scholarly journals Characterization of Phospholipase Activity of the Pseudomonas aeruginosa Type III Cytotoxin, ExoU

2005 ◽  
Vol 187 (3) ◽  
pp. 1192-1195 ◽  
Author(s):  
Hiromi Sato ◽  
Jimmy B. Feix ◽  
Cecilia J. Hillard ◽  
Dara W. Frank
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Substrate Specificity ◽  
Yeast Extract ◽  
Enzyme Activation ◽  
Phospholipase Activity ◽  
Eukaryotic Protein ◽  
Type Iii

ABSTRACT Recombinant ExoU (rExoU) and yeast extract were used to optimize an in vitro phospholipase assay as a basis for identifying the mechanism for enzyme activation and substrate specificity. Our results support a model in which a eukaryotic protein cofactor or complex facilitates the interaction of rExoU with phospholipid substrates.

scholarly journals Bioprocess development for enhanced endoglucanase production by newly isolated bacteria, purification, characterization and in-vitro efficacy as anti-biofilm of Pseudomonas aeruginosa

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Atef M. Ibrahim ◽  
Ragaa A. Hamouda ◽  
Noura El-Ahmady El-Naggar ◽  
Fatma M. Al-Shakankery
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Yeast Extract ◽  
Statistical Design ◽  
Life Time ◽  
Maximum Activity ◽  
Bioprocess Development ◽  
Ammonium Sulfate Precipitation ◽  
Face Centered ◽  
Central Composite

AbstractEndoglucanase producing bacteria were isolated from Egyptian soils and the most active bacterial strain was identified as Bacillus subtilis strain Fatma/1. Plackett–Burman statistical design was carried out to assess the effect of seven process variables on endoglucanase production. Carboxymethyl cellulose (CMC), yeast extract and peptone were the most significant variables that enhanced the endoglucanase production and thus were selected for further optimization using face-centered central composite design. The highest yield of endoglucanase (32.37 U/mL) was obtained in run no. 9, using 18 g/L CMC, 8 g/L peptone, 7 g/L yeast extract and 0.1 g/L FeSO4.7H2O. The optimized medium showed about eightfold increase in endoglucanase production compared to the unoptimized medium. The produced crude enzyme was further purified by ammonium sulfate precipitation, then DEAE-Sepharose CL6B column. The purified enzyme was shown to have a molecular weight of 37 kDa. The enzyme showed maximum activity at pH 8.0, temperature of 50 °C, incubation time of 60 min. The half-life time (T1/2) was 139.53 min at 50 °C, while being 82.67 min at 60 °C. Endoglucanase at concentration of 12 U/mL effectively removed 84.61% of biofilm matrix of Pseudomonas aeruginosa with marked reduction in carbohydrate content of the biofilm from 63.4 to 7.9 μg.

scholarly journals Construction and Characterization of a Pseudomonas aeruginosa Mucoid Exopolysaccharide-Alginate Conjugate Vaccine

2003 ◽  
Vol 71 (7) ◽  
pp. 3875-3884 ◽  
Author(s):  
Christian Theilacker ◽  
Fadie T. Coleman ◽  
Simone Mueschenborn ◽  
Nicolas Llosa ◽  
Martha Grout ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Conjugate Vaccine ◽  
Significant Proportion ◽  
Vaccine Trial ◽  
Keyhole Limpet Hemocyanin ◽  
Opsonic Activity ◽  
Human Sera ◽  
Normal Human

