Abteilung für Transfusionsmedizin, Universitätsklinikum Tübingen, Otfried-Müller-Str. 4/1, D-72076 Tübingen, Germany1
Author for correspondence: Birgid Neumeister. Tel: +49 7071 29 81608. Fax: +49 7071 29 5240. e-mail: birgid.neumeister{at}med.uni-tuebingen.de
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ABSTRACT |
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Keywords: virulence, exotoxins, intracellular bacteria, phospholipase A, lysophospholipase A
Abbreviations: CS, culture supernatant; DPPC, dipalmitoylphosphatidylcholine; DPPG, dipalmitoylphosphatidylglycerol; FFA, free fatty acid; LPC, lysophosphatidylcholine; LPLA, lysophospholipase A; MPLPC, monopalmitoyllysophosphatidylcholine; PLA, phospholipase A; p-NPP, p-nitrophenylphosphate
This work was in part presented as a poster and published as an abstract at the 5th International Conference on Legionella.
a Present address: Department of Microbiology-Immunology, NorthWestern University Medical School, 320 E Superior St, Searle 6-541, Chicago, IL 60611, USA.
b Present address: Department of Microbiology and Immunology, Chandler Medical Center, University of Kentucky, Lexington, KY 40536, USA.
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INTRODUCTION |
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Enzyme activities of L. pneumophila secreted via the type II protein secretion pathway include protease, acid phosphatase, lipase-like activity, nuclease, and both phospholipase A (PLA) and lysophospholipase A (LPLA) activities (Liles et al., 1999 ; Hales & Shuman, 1999
; Aragon et al., 2000
; Flieger et al., 2001
; Rossier & Cianciotto, 2001
).
The well-described zinc metalloproteinase of L. pneumophila has been characterized as one of the virulence factors of the bacterium. It has been shown to be responsible for some of the lethal effects of the bacterium in guinea pigs, but is not essential for entry, survival or growth in Acanthamoeba or macrophages (Moffat et al., 1994 ; Szeto & Shuman, 1990
). The ability of the protease to degrade interleukin-2 and TNF-
, and to cleave CD4, suggests that it may contribute to the pathogenesis of Legionnaires disease by interfering with immune responses (Mintz et al., 1993
; Hell et al., 1993
). Immunogold labelling using anti-protease antibody showed that the enzyme localizes within phagosomes and is distributed throughout the macrophage (Rechnitzer et al., 1992
).
Another enzyme, the major acid phosphatase of L. pneumophila, has been shown to be unnecessary for entry and multiplication in macrophages and Hartmannella vermiformis (Aragon et al., 2001 ). Besides the major acid phosphatase, a minor tartrate-resistant phosphatase has also been found to be secreted by the bacterium, but its importance in virulence has not been evaluated (Aragon et al., 2001
).
Destruction of phospholipids by bacterial phospholipases and the subsequent change of membrane constituents, which can lead to cell damage, are thought to be a major virulence mechanism in infection. Enzymes such as PLA and LPLA hydrolyse phospholipids and have been shown to be secreted by L. pneumophila (Flieger et al., 2000a , 2001
). L. pneumophila cells and their culture supernatants (CSs) can cleave lung surfactant phospholipids and generate free fatty acids (FFAs) and cytotoxic lysophosphatidylcholine (LPC) (Flieger et al., 2000a
, b
).
Despite efforts to characterize possible virulence determinants, it is uncertain why L. pneumophila is the species most frequently isolated from patients suffering from Legionnaires disease. In addition, it has not been established whether some of the secreted enzymes or their levels of activity are unique to L. pneumophila. Therefore, we aimed to characterize the secretion kinetics of putative virulence factors of L. pneumophila and to compare the kinetics to those of non-pneumophila species during late log and early stationary phases of growth, when L. pneumophila switches to a virulent phenotype (Byrne & Swanson, 1998 ) and secretes maximal activities of PLA, protease and acid phosphatase activities (Byrne & Swanson, 1998
; Flieger et al., 2000a
; Aragon et al., 2000
).
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METHODS |
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SDS-PAGE buffer strips, 12·5% homogeneous SDS-PAGE ready gels and silver staining kits for protein were obtained from Amersham Pharmacia Biotech. Low molecular mass standards for SDS-PAGE were purchased from Bio-Rad.
