Malate synthase expression is deregulated in the Pseudomonas aeruginosa cystic fibrosis isolate FRD1

2011 ◽  
Vol 57 (3) ◽  
pp. 186-195 ◽  
Author(s):  
Jessica M. Hagins ◽  
Jessica Scoffield ◽  
Sang-Jin Suh ◽  
Laura Silo-Suh
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Carbon Sources ◽  
Malate Synthase ◽  
Current Evidence ◽  
Pathway Expression ◽  
Genes Encoding ◽  
Key Enzyme ◽  
Cystic Fibrosis Isolate ◽  
Glyoxylate Pathway

Pseudomonas aeruginosa causes chronic pulmonary infections, which can persist for decades, in patients with cystic fibrosis (CF). Current evidence suggests that the glyoxylate pathway is an important metabolic pathway for P. aeruginosa growing within the CF lung. In this study, we identified glcB, which encodes for the second key enzyme of the glyoxylate pathway, malate synthase, as a requirement for virulence of P. aeruginosa on alfalfa seedlings. While expression of glcB in PAO1, an acute isolate of P. aeruginosa, responds to some carbon sources that use the glyoxylate pathway, expression of glcB in FRD1, a CF isolate, is constitutively upregulated. Malate synthase activity is moderately affected by glcB expression and is nearly constitutive in both backgrounds, with slightly higher activity in FRD1 than in PAO1. In addition, RpoN negatively regulates glcB in PAO1 but not in FRD1. In summary, the genes encoding for the glyoxylate-specific enzymes appear to be coordinately regulated, even though they are not located within the same operon on the P. aeruginosa genome. Furthermore, both genes encoding for the glyoxylate enzymes can become deregulated during adaptation of the bacterium to the CF lung.

scholarly journals Isocitrate Lyase Supplies Precursors for Hydrogen Cyanide Production in a Cystic Fibrosis Isolate of Pseudomonas aeruginosa

2009 ◽  
Vol 191 (20) ◽  
pp. 6335-6339 ◽  
Author(s):  
Jessica M. Hagins ◽  
Robert Locy ◽  
Laura Silo-Suh
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Growth Medium ◽  
Hydrogen Cyanide ◽  
Isocitrate Lyase ◽  
Acid Dehydrogenase ◽  
Amino Acid Dehydrogenase ◽  
Cystic Fibrosis Isolate ◽  
Glyoxylate Pathway

ABSTRACT Pseudomonas aeruginosa colonizes and can persist in the lungs of cystic fibrosis (CF) patients for decades. Adaptation of P. aeruginosa to the CF lung environment causes various genotypic and phenotypic alterations in the bacterium that facilitate persistence. We showed previously that isocitrate lyase (ICL) activity is constitutively upregulated in the P. aeruginosa CF isolate FRD1. We show here that high ICL activity in FRD1 contributes to increased hydrogen cyanide (HCN) production by this isolate. Disruption of aceA, which encodes ICL, results in reduced cyanide production by FRD1 but does not affect cyanide production in the wound isolate PAO1. Cyanide production is restored to the FRD1aceA mutant by addition of glyoxylate, a product of ICL activity, or glycine to the growth medium. Conversion of glyoxylate to glycine may provide a mechanism for increased cyanide production by P. aeruginosa growing on compounds that activate the glyoxylate pathway. Consistent with this hypothesis, disruption of PA5304, encoding a putative d-amino acid dehydrogenase (DadA), led to decreased cyanide production by FRD1. Cyanide production was restored to the FRD1dadA mutant by the addition of glycine, but not glyoxylate, to the growth medium, suggesting that loss of the ability to convert glyoxylate to glycine was associated with the dadA mutation. This was supported by increased glycine production from toluene-treated FRD1 cells with the addition of glyoxylate compared to FRD1dadA cells. This study indicates a larger role for ICL in the physiology and virulence of chronic isolates of P. aeruginosa than previously recognized.

scholarly journals Contextual Flexibility in Pseudomonas aeruginosa Central Carbon Metabolism during Growth in Single Carbon Sources

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Stephen K. Dolan ◽  
Michael Kohlstedt ◽  
Stephen Trigg ◽  
Pedro Vallejo Ramirez ◽  
Clemens F. Kaminski ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Metabolic Flux ◽  
Antimicrobial Agents ◽  
Carbon Sources ◽  
Aerobic Denitrification ◽  
Human Pathogen ◽  
Content Type ◽  
Opportunistic Human Pathogen

