Steady-state hydrogen peroxide induces glycolysis in Staphylococcus aureus and Pseudomonas aeruginosa (original) (raw)
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Antioxidants
Hydrogen peroxide (H2O2) is a key defense component of host-microbe interaction. However, H2O2 concentrations generated by immune cells or epithelia are usually insufficient for bacterial killing and rather modulate bacterial responses. Here, we investigated the impact of sublethal H2O2 concentration on gene expression of E. coli BW25113 after 10 and 60 min of exposure. RNA-seq analysis revealed that approximately 12% of bacterial genes were strongly dysregulated 10 min following exposure to 2.5 mM H2O2. H2O2 exposure led to the activation of a specific antioxidant response and a general stress response. The latter was characterized by a transient down-regulation of genes involved in general metabolism, such as nucleic acid biosynthesis and translation, with a striking and coordinated down-regulation of genes involved in ribosome formation, and a sustained up-regulation of the SOS response. We confirmed the rapid transient and specific response mediated by the transcription factor O...
Impact of the pentose phosphate pathway on metabolism and pathogenesis of Staphylococcus aureus
PLOS Pathogens, 2023
Staphylococcus aureus is an important pathogen that leads to significant disease through multiple routes of infection. We recently published a transposon sequencing (Tn-seq) screen in a mouse acute pneumonia model and identified a hypothetical gene (SAUSA300_1902, pgl) with similarity to a lactonase of Escherichia coli involved in the pentose phosphate pathway (PPP) that was conditionally essential. Limited studies have investigated the role of the PPP in physiology and pathogenesis of S. aureus. We show here that mutation of pgl significantly impacts ATP levels and respiration. RNA-seq analysis of the pgl mutant and parent strains identified compensatory changes in gene expression for glucose and gluconate as well as reductions in the pyrimidine biosynthesis locus. These differences were also evident through unbiased metabolomics studies and 13 C labeling experiments that showed mutation of pgl led to reductions in pyrimidine metabolism including decreases in ribose-5P, UMP and GMP. These nucleotide reductions impacted the amount of extracellular DNA in biofilms and reduced biofilm formation. Mutation also limited the capacity of the strain to resist oxidant damage induced by hydrogen peroxide and paraquat and subsequent intracellular survival inside macrophages. Changes in wall teichoic acid impacted susceptibility to hydrogen peroxide. We demonstrated the importance of these changes on virulence in three different models of infection, covering respiratory, skin and septicemia, demonstrating the need for proper PPP function in all models. This work demonstrates the multifaceted role metabolism can play in multiple aspects of S. aureus pathogenesis.
Cell Host & Microbe, 2013
Thiol-group oxidation of active and allosteric cysteines is a widespread regulatory posttranslational protein modification. Pathogenic bacteria, including Pseudomonas aeruginosa and Staphylococcus aureus, use regulatory cysteine oxidation to respond to and overcome reactive oxygen species (ROS) encountered in the host environment. To obtain a proteome-wide view of oxidation-sensitive cysteines in these two pathogens, we employed a competitive activity-based protein profiling approach to globally quantify hydrogen peroxide (H 2 O 2) reactivity with cysteines across bacterial proteomes. We identified $200 proteins containing H 2 O 2-sensitive cysteines, including metabolic enzymes, transcription factors, and uncharacterized proteins. Additional biochemical and genetic studies identified an oxidation-responsive cysteine in the master quorumsensing regulator LasR and redox-regulated activities for acetaldehyde dehydrogenase ExaC, arginine deiminase ArcA, and glyceraldehyde 3-phosphate dehydrogenase. Taken together, our data indicate that pathogenic bacteria exhibit a complex, multilayered response to ROS that includes the rapid adaption of metabolic pathways to oxidative-stress challenge.
