurs independently of the role individual PP-IPs (such as 5-PP-IP5) play in activating specialized stress/starvation response pathways. Thus, the PP-IP second messengers simultaneously activate and tune the global response to stress and starvation signals.
2013-03-22 | GSE45370 | GEO
Project description:Cells respond to stress and starvation by adjusting their growth rate and enacting stress defense programs. In eukaryotes this involves inactivation of TORC1, which in turn triggers downregulation of ribosome and protein synthesis genes and upregulation of stress response genes. Here we report that the highly conserved inositol pyrophosphate second messengers (including 1-PP-IP5, 5-PP-IP4, and 5-PP-IP5) are also critical regulators of cell growth and the general stress response, acting in parallel to the TORC1 pathway to control the activity of the class I HDAC Rpd3L. In fact, yeast cells that cannot synthesize any of the PP-IPs mount little to no transcriptional response in osmotic, heat, or oxidative stress. Furthermore, PP-IP dependent regulation of Rpd3L occurs independently of the role individual PP-IPs (such as 5-PP-IP5) play in activating specialized stress/starvation response pathways. Thus, the PP-IP second messengers simultaneously activate and tune the global response to stress and starvation signals. 2-condition experiments. Includes the responses of wild-type (ACY 044) and mutant yeast strains (all are W303 background) to log growth and stress conditions. This series of microarrays were performed on null mutants of various genes in the inositol pyrophosphate synthesis pathway, including several members of the Rpd3L histone deacetylase complex. All mutants were made in W303 strain, MatA yeast, using standard techniques (homologous recombination). Several stress conditions were tested, including heat-shock, oxidative (H2O2), and osmotic stress (0.375M KCl). Cells in mid-log growth were subjected to stress for 20 minutes. In one instance the TOR inhibitor rapamycin was added to determine whether PP-IPs act above/at or below TORC1 in activating the ESR. Taken together, these microarrays show the role of the inositol pyrophosphate synthesis pathway in activating the ESR in stress.
2013-03-22 | E-GEOD-45370 | biostudies-arrayexpress
Project description:Background: During gut colonization, the enteric pathogen C. jejuni has to surmount the toxic effects of reactive oxygen species produced by its own metabolism, by the host immune system and by the intestinal microflora. Elucidation of C. jejuni oxidative stress defense mechanisms is critical for understanding Campylobacter pathophysiology. Results: The mechanisms of oxidative stress defenses in Campylobacter jejuni were characterized by transcriptional profiling, genes mutagenesis, and phenotypic analysis. The transcriptome changes, in response to H2O2, cumene hydroperoxide, or menadione exposure, were found to be oxidant specific and revealed the differential expression of genes belonging to a variety of biological pathways, from the classical oxidative stress defense systems, to the heat shock response, DNA repair and metabolism, fatty acid and capsule biosynthesis, and multidrug efflux pumps. To define the peroxide sensing regulator PerR, an isogenic mutant was constructed and its transcriptome profile compared to the wild-type strain. Sixty-six genes were found to belong to the PerR regulon. PerR appear to regulate gene expression both dependently and independently of the presence of iron and/or H2O2. The perR mutant was affected in its motility and attenuated in the chick colonization model. Mutagenic and phenotypic studies of the superoxide disumutase SodB, the alkyl-hydroxyperoxidase AhpC, and the catalase KatA, revealed their role in oxidative stress defenses and chick gut colonization. Conclusion: This study reveals the interplay between PerR, the iron metabolism and the oxidative stress defenses and highlights their role in the colonization and/or survival of C. jejuni in the chick cecum. Keywords: Transcriptional response to 3 oxidants (H2O2, menadione and cumene hydroperoxide) and characterization of the perR regulon (comparison of the transciptomes from the wild-type and perR mutant).
2010-09-07 | GSE13126 | GEO
Project description:In the intracellular pathogen Brucella spp., the activation of the stringent response, a global regulatory network providing rapid adaptation to a variety of growth-affecting stress conditions such as nutrient deficiency, is essential for replication in the host. A single, bi-functional enzyme Rsh catalyzes synthesis and hydrolysis of the alarmone (p)ppGpp, responsible for differential gene expression under stringent conditions. cDNA microarray analysis allowed characterization of the transcriptional profiles of the B. suis 1330 wild-type and rsh mutant in a synthetic minimal medium, partially mimicking the nutrient-poor environment of the intramacrophagic vacuole. A total of 379 genes (11.6% of the genome) were differentially expressed in a rsh-dependent manner, of which 52% were up-regulated and 48% were down-regulated. The pleiotropic character of the response was confirmed, as the genes encoded factors belonging to various functional groups, comprising an important number of transcriptional regulators, cell envelope proteins, stress factors, transport systems, and energy metabolism proteins. Several virulence genes were under the positive control of (p)ppGpp. Methionine was the only amino acid whose biosynthesis was absolutely dependent on stringent response in B. suis. The study illustrated the complexity of the processes involved in adaptation to nutrient starvation, and contributed to a better understanding of the correlation between stringent response and Brucella virulence. Most interestingly, it clearly indicated (p)ppGpp-dependent cross-talk between at least three stress responses playing a central role in Brucella adaptation to the host: nutrient, oxidative, and low-oxygen stress.