Bacteria.
Legionella strains used for this investigation are listed in Table 1.
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SDS-PAGE.
This was performed under reducing conditions (Laemmli, 1970 ). The molecular masses of the protein bands were determined using a low molecular mass calibration kit from Bio-Rad. Ten microlitre samples of the concentrated and reduced CSs were loaded onto the gels. Proteins were visualized using a silver staining kit for proteins (Amersham Pharmacia Biotech) according to the manufacturers instructions.
Detection of enzyme activities.
Hydrolysis of p-NPP by CSs was detected by incubating 20 mM p-NPP, 6 mM NaN3, 40 mM CaCl2, 1% (v/v) Triton X-100 in 100 mM MES, pH 6·0 and an equal volume of CS at 37 °C with continuous agitation. The absorbance was determined after 2 h at 410 nm.
Hydrolysis of azocasein was detected by incubating 100 µl 2% (w/v) azocasein in 20 mM Tris/HCl, pH 7·2, with 50 µl CS at 37 °C for 1 h with continuous agitation and then the mixture was treated as described by Prestidge et al. (1971) .
PLA and LPLA activities were detected by incubating different phospholipids with CSs in a mixture containing 6 mM lipid substrate (DPPG, DPPC or MPLPC), 3 mM NaN3, 0·5% (v/v) Triton X-100 and 20 mM Tris/HCl, pH 7·2. The lipid substrates were vortexed for 15 min and sonicated three times for 1 min at a power setting of 5 (B12-Sonifier; Branson Sonic Power). All reactions were performed for 5 h at 37 °C with continuous agitation. FFAs were detected using the NEFA-C-Kit (Wako Chemicals), according to the manufacturers instructions. Data were expressed as the difference between the amount of FFA in the samples and in the negative control (BYE broth). Additionally, lipids from incubation of CSs with DPPC were extracted according to Bligh & Dyer (1959) and generation of the reaction product LPC was analysed by TLC.
TLC.
Lipids were separated using the solvent mixture chloroform:methanol:water (65:25:4 by vol.). For visualization of the lipids, silica plates were sprayed with the reagent described by Touchstone et al. (1983) .
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RESULTS |
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Different Legionella species secreted differing amounts of each of the enzyme activities, but different isolates of L. pneumophila resembled each other in their secreted hydrolytic activity. In contrast to its frequent association with disease, apparently L. pneumophila is not the species which generally produced the highest amounts of the tested enzymic activities (Fig. 1).
L. pneumophila and L. gormanii secreted the highest amounts of protease (Fig. 1). L. gormanii, L. dumoffii and L. steigerwaltii secreted the highest amounts of phosphatase (Fig. 1
). L. gormanii, L. steigerwaltii and L. pneumophila exhibited the highest PLA activity and both L. gormanii and one of the L. pneumophila strains had the most prominent LPLA activity (Fig. 1
).
When the timing of secretion of PLA and LPLA was examined it was apparent that PLA activity reached its peak prior to LPLA only in L. pneumophila, whereas non-pneumophila species secreted maximal LPLA activity simultaneously with or prior to PLA activity (Fig. 1). Since PLA generates LPC and LPLA degrades LPC, the ratio and the timing of secretion of both activities may be important for the generation and enrichment of LPC, which has several functions that may contribute to pathogenesis.
Generation of LPC
To estimate the amount of LPC generated from CSs of different Legionella species, we incubated supernatants with DPPC and analysed the lipids by TLC. CSs from the exponential growth phase of both L. pneumophila and L. steigerwaltii generated considerable amounts of LPC (Fig. 2). LPC was also produced by L. gormanii but not before the bacteria entered stationary phase. Probably due to their low PLA activity, L. dumoffii and L. micdadei did not enrich for LPC. The accumulation of LPC by L. steigerwaltii and L. gormanii can be explained by their very high PLA activity and more moderate LPLA activity. LPLA activity is high in comparison to PLA activity in L. pneumophila (see Fig. 1b
). However, L. pneumophila might be able to generate LPC because secretion of maximal PLA precedes secretion of maximal LPLA.
