Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0023241 (Legionella)
 6,990 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have purified the major outer membrane protein (MOMP) of Legionella pneumophila, determined that it is associated with peptidoglycan, and characterized it as a porin. To purify the MOMP, we used a simple, rapid, three-step procedure that gave us the protein in high yield. The first step of the purification procedure involved selectively extracting the MOMP from whole bacterial cells with calcium and zwitterionic detergent. The second and third steps achieved purification by ion-exchange and molecular-sieve chromatography. The dissociation of the MOMP into monomers was dependent upon the presence of a reducing agent and was enhanced by treatment at 100 degrees C. To study the relationship of the MOMP to peptidoglycan, we extracted the protein by a modification of the Rosenbusch procedure. Like the Escherichia coli porins, the MOMP was peptidoglycan associated. The MOMP was at least partially dissociated from peptidoglycan in sodium dodecyl sulfate and a high salt concentration. To study the ion channel-forming properties of the MOMP, we reconstituted the MOMP in planar lipid membranes. The MOMP formed ion-permeable channels with a single-channel conductance size of 100 picoSiemens. The MOMP channels exhibited a fourfold selectivity for cations over anions and voltage-independent gating. These findings demonstrate that the MOMP is a porin with properties similar to those of E. coli porins.
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PMID:Purification of Legionella pneumophila major outer membrane protein and demonstration that it is a porin. 257 42

A procedure is described for assaying antibodies based on the application of antigen to nitrocellulose as a line with an ink pen point. The method requires no expensive apparatus, is easy to perform, and requires less than 0.25 micrograms of antigen per assay. More than 45 antigens can be assayed simultaneously with a single antibody. Antigens can be applied as purified proteins, extracts, or sodium dodecyl sulfate solubilized extracts. The application of the line blot assay for the detection of monoclonal antibodies which recognize heat-sensitive and insensitive epitopes on the typhus rickettsia surface protein antigen is described. A new serotyping assay for Gram-negative bacteria is also described in which sodium dodecyl sulfate solubilized antigens are applied as lines with and without prior proteinase K digestion. The value of the line blot serotyping assay is demonstrated with Proteus. Rickettsia, Rochalimaea, and Legionella antigens. The line blot immunoassay is a simple, but powerful and flexible, alternative to dot and cross-dot immunoassays.
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PMID:The line blot: an immunoassay for monoclonal and other antibodies. Its application to the serotyping of gram-negative bacteria. 260 66

The legionellae are facultative intracellular bacterial pathogens which multiply in host phagocytes. Legionella micdadei cells contain an acid phosphatase (ACP2) which blocks superoxide anion production by human neutrophils stimulated with formyl-Met-Leu-Phe (fMLP) [A. K. Saha, et al. (1985) Arch. Biochem. Biophys. 243, 150-160]. In the present study, we have purified the Legionella phosphatase to homogeneity as indicated by the finding of a single 68,000-Da band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We explored the possibility that ACP2 acts by interfering with polyphosphoinositide hydrolysis and the production of the intracellular second messengers, inositol trisphosphate (IP3) and diacylglycerol, following neutrophil stimulation. Phosphatidylinositol 4,5-bisphosphate (PIP2) was hydrolyzed rapidly by ACP2 in vitro. The rate of hydrolysis of PIP2 was higher at pH 7.0 (Km 2.0 microM; 4 X 10(3) units/mg protein; 1 unit equals 1 nmol of Pi released/h) than at lower pH. IP3 was also a good substrate for ACP2 in vitro. When human neutrophil phosphoinositides were prelabeled with 32Pi, subsequent incubation with ACP2 resulted in an 85% loss of the labeled PIP2 over 2 h. Following fMLP stimulation of [3H]inositol-labeled neutrophils, the quantity of IP3 produced by ACP2-treated cells was reduced by 44%. Prior treatment of neutrophils with ACP2 also reduced by 45% the amount of diacylglycerol they produced when stimulated by fMLP. These results indicate that the Legionella phosphatase may compromise the neutrophils' microbicidal response to the organism by hydrolyzing PIP2, the progenitor of IP3 and diacylglycerol, and by hydrolyzing IP3 itself.
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PMID:Legionella micdadei phosphatase catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate in human neutrophils. 284 4

