scholarly journals IntracellularStaphylococcus aureusModulates Host Central Carbon Metabolism To Activate Autophagy

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
pp. e00374-18 ◽  
Author(s):  
Natalia Bravo-Santano ◽  
James K. Ellis ◽  
Luis M. Mateos ◽  
Yolanda Calle ◽  
Hector C. Keun ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Host Cell ◽  
Carbon Metabolism ◽  
Central Carbon Metabolism ◽  
Metabolic Changes ◽  
Autophagic Flux ◽  
Content Type ◽  
Central Carbon ◽  
Infected Cells ◽  
Intracellular Proliferation

ABSTRACTStaphylococcus aureusis a facultative intracellular pathogen that invades and replicates within many types of phagocytic and nonphagocytic cells. During intracellular infection,S. aureusis capable of subverting xenophagy and escaping to the cytosol of the host cell. Furthermore, drug-induced autophagy facilitates the intracellular replication ofS. aureus, but the reasons behind this are unclear. Here, we have studied the host central carbon metabolism duringS. aureusintracellular infection. We found extensive metabolic rerouting and detected several distinct metabolic changes that suggested starvation-induced autophagic flux in infected cells. These changes included increased uptake but lower intracellular levels of glucose and low abundance of several essential amino acids, as well as markedly upregulated glutaminolysis. Furthermore, we show that AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase (ERK) phosphorylation levels are significantly increased in infected cells. Interestingly, while autophagy was activated in response toS. aureusinvasion, most of the autophagosomes detected in infected cells did not contain bacteria, suggesting thatS. aureusinduces the autophagic flux during cell invasion for energy generation and nutrient scavenging. Accordingly, AMPK inhibition haltedS. aureusintracellular proliferation.IMPORTANCEStaphylococcus aureusescapes from immune recognition by invading a wide range of human cells. Once the pathogen becomes intracellular, the most important last resort antibiotics are not effective. Therefore, novel anti-infective therapies against intracellularS. aureusare urgently needed. Here, we have studied the physiological changes induced in the host cells byS. aureusduring its intracellular proliferation. This is important, because the pathogen exploits the host cell’s metabolism for its own proliferation. We find thatS. aureusseverely depletes glucose and amino acid pools, which leads to increased breakdown of glutamine by the host cell in an attempt to meet its own metabolic needs. All of these metabolic changes activate autophagy in the host cell for nutrient scavenging and energy generation. The metabolic activation of autophagy could be used by the pathogen to sustain its own intracellular survival, making it an attractive target for novel anti-infectives.

scholarly journals HexR Controls Glucose-Responsive Genes and Central Carbon Metabolism in Neisseria meningitidis

2015 ◽  
Vol 198 (4) ◽  
pp. 644-654 ◽  
Author(s):  
Ana Antunes ◽  
Giacomo Golfieri ◽  
Francesca Ferlicca ◽  
Marzia M. Giuliani ◽  
Vincenzo Scarlato ◽  
...  
Keyword(s):  
Energy Metabolism ◽  
Neisseria Meningitidis ◽  
Microarray Analysis ◽  
Carbon Metabolism ◽  
Tricarboxylic Acid Cycle ◽  
Transcriptional Regulator ◽  
Central Carbon Metabolism ◽  
Binding Motif ◽  
Content Type ◽  
Central Carbon

