Phospholipase PlaB of Legionella pneumophila represents a novel lipase family: protein residues essential for lipolytic activity, substrate specificity, and hemolysis

Abstract

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.

FIGURE 1.

Phospholipase A and lysophospholipase A activities upon intracellular infection of macrophages by L. pneumophila wild-type, plaB mutant and complementing strain. Wild-type Corby, mutant plaB1, and complementing strain plaB1 (pKH192) were used to infect monolayers of U937 cells, and PLA activity toward DPPG (A) and LPLA activity toward MPLPC (B) were determined for cell lysates at various time points postinoculation and after an additional 3 h of lipid hydrolysis. As a control, PLA and LPLA activities were monitored from uninfected U937 cells (U937).

FIGURE 2.

Enzymatic activities of PlaB and its site-directed mutant derivatives and distribution of crucial amino acids within the PlaB protein of L. pneumophila and homologs. A, mutagenesis of several amino acids severely influences PlaB activity. Values are expressed as percentages of wild-type PlaB activity. B, PlaB can be divided into an N-terminal region (amino acids 1 to ∼300) containing the catalytic triad of Ser-85/Asp-203/His-251 and further amino acids important for hydrolytic activity and a C-terminal domain with unknown function (amino acids ∼300–474). C, sequence alignment of L. pneumophila PlaB to potential phospholipases or hypothetical proteins of several bacterial species with an expect value of <0.01. Amino acids comprising the catalytic triad are marked with an asterisk and are highlighted by a gray background; further amino acids important for L. pneumophila PlaB activity are labeled with black squares (■). The last digits indicate the number of amino acids representing the residual C-terminal domain and are marked with a triangle. The consensus sequence is based on the amino acids most frequently found at this position. L.pneu, L. pneumophila; L.spir, L. spiritensis; P.ing, Psychromonas ingrahamii; D.acet, Desulfuromonas acetoxidans; S.peal, Shewanella pealeana; M.algi, Marinobacter algicola; P.aeru, P. aeruginosa PA7; P.mari, Persephonella marina. EV, empty vector pBCKS.

FIGURE 3.

Contact-dependent hemolysis of human red blood cells upon incubation of E. coli strains expressing mutated variants of PlaB. Bacteria, obtained from 1-day-old agar plates, were set to an equal A₆₆₀ before added to human erythrocytes. Cell lysis was determined by measuring absorbance after 18 h of incubation at 37 °C. EV, empty vector pBCKS.

FIGURE 4.

Acyl chain length selectivity of wild-type L. pneumophila PlaB. Cell lysates of Legionella pneumophila strain Corby wild-type (empty vector pBCKS), plaB1 (empty vector pBCKS) mutant, or the complementing strain plaB1 (pJB04), diluted prior to incubation as indicated, were exposed to various lipids ranging from 8 (1-monooctanoyllysophosphatidylcholine) to 12 (1-monolauroyllysophosphatidylcholine) to 16 (MPLPC) carbon atoms in chain length, and the release of fatty acids was quantified. As an internal control, DPPG, DPPC, and MPLPG were carried along under equal conditions. EV, empty vector pBCKS.

FIGURE 5.

Cell-associated PLA activity within clinical isolates of L. pneumophila and PlaB-like enzyme properties of non-pneumophila Legionellae. A, cell lysates of various clinical isolates (Table 1) were incubated with PLA (DPPG and DPPC) and LPLA (MPLPG and MPLPC) substrates. Nine clinical isolates were compared with L. pneumophila Corby and with the water conduit strain Legionella 257. B, determination of released fatty acids by L. pneumophila 130b and 12 different non-pneumophila strains. Cells were grown to an A₆₆₀ of 1.5, pelleted, lysed, and added to different lipids, followed by quantification of released fatty acids. C, cell lysates of E. coli DH5α expressing either L. pneumophila or L. spiritensis plaB were incubated with different lipid substrates, and released fatty acids were quantified. DPPC hydrolysis of L. spiritensis PlaB was significantly decreased (p < 0.05) compared with L. pneumophila Corby PlaB. D, lysis of human red blood cells by E. coli strains as mentioned in C was measured at 415 nm after the bacteria-cell suspension was incubated for 4 h at 37 °C. EV, empty vector pBCKS.