Protein–lipopolysaccharide interactions. 1. The reaction of lysozyme with Pseudomonas aeruginosa LPS

1978 ◽  
Vol 24 (2) ◽  
pp. 196-199 ◽  
Author(s):  
D. F. Day ◽  
M. L. Marceau-Day ◽  
J. M. Ingram
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Ionic Strength ◽  
Model System ◽  
Monovalent Cations ◽  
Low Speed ◽  
Initial Stage

Lysozyme (EC 3.2.1.17) complexes with extracted Pseudomonas aeruginosa LPS in two distinct stages. The initial stage does not produce turbidity detectable by nephelometry (measured as nephelos units (N) per time) but does permit low-speed sedimentation of the lysozyme–lipopolysaccharide (LPS) complex. This association is 100% disrupted by the action of 0.1 M Mg2+. Monovalent cations at equal ionic strength to the Mg2+ concentration used for these studies failed to alter significantly the lysozyme–LPS complex, indicating that the role of Mg2+ was not strictly an ionic one. The study of lysozyme–LPS complexes may provide a model system for investigating in vivo protein–LPS interactions.

Ethanol Extract Of Centella Asiatica (L.) Urban Leaves Effectively Inhibit Streptococcus pyogenes and Pseudomonas aeruginosa by Invitro Test

2020 ◽  
Vol 2 (2) ◽  
pp. 69-76
Author(s):  
Dini Aulia Azmi ◽  
Nurlailah Nurlailah ◽  
Ratih Dewi Dwiyanti
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Streptococcus Pyogenes ◽  
Bacterial Infections ◽  
Herbal Medicines ◽  
Centella Asiatica ◽  
Ethanol Extract ◽  
Dilution Method ◽  
Initial Stage ◽  
Independent Variable

Streptococcus pyogenes and Pseudomonas aeruginosa are some of the causes of infectious diseases. Centella asiatica (L.) Urban has many benefits for humans, including overcoming fever, anti-bacterial, and anti-inflammatory. This study aims to determine the inhibition of Centella asiatica (L.) Urban leaves ethanol extract on the growth of Streptococcus pyogenes and Pseudomonas aeruginosa. This research is the initial stage of the development of herbal medicines to treat Streptococcus pyogenes and Pseudomonas aeruginosa infections. The independent variable was the concentration of ethanol extract of Centella asiatica (L.) Urban leaves and the dependent variable was the growth of Streptococcus pyogenes and Pseudomonas aeruginosa. The anti-bacterial activity test was carried out by the liquid dilution method. The concentrations used are 20%, 40%, 60%, 80%. 100% The results showed that the minimum inhibitory concentration (MIC) against Streptococcus pyogenes: 40% and Pseudomonas aeruginosa: 40%. Minimum bactericidal concentration (MBC) results for Streptococcus pyogenes: 60% and Pseudomonas aeruginosa: 60%. So it can be concluded that there is inhibition of the ethanol extract of Centella asiatica (L.) Urban leaves on the growth of Streptococcus pyogenes and Pseudomonas aeruginosa. Centella Asiatica (L.) Urban extract has potential as herbal medicine against bacterial infections but requires further research to determine its effect in vivo.

scholarly journals Role of LecA and LecB Lectins in Pseudomonas aeruginosa-Induced Lung Injury and Effect of Carbohydrate Ligands

2009 ◽  
Vol 77 (5) ◽  
pp. 2065-2075 ◽  
Author(s):  
Chanez Chemani ◽  
Anne Imberty ◽  
Sophie de Bentzmann ◽  
Maud Pierre ◽  
Michaela Wimmerová ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Acute Lung Injury ◽  
Lung Injury ◽  
Parental Strain ◽  
A549 Cells ◽  
Carbohydrate Ligands ◽  
Vitro Model

