scholarly journals Inducible expression of (pp)pGpp synthetases in Staphylococcus aureus is associated with activation of stress response genes

PLoS Genetics ◽  
2020 ◽  
Vol 16 (12) ◽  
pp. e1009282
Author(s):  
Petra Horvatek ◽  
Andrea Salzer ◽  
Andrew Magdy Fekry Hanna ◽  
Fabio Lino Gratani ◽  
Daniela Keinhörster ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Staphylococcus Aureus ◽  
Stress Response ◽  
Stringent Response ◽  
Trna Synthetase ◽  
Oxidative Stress Response ◽  
Stress Conditions ◽  
Additional Stress ◽  
Iron Storage

The stringent response is characterized by the synthesis of the messenger molecules pppGpp, ppGpp or pGpp (here collectively designated (pp)pGpp). The phenotypic consequences resulting from (pp)pGpp accumulation vary among species and can be mediated by different underlying mechanisms. Most genome-wide analyses have been performed under stress conditions, which often mask the immediate effects of (pp)pGpp-mediated regulatory circuits. In Staphylococcus aureus, (pp)pGpp can be synthesized via the RelA-SpoT-homolog, RelSau upon amino acid limitation or via one of the two small (pp)pGpp synthetases RelP or RelQ upon cell wall stress. We used RNA-Seq to compare the global effects in response to induction of the synthetase of rel-Syn (coding for the enzymatic region of RelSau) or relQ without the need to apply additional stress conditions. Induction of rel-Syn resulted in changes in the nucleotide pool similar to induction of the stringent response via the tRNA synthetase inhibitor mupirocin: a reduction in the GTP pool, an increase in the ATP pool and synthesis of pppGpp, ppGpp and pGpp. Induction of all three enzymes resulted in similar changes in the transcriptome. However, RelQ was less active than Rel-Syn and RelP, indicating strong restriction of its (pp)pGpp-synthesis activity in vivo. (pp)pGpp induction resulted in the downregulation of many genes involved in protein and RNA/DNA metabolism. Many of the (pp)pGpp up-regulated genes are part of the GTP sensitive CodY regulon and thus likely regulated through lowering of the GTP pool. New CodY independent transcriptional changes were detected including genes involved in the SOS response, iron storage (e.g. ftnA, dps), oxidative stress response (e.g., perR katA, sodA) and the psmα1–4 and psmß1-2 operons coding for cytotoxic, phenol soluble modulins (PSMs). Analyses of the ftnA, dps and psm genes in different regulatory mutants revealed that their (pp)pGpp-dependent regulation can occur independent of the regulators PerR, Fur, SarA or CodY. Moreover, psm expression is uncoupled from expression of the quorum sensing system Agr, the main known psm activator. The expression of central genes of the oxidative stress response protects the bacteria from anticipated ROS stress derived from PSMs or exogenous sources. Thus, we identified a new link between the stringent response and oxidative stress in S. aureus that is likely crucial for survival upon phagocytosis.

scholarly journals Inducible expression of (pp)pGpp synthetases in Staphylococcus aureus is associated with activation of stress response genes

2020 ◽  
Author(s):  
Petra Horvatek ◽  
Andrew Magdy Fekry Hanna ◽  
Fabio Lino Gratani ◽  
Daniela Keinhörster ◽  
Natalya Korn ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Staphylococcus Aureus ◽  
Stress Response ◽  
Stringent Response ◽  
Trna Synthetase ◽  
Oxidative Stress Response ◽  
Stress Conditions ◽  
Additional Stress ◽  
Iron Storage ◽  
Transcriptional Induction

