The virulence factor PlaB promotes lung colonization, tissue destruction, and intracellular replication of Legionella pneumophila, the causative agent of Legionnaires' disease. It is a highly active phospholipase exposed at the bacterial surface and shows an extraordinary activation mechanism by tetramer deoligomerization. To unravel the molecular basis for enzyme activation and localization, we determined the crystal structure of PlaB in its tetrameric form. We found that the tetramer is a dimer of identical dimers, and a monomer consists of an N-terminal alpha/beta-hydrolase domain expanded by two noncanonical two-stranded beta-sheets, beta-6/beta-7 and beta-9/beta-10. The C-terminal domain reveals a fold displaying a bilobed beta-sandwich with a hook structure required for dimer formation and structural complementation of the enzymatic domain in the neighboring monomer. This highlights the dimer as the active form. deltabeta-9/beta-10 mutants showed a decrease in the tetrameric fraction and altered activity profiles. The variant also revealed restricted binding to membranes resulting in mislocalization and bacterial lysis. Unexpectedly, we observed eight NAD(H) molecules at the dimer/dimer interface, suggesting that these molecules stabilize the tetramer and hence lead to enzyme inactivation. Indeed, addition of NAD(H) increased the fraction of the tetramer and concomitantly reduced activity. Together, these data reveal structural elements and an unprecedented NAD(H)-mediated tetramerization mechanism required for spatial and enzymatic control of a phospholipase virulence factor. The allosteric regulatory process identified here is suited to fine tune PlaB in a way that protects Legionella pneumophila from self-inflicted lysis while ensuring its activity at the pathogen-host interface.
Plasmodium parasites, the causative agents of malaria, display a well-regulated lipid metabolism required to ensure their survival in the human host as well as in the mosquito vector. The fine-tuning of lipid metabolic pathways is particularly important for the parasites during the rapid erythrocytic infection cycles, and thus enzymes involved in lipid metabolic processes represent prime targets for malaria chemotherapeutics. While plasmodial enzymes involved in lipid synthesis and acquisition have been studied in the past, to date not much is known about the roles of phospholipases for proliferation and transmission of the malaria parasite. These phospholipid-hydrolyzing esterases are crucial for membrane dynamics during host cell infection and egress by the parasite as well as for replication and cell signaling, and thus they are considered important virulence factors. In this review, we provide a comprehensive bioinformatic analysis of plasmodial phospholipases identified to date. We further summarize previous findings on the lipid metabolism of Plasmodium, highlight the roles of phospholipases during parasite life-cycle progression, and discuss the plasmodial phospholipases as potential targets for malaria therapy.
Bacterial sphingomyelinases and phospholipases are a heterogeneous group of esterases which are usually surface associated or secreted by a wide variety of Gram-positive and Gram-negative bacteria. These enzymes hydrolyze sphingomyelin and glycerophospholipids, respectively, generating products identical to the ones produced by eukaryotic enzymes which play crucial roles in distinct physiological processes, including membrane dynamics, cellular signaling, migration, growth, and death. Several bacterial sphingomyelinases and phospholipases are essential for virulence of extracellular, facultative, or obligate intracellular pathogens, as these enzymes contribute to phagosomal escape or phagosomal maturation avoidance, favoring tissue colonization, infection establishment and progression, or immune response evasion. This work presents a classification proposal for bacterial sphingomyelinases and phospholipases that considers not only their enzymatic activities but also their structural aspects. An overview of the main physiopathological activities is provided for each enzyme type, as are examples in which inactivation of a sphingomyelinase- or a phospholipase-encoding gene impairs the virulence of a pathogen. The identification of sphingomyelinases and phospholipases important for bacterial pathogenesis and the development of inhibitors for these enzymes could generate candidate vaccines and therapeutic agents, which will diminish the impacts of the associated human and animal diseases.
Legionella pneumophila is a bacterial pathogen that thrives in alveolar macrophages, causing a severe pneumonia. The virulence of L. pneumophila depends on its Dot/Icm type IV secretion system (T4SS), which delivers more than 300 effector proteins into the host, where they rewire cellular signaling to establish a replication-permissive niche, the Legionella-containing vacuole (LCV). Biogenesis of the LCV requires substantial redirection of vesicle trafficking and remodeling of intracellular membranes. In order to achieve this, several T4SS effectors target regulators of membrane trafficking, while others resemble lipases. Here, we characterized LpdA, a phospholipase D effector, which was previously proposed to modulate the lipid composition of the LCV. We found that ectopically expressed LpdA was targeted to the plasma membrane and Rab4- and Rab14-containing vesicles. Subcellular targeting of LpdA required a C-terminal motif, which is posttranslationally modified by S-palmitoylation. Substrate specificity assays showed that LpdA hydrolyzed phosphatidylinositol, -inositol-3- and -4-phosphate, and phosphatidylglycerol to phosphatidic acid (PA) in vitro. In HeLa cells, LpdA generated PA at vesicles and the plasma membrane. Imaging of different phosphatidylinositol phosphate (PIP) and organelle markers revealed that while LpdA did not impact on membrane association of various PIP probes, it triggered fragmentation of the Golgi apparatus. Importantly, although LpdA is translocated inefficiently into cultured cells, an L. pneumophila DeltalpdA mutant displayed reduced replication in murine lungs, suggesting that it is a virulence factor contributing to L. pneumophila infection in vivo.
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.
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: The global regulatory proteins LetA and RpoS control phospholipase A, lysophospholipase A, acyltransferase, and other hydrolytic activities of Legionella pneumophila JR32 Broich M, Rydzewski K, McNealy TL, Marre R, Flieger A Ref: Journal of Bacteriology, 188:1218, 2006 : PubMed
Legionella pneumophila possesses a variety of secreted and cell-associated hydrolytic activities that could be involved in pathogenesis. The activities include phospholipase A, lysophospholipase A, glycerophospholipid:cholesterol acyltransferase, lipase, protease, phosphatase, RNase, and p-nitrophenylphosphorylcholine (p-NPPC) hydrolase. Up to now, there have been no data available on the regulation of the enzymes in L. pneumophila and no data at all concerning the regulation of bacterial phospholipases A. Therefore, we used L. pneumophila mutants in the genes coding for the global regulatory proteins RpoS and LetA to investigate the dependency of hydrolytic activities on a global regulatory network proposed to control important virulence traits in L. pneumophila. Our results show that both L. pneumophila rpoS and letA mutants exhibit on the one hand a dramatic reduction of secreted phospholipase A and glycerophospholipid:cholesterol acyltransferase activities, while on the other hand secreted lysophospholipase A and lipase activities were significantly increased during late logarithmic growth phase. The cell-associated phospholipase A, lysophospholipase A, and p-NPPC hydrolase activities, as well as the secreted protease, phosphatase, and p-NPPC hydrolase activities were significantly decreased in both of the mutant strains. Only cell-associated phosphatase activity was slightly increased. In contrast, RNase activity was not affected. The expression of plaC, coding for a secreted acyltransferase, phospholipase A, and lysophospholipase A, was found to be regulated by LetA and RpoS. In conclusion, our results show that RpoS and LetA affect phospholipase A, lysophospholipase A, acyltransferase, and other hydrolytic activities of L. pneumophila in a similar way, thereby corroborating the existence of the LetA/RpoS regulation cascade.
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.