ABSTRACT Pseudomonas aeruginosa is a frequently encountered pathogen that is involved in acute and chronic lung infections. Lectin-mediated bacterium-cell recognition and adhesion are critical steps in initiating P. aeruginosa pathogenesis. This study was designed to evaluate the contributions of LecA and LecB to the pathogenesis of P. aeruginosa-mediated acute lung injury. Using an in vitro model with A549 cells and an experimental in vivo murine model of acute lung injury, we compared the parental strain to lecA and lecB mutants. The effects of both LecA- and Lec B-specific lectin-inhibiting carbohydrates (α-methyl-galactoside and α-methyl-fucoside, respectively) were evaluated. In vitro, the parental strain was associated with increased cytotoxicity and adhesion on A549 cells compared to the lecA and lecB mutants. In vivo, the P. aeruginosa-induced increase in alveolar barrier permeability was reduced with both mutants. The bacterial burden and dissemination were decreased for both mutants compared with the parental strain. Coadministration of specific lectin inhibitors markedly reduced lung injury and mortality. Our results demonstrate that there is a relationship between lectins and the pathogenicity of P. aeruginosa. Inhibition of the lectins by specific carbohydrates may provide new therapeutic perspectives.

A Novel Pre-Clinical Xenograft Model to Study TSLP-CRLF2 Interactions in B-ALL

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1498-1498
Author(s):  
Ruijun Su ◽  
Olivia Francis ◽  
Shannalee Martinez ◽  
Terrence Bennett ◽  
Ineavely Baez ◽  
...  
Keyword(s):  
Cell Lines ◽  
Molecular Mechanisms ◽  
Xenograft Model ◽  
Model System ◽  
Leukemia Cells ◽  
Receptor Complex ◽  
Cellular Delivery ◽  
Mouse Xenograft Model

Abstract Abstract 1498 B-cell precursor acute lymphoblastic leukemia arising from overexpression of CRLF2 (CRLF2 B-ALL) is high-risk with poor prognosis. CRLF2 B-ALL occurs 5 times more frequently among children of Hispanic/Latino ethnicity than others and thus represents one of the most significant biological components of childhood cancer health disparity identified to date. CRLF2, together with the IL-7Rα, forms a receptor complex that is activated by the cytokine, TSLP. The JAK-STAT5 pathway is phosphorylated downstream of this receptor complex activation. Activating JAK mutations are found in some CRLF2 B-ALL and their presence has led to speculation that TSLP stimulation is not a factor in CRLF2 B-ALL. In preliminary studies to address this question we evaluated the effect of TSLP on CRLF2 B-ALL cell lines that have JAK defects and which have been reported to exhibit constitutive JAK-STAT5 activation. Our data show that TSLP increases STAT5 phosphorylation in these cell lines and also in primary B-ALL cells that overexpress CRLF2. Our next step was to evaluate the role of TSLP-CRLF2 interaction in vivo in the human-mouse xenograft model. However, mouse TSLP is different from most other cytokines produced in mice in that it is species-specific and does not activate the human TSLP receptor complex that contains CRLF2. Thus, traditional xenograft models do not provide the TSLP-CRLF2 interaction that our data implicate as a potential contributor to pathogenesis in CRLF2 B-ALL. To overcome this obstacle we engineered immune deficient NOD/SCID IL-2Rγ null (NSG) mice to express human TSLP (hTSLP+ mice), as well as control mice that lack the TSLP cytokine (hTSLP– mice). ELISA assays show plasma hTSLP levels in the hTSLP+ mice that approximate the normal range in human plasma. We used this hTSLP+/– xenograft model system to study the in vivo effects of TSLP on CRLF2 B-ALL cells harboring a JAK defect (MUTZ5 cell line) and on primary CRLF2 B-ALL cells from a Hispanic patient. Mice were euthanized at 5 weeks and BM disease was evaluated. In recipients of MUTZ5 B-ALL cells the percentage of viable leukemia cells in hTSLP– mice was half that observed in hTSLP+ mice. Similarly, in recipients of primary CRLF2 B-ALL cells, the percentage of viable leukemia cells was reduced in hTSLP– mice as compared to hTSLP+ mice. In addition, the viable primary B-ALL cells present in the BM of hTSLP+ mice showed higher expression levels of the TSLPR components (CRLF2 and IL-7Rα) than those in the hTSLP– mice. These data provide evidence that the TSLP produced in this model is active and that it impacts primary CRLF2 B-ALL cells. The hTSLP+/– xenograft model described here provides the first data on the in vivo role of TSLP-CRLF2 interactions in CRLF2 B-ALL. This preliminary data suggests that TSLP provides a signal that promotes in vivo survival of CRLF2 B-ALL cells and that it could play a role in selection of CRLF2-HI clones during in vivo leukemogenesis. This pre-clinical model will allow us to evaluate TSLP-CRLF2 interactions as a target for therapy and to perform translational studies that identify molecular mechanisms and additional targets downstream of TSLP-induced signaling in CRLF2 B-ALL. This model system will be be particularly important for assessing and identifying therapies, including drug and cellular delivery systems, to effectively target CRLF2 B-ALL and to reduce cancer health disparities in Hispanic childhood B-ALL. This work is supported by a National Institutes of Health R21CA162259, a Loma Linda University Grant to Promote Collaborative and Translational Research and a St. Baldrick's Foundation Research Award (to K.J.P.). Disclosures: No relevant conflicts of interest to declare.