AbstractThe stringent response is characterized by the synthesis of the messenger molecules pppGpp, ppGpp or pGpp (here collectively designated (pp)pGpp). The phenotypic consequences resulting from (pp)pGpp accumulation vary among species and can be mediated by different underlying mechanisms. Most genome-wide analyses have been performed under stress conditions, which often mask the immediate effects of (pp)pGpp-mediated regulatory circuits. In Staphylococcus aureus, (pp)pGpp can be synthesized via the RelA-SpoT-homolog (RSHSau) upon amino acid limitation or via one of the two small (pp)pGpp synthetases RelP or RelQ, upon cell wall stress. We used RNA-Seq to compare the global effects in response to transcriptional induction of the synthetase domain of RSH (RSH-Syn), RelP or RelQ without the need to apply additional stress conditions. Enzyme expression resulted in changes in the nucleotide pool similar to induction of the stringent response via the tRNA synthetase inhibitor mupirocin: a reduction in the GTP pool, an increase in the ATP pool and synthesis of pppGpp, ppGpp and pGpp. Induction of all three enzymes resulted in similar changes in the transcriptome. However, RelQ was less active than RSH-Syn and RelP, indicating strong restriction of its (pp)pGpp-synthesis activity in vivo. Genes involved in the SOS response, iron storage (e.g. ftnA, dps), oxidative stress response (e.g., katA, sodA) and the the psmα1-4 and psmß1-2 operons coding for cytotoxic, phenole soluble modulins (PSMs) were highly upregulated upon (pp)pGpp synthesis. Analyses of the ftnA, dps and psm genes in different regulatory mutants revealed that their (pp)pGpp-dependent regulation can occur independent of the regulators PerR, Fur, SarA or CodY. Moreover, psm expression is uncoupled from expression of the quorum sensing system Agr, the main known psm activator. The expression of central genes of the oxidative stress response protects the bacteria from anticipated ROS stress derived from PSMs or exogenous sources. Thus, we identified a new link between the stringent response and oxidative stress in S. aureus that is likely crucial for survival upon phagocytosis.SignificanceMost bacteria make use of the second messenger (pp)pGpp to reprogram bacterial metabolism under nutrient-limiting conditions. In the human pathogen Staphylococcus aureus, (pp)pGpp plays an important role in virulence, phagosomal escape and antibiotic tolerance. Here, we analyzed the immediate consequences of (pp)pGpp synthesis upon transcriptional induction of the (pp)pGpp-producing enzymes RSH, RelP or RelQ. (pp)pGpp synthesis provokes immediate changes in the nucleotide pool and severely impacts the expression of hundreds of genes. A main consequence of (pp)pGpp synthesis in S. aureus is the induction of ROS-inducing toxic phenol-soluble modulins (PSMs) and simultaneous expression of the detoxifying system to protect the producer. This mechanism is likely of special advantage for the pathogen after phagocytosis.

scholarly journals Stringent Response Governs the Virulence and Oxidative Stress Resistance of Francisella tularensis

2019 ◽  
Author(s):  
Zhuo Ma ◽  
Kayla King ◽  
Maha Alqahtani ◽  
Madeline Worden ◽  
Parthasarthy Muthuraman ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stress Response ◽  
Francisella Tularensis ◽  
Stress Resistance ◽  
Stringent Response ◽  
Oxidative Stress Response ◽  
Stress Conditions ◽  
Gram Negative ◽  
And Oxidative Stress ◽  
Starvation Conditions

AbstractFrancisella tularensis is a Gram-negative bacterium responsible for causing tularemia in the northern hemisphere. F. tularensis has long been developed as a biological weapon due to its ability to cause severe illness upon inhalation of as few as ten organisms and based on its potential to be used as a bioterror agent is now classified as a Tier 1 Category A select agent by the CDC. The stringent response facilitates bacterial survival under nutritionally challenging starvation conditions. The hallmark of stringent response is the accumulation of the effector molecules ppGpp and (p)ppGpp known as stress alarmones. The relA and spoT gene products generate alarmones in several Gram-negative bacterial pathogens. RelA is a ribosome-associated ppGpp synthetase that gets activated under amino acid starvation conditions whereas, SpoT is a bifunctional enzyme with both ppGpp synthetase and ppGpp hydrolase activities. Francisella encodes a monofunctional RelA and a bifunctional SpoT enzyme. Previous studies have demonstrated that stringent response under nutritional stresses increases expression of virulence-associated genes encoded on Francisella Pathogenicity Island. This study investigated how stringent response governs the oxidative stress response of F. tularensis. We demonstrate that RelA/SpoT-mediated ppGpp production alters global gene transcriptional profile of F. tularensis in the presence of oxidative stress. The lack of stringent response in relA/spoT gene deletion mutants of F. tularensis makes bacteria more susceptible to oxidants, attenuates survival in macrophages, and virulence in mice. Mechanistically, we provide evidence that the stringent response in Francisella contributes to oxidative stress resistance by enhancing the production of antioxidant enzymes.ImportanceThe unique intracellular life cycle of Francisella in addition to nutritional stress also exposes the bacteria to oxidative stress conditions upon its brief residence in the phagosomes, and escape into the cytosol where replication takes place. However, the contribution of the stringent response in gene regulation and management of the oxidative stress response when Francisella is experiencing oxidative stress conditions is not known. Our results provide a link between the stringent and oxidative stress responses. This study further improves our understanding of the intracellular survival mechanisms of F. tularensis.

