2-Aminopyridine Analogs Inhibit Both Enzymes of the Glyoxylate Shunt in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen responsible for many hospital-acquired infections. P. aeruginosa can thrive in diverse infection scenarios by rewiring its central metabolism. An example of this is the production of biomass from C2 nutrient sources such as acetate via the glyoxylate shunt when glucose is not available. The glyoxylate shunt is comprised of two enzymes, isocitrate lyase (ICL) and malate synthase G (MS), and flux through the shunt is essential for the survival of the organism in mammalian systems. In this study, we characterized the mode of action and cytotoxicity of structural analogs of 2-aminopyridines, which have been identified by earlier work as being inhibitory to both shunt enzymes. Two of these analogs were able to inhibit ICL and MS in vitro and prevented growth of P. aeruginosa on acetate (indicating cell permeability). Moreover, the compounds exerted negligible cytotoxicity against three human cell lines and showed promising in vitro drug metabolism and safety profiles. Isothermal titration calorimetry was used to confirm binding of one of the analogs to ICL and MS, and the mode of enzyme inhibition was determined. Our data suggest that these 2-aminopyridine analogs have potential as anti-pseudomonal agents.

Download Full-text

Pseudomonas aeruginosa Twitching Motility-Mediated Chemotaxis towards Phospholipids and Fatty Acids: Specificity and Metabolic Requirements

Journal of Bacteriology ◽

10.1128/jb.00129-08 ◽

2008 ◽

Vol 190 (11) ◽

pp. 4038-4049 ◽

Cited By ~ 44

Author(s):

Rhea M. Miller ◽

Andrew P. Tomaras ◽

Adam P. Barker ◽

Dennis R. Voelker ◽

Edward D. Chan ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Alternative Energy ◽

Isocitrate Lyase ◽

Malate Synthase ◽

Glyoxylate Shunt ◽

Twitching Motility ◽

Long Chain Fatty Acid ◽

Type Iv ◽

Energy Taxis ◽

Lipid Metabolism Genes

ABSTRACT Pseudomonas aeruginosa demonstrates type IV pilus-mediated directional twitching motility up a gradient of phosphatidylethanolamine (PE). Only one of four extracellular phospholipases C of P. aeruginosa (i.e., PlcB), while not required for twitching motility per se, is required for twitching-mediated migration up a gradient of PE or phosphatidylcholine. Whether other lipid metabolism genes are associated with this behavior was assessed by analysis of transcription during twitching up a PE gradient in comparison to transcription during twitching in the absence of any externally applied phospholipid. Data support the hypothesis that PE is further degraded and that the long-chain fatty acid (LCFA) moieties of PE are completely metabolized via β-oxidation and the glyoxylate shunt. It was discovered that P. aeruginosa exhibits twitching-mediated chemotaxis toward unsaturated LCFAs (e.g., oleic acid), but not saturated LCFAs (e.g., stearic acid) of corresponding lengths. Analysis of mutants that are deficient in glyoxylate shunt enzymes, specifically isocitrate lyase (ΔaceA) and malate synthase (ΔaceB), suggested that the complete metabolism of LCFAs through this pathway was required for the migration of P. aeruginosa up a gradient of PE or unsaturated LCFAs. At this point, our data suggested that this process should be classified as energy taxis. However, further evaluation of the ability of the ΔaceA and ΔaceB mutants to migrate up a gradient of PE or unsaturated LCFAs in the presence of an alternative energy source clearly indicated that metabolism of LCFAs for energy is not required for chemotaxis toward these compounds.

Download Full-text

Glyoxylate detoxification is an essential function of malate synthase required for carbon assimilation inMycobacterium tuberculosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1617655114 ◽

2017 ◽

Vol 114 (11) ◽

pp. E2225-E2232 ◽

Cited By ~ 30

Author(s):

Susan Puckett ◽

Carolina Trujillo ◽

Zhe Wang ◽

Hyungjin Eoh ◽

Thomas R. Ioerger ◽

...

