title: Oligomerization inhibits Legionella pneumophila PlaB phospholipase A activity.
abstract: The intracellularly replicating lung pathogen Legionella pneumophila consists of an extraordinary variety of phospholipases, including at least 15 different phospholipases A (PLA). Among them, PlaB, the first characterized member of a novel lipase family, is a hemolytic virulence factor that exhibits the most prominent PLA activity in L. pneumophila. We analyzed here protein oligomerization, the importance of oligomerization for activity, addressed further essential regions for activity within the PlaB C terminus, and the significance of PlaB-derived lipolytic activity for L. pneumophila intracellular replication. We determined by means of analytical ultracentrifugation and small angle x-ray scattering analysis that PlaB forms homodimers and homotetramers. The C-terminal 5, 10, or 15 amino acids, although the individual regions contributed to PLA activity, were not essential for protein tetramerization. Infection of mouse macrophages with L. pneumophila wild type, plaB knock-out mutant, and plaB complementing or various mutated plaB-harboring strains showed that catalytic activity of PlaB promotes intracellular replication. We observed that PlaB was most active in the lower nanomolar concentration range but not at or only at a low level at concentration above 0.1 μm where it exists in a dimer/tetramer equilibrium. We therefore conclude that PlaB is a virulence factor that, on the one hand, assembles in inactive tetramers at micromolar concentrations. On the other hand, oligomer dissociation at nanomolar concentrations activates PLA activity. Our data highlight the first example of concentration-dependent phospholipase inactivation by tetramerization, which may protect the bacterium from internal PLA activity, but enzyme dissociation may allow its activation after export.
authors.affiliation: From the Robert Koch-Institut, 38855 Wernigerode.
authors.forename: Katja
authors.initials: K
authors.lastname: Kuhle
authors.affiliation: the Helmholtz Center for Infection Research, 38124 Braunschweig.
authors.forename: Joern
authors.initials: J
authors.lastname: Krausze
authors.affiliation: the Institute for Biophysical Chemistry, Hannover Medical School, 30625 Hannover.
authors.forename: Ute
authors.initials: U
authors.lastname: Curth
authors.affiliation: the European Molecular Biology Laboratory, 22603 Hamburg Branch, c/o DESY, Hamburg, and the Lübeck University of Applied Sciences, 23562 Lübeck, Germany.
authors.forename: Manfred
authors.initials: M
authors.lastname: Rössle
authors.affiliation: From the Robert Koch-Institut, 38855 Wernigerode.
authors.forename: Klaus
authors.initials: K
authors.lastname: Heuner
authors.affiliation: From the Robert Koch-Institut, 38855 Wernigerode.
authors.forename: Christina
authors.initials: C
authors.lastname: Lang
authors.affiliation: From the Robert Koch-Institut, 38855 Wernigerode, fliegera@rki.de.
authors.forename: Antje
authors.initials: A
authors.lastname: Flieger
mesh_terms: D000818:Animals; D055162:Biocatalysis; D002460:Cell Line; D042541:Intracellular Space / Q000382:microbiology; D016952:Legionella pneumophila / Q000201:enzymology*; D008066:Lipolysis; D008264:Macrophages / Q000166:cytology / Q000382:microbiology; D051379:Mice; D008958:Models, Molecular; D010740:Phospholipases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary
chemical_list: D010740:Phospholipases
keywords: Bacteria; Enzyme Purification; Pathogenesis; Phospholipase A; Protein Structure