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)

The major outer membrane protein of Legionella pneumophila exhibits an apparent molecular mass of 100 kDa. Previous studies revealed the oligomer to be composed of 28- and 31-kDa subunits; the latter subunit is covalently bound to peptidoglycan. These proteins exhibit cross-reactivity with polyclonal anti-31-kDa protein serum. In this study, we present evidence to confirm that the 31-kDa subunit is a 28-kDa subunit containing a bound fragment of peptidoglycan. Peptide maps of purified proteins were generated following cyanogen bromide cleavage or proteolysis with staphylococcal V8 protease. A comparison of the banding patterns resulting from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a common pattern. Selected peptide fragments were sequenced on a gas phase microsequencer, and the sequence was compared with the sequence obtained for the 28-kDa protein. While the amino terminus of the 31-kDa protein was blocked, peptide fragments generated by cyanogen bromide treatment exhibited a sequence identical to that of the amino terminus of the 28-kDa protein, but beginning at amino acid four (glycine), which is preceded by methionine at the third position. This sequence, (Gly-Thr-Met)-Gly-Pro-Val-Trp-Thr-Pro-Gly-Asn ... , confirms that these proteins have a common amino terminus. An oligonucleotide synthesized from the codons of the common N-terminal amino acid sequence was used to establish by Southern and Northern (RNA) blot analyses that a single gene coded for both proteins. With regard to the putative porin structure, we have identified two major bands at 70 kDa and at approximately 120 kDa by nonreducing SDS-PAGE. The former may represent the typical trimeric motif, while the latter may represent either a double trimer or an aggregate. Analysis of these two forms by two-dimensional SDS-PAGE (first dimensions, nonreducing; second dimensions, reducing) established that both were composed of 31- and 28-kDa subunits cross-linked via interchain disulfide bonds. These studies confirm that the novel L. pneumophila major outer protein is covalently bound to peptidoglycan via a modified 28-kDa subunit (31-kDa anchor protein) and cross-linked to other 28-kDa subunits via interchain disulfide bonds.
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PMID:Molecular characterization of the 28- and 31-kilodalton subunits of the Legionella pneumophila major outer membrane protein. 131 95

Legionella spp., the causative organism of legionnaires' disease, were isolated from more than 80% of water samples in cooling towers before washing. Therefore, we evaluated the effect of microbicide treatment of cooling tower water on Legionella spp., other bacteria and protozoa. 2-Bromo-2-nitropane-1,3-dial, 2,4-dibromo-5,5-dimethylhydantoin or silver nitrate-treated silica gel was added to cooling tower water. The isolation rate of Legionella spp. in the cooling tower water was 50% after microbiocide treatment with 2-bromo-2-nitropane-1,3-dial being the most effective. The microbicide treatment had no effect on other bacteria or protozoa. These findings indicated the importance of regular washing and water exchange of cooling tower water with microbicide treatment.
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PMID:[Isolation of Legionella spp. from cooling tower water and the effect of microbicides]. 258 55

We established a model of the bacteria-macrophage interaction to study the cellular basis of Legionella pneumophila pathogenesis and to characterize avirulent L. pneumophila. We found that U937 cells, which are derived from a human histiocytic lymphoma cell line, support intracellular growth of L. pneumophila with a doubling time of 6 h, and that sustained intracellular growth is associated with a cytopathic effect (CPE) that can be detected by microscopic examination and quantified with the vital stain 3-(4,5-dimethyl thiazol-2-yl)-2,5,-diphenyl tetrazolium bromide (MTT). An L. pneumophila isolate obtained directly from infected guinea-pig spleens can grow and produce CPE in these cells, destroying most of the cell layer after 72 h of growth. Only 10(6) organisms of this strain are required to kill 50% of guinea-pigs inoculated by the intraperitoneal route. In contrast, an avirulent isolate derived by 203 successive plate passages of the same strain can neither kill guinea-pigs at an intraperitoneal inoculum of 10(7) nor grow or produce CPE in U937 cells. Since the cells were able to differentiate between a virulent and an avirulent strain of L. pneumophila, we conclude that U937 cells are an appropriate model system for study of the bacteria-macrophage interaction.
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PMID:Growth of Legionella pneumophila in a human macrophage-like (U937) cell line. 323 54