ABSTRACTNeisseria meningitidis, an exclusively human pathogen and the leading cause of bacterial meningitis, must adapt to different host niches during human infection.N. meningitidiscan utilize a restricted range of carbon sources, including lactate, glucose, and pyruvate, whose concentrations vary in host niches. Microarray analysis ofN. meningitidisgrown in a chemically defined medium in the presence or absence of glucose allowed us to identify genes regulated by carbon source availability. Most such genes are implicated in energy metabolism and transport, and some are implicated in virulence. In particular, genes involved in glucose catabolism were upregulated, whereas genes involved in the tricarboxylic acid cycle were downregulated. Several genes encoding surface-exposed proteins, including the MafA adhesins andNeisseriasurface protein A, were upregulated in the presence of glucose. Our microarray analysis led to the identification of a glucose-responsivehexR-like transcriptional regulator that controls genes of the central carbon metabolism ofN. meningitidisin response to glucose. We characterized the HexR regulon and showed that thehexRgene is accountable for some of the glucose-responsive regulation;in vitroassays with the purified protein showed that HexR binds to the promoters of the central metabolic operons of the bacterium. Based on DNA sequence alignment of the target sites, we propose a 17-bp pseudopalindromic consensus HexR binding motif. Furthermore,N. meningitidisstrains lackinghexRexpression were deficient in establishing successful bacteremia in an infant rat model of infection, indicating the importance of this regulator for the survival of this pathogenin vivo.IMPORTANCENeisseria meningitidisgrows on a limited range of nutrients during infection. We analyzed the gene expression ofN. meningitidisin response to glucose, the main energy source available in human blood, and we found that glucose regulates many genes implicated in energy metabolism and nutrient transport, as well as some implicated in virulence. We identified and characterized a transcriptional regulator (HexR) that controls metabolic genes ofN. meningitidisin response to glucose. We generated a mutant lacking HexR and found that the mutant was impaired in causing systemic infection in animal models. SinceN. meningitidislacks known bacterial regulators of energy metabolism, our findings suggest that HexR plays a major role in its biology by regulating metabolism in response to environmental signals.

scholarly journals Dynamic Metabolic Rewiring Enables Efficient Acetyl Coenzyme A Assimilation in Paracoccus denitrificans

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Katharina Kremer ◽  
Muriel C. F. van Teeseling ◽  
Lennart Schada von Borzyskowski ◽  
Iria Bernhardsgrütter ◽  
Rob J. M. van Spanning ◽  
...  
Keyword(s):  
Growth Rates ◽  
Carbon Metabolism ◽  
Paracoccus Denitrificans ◽  
High Growth ◽  
Central Carbon Metabolism ◽  
High Biomass ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Content Type ◽  
Central Carbon

ABSTRACT During growth, microorganisms have to balance metabolic flux between energy and biosynthesis. One of the key intermediates in central carbon metabolism is acetyl coenzyme A (acetyl-CoA), which can be either oxidized in the citric acid cycle or assimilated into biomass through dedicated pathways. Two acetyl-CoA assimilation strategies in bacteria have been described so far, the ethylmalonyl-CoA pathway (EMCP) and the glyoxylate cycle (GC). Here, we show that Paracoccus denitrificans uses both strategies for acetyl-CoA assimilation during different growth stages, revealing an unexpected metabolic complexity in the organism’s central carbon metabolism. The EMCP is constitutively expressed on various substrates and leads to high biomass yields on substrates requiring acetyl-CoA assimilation, such as acetate, while the GC is specifically induced on these substrates, enabling high growth rates. Even though each acetyl-CoA assimilation strategy alone confers a distinct growth advantage, P. denitrificans recruits both to adapt to changing environmental conditions, such as a switch from succinate to acetate. Time-resolved single-cell experiments show that during this switch, expression of the EMCP and GC is highly coordinated, indicating fine-tuned genetic programming. The dynamic metabolic rewiring of acetyl-CoA assimilation is an evolutionary innovation by P. denitrificans that allows this organism to respond in a highly flexible manner to changes in the nature and availability of the carbon source to meet the physiological needs of the cell, representing a new phenomenon in central carbon metabolism. IMPORTANCE Central carbon metabolism provides organisms with energy and cellular building blocks during growth and is considered the invariable “operating system” of the cell. Here, we describe a new phenomenon in bacterial central carbon metabolism. In contrast to many other bacteria that employ only one pathway for the conversion of the central metabolite acetyl-CoA, Paracoccus denitrificans possesses two different acetyl-CoA assimilation pathways. These two pathways are dynamically recruited during different stages of growth, which allows P. denitrificans to achieve both high biomass yield and high growth rates under changing environmental conditions. Overall, this dynamic rewiring of central carbon metabolism in P. denitrificans represents a new strategy compared to those of other organisms employing only one acetyl-CoA assimilation pathway.