ABSTRACT Pseudomonas aeruginosa is an opportunistic human pathogen, particularly noted for causing infections in the lungs of people with cystic fibrosis (CF). Previous studies have shown that the gene expression profile of P. aeruginosa appears to converge toward a common metabolic program as the organism adapts to the CF airway environment. However, we still have only a limited understanding of how these transcriptional changes impact metabolic flux at the systems level. To address this, we analyzed the transcriptome, proteome, and fluxome of P. aeruginosa grown on glycerol or acetate. These carbon sources were chosen because they are the primary breakdown products of an airway surfactant, phosphatidylcholine, which is known to be a major carbon source for P. aeruginosa in CF airways. We show that the fluxes of carbon throughout central metabolism are radically different among carbon sources. For example, the newly recognized “EDEMP cycle” (which incorporates elements of the Entner-Doudoroff [ED] pathway, the Embden-Meyerhof-Parnas [EMP] pathway, and the pentose phosphate [PP] pathway) plays an important role in supplying NADPH during growth on glycerol. In contrast, the EDEMP cycle is attenuated during growth on acetate, and instead, NADPH is primarily supplied by the reaction catalyzed by isocitrate dehydrogenase(s). Perhaps more importantly, our proteomic and transcriptomic analyses revealed a global remodeling of gene expression during growth on the different carbon sources, with unanticipated impacts on aerobic denitrification, electron transport chain architecture, and the redox economy of the cell. Collectively, these data highlight the remarkable metabolic plasticity of P. aeruginosa; that plasticity allows the organism to seamlessly segue between different carbon sources, maximizing the energetic yield from each. IMPORTANCE Pseudomonas aeruginosa is an opportunistic human pathogen that is well known for causing infections in the airways of people with cystic fibrosis. Although it is clear that P. aeruginosa is metabolically well adapted to life in the CF lung, little is currently known about how the organism metabolizes the nutrients available in the airways. In this work, we used a combination of gene expression and isotope tracer (“fluxomic”) analyses to find out exactly where the input carbon goes during growth on two CF-relevant carbon sources, acetate and glycerol (derived from the breakdown of lung surfactant). We found that carbon is routed (“fluxed”) through very different pathways during growth on these substrates and that this is accompanied by an unexpected remodeling of the cell’s electron transfer pathways. Having access to this “blueprint” is important because the metabolism of P. aeruginosa is increasingly being recognized as a target for the development of much-needed antimicrobial agents.

scholarly journals The Gluconeogenic Enzyme Fructose-1,6-Bisphosphatase Is Dispensable for Growth of the Yeast Yarrowia lipolytica in Gluconeogenic Substrates

2008 ◽  
Vol 7 (10) ◽  
pp. 1742-1749 ◽  
Author(s):  
Raquel Jardón ◽  
Carlos Gancedo ◽  
Carmen-Lisset Flores
Keyword(s):  
Yarrowia Lipolytica ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Isocitrate Lyase ◽  
Carbon Sources ◽  
Subcellular Fractionation ◽  
Fructose 1,6 Bisphosphatase ◽  
Genes Encoding ◽  
Key Enzyme ◽  
Gluconeogenic Enzyme ◽  
Fructose 1,6 Bisphosphate

ABSTRACT The genes encoding gluconeogenic enzymes in the nonconventional yeast Yarrowia lipolytica were found to be differentially regulated. The expression of Y. lipolytica FBP1 (YlFBP1) encoding the key enzyme fructose-1,6-bisphosphatase was not repressed by glucose in contrast with the situation in other yeasts; however, this sugar markedly repressed the expression of YlPCK1, encoding phosphoenolpyruvate carboxykinase, and YlICL1, encoding isocitrate lyase. We constructed Y. lipolytica strains with two different disrupted versions of YlFBP1 and found that they grew much slower than the wild type in gluconeogenic carbon sources but that growth was not abolished as happens in most microorganisms. We attribute this growth to the existence of an alternative phosphatase with a high Km (2.3 mM) for fructose-1,6-bisphosphate. The gene YlFBP1 restored fructose-1,6-bisphosphatase activity and growth in gluconeogenic carbon sources to a Saccharomyces cerevisiae fbp1 mutant, but the introduction of the FBP1 gene from S. cerevisiae in the Ylfbp1 mutant did not produce fructose-1,6-bisphosphatase activity or growth complementation. Subcellular fractionation revealed the presence of fructose-1,6-bisphosphatase both in the cytoplasm and in the nucleus.

scholarly journals Structural analysis of the lipopolysaccharide core of a rough, cystic fibrosis isolate of Pseudomonas aeruginosa

2001 ◽  
Vol 268 (17) ◽  
pp. 4708-4719 ◽  
Author(s):  
Yuriy A. Knirel ◽  
Ol'ga V. Bystrova ◽  
Alexander S. Shashkov ◽  
Buko Lindner ◽  
Nina A. Kocharova ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Structural Analysis ◽  
Cystic Fibrosis Isolate

scholarly journals Complete Genome Sequence of Persistent Cystic Fibrosis Isolate Pseudomonas aeruginosa Strain RP73

2013 ◽  
Vol 1 (4) ◽  
Author(s):  
J. Jeukens ◽  
B. Boyle ◽  
I. Bianconi ◽  
I. Kukavica-Ibrulj ◽  
B. Tummler ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Genome Sequence ◽  
Complete Genome Sequence ◽  
Complete Genome ◽  
Cystic Fibrosis Isolate

scholarly journals Antimicrobial Susceptibility of Pseudomonas aeruginosa Isolated from Cystic Fibrosis Patients in Northern Europe