scholarly journals Dps and DpsL Mediate SurvivalIn VitroandIn Vivoduring the Prolonged Oxidative Stress Response in Bacteroides fragilis

2015 ◽  
Vol 197 (20) ◽  
pp. 3329-3338 ◽  
Author(s):  
Michael I. Betteken ◽  
Edson R. Rocha ◽  
C. Jeffrey Smith
Keyword(s):  
Oxidative Stress ◽  
Stress Response ◽  
Peritoneal Cavity ◽  
Bacteroides Fragilis ◽  
Oxidative Stress Response ◽  
Iron Storage ◽  
Content Type ◽  
Iron Storage Proteins

ABSTRACTBacteroides fragilisis a Gram-negative anaerobe and member of the human intestinal tract microbiome, where it plays many beneficial roles. However, translocation of the organism to the peritoneal cavity can lead to peritonitis, intra-abdominal abscess formation, bacteremia, and sepsis. During translocation,B. fragilisis exposed to increased oxidative stress from the oxygenated tissues of the peritoneal cavity and the immune response. In order to survive,B. fragilismounts a robust oxidative stress response consisting of an acute and a prolonged oxidative stress (POST) response. This report demonstrates that the ability to induce high levels of resistance totert-butyl hydroperoxide (tBOOH) after extended exposure to air can be linked to the POST response. Disk diffusion assays comparing the wild type to a Δdpsmutant and a ΔdpsΔbfrmutant showed greater sensitivity of the mutants to tBOOH after exposure to air, suggesting that Dps and DpsL play a role in the resistance phenotype. Complementation studies withdpsorbfr(encoding DpsL) restored tBOOH resistance, suggesting a role for both of these ferritin-family proteins in the response. Additionally, cultures treated with the iron chelator dipyridyl were not killed by tBOOH, indicating Dps and DpsL function by sequestering iron to prevent cellular damage. Anin vivoanimal model showed that the ΔdpsΔbfrmutant was attenuated, indicating that management of iron is important for survival within the abscess. Together, these data demonstrate a role for Dps and DpsL in the POST response which mediates survivalin vitroandin vivo.IMPORTANCEB. fragilisis the anaerobe most frequently isolated from extraintestinal opportunistic infections, but there is a paucity of information about the factors that allow this organism to survive outside its normal intestinal environment. This report demonstrates that the iron storage proteins Dps and DpsL protect against oxidative stress and that they contribute to survival bothin vitroandin vivo. Additionally, this work demonstrates an important role for the POST response inB. fragilissurvival and provides insight into the complex regulation of this response.

scholarly journals Analysis of Functional Domains in Rho5, the Yeast Homolog of Human Rac1 GTPase, in Oxidative Stress Response

2019 ◽  
Vol 20 (22) ◽  
pp. 5550 ◽  
Author(s):  
Carolin Sterk ◽  
Lauren Gräber ◽  
Hans-Peter Schmitz ◽  
Jürgen J. Heinisch
Keyword(s):  
Oxidative Stress ◽  
Stress Response ◽  
Hypervariable Region ◽  
Oxidative Stress Response ◽  
Small Gtpase ◽  
Rac1 Gtpase ◽  
Yeast Homolog ◽  
And Oxidative Stress ◽  
Polybasic Region

The small GTPase Rho5 of Saccharomyces cerevisiae is required for proper regulation of different signaling pathways, which includes the response to cell wall, osmotic, nutrient, and oxidative stress. We here show that proper in vivo function and intracellular distribution of Rho5 depends on its hypervariable region at the carboxyterminal end, which includes the CAAX box for lipid modification, a preceding polybasic region (PBR) carrying a serine residue, and a 98 amino acid–specific insertion only present in Rho5 of S. cerevisiae but not in its human homolog Rac1. Results from trapping GFP-Rho5 variants to the mitochondrial surface suggest that the GTPase needs to be activated at the plasma membrane prior to its translocation to mitochondria in order to fulfil its role in oxidative stress response. These findings are supported by heterologous expression of a codon-optimized human RAC1 gene, which can only complement a yeast rho5 deletion in a chimeric fusion with RHO5 sequences that restore the correct spatiotemporal distribution of the encoded protein.

scholarly journals Global Regulatory Impact of ClpP Protease of Staphylococcus aureus on Regulons Involved in Virulence, Oxidative Stress Response, Autolysis, and DNA Repair

