Pneumococcal H₂O₂ Reshapes Mitochondrial Function and Reprograms Host Cell Metabolism
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE301606 | GEO | 2025/09/30
REPOSITORIES: GEO
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Project description:The catalase-negative, facultative anaerobe Streptococcus pneumoniae D39 is naturally resistant to hydrogen peroxide (H2O2) produced endogenously by pyruvate oxidase (SpxB). Here, we investigate the adaptive response to endogenously produced H2O2. We show that lactate oxidase, which converts lactate to pyruvate, positively impacts pyruvate flux through SpxB and that ∆lctO mutants produce significantly lower H2O2. In addition, both the SpxB and pyruvate dehydrogenase complex (PDHC) pathways contribute to acetyl-CoA production during aerobic growth, and the pyruvate format lyase (PFL) pathway is the major acetyl-CoA pathway during anaerobic growth. Microarray analysis of the D39 strain cultured under aerobic vs. strict anaerobic conditions show up-regulation of spxB, a rhodanese-like protein (spd0091), tpxD, sodA, piuB, piuD and an Fe-S protein biogenesis operon under H2O2-producing conditions. Proteome profiling of H2O2-induced sulfenylation reveals that sulfenylation levels correlate with cellular H2O2 production, with endogenous sulfenylation of ≈50 proteins. Deletion of tpxD increases cellular sulfenylation 5-fold and has an inhibitory effect on ATP generation. Two major targets of protein sulfenylation are glyceraldehyde-3-phosphate dehydrogenase (GapA) and SpxB itself, but also include pyruvate kinase, LctO, AdhE and acetate kinase (AckA). Sulfenylation of GapA is inhibitory, while the effect on SpxB activity is negligible, consistent with this cell-abundant protein functioning as a “sink” for endogenous H2O2. Strikingly, four enzymes of capsular polysaccharide biosynthesis are sulfenylated, as are enzymes associated nucleotide biosynthesis via ribulose-5-phosphate. We propose that LctO/SpxB-generated H2O2 functions as a signaling molecule to down-regulate capsule production and drive altered flux through sugar utilization pathways.
2016-12-08 | GSE19791 | GEO
Project description:New antimalarial drugs are urgently needed to control drug resistant forms of the malaria parasite, Plasmodium falciparum. Although mitochondrial metabolism is the target of both existing drugs and new lead compounds, the role of the mitochondrial tricarboxylic acid (TCA) cycle remains poorly understood. Herein, we describe 11 genetic knockout parasite lines that delete six of the eight TCA cycle enzymes. Although all TCA knockouts grew normally in asexual blood stages, these metabolic deficiencies halted lifecycle progression in later stages. Specifically, aconitase knockout parasites arrested as late gametocytes, whereas α-ketoglutarate dehydrogenase deficient parasites failed to develop oocysts in the mosquitoes. Mass-spectrometry analysis of 13C isotope-labeled TCA mutant parasites showed that P. falciparum has significant flexibility in TCA metabolism. This flexibility manifested itself through changes in pathway fluxes and through altered exchange of substrates between cytosolic and mitochondrial pools. Our findings suggest that mitochondrial metabolic plasticity is essential for parasite development . Two parallel timecourses resulting in a total of 16 samples (8 wildtype, Isocitrate Dehydrogenase/alpha-Ketogluterate Dehydrogenase double knockout) were hybridized against a Cy3-labeled reference pool of 3D7 mixed stage parasites on a two-color array.