scholarly journals Metabolic Changes Induced by Deletion of Transcriptional Regulator GCR2 in Xylose-Fermenting Saccharomyces cerevisiae

2020 ◽  
Vol 8 (10) ◽  
pp. 1499
Author(s):  
Minhye Shin ◽  
Soo Rin Kim
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Pentose Phosphate Pathway ◽  
Carbon Metabolism ◽  
Transcriptional Regulator ◽  
Central Carbon Metabolism ◽  
Pentose Phosphate ◽  
Metabolic Changes ◽  
Regulatory Systems ◽  
Central Carbon

Glucose repression has been extensively studied in Saccharomyces cerevisiae, including the regulatory systems responsible for efficient catabolism of glucose, the preferred carbon source. However, how these regulatory systems would alter central metabolism if new foreign pathways are introduced is unknown, and the regulatory networks between glycolysis and the pentose phosphate pathway, the two major pathways in central carbon metabolism, have not been systematically investigated. Here we disrupted gcr2, a key transcriptional regulator, in S. cerevisiae strain SR7 engineered to heterologously express the xylose-assimilating pathway, activating genes involved in glycolysis, and evaluated the global metabolic changes. gcr2 deletion reduced cellular growth in glucose but significantly increased growth when xylose was the sole carbon source. Global metabolite profiling revealed differential regulation of yeast metabolism in SR7-gcr2Δ, especially carbohydrate and nucleotide metabolism, depending on the carbon source. In glucose, the SR7-gcr2Δ mutant showed overall decreased abundance of metabolites, such as pyruvate and sedoheptulose-7-phosphate, associated with central carbon metabolism including glycolysis and the pentose phosphate pathway. However, SR7-gcr2Δ showed an increase in metabolites abundance (ribulose-5-phosphate, sedoheptulose-7-phosphate, and erythrose-4-phosphate) notably from the pentose phosphate pathway, as well as alteration in global metabolism when compared to SR7. These results provide insights into how the regulatory system GCR2 coordinates the transcription of glycolytic genes and associated metabolic pathways.

scholarly journals Noncanonical Inhibition of mTORC1 byCoxiella burnetiiPromotes Replication within a Phagolysosome-Like Vacuole

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Charles L. Larson ◽  
Kelsi M. Sandoz ◽  
Diane C. Cockrell ◽  
Robert A. Heinzen
Keyword(s):  
Host Cell ◽  
Coxiella Burnetii ◽  
Secretion System ◽  
Effector Proteins ◽  
Nutrient Deprivation ◽  
Autophagic Flux ◽  
Content Type ◽  
Mechanistic Target Of Rapamycin ◽  
Infected Cells ◽  
Bacterial Replication