Keyword(s):

Fatty Acids ◽

Isocitrate Lyase ◽

Carbon Assimilation ◽

Malate Synthase ◽

Glyoxylate Shunt ◽

Growth And Survival ◽

Physiologic Function ◽

Chain Fatty Acids

The glyoxylate shunt is a metabolic pathway of bacteria, fungi, and plants used to assimilate even-chain fatty acids (FAs) and has been implicated in persistence ofMycobacterium tuberculosis(Mtb). Recent work, however, showed that the first enzyme of the glyoxylate shunt, isocitrate lyase (ICL), may mediate survival ofMtbduring the acute and chronic phases of infection in mice through physiologic functions apart from fatty acid metabolism. Here, we report that malate synthase (MS), the second enzyme of the glyoxylate shunt, is essential for in vitro growth and survival ofMtbon even-chain fatty acids, in part, for a previously unrecognized activity: mitigating the toxicity of glyoxylate excess arising from metabolism of even-chain fatty acids. Metabolomic profiling revealed that MS-deficientMtbcultured on fatty acids accumulated high levels of the ICL aldehyde endproduct, glyoxylate, and increased levels of acetyl phosphate, acetoacetyl coenzyme A (acetoacetyl-CoA), butyryl CoA, acetoacetate, and β-hydroxybutyrate. These changes were indicative of a glyoxylate-induced state of oxaloacetate deficiency, acetate overload, and ketoacidosis. Reduction of intrabacterial glyoxylate levels using a chemical inhibitor of ICL restored growth of MS-deficientMtb, despite inhibiting entry of carbon into the glyoxylate shunt. In vivo depletion of MS resulted in sterilization ofMtbin both the acute and chronic phases of mouse infection. This work thus identifies glyoxylate detoxification as an essential physiologic function ofMtbmalate synthase and advances its validation as a target for drug development.

Download Full-text

The In Vitro Antibiofilm Activity of Water Leaf Extract of Papaya (Carica papaya L.) against Pseudomonas aeruginosa

Current Biochemistry ◽

10.29244/cb.2.3.150-163 ◽

2017 ◽

Vol 2 (3) ◽

pp. 150-163

Author(s):

Ekajayanti Kining ◽

Syamsul Falah ◽

Novik Nurhidayat

Keyword(s):

Pseudomonas Aeruginosa ◽

Contact Time ◽

Cell Attachment ◽

Water Extract ◽

Opportunistic Pathogen ◽

Bacterial Biofilm ◽

Antibiofilm Activity ◽

Bacterial Survival ◽

Optimum Conditions

Pseudomonas aeruginosa is one of opportunistic pathogen forming bacterial biofilm. The biofilm sustains the bacterial survival and infections. This study aimed to assess the activity of water extract of papaya leaves on inhibition of cells attachment, growth and degradation of the biofilm using crystal violet (CV) biofilm assay. Research results showed that water extract of papaya leaves contains alkaloids, tanins, flavonoids, and steroids/terpenoids and showed antibacterial activity and antibiofilm against P. aeruginosa. Addition of extract can inhibit the cell attachment and was able to degrade the biofilm of 40.92% and 48.058% respectively at optimum conditions: extract concentration of 25% (v/v), temperature 37.5 °C and contact time 45 minutes. With a concentration of 25% (v/v), temperature of 50 °C and the contact time of 3 days, extract of papaya leaves can inhibit the growth of biofilms of 39.837% v/v.

Download Full-text

Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt

Microbial Cell Factories ◽

10.1186/s12934-021-01529-y ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Claudia Durall ◽

Kateryna Kukil ◽

Jeffrey A. Hawkes ◽

Alessia Albergati ◽

Peter Lindblad ◽

...

