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 diacylphosphoand 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.Legionella pneumophila is an inhabitant of fresh water, where it intracellularly colonizes protozoa (20). When bacteria-laden aerosols are inhaled by humans, L. pneumophila exploits alveolar macrophages and epithelial cells for its multiplication, leading to a severe pneumonia characterized by destruction of alveolar cells (60). The cytopathology of Legionnaires' disease involves several cytotoxic or hemolytic factors produced by L. pneumophila, for example, the zinc metalloprotease ProA, the legiolysin Lly, and several pore-forming toxins, one of which is an RTX (repeats in structural toxin) protein (12,26,31,35,36,47,61). The zinc metalloprotease ProA is the major extracellular protease of L. pneumophila and its export depends on the L. pneumophila type II protein secretion system (28, 37). The enzyme hydrolyzes a broad spectrum of protein substrates and confers hemolytic as well as cytolytic activities (47, 53). Additionally, ProA has been shown to contribute to bacterial pathogenesis in a guinea pig model of pneumonia (38). Another hemolytic, but not cytotoxic, protein is the L. pneumophila legiolysin Lly, which is also responsible for color production and fluorescence of the bacterium (61). Pore-forming activities of L. pneumophila confer contact-dependent hemolytic and cytotoxic activities toward a variety of cells, especially at high bacterial nu...

Legionella oakridgensis ATCC 33761 genome sequence and phenotypic characterization reveals its replication capacity in amoebae

“…Transformation and infection of U937 cells was done as previously described with modifications (Liles et al, 1999, Cianciotto et al, 1992, Flieger et al, 2004. For differentiation into macrophage-like cells, U937 cells were adjusted to 3 x 10 5 cells/ml and transferred in 100 ml fresh RPMI medium containing 10% fetal calf serum (10% FCS) and PMA (phorbol-12-myristate-13-acetate, Stock 1 mg/ml in dH 2 O [P-8139; Sigma-Aldrich])…”

Phospholipase PlaB of Legionella pneumophila Represents a Novel Lipase Family

“…Bacterial Strains and Growth Conditions-L. pneumophila, environmental and clinical isolates, and different E. coli strains used in this study are listed in Table 1 and were grown as described previously (9,18).…”

“…Accordingly, activity of PlaB fits into the picture of Legionnaires' disease, the severe pneumonia caused by L. pneumophila bacteria, showing loss of a functional epithelial cell layer in the lung as well as degradation of lung macrophages in infected individuals (10,11). Now it is indeed clear that PlaB contributes to pathogenesis in a guinea pig infection model (1), 3 although it is dispensable for intracellular replication in macrophage and amoeba host cells (1,9). 3 This clearly underlines the importance of PlaB as a virulence factor of L. pneumophila; however, its mechanism of action has not yet been studied in detail.…”

“…We recently identified PlaB, the major cell-associated PLA/LPLA of Legionella pneumophila, to be hemolytic and thereby a membrane-destroying enzyme (9). Accordingly, activity of PlaB fits into the picture of Legionnaires' disease, the severe pneumonia caused by L. pneumophila bacteria, showing loss of a functional epithelial cell layer in the lung as well as degradation of lung macrophages in infected individuals (10,11).…”

“…PlaB was initially detected by screening an L. pneumophila genomic library in Escherichia coli, thus yielding a clone with remarkable hemolytic activity on human red blood agar (9). Subsequently, plaB insertion mutagenesis revealed that the gene encodes the most prominent cell-associated PLA/LPLA activity, being ϳ100-fold more active than secreted phospholipolytic activities of L. pneumophila.…”