Hybrid cell lines producing monoclonal antibodies against Bordetella pertussis filamentous hemagglutinin (FHA) were established. The specificity of the antibodies was ascertained by enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroblotting. The monoclonal antibody-based sandwich ELISA was developed for detection of B. pertussis FHA. The assay had a detection limit of B. pertussis FHA in concentrations ranging from 7 to 15 ng/ml. The assay was also able to detect whole B. pertussis, Bordetella parapertussis, and Bordetella bronchiseptica bacteria. No cross-reactions were observed with strains of Branhamella catarrhalis, Neisseria meningitidis, Haemophilus influenzae, Klebsiella pneumoniae, Legionella pneumophila, Streptococcus miteor, or Streptococcus pneumoniae. The monoclonal antibodies might be useful for the detection of soluble antigens and whole bacteria in clinical samples and for studies of the immunochemical structure of B. pertussis FHA.
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PMID:Monoclonal antibody-based sandwich enzyme-linked immunosorbent assay for detection of Bordetella pertussis filamentous hemagglutinin. 290 74

A preliminary screening of selected Legionella species for proteolytic and hemolytic phenotypes suggested a correlation between these activities. To investigate the relationship of these phenotypes, a mutant strain of Legionella pneumophila deficient in the expression of a 38-kilodalton (kDa) exoprotease was isolated and characterized. This strain, designated PRT8, was also found to be nonhemolytic when screened on blood agar composed of 5% canine or guinea pig erythrocytes. Strain PRT8 was serologically and biochemically identical to the parental strain with the exception of the expression of the exoprotease. Immunoblot analysis of concentrated culture filtrates from PRT8 probed with polyclonal anti-38-kDa exoprotease serum revealed no cross-reactive peptides. To resolve the role of the exoprotease in the hemolytic phenotype, the exoprotease was purified from the culture supernatant of the parental strain by a combination of ion-exchange and hydrophobic interaction chromatography steps. The hemolytic activity was found to copurify with the proteolytic activity, and analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot revealed a single protein species exhibiting an apparent molecular mass of 38 kDa. Finally, the purified exoprotease and concentrated culture supernatant from the parental strain, but not from PRT8, exhibited cytotoxicity (minimum cytotoxic activity of 0.17 U of protease activity) in a Chinese hamster ovary cell assay. These data suggest that the exoprotease is responsible for the hemolytic and cytotoxic phenotypes described for this species and therefore may be one of several determinants associated with virulence.
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PMID:Characterization of a Legionella pneumophila extracellular protease exhibiting hemolytic and cytotoxic activities. 291 85

We have examined the capacity of the major secretory protein (MSP) of Legionella pneumophila to induce humoral, cell-mediated, and protective immunity in a guinea pig model of Legionnaires' disease. MSP was purified to homogeneity by ammonium sulfate precipitation, molecular sieve chromatography, and ion-exchange chromatography. The purified MSP was nonlethal and nontoxic to guinea pigs upon subcutaneous administration. Guinea pigs immunized with a sublethal dose of aerosolized L. pneumophila or a subcutaneous dose of MSP developed a strong cell-mediated immune response to MSP. Such guinea pigs exhibited marked splenic lymphocyte proliferation and cutaneous delayed-type hypersensitivity to MSP in comparison with control animals. Guinea pigs immunized with MSP also developed a strong humoral immune response to MSP, as assayed by an ELISA. The median reciprocal antibody titer was 362 (range 45 to greater than 2,048) for immunized animals compared with less than 8 for controls. In contrast, guinea pigs immunized with a sublethal dose of L. pneumophila failed to develop anti-MSP antibody. Guinea pigs immunized with MSP and then challenged with a lethal aerosol dose of L. pneumophila exhibited highly significant protective immunity in each of five consecutive experiments. MSP induced protective immunity in dose-dependent fashion (40 greater than 10 greater than 2.5 greater than 0.6 micrograms MSP); vaccination with two doses of as little as 2.5 micrograms MSP induced significant protective immunity (p = 0.01, Fisher's Exact Test, two-tailed). Altogether, 21 (81%) of 26 animals immunized with 40 micrograms MSP survived challenge compared with 0 (0%) of 26 sham-immunized control animals (p = 7 x 10(-10), Fisher's Exact Test, two-tailed). MSP-immunized but not control guinea pigs were able to limit L. pneumophila multiplication in their lungs. This study demonstrates that (a) guinea pigs sublethally infected with L. pneumophila develop a strong cell-mediated immune response to MSP; (b) guinea pigs immunized with MSP develop a strong humoral and cell-mediated immune response to MSP; (c) guinea pigs immunized with MSP develop a very high level of protective immunity to lethal aerosol challenge with L. pneumophila; and (d) MSP-immunized animals are able to limit L. pneumophila multiplication in their lungs. MSP, an extracellular protein of an intracellular pathogen, has potential as a vaccine for the prevention of Legionnaires' disease. Secretory molecules of other intracellular pathogens may also have vaccine potential.
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PMID:Vaccination with the major secretory protein of Legionella pneumophila induces cell-mediated and protective immunity in a guinea pig model of Legionnaires' disease. 292 24