Journal of Bacteriology, 2013
Pseudomonas aeruginosa, a human opportunistic pathogen, possesses a number of antioxidant 28 defense enzymes under the control of multiple regulatory systems. We recently reported that 29 inactivation of the P. aeruginosa stringent response (SR), a starvation stress response controlled 30 by the alarmone (p)ppGpp, caused impaired antioxidant defenses and antibiotic tolerance. Since 31 catalases are key antioxidant enzymes in P. aeruginosa, we compared the H 2 O 2 susceptibility 32 and catalase activity in wild-type and ΔrelA ΔspoT (ΔSR) mutant cells. We found that the SR 33 was required for optimal catalase activity and mediated H 2 O 2 tolerance during both planktonic 34 and biofilm growth. Upon amino acid starvation, induction of the SR upregulated catalase 35 activity. Full expression of katA and katB also required the SR, and this regulation occurred 36 through both RpoS-independent and RpoS-dependent mechanisms. Furthermore, overexpression 37 of katA was sufficient to restore H 2 O 2 tolerance, and to partially rescue the antibiotic tolerance of 38 ΔSR cells. Altogether, these results suggest that the SR regulates catalases, and this is an 39 important mechanism in protecting nutrient-starved and biofilm bacteria from H 2 O 2 and 40 antibiotic mediated killing. 41 42 43 on December 10, 2016 by guest http://jb.asm.org/ Downloaded from Stringent response control of catalases in P. aeruginosa 44 Reactive oxygen species (ROS) are spontaneously produced during aerobic respiration. During 45 infections, bacteria are also challenged by ROS produced by host phagocytic cells. Given that 46 ROS can readily damage membranes, DNA and proteins (1, 2), bacteria possess multiple 47 antioxidant defenses to survive during aerobic growth and in vivo. While it is expected that 48 oxidative stress induces antioxidant defenses, interestingly, other stresses such as nutrient 49 limitation also elicit antioxidant responses. For example, carbon and nitrogen starvation 50 increases resistance to H 2 O 2 in different bacterial species, but the mechanism remains poorly 51 understood (3-6). Stationary phase and biofilm bacteria are also nutrient limited, and exhibit high 52 oxidant tolerance (7-9). Does nutrient limitation induce starvation responses that also confer 53 protection against oxidative stress? To investigate the contribution of starvation responses in 54 inducing anti-oxidant defenses, we examined the role of the stringent response.
Frontiers in Immunology, 2021
Reactive oxygen species (ROS) play a crucial role in the cellular defense against S. aureus, as evidenced by the importance of this pathogen in patients lacking the ROS-generating phagocyte NADPH oxidase NOX2. ROS concentrations required to kill S. aureus in vitro are much higher than those found in the phagosome. We therefore hypothesized that sublethal ROS concentrations may play a role in S. aureus gene dysregulation and investigated the in vitro transcriptomic response of S. aureus to sublethal concentrations of hydrogen peroxide (H2O2). A striking observation of these experiments was a coordinated and massive downregulation of genes involved in pyrimidine metabolism. Using transposon insertion mutants, we demonstrated that deletion of carA, a gene involved in pyrimidine synthesis, led to a significant growth defect and to an increased sensitivity of S. aureus to added H2O2. The phenotype of the carA mutant could be reversed through supplementation with the pyrimidine precursor ...
Infection and Immunity, 2010
The Gram-positive bacterium Staphylococcus aureus contains two glyceraldehyde-3-phosphate dehydrogenase (GAPDH) homologues known as GapA and GapB. GapA has been characterized as a functional GAPDH protein, but currently there is no biological evidence for the role of GapB in metabolism in S. aureus. In this study we show through a number of complementary methods that S. aureus GapA is essential for glycolysis while GapB is essential in gluconeogenesis. These proteins are reciprocally regulated in response to glucose concentrations, and both are influenced by the glycolysis regulator protein GapR, which is the first demonstration of the role of this regulator in S. aureus and the first indication that GapR homologues control genes other than those within the glycolytic operon. Furthermore, we show that both GapA and GapB are important in the pathogenesis of S. aureus in a Galleria mellonella model of infection, showing for the first time in any bacteria that both glycolysis and gluconeogenesis have important roles in virulence.