scholarly journals Characterization and a role of Pseudomonas aeruginosa spermidine dehydrogenase in polyamine catabolism

Microbiology ◽  
2006 ◽  
Vol 152 (8) ◽  
pp. 2265-2272 ◽  
Author(s):  
Veeranki Venkata Dasu ◽  
Yuji Nakada ◽  
Mayumi Ohnishi-Kameyama ◽  
Keitarou Kimura ◽  
Yoshifumi Itoh
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Specific Activity ◽  
Signal Sequence ◽  
Inducible Promoter ◽  
Polyamine Catabolism ◽  
Amino Terminal ◽  
Unknown Gene ◽  
Membrane Location

Pseudomonas aeruginosa PAO1 has two possible catabolic pathways of spermidine and spermine; one includes the spuA and spuB products with unknown functions and the other involves spermidine dehydrogenase (SpdH; EC 1.5.99.6) encoded by an unknown gene. The properties of SpdH in P. aeruginosa PAO1 were characterized and the corresponding spdH gene in this strain identified. The deduced SpdH (620 residues, calculated M r of 68 861) had a signal sequence of 28 amino acids at the amino terminal and a potential transmembrane segment between residues 76 and 92, in accordance with membrane location of the enzyme. Purified SpdH oxidatively cleaved spermidine into 1,3-diaminopropane and 4-aminobutyraldehyde with a specific activity of 37 units (mg protein)−1 and a K m value of 36 μM. The enzyme also hydrolysed spermine into spermidine and 3-aminopropanaldehyde with a specific activity of 25 units (mg protein)−1 and a K m of 18 μM. Knockout of spdH had no apparent effect on the utilization of both polyamines, suggesting that this gene is minimally involved in polyamine catabolism. However, when spdH was fused to the polyamine-inducible promoter of spuA, it fully restored the ability of a spuA mutant to utilize spermidine. It is concluded that SpdH can perform a catabolic role in vivo, but P. aeruginosa PAO1 does not produce sufficient amounts of the enzyme to execute this function.

scholarly journals Convergent Metabolic Specialization through Distinct Evolutionary Paths inPseudomonas aeruginosa

mBio ◽  
2018 ◽  
Vol 9 (2) ◽  
Author(s):  
Ruggero La Rosa ◽  
Helle Krogh Johansen ◽  
Søren Molin
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Slow Growth ◽  
Model System ◽  
Growth Potential ◽  
Cell Physiology ◽  
Content Type ◽  
Successful Adaptation ◽  
Insight Into