A highly sensitive nested polymerase chain reaction (PCR) method was evaluated for detection of Legionella pneumophila in water. Two sets of primers homologous to the coding region of the L. pneumophila macrophage infectivity potentiator (mip) gene were used. Even when starting from minute amounts of L. pneumophila DNA, the double PCR products were readily detected by direct visualization in ethidium-bromide-stained agarose gels. The method was tested on 34 potable water samples from a hospital building and compared with standard culture isolation. L. pneumophila was isolated in only twelve samples, whereas, by nested PCR, 19 samples were positive, 12 of them coincidental with culturing. These results indicate that nested PCR permits detection of L. pneumophila in samples where culturing fails, with the advantage of a rapid turnaround time, simplicity and the ability to detect non-culturable cells, fulfilling the requirements of sensitivity and specificity for routine use in an environmental laboratory.
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PMID:Nested polymerase chain reaction for detection of Legionella pneumophila in water. 787 Dec 39

A rapid colorimetric technique for in vitro quantitation of Legionella pneumophila intracellular proliferation in macrophages is described. The assay is based on the electron transport activity of metabolically active L. pneumophila. The yellow tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is cleaved by the mitochondrial activity of viable L. pneumophila, forming a dark formazan derivative with an absorption spectrum different from that of the native compound. The MTT method for measuring intracellular growth of L. pneumophila closely correlated with the CFU assay. The ability of macrophages from the A/J mouse strain to support intracellular growth of L. pneumophila and the ability of desferrioxamine to restrict L. pneumophila intracellular proliferation were confirmed by both methods. The MTT assay offers the advantages of rapidity, simplicity, and cost efficiency over the CFU assay, since it can be performed in the same flat-bottom microtiter plate with measurement in an enzyme-linked immunosorbent assay reader, allowing efficient processing of large numbers of samples.
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PMID:A rapid colorimetric assay for evaluating Legionella pneumophila growth in macrophages in vitro. 812 66

We developed a new simple assay for the quantitation of the activities of drugs against intracellular Legionella pneumophila. The cells of a murine macrophage-like cell line (J774.1 cells) allowed the intracellular growth and replication of the bacteria, which ultimately resulted in cell death. The infected J774.1 cell monolayers in 96-well microplates were first treated with antibiotics and were further cultured for 72 h. The number of viable J774.1 cells in each well was quantified by a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and an enzyme-linked immunosorbent assay reader. The number of growing bacteria in each well was also determined by counting the numbers of CFU on buffered charcoal yeast extract-alpha agar plates. Viable J774.1 cell counts, determined by the colorimetric assay, were inversely proportional to the number of intracellular replicating bacteria. The minimum extracellular concentrations (MIECs) of 24 antibiotics causing inhibition of intracellular growth of L. pneumophila were determined by the colorimetric assay system. The MIECs of beta-lactams and aminoglycosides were markedly higher than the MICs in buffered yeast extract-alpha broth. The MIECs of macrolides, fluoroquinolones, rifampin, and minocycline were similar to the respective MICs. According to their intracellular activities, clarithromycin and sparfloxacin were the most potent among the macrolides or fluoroquinolones tested in this study. Our results indicated that the MTT assay system allows comparative and quantitative evaluations of the intracellular activities of antibiotics and efficient processing of a large number of samples.
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PMID:Simplified quantitative assay system for measuring activities of drugs against intracellular Legionella pneumophila. 957 12

We determined the MICs of ampicillin, ciprofloxacin, erythromycin, imipenem, and rifampin for two clinical isolates of Legionella pneumophila serogroup 1 by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction assay and by quantitative culture. To test the influence of subinhibitory concentrations (sub-MICs) of antimicrobial agents on Legionella uptake into Acanthamoeba castellanii and U937 macrophage-like cells, both strains were pretreated with 0.25 MICs of the antibiotics for 24 h. In comparison to that for the untreated control, subinhibitory concentrations of antibiotics significantly reduced Legionella uptake into the host cells. Measurement of the binding of monoclonal antibodies against several Legionella antigens by enzyme-linked immunoassays indicated that sub-MIC antibiotic treatment reduced the expression of the macrophage infectivity potentiator protein (Mip), the Hsp 60 protein, the outer membrane protein (OmpM), an as-yet-uncharacterized protein of 55 kDa, and a few lipopolysaccharide (LPS) epitopes. In contrast, the expression of some LPS epitopes recognized by monoclonal antibodies 8/5 and 30/4 as well as a 45-kDa protein, a 58-kDa protein, and the major outer membrane protein (OmpS) remained unaffected.
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PMID:Subinhibitory concentrations of antimicrobial agents reduce the uptake of Legionella pneumophila into Acanthamoeba castellanii and U937 cells by altering the expression of virulence-associated antigens. 979 18

