Novel phospholipase A activity secreted by Legionella species - PubMed (original) (raw)

Novel phospholipase A activity secreted by Legionella species

A Flieger et al. J Bacteriol. 2000 Mar.

Abstract

Bacterial phospholipases are regarded as a major virulence factor in infection. In bacteria associated with pneumonia, destruction of lung surfactant and host cell membranes by bacterial phospholipases secreted during infection is thought to contribute to the disease. Phospholipase C (PLC) activity has been described in several Legionella species (W. B. Baine, J. Gen. Microbiol. 134:489-498, 1988; W. B. Baine, J. Gen. Microbiol. 131:1383-1391, 1985). By using detection methods such as thin-layer chromatography and mass spectrometry, PLC activity could not be detected in several strains of Legionella pneumophila. Instead, phospholipid degradation was identified to be caused by a novel PLA activity. We could demonstrate that PLA secretion starts at the mid-exponential-growth phase when bacteria were grown in liquid culture. Several Legionella species secreted different amounts of PLA. Legionella PLA may act as a powerful agent in the mediation of pathogenicity due to destruction of lung surfactant and epithelial cells.

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Figures

FIG. 1

Apolar and polar lipids after incubation of PC with CCCS of L. pneumophila. PC was incubated with the CCCS of different L. pneumophila strains for 16 h. Apolar (A) and polar (B) lipids were characterized by TLC. A representative experiment is shown. Abbreviations: St, standard (1A, 1,2-dipalmitoylglycerol, palmitic acid; 1B, LPC, dipalmitoylphosphatidylcholine); C, negative control (concentrated BYE broth or concentrated −P broth, respectively); P, positive control (PLC from C. perfringens); 1, Lp1-MM6; 2, Lp1-MRC; 3, Lp2-ATCC; 4, Lp6-c; 5, Lp12-ATCC.

FIG. 2

Characterization of AEC fractions. CCCS of Lp6-c was applied onto an AEC column. Fractions eluted by 0.1 to 0.7 M sodium chloride were investigated for protein content, hydrolysis of _p_-NPPC (OD410), and hydrolysis of phosphomonoesters (Pi content). A representative experiment is shown.

FIG. 3

SDS-PAGE of AEC fractions causing hydrolysis of _p_-NPPC. St, molecular weight standard in kilodaltons; 19–21, AEC fractions. The 54-kDa protein band is designated by the arrow. A representative experiment is shown.

FIG. 4

Apolar lipids after incubation of Alveofact with AEC fractions containing the 54-kDa protein. Alveofact was incubated with AEC fractions containing the 54-kDa protein for 16 h. Apolar lipids were characterized by TLC. A representative experiment is shown. Abbreviations: C, negative control (20 mM Tris-HCl, pH 7.2); P, positive control (PLC from C. perfringens); St, standard (1,2-dipalmitoylglycerol, palmitic acid).

FIG. 5

ESI-MS. CCCS of Lp6-c was incubated with Alveofact for 16 h. Lipids were separated from the water-soluble substances by lipid extraction. Water-soluble substances were analyzed by ESI-MS. A representative experiment is shown.

FIG. 6

Kinetics of PLA secretion in different Legionella species. Legionellae were grown in BYE broth. PLA activity was determined after incubation of the culture supernatant with Alveofact for 24 h by detection of FFA release. Data are mean values. For a better survey, standard deviations which were maximally ±0.1 mM were not added to the diagram.

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Novel phospholipase A activity secreted by Legionella species - PubMed (original) (raw)