2014-08-01 | E-GEOD-59015 | biostudies-arrayexpress
Project description:Activation of glycolytic genes by HIF-1 is considered critical for metabolic adaptation to hypoxia. We found that HIF-1 also actively suppresses glucose metabolism through the tricarboxylic acid cycle (TCA) by directly trans-activating the gene encoding pyruvate dehydrogenase kinase 1 (PDK1). PDK1 inactivates the TCA cycle enzyme, pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA. Forced PDK1 expression in hypoxic HIF-1α-null cells increases ATP levels, attenuates hypoxic ROS generation and rescues these cells from hypoxia-induced apoptosis. These studies reveal a novel hypoxia-induced metabolic switch that shunts glucose metabolites from the mitochondria to glycolysis to maintain ATP production and to prevent toxic ROS production. Experiment Overall Design: We sought to determine by microarray analysis of gene expression the genes responsive to hypoxia using the human B lymphocyte cell line, P493-6. Hypoxia-responsive genes were globally assessed in cells incubated in 0.1% O2 for 29 hours at which the highest HIF-1 levels were obtained
2008-06-12 | E-GEOD-4086 | biostudies-arrayexpress
Project description:Activation of glycolytic genes by HIF-1 is considered critical for metabolic adaptation to hypoxia. We found that HIF-1 also actively suppresses glucose metabolism through the tricarboxylic acid cycle (TCA) by directly trans-activating the gene encoding pyruvate dehydrogenase kinase 1 (PDK1). PDK1 inactivates the TCA cycle enzyme, pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA. Forced PDK1 expression in hypoxic HIF-1α-null cells increases ATP levels, attenuates hypoxic ROS generation and rescues these cells from hypoxia-induced apoptosis. These studies reveal a novel hypoxia-induced metabolic switch that shunts glucose metabolites from the mitochondria to glycolysis to maintain ATP production and to prevent toxic ROS production. Keywords: Hypoxia responsive, case control
2006-03-16 | GSE4086 | GEO
Project description:Pyruvate oxidase encoded by spxB is a major virulence factor in the human respiratory pathogen Streptococcus pneumoniae. During aerobic growth, SpxB synthesizes large amounts of H2O2 and acetyl phosphate, which can serve as a phosphoryl group donor to response regulators and be converted to ATP. SpxB is the main source of the millimolar concentrations of H2O2 produced and tolerated by pneumococcus, despite its lack of a catalase. We report here the first cis- and trans-acting regulatory elements for spxB transcription. These elements were identified in a genetic screen, similar to those used previously for phase variants, for spontaneous mutations that caused colonies of virulent serotype 2 strain D39 to change from a transparent to an opaque appearance. Six of the seven opaque colonies recovered (frequency of 3 x 10-5) were impaired for SpxB function. Modeling suggested that two mutations changed amino acids in SpxB required for FAD cofactor or subunit binding. One mutation deleted a cis-acting adjacent direct repeat required for optimal spxB transcription. The other three independent mutations created the same frameshift near the start of a trans-acting regulatory gene designated as spxR. The SpxR protein contains helix-turn-helix, CBS, and HotDog domains implicated in DNA, adenosine, and CoA compound binding, respectively, consistent with the idea that SpxR positively regulates spxB transcript amount in response to energy and metabolic state rather than oxidative state. Finally, microarray analyses of a null spxB or a spxR mutant revealed the presence of a new oxidative stress response in pneumococcus and unexpectedly demonstrated that SpxR strongly positively regulates the transcript amount of the strH exoglycosidase gene, which like spxB, has been implicated in host colonization. Keywords: genetic modification Bacterial strains were grown exponentially in rich (BHI) media at 37C and an atmosphere of 5% CO2, and were processed as described in the related Sample records. Samples were collected from three independent biological replicates and included one dye swap. Data were normalized using the Lowess (subgrid) method without background subtraction.
2010-05-26 | E-GEOD-7109 | biostudies-arrayexpress
Project description:spxB-encoded pyruvate oxidase is a major virulence factor of Streptococcus pneumoniae. During aerobic growth, SpxB synthesizes H2O2 and acetyl phosphate, which play roles in metabolism, signaling, and oxidative stress. We report here the first cis- and trans-acting regulatory elements for spxB transcription. These elements were identified in a genetic screen for spontaneous mutations that caused colonies of strain D39 to change from a semi-transparent to an opaque appearance. Six of the seven opaque colonies recovered (frequency 3x10-5) were impaired for SpxB function or expression. Two mutations changed amino acids in SpxB likely required for cofactor or subunit binding. One mutation defined a cis-acting adjacent direct repeat required for optimal spxB transcription. The other three spontaneous mutations created the same frameshift near the start of the trans-acting spxR regulatory gene. The SpxR protein contains helix-turn-helix, CBS, and HotDog domains implicated in binding DNA, adenosyl compounds, and CoA-containing compounds, respectively, and suggest that SpxR positively regulates spxB transcription in response to energy and metabolic state. Finally, microarray analyses of a spxR mutant demonstrated that SpxR positively regulates the strH exoglycosidase gene, which like spxB, has been implicated in colonization. Keywords: bacterial genetic modification
2008-01-04 | GSE9284 | GEO