ABSTRACTThe Q fever agentCoxiella burnetiiis a Gram-negative bacterium that invades macrophages and replicates inside a specialized lysosomal vacuole. The pathogen employs a type 4B secretion system (T4BSS) to deliver effector proteins into the host cell that modify theCoxiella-containing vacuole (CCV) into a replication-permissive niche. Mature CCVs are massive degradative organelles that acquire lysosomal proteins. Inhibition of mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) kinase by nutrient deprivation promotes autophagy and lysosome fusion, as well as activation of the transcription factors TFE3 and TFEB (TFE3/B), which upregulates expression of lysosomal genes. Here, we report thatC. burnetiiinhibits mTORC1 as evidenced by impaired localization of mTORC1 to endolysosomal membranes and decreased phosphorylation of elF4E-binding protein 1 (4E-BP1) and S6 kinase 1 in infected cells. Infected cells exhibit increased amounts of autophagy-related proteins protein 1A/1B-light chain 3 (LC3) and p62 as well as of activated TFE3. However,C. burnetiidid not accelerate autophagy or block autophagic flux triggered by cell starvation. Activation of autophagy or transcription by TFE3/B increased CCV expansion without enhancing bacterial replication. By contrast, knockdown of tuberous sclerosis complex 1 (TSC1) or TSC2, which hyperactivates mTORC1, impaired CCV expansion and bacterial replication. Together, these data demonstrate that specific inhibition of mTORC1 byC. burnetii, but not amplified cell catabolism via autophagy, is required for optimal pathogen replication. These data reveal a complex interplay between lysosomal function and host cell metabolism that regulatesC. burnetiiintracellular growth.IMPORTANCECoxiella burnetiiis an intracellular pathogenic bacterium that replicates within a lysosomal vacuole. Biogenesis of theCoxiella-containing vacuole (CCV) requires effector proteins delivered into the host cell cytosol by the type 4B secretion system (T4BSS). Modifications to lysosomal physiology required for pathogen replication within the CCV are poorly understood. Mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) is a master kinase that regulates lysosome structure and function. Nutrient deprivation inhibits mTORC1, which promotes cell catabolism in the form of accelerated autophagy and increased lysosome biosynthesis. Here, we report thatC. burnetiigrowth is enhanced by T4BSS-dependent inhibition of mTORC1 that does not activate autophagy. Canonical inhibition of mTORC1 by starvation or inhibitor treatment that induces autophagic flux does not benefitC. burnetiigrowth. Furthermore, hyperactivation of mTORC1 impairs bacterial replication. These findings indicate thatC. burnetiiinhibition of mTORC1 without accelerated autophagy promotes bacterial growth.

scholarly journals The Functional Structure of Central Carbon Metabolism in Pseudomonas putida KT2440

2014 ◽  
Vol 80 (17) ◽  
pp. 5292-5303 ◽  
Author(s):  
Suresh Sudarsan ◽  
Sarah Dethlefsen ◽  
Lars M. Blank ◽  
Martin Siemann-Herzberg ◽  
Andreas Schmid
Keyword(s):  
Pseudomonas Putida ◽  
Carbon Metabolism ◽  
Carbon Sources ◽  
Central Carbon Metabolism ◽  
Transcriptional Level ◽  
Regulation Mechanism ◽  
Central Metabolism ◽  
Content Type ◽  
Central Carbon ◽  
Definition Of

ABSTRACTWhat defines central carbon metabolism? The classic textbook scheme of central metabolism includes the Embden-Meyerhof-Parnas (EMP) pathway of glycolysis, the pentose phosphate pathway, and the citric acid cycle. The prevalence of this definition of central metabolism is, however, equivocal without experimental validation. We address this issue using a general experimental approach that combines the monitoring of transcriptional and metabolic flux changes between steady states on alternative carbon sources. This approach is investigated by using the model bacteriumPseudomonas putidawith glucose, fructose, and benzoate as carbon sources. The catabolic reactions involved in the initial uptake and metabolism of these substrates are expected to show a correlated change in gene expressions and metabolic fluxes. However, there was no correlation for the reactions linking the 12 biomass precursor molecules, indicating a regulation mechanism other than mRNA synthesis for central metabolism. This result substantiates evidence for a (re)definition of central carbon metabolism including all reactions that are bound to tight regulation and transcriptional invariance. Contrary to expectations, the canonical Entner-Doudoroff and EMP pathwayssensu strictoare not a part of central carbon metabolism inP. putida, as they are not regulated differently from the aromatic degradation pathway. The regulatory analyses presented here provide leads on a qualitative basis to address the use of alternative carbon sources by deregulation and overexpression at the transcriptional level, while rate improvements in central carbon metabolism require careful adjustment of metabolite concentrations, as regulation resides to a large extent in posttranslational and/or metabolic regulation.

scholarly journals The Metabolite Repair Enzyme Phosphoglycolate Phosphatase Regulates Central Carbon Metabolism and Fosmidomycin Sensitivity in Plasmodium falciparum

mBio ◽  
2019 ◽  
Vol 10 (6) ◽  
Author(s):  
Laure Dumont ◽  
Mark B. Richardson ◽  
Phillip van der Peet ◽  
Danushka S. Marapana ◽  
Tony Triglia ◽  
...  
Keyword(s):  
Pentose Phosphate Pathway ◽  
Carbon Metabolism ◽  
Central Carbon Metabolism ◽  
Pentose Phosphate ◽  
Repair Enzyme ◽  
Content Type ◽  
Phosphoglycolate Phosphatase ◽  
Central Carbon ◽  
Metabolite Repair ◽  
By Products