Keyword(s):

Phosphoenolpyruvate Carboxylase ◽

Isocitrate Lyase ◽

Tricarboxylic Acid Cycle ◽

Cell Metabolism ◽

Tca Cycle ◽

Malate Synthase ◽

Glyoxylate Shunt ◽

Control Strain ◽

Genes Encoding ◽

Pcc 6803

Abstract Background Cyanobacteria are promising hosts for the production of various industrially important compounds such as succinate. This study focuses on introduction of the glyoxylate shunt, which is naturally present in only a few cyanobacteria, into Synechocystis PCC 6803. In order to test its impact on cell metabolism, engineered strains were evaluated for succinate accumulation under conditions of light, darkness and anoxic darkness. Each condition was complemented by treatments with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase enzyme, and acetate, both in nitrogen replete and deplete medium. Results We were able to introduce genes encoding the glyoxylate shunt, aceA and aceB, encoding isocitrate lyase and malate synthase respectively, into a strain of Synechocystis PCC 6803 engineered to overexpress phosphoenolpyruvate carboxylase. Our results show that complete expression of the glyoxylate shunt results in higher extracellular succinate accumulation compared to the wild type control strain after incubation of cells in darkness and anoxic darkness in the presence of nitrate. Addition of the inhibitor 2-thenoyltrifluoroacetone increased succinate titers in all the conditions tested when nitrate was available. Addition of acetate in the presence of the inhibitor further increased the succinate accumulation, resulting in high levels when phosphoenolpyruvate carboxylase was overexpressed, compared to control strain. However, the highest succinate titer was obtained after dark incubation of an engineered strain with a partial glyoxylate shunt overexpressing isocitrate lyase in addition to phosphoenolpyruvate carboxylase, with only 2-thenoyltrifluoroacetone supplementation to the medium. Conclusions Heterologous expression of the glyoxylate shunt with its central link to the tricarboxylic acid cycle (TCA) for acetate assimilation provides insight on the coordination of the carbon metabolism in the cell. Phosphoenolpyruvate carboxylase plays an important role in directing carbon flux towards the TCA cycle.

Download Full-text

Role of pili in adherence of Pseudomonas aeruginosa to mammalian buccal epithelial cells

Infection and Immunity ◽

10.1128/iai.29.3.1146-1151.1980 ◽

1980 ◽

Vol 29 (3) ◽

pp. 1146-1151 ◽

Cited By ~ 1

Author(s):

D E Woods ◽

D C Straus ◽

W G Johanson ◽

V K Berry ◽

J A Bass

Keyword(s):

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Opportunistic Pathogen ◽

In Vitro System ◽

Heterologous Antiserum ◽

Buccal Epithelial Cells ◽

Serological Studies ◽

Seriously Ill Patients

Adherence of Pseudomonas aeruginosa organisms to the upper respiratory epithelium of seriously ill patients in vitro is correlated with subsequent colonization of the respiratory tract by this opportunistic pathogen. The role of pili in the attachment to epithelial cells of P. aeruginosa was studied in an in vitro system employing human buccal epithelial cells and P. aeruginosa pretreated by various means. Pretreatment of the bacteria with proteases, heat, or Formalin caused a significant decrease in adherence. A decrease when compared with controls was also noted in the adherence of P. aeruginosa organisms to buccal epithelial cells preincubated with purified pili prepared from the strain used for adherence testing; however, pili prepared from a heterologous strain failed to block adherence. Similar results were obtained in serological studies when antisera to purified pili prepared from the strain used for adherence testing decreased adherence, whereas heterologous antiserum to pili did not decrease adherence. From these results it appears that pili mediate the adherence of P. aeruginosa organisms to human buccal epithelial cells.

Download Full-text

Increase in glyoxylate shunt enzymes during cellular morphogenesis in Myxococcus xanthus

Canadian Journal of Microbiology ◽

10.1139/m71-036 ◽

1971 ◽

Vol 17 (2) ◽

pp. 209-211 ◽

Cited By ~ 5

Author(s):

J. Bland ◽

Wu-Kuang Yeh ◽

D. White ◽

A. Hendricks

Keyword(s):

Actinomycin D ◽

Isocitrate Lyase ◽

Myxococcus Xanthus ◽

Malate Synthase ◽

Glyoxylate Shunt ◽

Cellular Morphogenesis

Isocitrate lyase (EC. 4.1.3.1) and malate synthase (EC. 4.1.3.2) increase during microcyst formation in Myxococcus xanthus. The increase in activity is inhibited by chloramphenicol and actinomycin-D.