Project description:Pyruvate oxidase encoded by spxB is a major virulence factor in the human respiratory pathogen Streptococcus pneumoniae. During aerobic growth, SpxB synthesizes large amounts of H2O2 and acetyl phosphate, which can serve as a phosphoryl group donor to response regulators and be converted to ATP. SpxB is the main source of the millimolar concentrations of H2O2 produced and tolerated by pneumococcus, despite its lack of a catalase. We report here the first cis- and trans-acting regulatory elements for spxB transcription. These elements were identified in a genetic screen, similar to those used previously for phase variants, for spontaneous mutations that caused colonies of virulent serotype 2 strain D39 to change from a transparent to an opaque appearance. Six of the seven opaque colonies recovered (frequency of 3 x 10-5) were impaired for SpxB function. Modeling suggested that two mutations changed amino acids in SpxB required for FAD cofactor or subunit binding. One mutation deleted a cis-acting adjacent direct repeat required for optimal spxB transcription. The other three independent mutations created the same frameshift near the start of a trans-acting regulatory gene designated as spxR. The SpxR protein contains helix-turn-helix, CBS, and HotDog domains implicated in DNA, adenosine, and CoA compound binding, respectively, consistent with the idea that SpxR positively regulates spxB transcript amount in response to energy and metabolic state rather than oxidative state. Finally, microarray analyses of a null spxB or a spxR mutant revealed the presence of a new oxidative stress response in pneumococcus and unexpectedly demonstrated that SpxR strongly positively regulates the transcript amount of the strH exoglycosidase gene, which like spxB, has been implicated in host colonization. Keywords: genetic modification
2008-02-22 | GSE7109 | GEO
Project description:We studied the transcriptomic response of S. pneumoniae to moxifloxacin, a fluoroquinolone that inhibits DNA gyrase. At 10 × MIC, 30 and 140 responsive genes were detected at 15 and 30 minutes, respectively. Several pathways leading to an increase in pyruvate showed up-regulation. These included 2 genes introducing P-sugars into the glycolysis and 3 out of 7 genes of the glycolysis pathway, which converts fructose-6P to pyruvate. In addition, an increase in acetyl-coA would be expected from the down-regulation of the genes coding for the acetyl-coA carboxylase, and in turn, to a further increase in pyruvate by the up-regulation of formate acetyltransferase. Since pyruvate is converted to hydrogen peroxide (H2O2) by pyruvate oxidase (SpxB), its increase would lead to an equivalent increase in the intracellular amount of H2O2, and in turn, in those of hydroxyl radicals resulting from the Fenton reaction, which damage DNA, lipids and proteins. We observed similar increases in the production of H2O2 and hydroxyl radicals under moxifloxacin treatment. These reactive oxygen species contributed to the lethality of the drug, as showed by the attenuation of its lethality in a strain lacking SpxB. These results support the production of redox alterations by fluoroquinolones and are in agreement with our previous findings showing that levofloxacin, an inhibitor of topoisomerase IV, triggers the transcriptional activation of iron transport genes. Both fluoroquinolones stimulate the Fenton reaction in their mechanism of action, by increasing the amounts of any of their two components: iron by levofloxacin or H2O2 by moxifloxacin.
2016-02-05 | GSE68947 | GEO
Project description:New antimalarial drugs are urgently needed to control drug resistant forms of the malaria parasite, Plasmodium falciparum. Although mitochondrial metabolism is the target of both existing drugs and new lead compounds, the role of the mitochondrial tricarboxylic acid (TCA) cycle remains poorly understood. Herein, we describe 11 genetic knockout parasite lines that delete six of the eight TCA cycle enzymes. Although all TCA knockouts grew normally in asexual blood stages, these metabolic deficiencies halted lifecycle progression in later stages. Specifically, aconitase knockout parasites arrested as late gametocytes, whereas α-ketoglutarate dehydrogenase deficient parasites failed to develop oocysts in the mosquitoes. Mass-spectrometry analysis of 13C isotope-labeled TCA mutant parasites showed that P. falciparum has significant flexibility in TCA metabolism. This flexibility manifested itself through changes in pathway fluxes and through altered exchange of substrates between cytosolic and mitochondrial pools. Our findings suggest that mitochondrial metabolic plasticity is essential for parasite development .
2014-08-01 | GSE59015 | GEO
Project description:Transcriptional profiling of the spxA1-null mutant of Streptococcus sanguinis SK36 compared with wild type. The spxA1 gene was inactivated in Streptococcus sanguinis SK36, and the mutant demonstrated opaque colony morphology, reduced hydrogen peroxide (H2O2) production, and reduced antagonistic activity against Streptococcus S. mutans UA159 both on plates and in liquid media. The mutant also showed decreased tolerance to high temperature, and acidic and oxidative stresses. Complementation of the ΔspxA1 mutant with spxA1 restored colony morphology, H2O2 production and stress tolerance to the ΔspxA1 mutant. The mutant also exhibited an ~5-fold reduction in competitiveness in an animal model of endocarditis, indicating the involvement of SpxA1 in endocarditis virulence. Microarray studies revealed that a number of SpxA1-upregulated genes are involved in oxidative stress. The expression of spxB and nox (which encode pyruvate oxidase and NADH oxidase, respectively, and are involved in H2O2 production and nox involved virulence) significantly decreased in ΔspxA1 compared with the wild type. This may be at least partly responsible for the decreased H2O2 production and reduced virulence in the ΔspxA1 mutant because spxB and nox were involved in H2O2 production and nox involved virulence.
2011-12-08 | GSE34203 | GEO