Purification of Pseudomonas aeruginosa exoenzyme S

Woods, Donald E.; Que, Johnu .

doi:10.1128/iai.55.3.579-586.1987

Cited by 32 publications

(13 citation statements)

References 28 publications

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“…Exoenzyme S and antibodies to exoenzyme S. Exoenzyme S was purified from P. aeruginosa DG1 as described previously (26). The composition of the preparations used in the GSL binding studies varied because some samples contained lipopolysaccharide (LPS; see Fig.…”

Section: Methodsmentioning

confidence: 99%

“…The preparation used in the adherence inhibition assays produced a single protein band on sodium dodecyl sulfate (SDS)-polyacrylamide gels as visualized by Coomassie blue staining. Polyclonal antiserum to exoenzyme S raised in New Zealand rabbits was prepared as described previously (26). Western blotting (immunoblotting) with this antibody preparation revealed reactivity with exoenzyme S and LPS.…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, antiserum used in gold labeling studies was absorbed with P. aeruginosa DG1-ExS5, a mutant strain which is not totally exoenzyme S deficient but which produces significantly lower amounts of exoenzyme S than the parent strain (27). The production of monoclonal antibodies to exoenzyme S has been described previously (26).…”

Section: Methodsmentioning

confidence: 99%

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Pseudomonas aeruginosa exoenzyme S is an adhesion

et al. 1991

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Exoenzyme S from Pseudomonas aeruginosa has been studied as an adhesin for glycosphingolipids and buccal cells. Binding of exoenzyme S to gangliotriosylceramide (GalNAcfll-4Gal,l-4Glci3l-1Cer), gangliotetraosylceramide (Gall-3GaINAcTIIl-4Gal1l-4Glcil-lCer), and lactosylceramide (Gal31-4Glco1-lCer) separated on thin-layer chromatograms was observed. Binding curves for exoenzyme S with dilutions of gangliotetraosylceramide immobilized on plastic plates were similar to previously reported results for the intact bacteria. Binding of exoenzyme S to sialylated counterparts of these glycosphingolipids was not seen, indicating that the addition of a sialic acid residue interferes with binding. Exoenzyme S and monoclonal antibody to exoenzyme S inhibit the binding of P. aeruginosa to buccal cells. The presence of exoenzyme S on the surface of P. aeruginosa was detected by immunogold labeling of bacteria with antibodies to exoenzyme S. Results of these studies led us to conclude that exoenzyme S is an important adhesin of P. aeruginosa. Pseudomonas aeruginosa is an important opportunistic pathogen of immunocompromised patients (7). Infections of the respiratory tract are of particular concern because they are associated with high mortality rates (2). Cystic fibrosis patients are at high risk for lung infections due to P. aeruginosa which lead to significant morbidity and contribute to death of the patients (15, 17). Identification of the factors responsible for colonization of the lungs of cystic fibrosis patients by P. aeruginosa is particularly important since the organism is virtually impossible to eradicate once a chronic infection has been established.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Pseudomonas aeruginosa exoenzyme S is an adhesion

et al. 1991

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“…Exoenzyme S is an ADP-ribosyltransferase and is one of the virulence factors of Pseudomonas aeruginosa involved in the pathogenesis of this organism (30). We have recently found (14) that this enzyme also specifically binds to the H. pylori receptor glycerolipid.…”

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The glycerolipid receptor for Helicobacter pylori (and exoenzyme S) is phosphatidylethanolamine