ABSTRACT Deterioration of lung function in patients with cystic fibrosis (CF) is closely associated with chronic pulmonary infection with mucoid Pseudomonas aeruginosa. The mucoid exopolysaccharide (MEP) from P. aeruginosa has been shown to induce opsonic antibodies in mice that are protective against this chronic infection. MEP-specific opsonic antibodies are also commonly found in the sera of older CF patients lacking detectable P. aeruginosa infection. When used in a human vaccine trial, however, MEP only minimally induced opsonic antibodies. To evaluate whether conjugation of MEP to a carrier protein could improve its immunogenicity, we bound thiolated MEP to keyhole limpet hemocyanin (KLH) by using succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) as a linker. In contrast to the native MEP polymer, the MEP-KLH conjugate vaccine induced high titers of MEP-specific immunoglobulin G (IgG) in C3H-HeN mice and in a rabbit. Sera from mice immunized with MEP-KLH conjugate, but not from animals immunized with comparable doses of native MEP, demonstrated opsonic killing activity. Vaccination with MEP-KLH conjugate induced opsonic antibodies broadly cross-reactive to heterologous mucoid strains of P. aeruginosa. Preexisting nonopsonic antibodies to MEP are found in normal human sera, including young CF patients, and their presence impedes the induction of opsonic antibodies. Induction of nonopsonic antibodies by either intraperitoneal injection of MEP or injection or feeding of the cross-reactive antigen, seaweed alginate, reduced the level of overall IgG elicited by follow-up immunization with the MEP-KLH conjugate. However, the opsonic activity was lower only in the sera of MEP-KLH conjugate-immunized mice with preexisting antibodies induced by MEP but not with antibodies induced by seaweed alginate. Immunization with MEP-KLH elicited a significant proportion of antibodies specific to epitopes involving O-acetate residues, and this subpopulation of antibodies mediated opsonic killing of mucoid P. aeruginosa in vitro. These results indicate that conjugation of MEP to KLH significantly enhances its immunogenicity and the elicitation of opsonic antibodies in mice and rabbits, that the conjugate induces opsonic antibodies in the presence of preexisting nonopsonic antibodies, and that opsonic antibodies to MEP are directed at epitopes that include acetate residues on the uronic acid polymer.

scholarly journals Effect of Metabolic Imbalance on Expression of Type III Secretion Genes in Pseudomonas aeruginosa

2004 ◽  
Vol 72 (3) ◽  
pp. 1383-1390 ◽  
Author(s):  
Arne Rietsch ◽  
Matthew C. Wolfgang ◽  
John J. Mekalanos
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Cell Contact ◽  
Insertion Mutant ◽  
Insertion Mutation ◽  
Type Iii ◽  
Transposon Insertion ◽  
Metabolic Imbalance ◽  
Cell Expression

ABSTRACT The type III secretion system is a dedicated machinery used by many pathogens to deliver toxins directly into the cytoplasm of a target cell. Expression and secretion of the type III effectors are triggered by cell contact. In Pseudomonas aeruginosa and Yersinia spp., expression can be triggered in vitro by removing calcium from the medium. The mechanism underlying either mode of regulation is unclear. Here we characterize a transposon insertion mutant of P. aeruginosa PAO1 that displays a marked defect in cytotoxicity. The insertion is located upstream of several genes involved in histidine utilization and impedes the ability of PAO1 to intoxicate eukaryotic cells effectively in a type III-dependent fashion. This inhibition depends on the presence of histidine in the medium and appears to depend on the excessive uptake and catabolism of histidine. The defect in cytotoxicity is mirrored by a decrease in exoS expression. Other parameters such as growth or piliation are unaffected. The cytotoxicity defect is partially complemented by an insertion mutation in cbrA that also causes overexpression of cbrB. The cbrAB two-component system has been implicated in sensing and responding to a carbon-nitrogen imbalance. Taken together, these results suggest that the metabolic state of the cell influences expression of the type III regulon.

scholarly journals Interactions between effector proteins of the Pseudomonas aeruginosa type III secretion system do not significantly affect several measures of disease severity in mammals

Microbiology ◽  
2006 ◽  
Vol 152 (1) ◽  
pp. 143-152 ◽  
Author(s):  
Ciara M. Shaver ◽  
Alan R. Hauser
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Disease Severity ◽  
Type Iii Secretion ◽  
Disease Process ◽  
Effector Proteins ◽  
Host Cells ◽  
Secreted Proteins ◽  
Secretion Systems ◽  
Type Iii