2006 ◽  
Vol 188 (16) ◽  
pp. 5783-5796 ◽  
Author(s):  
Antje Michel ◽  
Franziska Agerer ◽  
Christof R. Hauck ◽  
Mathias Herrmann ◽  
Joachim Ullrich ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Staphylococcus Aureus ◽  
Dna Repair ◽  
Stress Response ◽  
Oxidative Stress Response ◽  
Transcriptional Analysis ◽  
Expression Of Genes ◽  
Wide Range ◽  
Genes Encoding ◽  
Regulatory Impact

ABSTRACT Staphylococcus aureus is an important pathogen, causing a wide range of infections including sepsis, wound infections, pneumonia, and catheter-related infections. In several pathogens ClpP proteases were identified by in vivo expression technologies to be important for virulence. Clp proteolytic complexes are responsible for adaptation to multiple stresses by degrading accumulated and misfolded proteins. In this report clpP, encoding the proteolytic subunit of the ATP-dependent Clp protease, was deleted, and gene expression of ΔclpP was determined by global transcriptional analysis using DNA-microarray technology. The transcriptional profile reveals a strong regulatory impact of ClpP on the expression of genes encoding proteins that are involved in the pathogenicity of S. aureus and adaptation of the pathogen to several stresses. Expression of the agr system and agr-dependent extracellular virulence factors was diminished. Moreover, the loss of clpP leads to a complete transcriptional derepression of genes of the CtsR- and HrcA-controlled heat shock regulon and a partial derepression of genes involved in oxidative stress response, metal homeostasis, and SOS DNA repair controlled by PerR, Fur, MntR, and LexA. The levels of transcription of genes encoding proteins involved in adaptation to anaerobic conditions potentially regulated by an Fnr-like regulator were decreased. Furthermore, the expression of genes whose products are involved in autolysis was deregulated, leading to enhanced autolysis in the mutant. Our results indicate a strong impact of ClpP proteolytic activity on virulence, stress response, and physiology in S. aureus.

Multiple methionine sulfoxide reductase genes in Staphylococcus aureus: expression of activity and roles in tolerance of oxidative stress

Microbiology ◽  
2003 ◽  
Vol 149 (10) ◽  
pp. 2739-2747 ◽  
Author(s):  
Vineet K. Singh ◽  
Jackob Moskovitz
Keyword(s):  
Oxidative Stress ◽  
Staphylococcus Aureus ◽  
Stress Response ◽  
Double Mutant ◽  
Mutational Analysis ◽  
Physiological Role ◽  
Methionine Sulfoxide ◽  
Oxidative Stress Response ◽  
Methionine Sulfoxide Reductase ◽  
Activity Data

Staphylococcus aureus contains three genes encoding MsrA-specific methionine sulfoxide reductase (Msr) activity (msrA1, msrA2 and msrA3) and an additional gene that encodes MsrB-specific Msr activity. Data presented here suggest that MsrA1 is the major contributor of the MsrA activity in S. aureus. In mutational analysis, while the total Msr activity in msrA2 mutant was comparable to that of the parent, Msr activity was significantly up-regulated in the msrA1 or msrA1 msrA2 double mutant. Assessment of substrate specificity together with increased reactivity of the cell-free protein extracts of the msrA1 mutants to anti-MsrB polyclonal antibodies in Western analysis provided evidence that increased Msr activity was due to elevated synthesis of MsrB in the MsrA1 mutants. Previously, it was reported that oxacillin treatment of S. aureus cells led to induced synthesis of MsrA1 and a mutation in msrA1 increased the susceptibility of the organism to H2O2. A mutation in the msrA2 gene, however, was not significant for the bacterial oxidative stress response. In complementation assays, while the msrA2 gene was unable to complement the msrA1 msrA2 double mutant for H2O2 resistance, the same gene restored H2O2 tolerance in the double mutant when placed under the control of the msrA1 promoter. However, msrA1 which was able to complement the oxidative stress response in msrA1 mutants could not restore the tolerance of the msrA1 msrA2 mutants to H2O2 when placed under the control of the msrA2 promoter. Additionally, although the oxacillin minimum inhibitory concentration of the msrA1 mutant was comparable to that of the wild-type parent, in shaking liquid culture, the msrA1 mutant responded more efficiently to sublethal doses of oxacillin. The data suggest complex regulation of Msr proteins and a more significant physiological role for msrA1/msrB in S. aureus.

scholarly journals Proteomics of autophagy deficient macrophages reveals enhanced antimicrobial immunity via the oxidative stress response

