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Chapter 59 : Characterization of GDSL-Hydrolases of the Lung Pathogen

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Characterization of GDSL-Hydrolases of the Lung Pathogen , Page 1 of 2

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Abstract:

possesses secreted phospholipase A (PLA) and lysophospholipase A (LPLA) activities which might be involved in bacterial virulence, because they represent tools for modification and lysis of host cell membranes and for the interference with the host signal transduction pathways. The genomes Philadelphia-1, Paris, and Lens code for three members of the GDSL family of lipolytic proteins, namely PlaA, PlaC, and PlaD. The authors aimed to identify the factor which directly activates PlaC. Furthermore, they sought to characterize PlaD, the third GDSL enzyme, and to compare the lipolytic properties as well as the capacity of mutants to multiply intracellularly with wild type and and mutants. The PlaC-activating factor, characterized by its ability to enhance glycerophospholipid:cholesterol acyltransferase (GCAT) activity of PlaC expressed in , was biochemically purified from the culture supernatant of an 130b mutant by anion exchange chromatography (AEX). The main protein in the activating fractions consistently was the zinc metalloprotease ProA. To investigate whether Corby could confer PLA and LPLA activities to , cell lysates as well as the culture supernatants from clones carrying in were examined for hydrolysis of PLA, LPLA, and lipase substrates.

Citation: Banerji S, Rastew E, Hermes B, Flieger A. 2006. Characterization of GDSL-Hydrolases of the Lung Pathogen , p 238-241. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch59

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Figures

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FIGURE 1

Analysis of AEX fractions from culture supernatant of an 130b mutant with respect to PlaC activating properties and protease activity. Culture supernatant from an 130b mutant was purified by AEX, and the fractions (1 to 17) were assayed for induction of GCAT activity detected by the formation of cholesterol ester by harboring () and for protease activity (). Fractions were also analyzed by reducing sodium dodecylsulfate-polyacrylamide gel electrophoresis (). Similar results were obtained on one more occasion. AEX, anion exchange chromatography; Chol, cholesterol; CholE, cholesterol ester; FFA, free fatty acid; St, protein mass standard.

Citation: Banerji S, Rastew E, Hermes B, Flieger A. 2006. Characterization of GDSL-Hydrolases of the Lung Pathogen , p 238-241. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch59
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Image of FIGURE 2
FIGURE 2

Lipolytic activities of Corby culture supernatants from wild type and mutants. Corby culture supernatants from wild type, , and knockout mutants were obtained at late logarithmic growth phase and incubated with dipalmitoylphosphatidylcholine (DPPC), di-palmitoyllysophosphatidylglycerol (DPPG), 1-monopalmitoylglycerol (1-MPG), monopalmitoyllysophosphatidylcholine (MPLPC), and monopalmitoyllysophos-phatidylglycerol (MPLPG) at 37°C. Subsequently, the release of fatty acids was quantified. Similar results were obtained on two more occasions. plaA-A3 and plaA-B7 represent two independent Corby mutants. Likewise, plaD-A4 and plaD-C2 represent two independent Corby mutants.

Citation: Banerji S, Rastew E, Hermes B, Flieger A. 2006. Characterization of GDSL-Hydrolases of the Lung Pathogen , p 238-241. In Cianciotto N, Kwaik Y, Edelstein P, Fields B, Geary D, Harrison T, Joseph C, Ratcliff R, Stout J, Swanson M (ed), . ASM Press, Washington, DC. doi: 10.1128/9781555815660.ch59
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References

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5. Flieger A.,, B. Neumeister, and, N. P. Cianciotto. 2002. Characterization of the gene encoding the major secreted lysophospholipase A of Legionella pneumophila and its role in detoxification of lysophosphatidylcholine. Infect. Immun. 70:60946106.
6. Lee K. K., and, A. E. Ellis. 1990. Glycerophos-pholipid:cholesterol acyltransferase complexed with lipopolysaccharide (LPS) is a major lethal exo-toxin and cytolysin of Aeromonas salmonicida: LPS stabilizes and enhances toxicity of the enzyme.J. Bacteriol. 172:53825393.
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