Download Full-text

Rapid diversification of Pseudomonas aeruginosa in cystic fibrosis lung-like conditions

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1721270115 ◽

2018 ◽

Vol 115 (42) ◽

pp. 10714-10719 ◽

Cited By ~ 20

Author(s):

Alana Schick ◽

Rees Kassen

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Chronic Infection ◽

Opportunistic Pathogen ◽

Chronic Infections ◽

Evolutionary Diversification ◽

Nutritional Conditions ◽

Patient Health ◽

Rapid Diversification

Chronic infection of the cystic fibrosis (CF) airway by the opportunistic pathogen Pseudomonas aeruginosa is the leading cause of morbidity and mortality for adult CF patients. Prolonged infections are accompanied by adaptation of P. aeruginosa to the unique conditions of the CF lung environment, as well as marked diversification of the pathogen into phenotypically and genetically distinct strains that can coexist for years within a patient. Little is known, however, about the causes of this diversification and its impact on patient health. Here, we show experimentally that, consistent with ecological theory of diversification, the nutritional conditions of the CF airway can cause rapid and extensive diversification of P. aeruginosa. Mucin, the substance responsible for the increased viscosity associated with the thick mucus layer in the CF airway, had little impact on within-population diversification but did promote divergence among populations. Furthermore, in vitro evolution recapitulated traits thought to be hallmarks of chronic infection, including reduced motility and increased biofilm formation, and the range of phenotypes observed in a collection of clinical isolates. Our results suggest that nutritional complexity and reduced dispersal can drive evolutionary diversification of P. aeruginosa independent of other features of the CF lung such as an active immune system or the presence of competing microbial species. We suggest that diversification, by generating extensive phenotypic and genetic variation on which selection can act, may be a key first step in the development of chronic infections.

Download Full-text

cAMP and Vfr Control Exolysin Expression and Cytotoxicity of Pseudomonas aeruginosa Taxonomic Outliers

Journal of Bacteriology ◽

10.1128/jb.00135-18 ◽

2018 ◽

Vol 200 (12) ◽

Cited By ~ 9

Author(s):

Alice Berry ◽

Kook Han ◽

Julian Trouillon ◽

Mylène Robert-Genthon ◽

Michel Ragno ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Epithelial Cells ◽

Transcriptional Activation ◽

Opportunistic Pathogen ◽

Secretion System ◽

Intracellular Camp ◽

Virulence Determinants ◽

Cell Infection ◽

Content Type

ABSTRACT The two-partner secretion system ExlBA, expressed by strains of Pseudomonas aeruginosa belonging to the PA7 group, induces hemorrhage in lungs due to disruption of host cellular membranes. Here we demonstrate that the exlBA genes are controlled by a pathway consisting of cAMP and the virulence factor regulator (Vfr). Upon interaction with cAMP, Vfr binds directly to the exlBA promoter with high affinity (equilibrium binding constant [ K eq ] of ≈2.5 nM). The exlB and exlA expression was diminished in the Vfr-negative mutant and upregulated with increased intracellular cAMP levels. The Vfr binding sequence in the exlBA promoter was mutated in situ , resulting in reduced cytotoxicity of the mutant, showing that Vfr is required for the exlBA expression during intoxication of epithelial cells. Vfr also regulates function of type 4 pili previously shown to facilitate ExlA activity on epithelial cells, which indicates that the cAMP/Vfr pathway coordinates these two factors needed for full cytotoxicity. As in most P. aeruginosa strains, the adenylate cyclase CyaB is the main provider of cAMP for Vfr regulation during both in vitro growth and eukaryotic cell infection. We discovered that the absence of functional Vfr in the reference strain PA7 is caused by a frameshift in the gene and accounts for its reduced cytotoxicity, revealing the conservation of ExlBA control by the CyaB-cAMP/Vfr pathway in P. aeruginosa taxonomic outliers. IMPORTANCE The human opportunistic pathogen Pseudomonas aeruginosa provokes severe acute and chronic human infections associated with defined sets of virulence factors. The main virulence determinant of P. aeruginosa taxonomic outliers is exolysin, a membrane-disrupting pore-forming toxin belonging to the two-partner secretion system ExlBA. In this work, we demonstrate that the conserved CyaB-cAMP/Vfr pathway controls cytotoxicity of outlier clinical strains through direct transcriptional activation of the exlBA operon. Therefore, despite the fact that the type III secretion system and exolysin are mutually exclusive in classical and outlier strains, respectively, these two major virulence determinants share similarities in their mechanisms of regulation.

