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 mum 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.
Legionella oakridgensis is able to cause Legionnaires' disease, but is less virulent compared to L. pneumophila strains and very rarely associated with human disease. L. oakridgensis is the only species of the family legionellae which is able to grow on media without additional cysteine. In contrast to earlier publications, we found that L. oakridgensis is able to multiply in amoebae. We sequenced the genome of L. oakridgensis type strain OR-10 (ATCC 33761). The genome is smaller than the other yet sequenced Legionella genomes and has a higher G+C-content of 40.9%. L. oakridgensis lacks a flagellum and it also lacks all genes of the flagellar regulon except of the alternative sigma-28 factor FliA and the anti-sigma-28 factor FlgM. Genes encoding structural components of type I, type II, type IV Lvh and type IV Dot/Icm, Sec- and Tat-secretion systems could be identified. Only a limited set of Dot/Icm effector proteins have been recognized within the genome sequence of L. oakridgensis. Like in L. pneumophila strains, various proteins with eukaryotic motifs and eukaryote-like proteins were detected. We could demonstrate that the Dot/Icm system is essential for intracellular replication of L. oakridgensis. Furthermore, we identified new putative virulence factors of Legionella.
Title: Phospholipase PlaB of Legionella pneumophila represents a novel lipase family: protein residues essential for lipolytic activity, substrate specificity, and hemolysis Bender J, Rydzewski K, Broich M, Schunder E, Heuner K, Flieger A Ref: Journal of Biological Chemistry, 284:27185, 2009 : PubMed
Legionella pneumophila possesses several phospholipases capable of host cell manipulation and lung damage. Recently, we discovered that the major cell-associated hemolytic phospholipase A (PlaB) shares no homology to described phospholipases and is dispensable for intracellular replication in vitro. Nevertheless, here we show that PlaB is the major lipolytic activity in L. pneumophila cell infections and that PlaB utilizes a typical catalytic triad of Ser-Asp-His for effective hydrolysis of phospholipid substrates. Crucial residues were found to be located within the N-terminal half of the protein, and amino acids embedding these active sites were unique for PlaB and homologs. We further showed that catalytic activity toward phosphatidylcholine but not phosphatidylglycerol is directly linked to hemolytic potential of PlaB. Although the function of the prolonged PlaB C terminus remains to be elucidated, it is essential for lipolysis, since the removal of 15 amino acids already abolishes enzyme activity. Additionally, we determined that PlaB preferentially hydrolyzes long-chain fatty acid substrates containing 12 or more carbon atoms. Since phospholipases play an important role as bacterial virulence factors, we examined cell-associated enzymatic activities among L. pneumophila clinical isolates and non-pneumophila species. All tested clinical isolates showed comparable activities, whereas of the non-pneumophila species, only Legionella gormanii and Legionella spiritensis possessed lipolytic activities similar to those of L. pneumophila and comprised plaB-like genes. Interestingly, phosphatidylcholine-specific phospholipase A activity and hemolytic potential were more pronounced in L. pneumophila. Therefore, hydrolysis of the eukaryotic membrane constituent phosphatidylcholine triggered by PlaB could be an important virulence tool for Legionella pathogenicity.
Title: Cloning and characterization of the gene encoding the major cell-associated phospholipase A of Legionella pneumophila, plaB, exhibiting hemolytic activity Flieger A, Rydzewski K, Banerji S, Broich M, Heuner K Ref: Infect Immun, 72:2648, 2004 : PubMed
Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular pathogen of amoebae, macrophages, and epithelial cells. The pathology of Legionella infections involves alveolar cell destruction, and several proteins of L. pneumophila are known to contribute to this ability. By screening a genomic library of L. pneumophila, we found an additional L. pneumophila gene, plaB, which coded for a hemolytic activity and contained a lipase consensus motif in its deduced protein sequence. Moreover, Escherichia coli harboring the L. pneumophila plaB gene showed increased activity in releasing fatty acids predominantly from diacylphospho- and lysophospholipids, demonstrating that it encodes a phospholipase A. It has been reported that culture supernatants and cell lysates of L. pneumophila possess phospholipase A activity; however, only the major secreted lysophospholipase A PlaA has been investigated on the molecular level. We therefore generated isogenic L. pneumophila plaB mutants and tested those for hemolysis, lipolytic activities, and intracellular survival in amoebae and macrophages. Compared to wild-type L. pneumophila, the plaB mutant showed reduced hemolysis of human red blood cells and almost completely lost its cell-associated lipolytic activity. We conclude that L. pneumophila plaB is the gene encoding the major cell-associated phospholipase A, possibly contributing to bacterial cytotoxicity due to its hemolytic activity. On the other hand, in view of the fact that the plaB mutant multiplied like the wild type both in U937 macrophages and in Acanthamoeba castellanii amoebae, plaB is not essential for intracellular survival of the pathogen.