2016 ◽  
Vol 60 (11) ◽  
pp. 6735-6741 ◽  
Author(s):  
Muhammad-Hariri Mustafa ◽  
Hussein Chalhoub ◽  
Olivier Denis ◽  
Ariane Deplano ◽  
Anne Vergison ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Multidrug Resistant ◽  
Sequence Type ◽  
Content Type ◽  
The United Kingdom ◽  
Genes Encoding ◽  
Control Criteria ◽  
Susceptibility Profiles ◽  
And Control

ABSTRACTPseudomonas aeruginosais a major cause of morbidity and mortality in cystic fibrosis patients. This study compared the antimicrobial susceptibilities of 153P. aeruginosaisolates from the United Kingdom (UK) (n =58), Belgium (n =44), and Germany (n =51) collected from 118 patients during routine visits over the period from 2006 to 2012. MICs were measured by broth microdilution. Genes encoding extended-spectrum β-lactamases (ESBL), metallo-β-lactamases, and carbapenemases were detected by PCR. Pulsed-field gel electrophoresis and multilocus sequence typing were performed on isolates resistant to ≥3 antibiotic classes among the penicillins/cephalosporins, carbapenems, fluoroquinolones, aminoglycosides, and polymyxins. Based on EUCAST/CLSI breakpoints, susceptibility rates were ≤30%/≤40% (penicillins, ceftazidime, amikacin, and ciprofloxacin), 44 to 48%/48 to 63% (carbapenems), 72%/72% (tobramycin), and 92%/78% (colistin) independent of patient age. Sixty percent of strains were multidrug resistant (MDR; European Centre for Disease Prevention and Control criteria). Genes encoding the most prevalent ESBL (BEL, PER, GES, VEB, CTX-M, TEM, SHV, and OXA), metallo-β-lactamases (VIM, IMP, and NDM), or carbapenemases (OXA-48 and KPC) were not detected. The Liverpool epidemic strain (LES) was prevalent in UK isolates only (75% of MDR isolates). Four MDR sequence type 958 (ST958) isolates were found to be spread over the three countries. The other MDR clones were evidenced in ≤3 isolates and localized in a single country. A new sequence type (ST2254) was discovered in one MDR isolate in Germany. Clonal and nonclonal isolates with different susceptibility profiles were found in 20 patients. Thus, resistance and MDR are highly prevalent in routine isolates from 3 countries, with meropenem, tobramycin, and colistin remaining the most active drugs.

scholarly journals Adenylate Kinase as a Virulence Factor ofPseudomonas aeruginosa

2001 ◽  
Vol 183 (11) ◽  
pp. 3345-3352 ◽  
Author(s):  
Adam Markaryan ◽  
Olga Zaborina ◽  
Vasu Punj ◽  
A. M. Chakrabarty
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Cell Death ◽  
Virulence Factor ◽  
Adenylate Kinase ◽  
Adenine Nucleotides ◽  
Macrophage Cell ◽  
Cystic Fibrosis Isolate ◽  
Macrophage Killing

ABSTRACT Adenylate kinase (AK; ATP:AMP phosphotransferase, EC 2.7.4.3 ) is a ubiquitous enzyme that contributes to the homeostasis of adenine nucleotides in eukaryotic and prokaryotic cells. AK catalyzes the reversible reaction Mg · ATP + AMP ↔ Mg · ADP + ADP. In this study we show that AK secreted by the pathogenic strains of Pseudomonas aeruginosa appears to play an important role in macrophage cell death. We purified and characterized AK from the growth medium of a cystic fibrosis isolate strain of P. aeruginosa 8821 and hyperproduced it as a fusion protein with glutathione S-transferase. We demonstrated enhanced macrophage cell death in the presence of both the secreted and recombinant purified AK and its substrates AMP plus ATP or ADP. These data suggested that AK converts its substrates to a mixture of AMP, ADP, and ATP, which are potentially more cytotoxic than ATP alone. In addition, we observed increased macrophage killing in the presence of AK and ATP alone. Since the presence of ATPase activity on the macrophages was confirmed in the present work, external macrophage-effluxed ATP is converted to ADP, which in turn can be transformed by AK into a cytotoxic mixture of three adenine nucleotides. Evidence is presented in this study that secreted AK was detected in macrophages during infection with P. aeruginosa. Thus, the possible role of secreted AK as a virulence factor is in producing and keeping an intact pool of toxic mixtures of AMP, ADP, and ATP, which allows P. aeruginosa to exert its full virulence.

Virulence determinants from a cystic fibrosis isolate of Pseudomonas aeruginosa include isocitrate lyase

Microbiology ◽  
2008 ◽  
Vol 154 (6) ◽  
pp. 1616-1627 ◽  
Author(s):  
Tamishia L. Lindsey ◽  
Jessica M. Hagins ◽  
Pamela A. Sokol ◽  
Laura A. Silo-Suh
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Isocitrate Lyase ◽  
Virulence Determinants ◽  
Cystic Fibrosis Isolate
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