Download Full-text

Design of Epitope-Based Peptide Vaccine against Pseudomonas aeruginosa Fructose Bisphosphate Aldolase Protein Using Immunoinformatics

Journal of Immunology Research ◽

10.1155/2020/9475058 ◽

2020 ◽

Vol 2020 ◽

pp. 1-11

Author(s):

Mustafa Elhag ◽

Ruaa Mohamed Alaagib ◽

Nagla Mohamed Ahmed ◽

Mustafa Abubaker ◽

Esraa Musa Haroun ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Peptide Vaccine ◽

Multidrug Resistant ◽

Fructose Bisphosphate ◽

Mhc I ◽

Mhc Ii ◽

Fructose Bisphosphate Aldolase ◽

Hospital Acquired

Pseudomonas aeruginosa is a common pathogen that is responsible for serious hospital-acquired infections, ventilator-associated pneumonia, and various sepsis syndromes. Also, it is a multidrug-resistant pathogen recognized for its ubiquity and its intrinsically advanced antibiotic-resistant mechanisms. It usually affects immunocompromised individuals but can also infect immunocompetent individuals. There is no vaccine against it available till now. This study predicts an effective epitope-based vaccine against fructose bisphosphate aldolase (FBA) of Pseudomonas aeruginosa using immunoinformatics tools. The protein sequences were obtained from NCBI, and prediction tests were undertaken to analyze possible epitopes for B and T cells. Three B cell epitopes passed the antigenicity, accessibility, and hydrophilicity tests. Six MHC I epitopes were found to be promising, while four MHC II epitopes were found promising from the result set. Nineteen epitopes were shared between MHC I and II results. For the population coverage, the epitopes covered 95.62% worldwide excluding certain MHC II alleles. We recommend in vivo and in vitro studies to prove its effectiveness.

Download Full-text

Selection of Bacteriophages to Control In Vitro 24 h Old Biofilm of Pseudomonas aeruginosa Isolated from Drinking and Thermal Water

Viruses ◽

10.3390/v11080749 ◽

2019 ◽

Vol 11 (8) ◽

pp. 749 ◽

Cited By ~ 1

Author(s):

Vanessa Magin ◽

Nathalie Garrec ◽

Yves Andrés

Keyword(s):

Pseudomonas Aeruginosa ◽

Opportunistic Pathogen ◽

Planktonic Cell ◽

Test Method ◽

Public Healthcare ◽

Bacterial Strains ◽

Promising Alternative ◽

Standard Culture ◽

Stainless Steel Coupon

Pseudomonas aeruginosa is an opportunistic pathogen that causes public healthcare issues. In moist environments, this Gram-negative bacterium persists through biofilm-associated contamination on surfaces. Bacteriophages are seen as a promising alternative strategy to chemical biocides. This study evaluates the potential of nine lytic bacteriophages as biocontrol treatments against nine environmental P. aerginosa isolates. The spot test method is preliminarily used to define the host range of each virus and to identify their minimum infectious titer, depending on the strain. Based on these results, newly isolated bacteriophages 14.1, LUZ7, and B1 are selected and assessed on a planktonic cell culture of the most susceptible isolates (strains MLM, D1, ST395E, and PAO1). All liquid infection assays are achieved in a mineral minimum medium that is much more representative of real moist environments than standard culture medium. Phages 14.1 and LUZ7 eliminate up to 90% of the PAO1 and D1 bacterial strains. Hence, their effectiveness is evaluated on the 24 h old biofilms of these strains, established on a stainless steel coupon that is characteristic of materials found in thermal and industrial environments. The results of quantitative PCR viability show a maximum reduction of 1.7 equivalent Log CFU/cm2 in the coupon between treated and untreated surfaces and shed light on the importance of considering the entire virus/host/environment system for optimizing the treatment.

Download Full-text