ABSTRACTEvolution by natural selection under complex and dynamic environmental conditions occurs through intricate and often counterintuitive trajectories affecting many genes and metabolic solutions. To study short- and long-term evolution of bacteriain vivo, we used the natural model system of cystic fibrosis (CF) infection. In this work, we investigated how and through which trajectories evolution ofPseudomonas aeruginosaoccurs when migrating from the environment to the airways of CF patients, and specifically, we determined reduction of growth rate and metabolic specialization as signatures of adaptive evolution. We show that central metabolic pathways of three distinctPseudomonas aeruginosalineages coevolving within the same environment become restructured at the cost of versatility during long-term colonization. Cell physiology changes from naive to adapted phenotypes resulted in (i) alteration of growth potential that particularly converged to a slow-growth phenotype, (ii) alteration of nutritional requirements due to auxotrophy, (iii) tailored preference for carbon source assimilation from CF sputum, (iv) reduced arginine and pyruvate fermentation processes, and (v) increased oxygen requirements. Interestingly, although convergence was evidenced at the phenotypic level of metabolic specialization, comparative genomics disclosed diverse mutational patterns underlying the different evolutionary trajectories. Therefore, distinct combinations of genetic and regulatory changes converge to common metabolic adaptive trajectories leading to within-host metabolic specialization. This study gives new insight into bacterial metabolic evolution during long-term colonization of a new environmental niche.IMPORTANCEOnly a few examples of real-time evolutionary investigations in environments outside the laboratory are described in the scientific literature. Remembering that biological evolution, as it has progressed in nature, has not taken place in test tubes, it is not surprising that conclusions from our investigations of bacterial evolution in the CF model system are different from what has been concluded from laboratory experiments. The analysis presented here of the metabolic and regulatory driving forces leading to successful adaptation to a new environment provides an important insight into the role of metabolism and its regulatory mechanisms for successful adaptation of microorganisms in dynamic and complex environments. Understanding the trajectories of adaptation, as well as the mechanisms behind slow growth and rewiring of regulatory and metabolic networks, is a key element to understand the adaptive robustness and evolvability of bacteria in the process of increasing theirin vivofitness when conquering new territories.

scholarly journals The NtrC Family Regulator AlgB, Which Controls Alginate Biosynthesis in Mucoid Pseudomonas aeruginosa, Binds Directly to the algD Promoter

2007 ◽  
Vol 190 (2) ◽  
pp. 581-589 ◽  
Author(s):  
Andrew J. Leech ◽  
April Sprinkle ◽  
Lynn Wood ◽  
Daniel J. Wozniak ◽  
Dennis E. Ohman
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Response Regulator ◽  
Mobility Shift ◽  
Alginate Production ◽  
Mobility Shift Assay ◽  
Exponential Enrichment ◽  
Microarray Analyses ◽  
Phosphorylation Domain

ABSTRACT Alginate production in mucoid (MucA-defective) Pseudomonas aeruginosa is dependent upon several transcriptional regulators, including AlgB, a two-component response regulator belonging to the NtrC family. This role of AlgB was apparently independent of its sensor kinase, KinB, and even the N-terminal phosphorylation domain of AlgB was dispensable for alginate biosynthetic gene (i.e., algD operon) activation. However, it remained unclear whether AlgB stimulated algD transcription directly or indirectly. In this study, microarray analyses were used to examine a set of potential AlgB-dependent, KinB-independent genes in a PAO1 mucA background that overlapped with genes induced by d-cycloserine, which is known to activate algD expression. This set contained only the algD operon plus one other gene that was shown to be uninvolved in alginate production. This suggested that AlgB promotes alginate production by directly binding to the algD promoter (PalgD). Chromosome immunoprecipitation revealed that AlgB bound in vivo to PalgD but did not bind when AlgB had an R442E substitution that disrupted the DNA binding domain. AlgB also showed binding to PalgD fragments in an electrophoretic mobility shift assay at pH 4.5 but not at pH 8.0. A direct systematic evolution of ligands by exponential enrichment approach showed AlgB binding to a 50-bp fragment located at bp −224 to −274 relative to the start of PalgD transcription. Thus, AlgB belongs to a subclass of NtrC family proteins that can activate promoters which utilize a sigma factor other than σ54, in this case to stimulate transcription from the σ22-dependent PalgD promoter.

scholarly journals Pseudomonas aeruginosa as a cause of infectious diarrhoea

1998 ◽  
Vol 121 (1) ◽  
pp. 237-241 ◽  
Author(s):  
P. A. ADLARD ◽  
S. M. KIROV ◽  
K. SANDERSON ◽  
G. E. COX
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Signs And Symptoms ◽  
Free Cell ◽  
Persistent Infections ◽  
Infectious Diarrhoea ◽  
Type Iv ◽  
Underlying Illness

