Volume 954 of the series Methods in Molecular Biology pp 355-365


Legionella Phospholipases Implicated in Infection: Determination of Enzymatic Activities

  • Kathleen SeipelAffiliated withDivision of Bacterial Infections (FG11), Robert Koch-Institut
  • , Antje FliegerAffiliated withDivision of Bacterial Infections (FG11), Robert Koch-Institute Email author 

* Final gross prices may vary according to local VAT.

Get Access


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.

Key words

Legionella Phospholipase A (PLA) Lysophospholipase A (LPLA) Glycerophospholipid:cholesterol acyltransferase (GCAT) Lipid hydrolysis Lipid extraction Thin-layer chromatography