2020 ◽  
Author(s):  
Timurs Maculins ◽  
Erik Verschueren ◽  
Trent Hinkle ◽  
Patrick Chang ◽  
Cecile Chalouni ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stress Response ◽  
Shigella Flexneri ◽  
Myeloid Cell ◽  
Oxidative Stress Response ◽  
Intracellular Pathogens ◽  
Loss Of Function ◽  
Autophagy Gene ◽  
Antimicrobial Immunity

AbstractDefective autophagy is associated with chronic inflammation. Loss-of-function of the core autophagy gene Atg16l1 increases risk for Crohn’s disease by enhancing innate immunity in macrophages. However, autophagy also mediates clearance of intracellular pathogens. These divergent observations prompted a re-evaluation of ATG16L1 in antimicrobial immunity. In this study, we found that loss of Atg16l1 in macrophages enhanced the killing of virulent Shigella flexneri (S.flexneri), an enteric bacterium that resides within the cytosol by escaping all membrane-bound compartments. Quantitative multiplexed proteomics revealed that ATG16L1 deficiency significantly upregulated proteins involved in the glutathione-mediated antioxidant response to compensate for elevated oxidative stress, which also promoted S.flexneri killing. Consistently, myeloid cell-specific deletion of Atg16l1 accelerated bacterial clearance in vivo. Finally, pharmacological modulation of oxidative stress by suppression of cysteine import conferred enhanced microbicidal properties to wild type macrophages. These findings demonstrate that control of oxidative stress by ATG16L1 regulates antimicrobial immunity against intracellular pathogens.Impact statementMaculins et al utilize multiplexed mass spectrometry to show that loss of the autophagy gene Atg16l1 in macrophages enhances antimicrobial immunity against intracellular pathogens via the oxidative stress response.

scholarly journals Nonthermal Plasma Induces the Viable-but-Nonculturable State in Staphylococcus aureus via Metabolic Suppression and the Oxidative Stress Response

2019 ◽  
Vol 86 (5) ◽  
Author(s):  
Xinyu Liao ◽  
Donghong Liu ◽  
Tian Ding
Keyword(s):  
Oxidative Stress ◽  
Staphylococcus Aureus ◽  
Stress Response ◽  
Nonthermal Plasma ◽  
Energy Allocation ◽  
Oxidative Stress Response ◽  
Vbnc State ◽  
Viable But Nonculturable ◽  
Content Type ◽  
Energy Dependent

ABSTRACT As a novel nonthermal technology, nonthermal plasma (NTP) has attracted a lot of attention. However, it could induce microorganisms into a viable but nonculturable (VBNC) state, posing a potential risk to food safety and public health. In this study, the molecular mechanisms of VBNC Staphylococcus aureus induced by NTP were investigated. With the use of a propidium monoazide quantitative PCR (PMA-qPCR) technique combined with a plate count method, we confirmed that 8.1 to 24.3 kJ NTP induced S. aureus into a VBNC state at a level of 7.4 to 7.6 log10 CFU/ml. The transcriptomic analysis was conducted and revealed that most energy-dependent physiological activities (e.g., metabolism) were arrested in VBNC S. aureus, while the oxidative stress response-related genes (katA, dps, msrB, msrA, and trxA) were significantly upregulated. In addition, this study showed that the ATP depletion by carbonyl cyanide m-chlorophenyl hydrazone (CCCP) pretreatment could accelerate the formation of VBNC S. aureus. The NTP-generated oxidative stress triggers the staphylococcal oxidative stress response, which consumes part of cellular energy (e.g., ATP). The energy allocation is therefore changed, and the energy assigned for other energy-dependent physiological activities (cell growth and division, etc.) is reduced, subsequently forcing S. aureus into a VBNC state. Therefore, the alterations of energy allocation should be some of the major contributors to the induction of VBNC S. aureus with NTP exposure. This study provides valuable knowledge for controlling the formation of VBNC S. aureus during NTP treatment. IMPORTANCE In recent years, nonthermal plasma (NTP) technology has received a lot of attention as a promising alternative to thermal pasteurization in the food industry. However, little is known about the microbial stress response toward NTP, which could be a potential risk to food safety and impede the development of NTP. A viable but nonculturable (VBNC) state is one of the most common survival strategies employed by microorganisms against external stress. This study investigated the mechanisms of the formation of VBNC Staphylococcus aureus by NTP in a more comprehensive and systematic aspect than had been done before. Our work confirmed that the NTP-generated oxidative stress induced changes in energy allocation as a driving force for the formation of VBNC S. aureus. This study could provide better knowledge for controlling the occurrence of VBNC S. aureus induced by NTP, which could lead to more rational design and ensure the development of safe foods.

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