Pseudomonas aeruginosa is not generally considered a cause of infectious diarrhoea. However, it was the predominant organism isolated from the faeces of 23 unrelated, hospital outpatients investigated in the course of a year for persistent (>1 week duration) diarrhoea. To investigate the possible aetiological role of P. aeruginosa, these patient histories were reviewed and a selection of their faecal isolates were investigated in vitro (n[ges ]10) and in vivo (n=2) for virulence. The patients had a mean age of 60 years, were receiving antibiotics and/or had an underlying illness. Extensive microbiological investigations identified no other potential or recognized enteropathogen in the faeces of 20 of these patients. More than 40% of the isolates tested were able to adhere to HEp-2 cells and exhibited twitching motility (type IV pili), properties indicative of their ability to colonize the human intestine. Cytotoxic activity was demonstrated in bacterium-free cell supernatants of over 80% of isolates; supernatants of four isolates tested in infant mice were weakly enterotoxigenic. Two isolates intragastrically inoculated into clindamycin pre-treated rats established persistent infections and induced signs and symptoms of enteritis. Overall these findings suggest that P. aeruginosa can cause diarrhoea particularly in immunodeficient individuals.

scholarly journals Pseudomonas aeruginosa Aggregate Formation in an Alginate Bead Model System Exhibits In Vivo-Like Characteristics

2017 ◽  
Vol 83 (9) ◽  
Author(s):  
Majken Sønderholm ◽  
Kasper Nørskov Kragh ◽  
Klaus Koren ◽  
Tim Holm Jakobsen ◽  
Sophie E. Darch ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Electron Acceptor ◽  
Chronic Wounds ◽  
Alginate Bead ◽  
Alginate Beads ◽  
Model System ◽  
Chronic Infections ◽  
Content Type

ABSTRACT Alginate beads represent a simple and highly reproducible in vitro model system for diffusion-limited bacterial growth. In this study, alginate beads were inoculated with Pseudomonas aeruginosa and followed for up to 72 h. Confocal microscopy revealed that P. aeruginosa formed dense clusters similar in size to in vivo aggregates observed ex vivo in cystic fibrosis lungs and chronic wounds. Bacterial aggregates primarily grew in the bead periphery and decreased in size and abundance toward the center of the bead. Microsensor measurements showed that the O2 concentration decreased rapidly and reached anoxia ∼100 μm below the alginate bead surface. This gradient was relieved in beads supplemented with NO3 − as an alternative electron acceptor allowing for deeper growth into the beads. A comparison of gene expression profiles between planktonic and alginate-encapsulated P. aeruginosa confirmed that the bacteria experienced hypoxic and anoxic growth conditions. Furthermore, alginate-encapsulated P. aeruginosa exhibited a lower respiration rate than the planktonic counterpart and showed a high tolerance toward antibiotics. The inoculation and growth of P. aeruginosa in alginate beads represent a simple and flexible in vivo-like biofilm model system, wherein bacterial growth exhibits central features of in vivo biofilms. This was observed by the formation of small cell aggregates in a secondary matrix with O2-limited growth, which was alleviated by the addition of NO3 − as an alternative electron acceptor, and by reduced respiration rates, as well as an enhanced tolerance to antibiotic treatment. IMPORTANCE Pseudomonas aeruginosa has been studied intensively for decades due to its involvement in chronic infections, such as cystic fibrosis and chronic wounds, where it forms biofilms. Much research has been dedicated to biofilm formation on surfaces; however, in chronic infections, most biofilms form small aggregates of cells not attached to a surface, but embedded in host material. In this study, bacteria were encapsulated in small alginate beads and formed aggregates similar to what is observed in chronic bacterial infections. Our findings show that aggregates are exposed to steep oxygen gradients, with zones of oxygen depletion, and that nitrate may serve as an alternative to oxygen, enabling growth in oxygen-depleted zones. This is important, as slow growth under low-oxygen conditions may render the bacteria tolerant toward antibiotics. This model provides an alternative to surface biofilm models and adds to the comprehension that biofilms do not depend on a surface for formation.

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