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DISCUSSION |
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L. pneumophila becomes infectious, cytotoxic, sodium sensitive, motile and capable of evading macrophage lysosomes in response to starvation (Byrne & Swanson, 1998 ). In vitro this occurs when bacteria exit the exponential growth phase (Byrne & Swanson, 1998
). We found the highest levels of secreted protease, acid phosphatase, PLA and LPLA activities in several Legionella species as they entered stationary phase, suggesting a possible role for these activities in the switch to a virulent phenotype. In accordance with previous studies, we failed to detect extracellular protease activity in L. longbeachae and L. micdadei (McIntyre et al., 1991
; Berdal, 1983
). As six discrete protease activities have been recovered from CSs of L. pneumophila by ion-exchange chromatography in previous experiments, legionellae might secrete other proteolytic enzymes besides the zinc metalloproteinase (Conlan et al., 1986
).
PLA activity was detected in culture supernatants from both L. pneumophila and a variety of non-pneumophila species, but not in those from L. longbeachae or L. micdadei. This has already been noted for two different isolates of L. micdadei (Flieger et al., 2000a ). However, in previous experiments, PLA activity was found in CSs of another isolate of L. longbeachae which, in contrast to the isolate tested in the present studies, had been passaged less than three times on BCYE
agar (Flieger et al., 2000a
). Attenuation of Legionella species by multiple passages on artificial media (Nagl et al., 2000
) may be related to the reduction of PLA secretion, although differences in PLA secretion by different isolates of L. steigerwaltii have been described and might also occur in other species (Flieger et al., 2000a
).
Previously, it was not known whether non-pneumophila species export acid phosphatase or LPLA activity. Like the other activities examined, their secretion was not restricted to L. pneumophila. Acid phosphatase and LPLA activity were found in all Legionella species tested, except in L. longbeachae and L. micdadei. The absence of the type II secreted activities in L. micdadei suggests that this species does not possess the genes for the enzymes, is not able to express these genes or lacks the secretion apparatus. Since PLA activity has been found in another isolate of L. longbeachae (Flieger et al. 2000a ), the L. longbeachae strain investigated in this study might not express the genes for the secretion system or the secretion system may be inactivated by the artificial growth conditions.
Aside from L. pneumophila, only certain non-pneumophila species (L. steigerwaltii and L. gormanii) exhibiting PLA activity were able to accumulate LPC when their CSs were incubated with DPPC. Enrichment of LPC occurred when PLA activity was high in comparison to LPLA activity and/or when LPLA was secreted in a delayed manner. LPC is known to contribute to induction of inflammation, impairment of lung function, cytotoxicity, pore formation and signal transduction (Aronson & Johns, 1977 ; Dennis, 1997
; Flieger et al., 2000b
; Holm et al., 1991
; Kume et al., 1992
; Lindahl et al., 1986
; Niewoehner et al., 1987
; Prokazova et al., 1998
; Weltzien, 1979
). Accumulation of LPC could therefore trigger important mechanisms in the development of Legionnaires disease.
In the search for reasons for the more frequent association of L. pneumophila with Legionnaires disease compared to non-pneumophila species, another observation has to be considered. In this and another recent study it has been shown that PLA secretion commences during the mid-exponential growth phase and peaks upon entry into stationary phase (Flieger et al., 2000a ). Since L. pneumophila is the only species examined that is known to replicate efficiently in macrophages, the non-pneumophila species investigated might not reach mid-log growth phase in their host (Neumeister et al., 1997
). Therefore it is conceivable that L. steigerwaltii and L. gormanii may not secrete PLA and accordingly may not produce dangerous amounts of LPC in vivo. Thus, future experiments should include the detection of enzyme secretion within the specialized Legionella phagosome.
In conclusion, we have shown that the secreted protease, acid phosphatase, PLA and LPLA activities were neither unique nor extraordinarily high in L. pneumophila. However, a different pattern of secretion of PLA and LPLA was found that was unique to L. pneumophila, and this is likely to be important in the timing of LPC enrichment.
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ACKNOWLEDGEMENTS |
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Antje Flieger was supported by grants of Boehringer-Ingelheim-Pharma-KG (Biberach, Germany), Boehringer-Ingelheim-Foundation (Mainz, Germany) and fortuene Fund (179-1 and 179-2, University Hospital of Tübingen, Germany). Shimei Gong was supported by fortuene Fund (305-1 and 305-2, University Hospital of Tübingen, Germany).
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Received 23 February 2001;
revised 31 May 2001;
accepted 6 July 2001.