Beta-dolabrin, gamma-thujaplicin, and 4-acetyltropolone, the components of Aomori Hiba (Thujopsis dolabrata SIEB. et ZUCC. var. hondai MAKINO), showed antifungal activity on seven kinds of plant-pathogenic fungi, antibacterial activity against two kinds of Legionella sp., and in vitro cytotoxic effect on murine P388 lymphocytic leukemia cell line. Firstly, beta-dolabrin, gamma-thujaplicin and 4-acetyltropolone had clear antifungal activity against seven kinds of plant-pathogenic fungi tested. In particular, beta-dolabrin and 4-acetyltropolone showed strong antifungal activity against Pythium aphanidermatum IFO 32440, with minimum inhibitory concentration (MIC) values of 6.0 microg/ml. Secondly, beta-dolabrin, gamma-thujaplicin and 4-acetyltropolone had obvious growth-inhibitory effect on two kinds of Legionella sp. 4-Acetyltropolone especially had strong antibacterial activity toward Legionella pneumophila SG 1, and its MIC value was 3.1 microg/ml. These three compounds showed cytotoxic effects against murine P388 lymphocytic leukemia cell line in vitro. The cytotoxic effect of three compounds in the murine P388 lymphocytic leukemia cell line were clear when cell growth was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. At 48 h after treatment, gamma-thujaplicin and 4-acetyltropolone at 0.63 microg/ml inhibited cell growth of murine P388 lymphocytic leukemia by 85% and 65%, respectively. At the same time after treatment, the growth of the murine P388 lymphocytic leukemia cell line was completely suppressed by the three compounds at concentrations higher than 5.0 microg/ml. Among these three compounds, gamma-thujaplicin had the strongest cytotoxic activity on the growth of this tumor cell line in vitro.
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PMID:Biological activity of beta-dolabrin, gamma-thujaplicin, and 4-acetyltropolone, hinokitiol-related compounds. 1546 16

PCR-based methods have been developed to rapidly screen for Legionella pneumophila in water as an alternative to time-consuming culture techniques. However, these methods fail to discriminate between live and dead bacteria. Here, we report a viability assay (viability PCR [v-PCR]) for L. pneumophila that combines ethidium monoazide bromide with quantitative real-time PCR (qPCR). The ability of v-PCR to differentiate viable from nonviable L. pneumophila cells was confirmed with permeabilizing agents, toluene, or isopropanol. v-PCR suppressed more than 99.9% of the L. pneumophila PCR signal in nonviable cultures and was able to discriminate viable cells in mixed samples. A wide range of physiological states, from culturable to dead cells, was observed with 64 domestic hot-water samples after simultaneous quantification of L. pneumophila cells by v-PCR, conventional qPCR, and culture methods. v-PCR counts were equal to or higher than those obtained by culture and lower than or equal to conventional qPCR counts. v-PCR was used to successfully monitor in vitro the disinfection efficacy of heating to 70 degrees C and glutaraldehyde and chlorine curative treatments. The v-PCR method appears to be a promising and rapid technique for enumerating L. pneumophila bacteria in water and, in comparison with conventional qPCR techniques used to monitor Legionella, has the advantage of selectively amplifying only viable cells.
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PMID:Viability PCR, a culture-independent method for rapid and selective quantification of viable Legionella pneumophila cells in environmental water samples. 1936 80

A total of 57 isolates of Legionella pneumophila were randomly selected from the German National Legionella strain collection and typed by monoclonal antibody subgrouping, seven-gene locus sequence-based typing (SBT) scheme and a newly developed variable element typing (VET) system based on the presence or absence of ten variable genetic elements. These elements were detected while screening a genomic library of strain Corby, as well as being taken from published data for PAI-1 (pathogenicity island) from strain Philadelphia. Specific primers were designed and used in gel-based polymerase chain reaction (PCR) assays. PCR amplification of the mip gene served as a control. The end-point was the presence/absence of a PCR product on an ethidium bromide-strained gel. In the present study, the index of discrimination was somewhat lower than that of the SBT (0.87 versus 0.97). Nevertheless, the results obtained showed as a 'proof of principle' that this simple and quick typing assay might be useful for the epidemiological characterisation of L. pneumophila strains.
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PMID:Variable genetic element typing: a quick method for epidemiological subtyping of Legionella pneumophila. 2011 76


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