1992

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We have previously shown that Helicobacter pylori specifically binds to a glycerolipid species preferentially found in the antrum of the human stomach. We now show by high-pressure liquid chromatographic analysis that this species is a form of phosphatidylethanolamine and that H. pylori specifically binds to bona fide phosphatidylethanolamine as detected by a thin-layer chromatogram overlay procedure. Considerable variation in the binding of H. pyloyi to phosphatidylethanolamine from different sources was observed, however, suggesting the importance of the nature of the long-chain hydrophobic moiety. A similar binding specificity was shown by exoenzyme S from Pseudonwnas aeruginosa, consistent with our hypothesis that that an exoenzyme S-like adhesin is responsible for the binding of H. pylori to its lipid receptors.Colonization of the human stomach with Helicobacter pylori has been implicated as the major etiological agent in the development of gastritis and possibly subsequent duodenal ulcer (3, 6, 27) and gastric carcinoma (17,19,20). Because of the known tropism of this organism for colonization of gastric epithelium, even when mislocated elsewhere in the gastrointestinal tract (31), we previously investigated the presence of gastric membrane species able to specifically bind to this organism, as monitored by the solid-phase thin-layer chromatography (TLC) overlay procedure (13). We isolated a charged glycerolipid species from the antrum of human stomach and from human erythrocytes which was specifically recognized by this organism, using this procedure.Exoenzyme S is an ADP-ribosyltransferase and is one of the virulence factors of Pseudomonas aeruginosa involved in the pathogenesis of this organism (30). We have recently found (14) that this enzyme also specifically binds to the H. pylori receptor glycerolipid.We now report on the partial structural characterization of this receptor species.MATERUILS AND METHODS Phosphatidylethanolamine (PE) from various tissues; phosphatidic acid, phosphatidylserine, and phosphatidylcholine from egg yolk; and phosphatidylglycerol from bovine brain were purchased from Sigma. Plastic-backed silica gel (SIL G) TLC plates were from Brinkman Inc. Goat antirabbit-horseradish peroxidase conjugate was from Bio-Rad.Purification of H. pylori receptor. The Helicobacter glycerolid receptor was purified as previously described from pooled human erythrocytes (13). Briefly, the cells were extracted with chloroform-methanol (2:1, vol/vol), and lower-phase lipids were separated by silicic acid chromatography. The column was eluted in sequence with chloroform, acetone-methanol (3:1), and finally methanol. The methanol fraction was reapplied on a fresh column which was eluted with a linear polarity gradient of chloroform methanol (10:1 to 2:1). * Corresponding author.Receptor binding. H. pylori was grown on Skirrow's medium and transferred to brucella broth supplemented with 10% fetal calf serum before binding (13). Only highly motile cultures were used for binding experiments. Purified exoen...

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“…Purification was performed as previously described [22]. Briefly, ammonium sulfate precipitation was performed on culture supernatants from P. aeruginosa strain DG1.…”

Section: Purification Of P Aeruginosa Exoenzyme Smentioning

confidence: 99%

Exoenzyme S from Pseudomonas aeruginosa induces apoptosis in T lymphocytes

Bruno

Woods

Mody

2000

Journal of Leukocyte Biology

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Exoenzyme S from Pseudomonas aeruginosa is a unique T cell mitogen; it is a powerful immunostimulus that activates a large proportion of T cells, but results in delayed and reduced lymphocyte proliferation. This study was performed to explain the discrepancy between early T cell activation and subsequent proliferation. Studies revealed that exoenzyme S induced rapid and unsustained surface expression of CD69, but could not induce interleukin-2 receptor ␣ (IL-2R␣) upregulation on T cells. IL-2 was undetectable in supernatants and addition of rIL-2 could not reverse the unresponsiveness, indicating that anergy was not involved. Exoenzyme S induced membrane phosphatidylserine translocation, DNA hypodiploidy, and DNA fragmentation, implicating apoptosis as the mechanism for the unresponsiveness.

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Purification of Pseudomonas aeruginosa exoenzyme S

Cited by 32 publications

References 28 publications

Pseudomonas aeruginosa exoenzyme S is an adhesion

Pseudomonas aeruginosa exoenzyme S is an adhesion

The glycerolipid receptor for Helicobacter pylori (and exoenzyme S) is phosphatidylethanolamine

Exoenzyme S from Pseudomonas aeruginosa induces apoptosis in T lymphocytes

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