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Legionella Phospholipases Implicated in Infection: Determination of Enzymatic Activities

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Legionella

Part of the book series: Methods in Molecular Biology ((MIMB,volume 954))

Abstract

The intracellularly replicating lung pathogen Legionella pneumophila expresses a multitude of different phospholipases which are important virulence tools during host cell infection. To study the lipolytic properties including substrate specificities of potential L. pneumophila phospholipases A (PLA), we used different assays to monitor lipid hydrolysis. Here we describe methods for quantitative analysis of liberated fatty acids via a photometric assay and for identification of specific lipids which are generated by PLA action by means of lipid extraction and thin-layer chromatography. The latter approach also identifies glycerophospholipid:cholesterol acyltransferase activity which may be associated with PLA activity and is responsible for the transfer of fatty acids derived from a phospholipid to an acceptor molecule, such as cholesterol. These methods applied for specific L. pneumophila enzyme knockout mutants compared to the wild type or for recombinantly expressed protein allow to conclude on substrate specificity and/or contribution of a specific enzyme to the total lipolytic activity. Further, via analysis of separated cellular fractions, such as culture supernatants and cell lysates, information on the localization of the enzymes will be obtained.

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Author information

Authors and Affiliations

  1. Division of Bacterial Infections (FG11), Robert Koch-Institut, Wernigerode, Germany

    Kathleen Seipel

  2. Division of Bacterial Infections (FG11), Robert Koch-Institute, Wernigerode, Germany

    Antje Flieger

Authors
  1. Kathleen Seipel
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  2. Antje Flieger
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Corresponding author

Correspondence to Antje Flieger .

Editor information

Editors and Affiliations

  1. , Biology of Intracellular Bacteria, Institut Pasteur, Rue du Dr. Roux 28, Paris, 75724, France

    Carmen Buchrieser

  2. Max von Pettenkofer Institute, Ludwig-Maximilians-University, Pettenkoferstrasse 9a, Munich, 80336, Germany

    Hubert Hilbi

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Seipel, K., Flieger, A. (2013). Legionella Phospholipases Implicated in Infection: Determination of Enzymatic Activities. In: Buchrieser, C., Hilbi, H. (eds) Legionella. Methods in Molecular Biology, vol 954. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-161-5_22

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  • DOI: https://doi.org/10.1007/978-1-62703-161-5_22

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-160-8

  • Online ISBN: 978-1-62703-161-5

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