2013-07-08 | GSE44688 | GEO
Project description:Background: During gut colonization, the enteric pathogen C. jejuni has to surmount the toxic effects of reactive oxygen species produced by its own metabolism, by the host immune system and by the intestinal microflora. Elucidation of C. jejuni oxidative stress defense mechanisms is critical for understanding Campylobacter pathophysiology. Results: The mechanisms of oxidative stress defenses in Campylobacter jejuni were characterized by transcriptional profiling, genes mutagenesis, and phenotypic analysis. The transcriptome changes, in response to H2O2, cumene hydroperoxide, or menadione exposure, were found to be oxidant specific and revealed the differential expression of genes belonging to a variety of biological pathways, from the classical oxidative stress defense systems, to the heat shock response, DNA repair and metabolism, fatty acid and capsule biosynthesis, and multidrug efflux pumps. To define the peroxide sensing regulator PerR, an isogenic mutant was constructed and its transcriptome profile compared to the wild-type strain. Sixty-six genes were found to belong to the PerR regulon. PerR appear to regulate gene expression both dependently and independently of the presence of iron and/or H2O2. The perR mutant was affected in its motility and attenuated in the chick colonization model. Mutagenic and phenotypic studies of the superoxide disumutase SodB, the alkyl-hydroxyperoxidase AhpC, and the catalase KatA, revealed their role in oxidative stress defenses and chick gut colonization. Conclusion: This study reveals the interplay between PerR, the iron metabolism and the oxidative stress defenses and highlights their role in the colonization and/or survival of C. jejuni in the chick cecum. Keywords: Transcriptional response to 3 oxidants (H2O2, menadione and cumene hydroperoxide) and characterization of the perR regulon (comparison of the transciptomes from the wild-type and perR mutant). To investigate the transcriptional responses of C. jejuni to oxidant exposure, hydrogen peroxide (H2O2), cumene hydroperoxide (CHP), or menadione sodium bisulfite (MND) was added to the 50 ml broth at a final concentration of 1 mM. The same amount of water or DMSO was added to the bacterial culture that served as reference samples for the transcriptional profile study in response to H2O2, MND or CHP. Furthermore, to investigate the transcriptional response of C. jejuni to H2O2 exposure in the presence of excess iron, ferrous sulfate was added to the bacterial culture at a final concentration of 40 µM, 15 min prior to H2O2 exposure. Ten minutes after the addition of the oxidant, total RNA was extracted and processed for microarray hybridization. To identify the PerR regulon, the wild-type strain C. jejuni NCTC 11168 and the perR mutant were grown in 500 ml flasks containing 250 ml of MEMα medium. At mid-log phase, 50 ml of the cultures were transferred to 100 ml flasks and ferrous sulfate and/or H2O2 were added . Ten minutes following the addition of H2O2 the cells were collected and the total RNA extracted.
2010-09-07 | E-GEOD-13126 | biostudies-arrayexpress
Project description:ppGpp accumulation caused by ectopic expression of RelA in Saccharomyces cerevisiae gave rise to marked changes in gene expression with both upregulation and downregulation, including changes in mitochondrial gene expression. The most prominent upregulation (38-fold) was detected in the function-unknown hypothetical gene YBR072C-A, followed by many other known stress-responsive genes. ppGpp acuumulation resulted in enhancement of tolerance against various stress stimuli, such as osmotic stress, ethanol, hydrogen peroxide, and high temperature. A two chip study using total RNA recoverd from the Saccharomyces cerevisiae TN2080 (accumulating ppGpp) and TN2077 (vector control) grown to mid-growth phase (8h) in SC-uracil medium. The plasmid pYC2/CT (V5-epitope tag vector) was used as a vector to express Sj-RSH.
2012-05-30 | E-GEOD-38239 | biostudies-arrayexpress
Project description:The stringent response is a conserved stress response employed by various bacteria to respond to and cope with conditions of amino acid starvation, carbon-source, fatty acid, oxygen/iron limitation, heat shock, antimicrobial challenge, and/or other environmental stressors. In S. aureus, the stringent response is mediated in part by enzyme RSH (relA spoT homologue) which synthesizes two nucleotide-based molecules, guanosine tetraphosphate (ppGpp) and pentaphosphate (pppGpp), collectively known as (p) ppGpp. This response is known to result in a number of diverse physiological and metabolic changes which we further characterized in the present study. Specifically, the global transcriptome of the RSH synthase mutant (rshsyn) was compared to the WT parent S. aureus strain (NCTC 8325) by RNA-Seq analysis under both normal and nutrient-limited media conditions. In order to mimic nutrient limitation, bacteria were treated with mupirocin, an antibiotic and isoleucine analogue known to lead to isoleucine starvation. In the WT strain, in response to mupirocin-induced isoleucine starvation, genes encoding protein synthesis machinery were suppressed while genes encoding amino acid biosynthetic machinery were upregulated. This response likely employed to compensate for amino acid deprivation, was not induced in the stringent response mutant. Furthermore, under starvation conditions, biofilm-related genes (i.e. icaA, icaC, fnbA, clfB, emp) were more upregulated in the WT than the stringent response mutant. Moreover, the effect of mupirocin caused a stronger upregulation of several proteases (i.e. sspA, sspP, sppB, aur), lipases (lip, geh) and phenol soluble modulin toxins in the WT as compared to the mutant. Overall, these results suggest that under starvation conditions, RSH plays an important role in helping the cell respond to nutritional stress as well as regulates the expression of numerous colonization and virulence factors.
2019-02-02 | GSE110170 | GEO