Cloning and characterization of the gene encoding the major cell-associated phospholipase A of Legionella pneumophila, plaB, exhibiting hemolytic activity

Abstract

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.

FIG. 1.

plaB locus in L. pneumophila and recombinant E. coli. (Top) Diagram represents the L. pneumophila chromosome region that contains the phospholipase A gene plaB, along with the locations of the relevant restriction enzyme sites (B, BamHI; H, HindIII; Ps, PstI; S, Sau3A1; ScI, SacI; ScII, SacII). (Center) Horizontal arrows depict the relative location, size, and orientation of plaB and neighboring ORFs. (Bottom) Lines represent the segments of Legionella DNA that were cloned into plasmid vectors. Plasmids pKH194 and pKH195 contained a Km^r gene cassette. Plus and minus signs indicate whether recombinant E. coli exhibited hemolytic activity on human red blood agar plates.

FIG. 2.

Hemolysis of human red blood cells by recombinant E. coli containing L. pneumophila plaB. (A) Human blood agar was inoculated with recombinant E. coli harboring different plasmids and then incubated for 48 h at 37°C. Plasmids contained the following inserts: pKH190 and pKH192, intact plaB; pKH194, disrupted plaB; pUC19, empty vector; phlyCABD, hemolysin-encoding operon of E. coli 536 in pUC18 (compare Fig. 1). (B) Recombinant E. coli containing either plain pBCKS+ or pBCKS+ with L. pneumophila plaB (pKH192) was grown to the logarithmic-growth phase and subsequently treated with IPTG. Afterwards, human red blood cells were incubated with the bacteria for 20 h and then quantitatively assessed for hemolysis. Results are means and standard deviations from triplicate cultures and are representative of three independent experiments.

FIG. 3.

Lipolytic activities of recombinant E. coli containing L. pneumophila plaB. Culture supernatants (A) and cell lysates (B) of E. coli containing pBCKS or its derivative pKH192, or pUC18 or its derivative pKH190 or pKH194, were mixed with DPPG, DPPC, MPLPG, MPLPC, or 1-MPG. In some cases, culture supernatants or cell lysates were diluted prior to incubation with lipids as indicated. After a 2-h (A) or 50-min (B) incubation at 37°C, the release of FFA was quantified. Data are expressed as differences between the amount of FFA released by the culture supernatant or cell lysate and the amount released by uninoculated LB broth or Tris-HCl buffer, respectively. Results are means and standard deviations of duplicate cultures and are representative of three independent experiments.

FIG. 4.

Lipolytic activities of wild type, plaB mutant, and genetically complemented L. pneumophila strains. Culture supernatants (A) and cell lysates (B) of wild type, plaB mutant, and genetically complemented L. pneumophila strains were incubated with DPPG, DPPC, MPLPG, MPLPC, or 1-MPG for 2 h (A) or 30 min (B) at 37°C, and then the release of FFA was quantified. In some cases, culture supernatants or cell lysates were diluted with lipids as indicated prior to incubation. Data are expressed as differences between the amount of FFA released by the culture supernatant or cell lysate and the amount released by uninoculated BYE broth or Tris-HCl buffer, respectively. Results are means and standard deviations from duplicate cultures and are representative of three independent experiments.

FIG. 5.

TLC analysis of lipid hydrolysis by cell lysates of wild-type, plaB mutant, and genetically complemented L. pneumophila strains. Cell lysates of wild-type L. pneumophila (L), plaB mutants plaB60 (6) and plaB1 (1), or genetically complemented L. pneumophila (wild type and plaB60 mutant harboring the empty vector [L_E and 6_E, respectively] and wild type and plaB60 mutant harboring pKH192, containing intact plaB [L_L and 6_L, respectively]) were incubated with DPPC (PC) or DPPG (PG) (A and B) or with 1-MPG (MG), 1,2-DG (DG), or TG (C) for 24 h at 37°C, and then lipids were extracted and subjected to TLC. A polar or apolar solvent mixture was used for the separation of the polar (A) or apolar (B and C) lipids, respectively. A mixture of Tris-HCl buffer and the lipids was also incubated and served as a negative control (N). Furthermore, bacterial-cell lysates not incubated with any lipid were extracted and served as the bacterial lipid background control (designated by dotted lines). In all incubations, samples were examined for degradation of the lipid substrate (for DPPC and DPPG, polar TLC [A]; for 1-MPG, 1,2-DG, and TG, apolar TLC [C]), enrichment of FFA (apolar TLC [B and C]), and in the case of DPPC and DPPG, incubations for enrichment of the respective lysophospholipid (polar TLC [A]). For qualitative identification of the lipid spots, lanes containing lipid standards (St) were included. The observations depicted here were made on one more occasion.

FIG. 6.

Hemolysis of human red blood cells by recombinant wild-type, plaB mutant, and genetically complemented L. pneumophila strains. Wild-type (Corby), plaB mutant (plaB60), and genetically complemented L. pneumophila strains were grown for 24 h at 37°C on BCYE agar plates. Afterward, the bacteria were added to human red blood cells, centrifuged onto the cells or not, incubated for 7 h at 37°C, and then quantitatively assessed for hemolysis. SDS served as a positive control, which was able to lyse 100% of the red blood cells. As a negative control, PBS was added to the cells instead of bacteria. Results are means and standard deviations from triplicate cultures and are representative of three independent experiments.

FIG. 7.

Intracellular infection by wild-type and plaB mutant L. pneumophila. Strains Corby and plaB1 were used to infect monolayers of A. castellanii amoebae (A) or cultures of U937 macrophages (B) at a multiplicity of infection of 0.01 or 1, respectively. At various time points postinoculation, bacteria were quantitated by plating aliquots on BCYE agar. Results are means and standard deviations from triplicate samples and are representative of three independent experiments.