The effector proteins of the type III secretion systems of many bacterial pathogens act in a coordinated manner to subvert host cells and facilitate the development and progression of disease. It is unclear whether interactions between the type-III-secreted proteins of Pseudomonas aeruginosa result in similar effects on the disease process. We have previously characterized the contributions to pathogenesis of the type-III-secreted proteins ExoS, ExoT and ExoU when secreted individually. In this study, we extend our prior work to determine whether these proteins have greater than expected effects on virulence when secreted in combination. In vitro cytotoxicity and anti-internalization activities were not enhanced when effector proteins were secreted in combinations rather than alone. Likewise in a mouse model of pneumonia, bacterial burden in the lungs, dissemination and mortality attributable to ExoS, ExoT and ExoU were not synergistically increased when combinations of these effector proteins were secreted. Because of the absence of an appreciable synergistic increase in virulence when multiple effector proteins were secreted in combination, we conclude that any cooperation between ExoS, ExoT and ExoU does not translate into a synergistically significant enhancement of disease severity as measured by these assays.

scholarly journals Pseudolipasin A Is a Specific Inhibitor for Phospholipase A2 Activity of Pseudomonas aeruginosa Cytotoxin ExoU

2006 ◽  
Vol 75 (3) ◽  
pp. 1089-1098 ◽  
Author(s):  
Vincent T. Lee ◽  
Stefan Pukatzki ◽  
Hiromi Sato ◽  
Eriya Kikawada ◽  
Anastasia A. Kazimirova ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Specific Inhibitor ◽  
Effector Proteins ◽  
Host Cells ◽  
Cytotoxic Effects ◽  
Type Iii ◽  
Secretion Pathway ◽  
Phospholipase A2 Activity

ABSTRACT A number of bacterial pathogens utilize the type III secretion pathway to deliver effector proteins directly into the host cell cytoplasm. Certain strains of Pseudomonas aeruginosa associated with acute infections express a potent cytotoxin, exoenzyme U (ExoU), that is delivered via the type III secretion pathway directly into contacting host cells. Once inside the mammalian cell, ExoU rapidly lyses the intoxicated cells via its phospholipase A2 (PLA2) activity. A high-throughput cell-based assay was developed to screen libraries of compounds for those capable of protecting cells against the cytotoxic effects of ExoU. A number of compounds were identified in this screen, including one group that blocks the intracellular activity of ExoU. In addition, these compounds specifically inhibited the PLA2 activity of ExoU in vitro, whereas eukaryotic secreted PLA2 and cytosolic PLA2 were not inhibited. This novel inhibitor of ExoU-specific PLA2 activity, named pseudolipasin A, may provide a new lead for virulence factor-based therapeutic design.

scholarly journals Expression of ExsA in trans Confers Type III Secretion System-Dependent Cytotoxicity on Noncytotoxic Pseudomonas aeruginosa Cystic Fibrosis Isolates

2001 ◽  
Vol 69 (1) ◽  
pp. 538-542 ◽  
Author(s):  
Denis Dacheux ◽  
Ina Attree ◽  
Bertrand Toussaint
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Transcriptional Activation ◽  
Type Iii Secretion ◽  
Ex Vivo ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Type Iii ◽  
In Trans

ABSTRACT Twelve Pseudomonas aeruginosa cystic fibrosis isolates that are not able to exert a type III secretion system (TTSS)-dependent cytotoxicity towards phagocytes have been further studied. The strains, although possessing TTSS genes and exsA, which encodes a positive regulator of the TTSS regulon, showed no transcriptional activation of the exsCBA regulatory operon. The expression of exsA in trans restored the in vitro secretion of TTSS proteins and ex vivo cytotoxicity.

Evaluation of biofilm production and characterization of genes encoding type III secretion system among Pseudomonas aeruginosa isolated from burn patients

Burns ◽  
2012 ◽  
Vol 38 (8) ◽  
pp. 1192-1197 ◽  
Author(s):  
Fereshteh Jabalameli ◽  
Akbar Mirsalehian ◽  
Babak Khoramian ◽  
Marzieh Aligholi ◽  
Seyed Sajjad Khoramrooz ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Burn Patients ◽  
Type Iii ◽  
Biofilm Production ◽  
Genes Encoding
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