Glycine metabolism and anti-oxidative defence mechanisms in Pseudomonas fluorescens
Microbiological Research, 2015
The role of metabolism in anti-oxidative defence is only now beginning to emerge. Here, we show that the nutritionally-versatile microbe, Pseudomonas fluorescens, reconfigures its metabolism in an effort to generate NADPH, ATP and glyoxylate in order to fend off oxidative stress. Glyoxylate was produced predominantly via the enhanced activities of glycine dehydrogenase-NADP + (GDH), glycine transaminase (GTA) and isocitrate lyase (ICL) in a medium exposed to hydrogen peroxide (H 2 O 2 ). This ketoacid was utilized to produce ATP by substrate-level phosphorylation and to neutralize reactive oxygen species with the concomitant formation of formate. The latter was also a source of NADPH, a process mediated by formate dehydrogenase-NADP + (FDH). The increased activities of phosphoenolpyruvate carboxylase (PEPC) and pyruvate orthophosphate dikinase (PPDK) worked in tandem to synthesize ATP in the H 2 O 2challenged cells that had markedly diminished capacity for oxidative phosphorylation. These metabolic networks provide an effective means of combating ROS and reveal therapeutic targets against microbes resistant to oxidative stress.
Pseudomonas fluorescens invoked a metabolic reconfiguration that resulted in enhanced production of pyruvate under the challenge of hydrogen peroxide (H₂O₂). Although this stress led to a sharp reduction in the activities of numerous tricarboxylic acid (TCA) cycle enzymes, there was a marked increase in the activities of catalase and various NADPH-generating enzymes to counter the oxidative burden. The upregulation of phosphoenolpyruvate synthase (PEPS) and pyruvate kinase (PK) coupled with the reduction of pyruvate dehydrogenase (PDH) in the H₂O₂-challenged cells appear to be important contributors to the elevated levels of pyruvate found in these bacteria. Increased pyruvate synthesis was evident in the presence of a variety of carbon sources including d-glucose. Intact cells rapidly consumed d-glucose with the concomitant formation of this monocarboxylic acid. At least a 12-fold increase in pyruvate production within 1h was observed in the stressed cells. These findings may be exploited in the development of technologies aimed at the conversion of carbohydrates into pyruvate.
Non-lethal exposure to H2O2 boosts bacterial survival and evolvability against oxidative stress
Unicellular organisms have the prevalent challenge to survive under oxidative stress of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2). ROS are present as by-products of photosynthesis and aerobic respiration. These reactive species are even employed by multicellular organisms as potent weapons against microbes. Although bacterial defences against lethal and sub-lethal oxidative stress have been studied in model bacteria, the role of fluctuating H2O2 concentrations remains unexplored. It is known that sub-lethal exposure of Escherichia coli to H2O2 results in enhanced survival upon subsequent exposure. Here we investigate the priming response to H2O2 at physiological concentrations. The basis and the duration of the response (memory) were also determined by time-lapse quantitative proteomics. We found that a low level of H2O2 induced several scavenging enzymes showing a long half-life, subsequently protecting cells from future exposure. We then asked if the phenotypi...
Journal of Bacteriology, 2001
Hydrogen peroxide is generated during aerobic metabolism and is capable of damaging critical biomolecules. However, mutants of Escherichia coli that are devoid of catalase typically exhibit no adverse phenotypes during growth in aerobic media. We discovered that catalase mutants retain the ability to rapidly scavenge H 2 O 2 whether it is formed internally or provided exogenously. Analysis of candidate genes revealed that the residual activity is due to alkyl hydroperoxide reductase (Ahp). Mutants that lack both Ahp and catalase could not scavenge H 2 O 2 . These mutants excreted substantial amounts of H 2 O 2 , and they grew poorly in air. Ahp is kinetically a more efficient scavenger of trace H 2 O 2 than is catalase and therefore is likely to be the primary scavenger of endogenous H 2 O 2 . Accordingly, mutants that lack Ahp accumulated sufficient hydrogen peroxide to induce the OxyR regulon, whereas the OxyR regulon remained off in catalase mutants. Catalase still has an importa...