ABSTRACT Members of the haloacid dehalogenase (HAD) family of metabolite phosphatases play an important role in regulating multiple pathways in Plasmodium falciparum central carbon metabolism. We show that the P. falciparum HAD protein, phosphoglycolate phosphatase (PGP), regulates glycolysis and pentose pathway flux in asexual blood stages via detoxifying the damaged metabolite 4-phosphoerythronate (4-PE). Disruption of the P. falciparum pgp gene caused accumulation of two previously uncharacterized metabolites, 2-phospholactate and 4-PE. 4-PE is a putative side product of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase, and its accumulation inhibits the pentose phosphate pathway enzyme, 6-phosphogluconate dehydrogenase (6-PGD). Inhibition of 6-PGD by 4-PE leads to an unexpected feedback response that includes increased flux into the pentose phosphate pathway as a result of partial inhibition of upper glycolysis, with concomitant increased sensitivity to antimalarials that target pathways downstream of glycolysis. These results highlight the role of metabolite detoxification in regulating central carbon metabolism and drug sensitivity of the malaria parasite. IMPORTANCE The malaria parasite has a voracious appetite, requiring large amounts of glucose and nutrients for its rapid growth and proliferation inside human red blood cells. The host cell is resource rich, but this is a double-edged sword; nutrient excess can lead to undesirable metabolic reactions and harmful by-products. Here, we demonstrate that the parasite possesses a metabolite repair enzyme (PGP) that suppresses harmful metabolic by-products (via substrate dephosphorylation) and allows the parasite to maintain central carbon metabolism. Loss of PGP leads to the accumulation of two damaged metabolites and causes a domino effect of metabolic dysregulation. Accumulation of one damaged metabolite inhibits an essential enzyme in the pentose phosphate pathway, leading to substrate accumulation and secondary inhibition of glycolysis. This work highlights how the parasite coordinates metabolic flux by eliminating harmful metabolic by-products to ensure rapid proliferation in its resource-rich niche.

scholarly journals A Vector Library for Silencing Central Carbon Metabolism Genes with Antisense RNAs in Escherichia coli

2013 ◽  
Vol 80 (2) ◽  
pp. 564-573 ◽  
Author(s):  
Nobutaka Nakashima ◽  
Satoshi Ohno ◽  
Katsunori Yoshikawa ◽  
Hiroshi Shimizu ◽  
Tomohiro Tamura
Keyword(s):  
Escherichia Coli ◽  
Carbon Metabolism ◽  
Target Genes ◽  
Central Carbon Metabolism ◽  
Accurate Information ◽  
Content Type ◽  
E Coli ◽  
Antisense Rnas ◽  
Central Carbon ◽  
Multiple Gene Silencing

ABSTRACTWe describe here the construction of a series of 71 vectors to silence central carbon metabolism genes inEscherichia coli. The vectors inducibly express antisense RNAs called paired-terminus antisense RNAs, which have a higher silencing efficacy than ordinary antisense RNAs. By measuring mRNA amounts, measuring activities of target proteins, or observing specific phenotypes, it was confirmed that all the vectors were able to silence the expression of target genes efficiently. Using this vector set, each of the central carbon metabolism genes was silenced individually, and the accumulation of metabolites was investigated. We were able to obtain accurate information on ways to increase the production of pyruvate, an industrially valuable compound, from the silencing results. Furthermore, the experimental results of pyruvate accumulation were compared toin silicopredictions, and both sets of results were consistent. Compared to the gene disruption approach, the silencing approach has an advantage in that anyE. colistrain can be used and multiple gene silencing is easily possible in any combination.