Serospecific antigens isolated by EDTA extraction from four serogroups of Legionella pneumophila were analyzed for their chemical composition, molecular heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological properties. The antigens were shown to be lipopolysaccharides and to differ from the lipopolysaccharides of other gram-negative bacteria. The serospecific antigens contained rhamnose, mannose, glucosamine, and two unidentified sugars together with 2-keto-3-deoxyoctonate, phosphate, and fatty acids. The fatty acid composition was predominantly branched-chain acids with smaller amounts of 3-hydroxymyristic acid. The antigens contain periodate-sensitive groups; mannosyl residues were completely cleaved by periodate oxidation. Hydrolysis of the total lipopolysaccharide by acetic acid resulted in the separation of a lipid A-like material that cross-reacted with the antiserum to lipid A from Salmonella minnesota but did not comigrate with it on sodium dodecyl sulfate gels. None of the four antigens contained heptose. All of the antigen preparations showed endotoxicity when tested by the Limulus amebocyte lysate assay. The results of this study indicate that the serogroup-specific antigens of L. pneumophila are lipopolysaccharides containing an unusual lipid A and core structure and different from those of other gram-negative bacteria.
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PMID:Serospecific antigens of Legionella pneumophila. 301 18

A genuswide protein antigen extracted from Legionella pneumophila serogroup 1 (strain Philadelphia 1) cells was enriched by differential pelleting and ammonium sulfate precipitation and subsequently purified with a combination of high-performance size-exclusion and ion-exchange chromatography. The protein has an apparent molecular weight of 650,000 before and 63,000 after urea (5 M) treatment, as determined by size-exclusion chromatography. These proteins resolved to a single band of 60,000 after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The urea-treated protein had an isoelectric point of 5.8. This purified 60-kilodalton protein reacted with a convalescent-phase serum sample from a patient with legionellosis and rabbit immune sera prepared against each of 23 Legionella species. The 60-kilodalton protein may be useful in developing diagnostic tests for legionellosis.
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PMID:Purification, partial characterization, and seroreactivity of a genuswide 60-kilodalton Legionella protein antigen. 334 16

Outer membranes were isolated from eight serogroups of L. pneumophila and five other Legionella species. The protein composition of the membranes was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single, disulfide stabilized protein with a molecular size of 29,000 to 30,000 daltons was found to be the major outer membrane protein (MOMP) of all the serogroups. The equivalent of the L. pneumophila MOMP was not observed in any of the other Legionella species examined. Silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels revealed distinctive patterns for each serogroup and other Legionella species that were not observed by staining with Coomassie blue and may result from the presence of lipopolysaccharide in the membrane preparations. The MOMP from serogroup 1 was isolated by exposing crude peptidoglycan to detergent in the presence of heat and reducing agent and was found to be tightly associated with lipopolysaccharide. Antibodies to this complex were used to probe the outer membranes of the remaining, L. pneumophila serogroups and other Legionella species by Western blotting. Serogroup 1 anti-MOMP antibodies were found to react with the MOMP from the remaining seven serogroups examined, whereas antibodies directed against the lipopolysaccharide of serogroup 1 only reacted with lipopolysaccharide from two of the remaining seven serogroups.
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PMID:Outer membrane proteins from Legionella pneumophila serogroups and other Legionella species. 351 Jan 78

An extracellular proteolytic enzyme of Legionella pneumophila was purified by sequential batch separation with DEAE-cellulose, hydrophobic interaction chromatography with octyl-Sepharose, and ion-exchange chromatography with DEAE-Bio-Gel A (Bio-Rad Laboratories, Richmond, Calif.). The resulting protease preparation was determined to be homogeneous by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Although free of contaminating proteins, the purified protease separated into two antigenically indistinguishable proteins both of which possessed proteolytic activity. The apparent masses of the proteins were 38 and 40 kilodaltons (kDa) as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, whereas gel filtration chromatography revealed a single mass of 34 kDa. Immunoblot analysis indicated that the 38-kDa protein probably originated from the 40-kDa protein during purification. The isoelectric points of the two protease species were 4.20 and 4.42. Enzyme activity, which was optimum between pH 5.5 and 7.5, was inhibited by various metal chelators; however, no effect was observed after treatment with phenylmethylsulfonyl fluoride, chymostatin, trypsin inhibitor, or dithiothreitol. Enzyme activity inhibited by metal chelators was restored upon the addition of various metal ions, including Zn2+, Fe2+, Mn2+, Cu2+, and Fe3+, but was not restored by Mg2+ or Ca2+. Atomic absorption analysis of the purified protease revealed a single gram-atom of zinc per mole of enzyme. Our findings indicate that the L. pneumophila protease resembles neutral zinc-containing metalloproteases similar to those found in other bacterial species.
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PMID:Purification and characterization of an extracellular protease of Legionella pneumophila. 351 31


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