scholarly journals Cofactor Specificity of Glucose-6-Phosphate Dehydrogenase Isozymes in Pseudomonas putida Reveals a General Principle Underlying Glycolytic Strategies in Bacteria

mSystems ◽  
2021 ◽  
Vol 6 (2) ◽  
Author(s):  
Daniel Christoph Volke ◽  
Karel Olavarría ◽  
Pablo Iván Nikel
Keyword(s):  
Gene Duplication ◽  
Pseudomonas Putida ◽  
Carbon Metabolism ◽  
Redox Balance ◽  
Central Carbon Metabolism ◽  
Evolutionary Strategy ◽  
Content Type ◽  
Cofactor Specificity ◽  
Central Carbon ◽  
Glucose 6 Phosphate Dehydrogenase

ABSTRACT Glucose-6-phosphate dehydrogenase (G6PDH) is widely distributed in nature and catalyzes the first committing step in the oxidative branch of the pentose phosphate (PP) pathway, feeding either the reductive PP or the Entner-Doudoroff pathway. Besides its role in central carbon metabolism, this dehydrogenase provides reduced cofactors, thereby affecting redox balance. Although G6PDH is typically considered to display specificity toward NADP+, some variants accept NAD+ similarly or even preferentially. Furthermore, the number of G6PDH isozymes encoded in bacterial genomes varies from none to more than four orthologues. On this background, we systematically analyzed the interplay of the three G6PDH isoforms of the soil bacterium Pseudomonas putida KT2440 from genomic, genetic, and biochemical perspectives. P. putida represents an ideal model to tackle this endeavor, as its genome harbors gene orthologues for most dehydrogenases in central carbon metabolism. We show that the three G6PDHs of strain KT2440 have different cofactor specificities and that the isoforms encoded by zwfA and zwfB carry most of the activity, acting as metabolic “gatekeepers” for carbon sources that enter at different nodes of the biochemical network. Moreover, we demonstrate how multiplication of G6PDH isoforms is a widespread strategy in bacteria, correlating with the presence of an incomplete Embden-Meyerhof-Parnas pathway. The abundance of G6PDH isoforms in these species goes hand in hand with low NADP+ affinity, at least in one isozyme. We propose that gene duplication and relaxation in cofactor specificity is an evolutionary strategy toward balancing the relative production of NADPH and NADH. IMPORTANCE Protein families have likely arisen during evolution by gene duplication and divergence followed by neofunctionalization. While this phenomenon is well documented for catabolic activities (typical of environmental bacteria that colonize highly polluted niches), the coexistence of multiple isozymes in central carbon catabolism remains relatively unexplored. We have adopted the metabolically versatile soil bacterium Pseudomonas putida KT2440 as a model to interrogate the physiological and evolutionary significance of coexisting glucose-6-phosphate dehydrogenase (G6PDH) isozymes. Our results show that each of the three G6PDHs in this bacterium display distinct biochemical properties, especially at the level of cofactor preference, impacting bacterial physiology in a carbon source-dependent fashion. Furthermore, the presence of multiple G6PDHs differing in NAD+ or NADP+ specificity in bacterial species strongly correlates with their predominant metabolic lifestyle. Our findings support the notion that multiplication of genes encoding cofactor-dependent dehydrogenases is a general evolutionary strategy toward achieving redox balance according to the growth conditions.

scholarly journals Copper stress in Staphylococcus aureus leads to adaptive changes in central carbon metabolism

Metallomics ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 183-200 ◽  
Author(s):  
Emma Tarrant ◽  
Gustavo P. Riboldi ◽  
Matthew R. McIlvin ◽  
Jack Stevenson ◽  
Anna Barwinska-Sendra ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Carbon Metabolism ◽  
Central Carbon Metabolism ◽  
Glycolytic Enzyme ◽  
Copper Stress ◽  
Specific Inhibition ◽  
Adaptive Changes ◽  
Central Carbon

Pathogenic Staphylococcus aureus respond to copper stress by altering central carbon metabolism in response to a specific inhibition of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase.

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