Department of Microbiology-Immunology, Northwestern University Medical School, 320 East Superior Street, Chicago, IL 60611-3010, USA1
Author for correspondence: Nick Cianciotto. Tel: +1 312 503 0385. Fax: +1 312 503 1339. e-mail: n-cianciotto{at}northwestern.edu
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ABSTRACT |
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Keywords: Legionaires disease, type II secretion, lipolytic enzymes, macrophages, intracellular bacteria
Abbreviations: 1,2-DG, 1,2-dipalmitoylglycerol; FFA, free fatty acid; 1-MG, 1-monopalmitoylglycerol; PLC, phospholipase C; PNP, p-nitrophenol; pNPPC, p-nitrophenylphosphorylcholine
a The GenBank accession numbers for the L. pneumophila lipA, lipB and plcA sequences are AF454863, AF454864 and AF454865, respectively.
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INTRODUCTION |
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Mutational analysis of the L. pneumophila lsp genes has determined that the Legionella type II protein secretion system is required for intracellular infection (Hales & Shuman, 1999 ; Polesky et al., 2001
; Rossier & Cianciotto, 2001
). Although necessary for optimal infection of human macrophages, the L. pneumophila type II system appears to be most critical for growth within protozoa (Rossier & Cianciotto, 2001
). One of six protein secretion systems that can occur in Gram-negative bacteria, type II secretion is a two-step process (Koster et al., 2000
; Lee & Schneewind, 2001
; Sandkvist, 2001a
; Thanassi & Hultgren, 2000
). In the first step, the proteins destined for export are transported across the inner membrane and into the periplasmic space via the general secretory (Sec) pathway, and then, in the second step, the proteins translocate the outer membrane by interacting with the multiprotein type II secretion apparatus (Nunn, 1999
; Russel, 1998
; Sandkvist, 2001a
). Type II secretion is dependent upon the PilD prepilin peptidase, an inner membrane enzyme that processes, among other things, pseudopilins which help form the type II apparatus (Liles et al., 1998
; Nunn, 1999
). In L. pneumophila, the type II- and PilD-dependent enzymic activities include a zinc metalloprotease, phospholipase A, lysophospholipase A, lipase, p-nitrophenylphosphorylcholine (pNPPC) hydrolase, and tartrate-sensitive and tartrate-resistant acid phosphatase (Aragon et al., 2000
, 2001
; Flieger et al., 2001
; Hales & Shuman, 1999
; Liles et al., 1999
; Rossier & Cianciotto, 2001
). Since the L. pneumophila system is the only type II secretion pathway that has been implicated in intracellular infection (Lee & Schneewind, 2001
; Rossier & Cianciotto, 2001
; Sandkvist, 2001b
), identifying the biological significance of each of the Legionella type II exoenzymes is an important goal. Thus far, mutational analysis has determined that the L. pneumophila metalloprotease and tartrate-sensitive acid phosphatase are not required for intracellular infection (Aragon et al., 2001
; Moffat et al., 1994
; Szeto & Shuman, 1990
).
For three reasons, we chose the lipase and pNPPC hydrolase activities for further study. First, bacterial lipases have been implicated in a variety of pathogenic processes, including the inhibition of phagocyte function (Gribbon et al., 1993 ; Jaeger et al., 1994
; Konig et al., 1996
; Pratt et al., 2000
; Rollof et al., 1988
). Second, pNPPC hydrolysis has been associated with the production of phospholipase C (PLC) enzymes in L. pneumophila and others, and in some of those other bacteria, the PLC is implicated in virulence (Baine, 1988
; Dowling et al., 1992
; Filloux et al., 1987
; Jepson et al., 1999
; Merino et al., 1999
; Schmiel & Miller, 1999
; Songer, 1997
; Strom et al., 1991
; Swanson & Hammer, 2000
; Terada et al., 1999
; Titball, 1998
; Weingart & Hooke, 1999
). Third, lipase- and PLC-like activities are conserved among clinical isolates of L. pneumophila as well as other pathogenic Legionella species (Baine, 1985
; Muller, 1981
; Nolte et al., 1982
; Thorpe & Miller, 1981
). Here, we demonstrate that L. pneumophila supernatants have activity against mono, di- and triacylglycerols, and that these lipase activities require at least two genes (lipA, lipB), whose predicted products share structural characteristics with known lipases. Furthermore, we identify a L. pneumophila gene (plcA), whose predicted product catalyses pNPPC hydrolysis and is homologous with a Pseudomonas fluorescens PLC. Mutants defective for lipA, lipB or plcA were examined for their relative ability to infect protozoan and macrophage hosts.
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METHODS |
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Lipase and phospholipase assays.
To test for the presence of secreted enzymes, supernatants from L. pneumophila BYE cultures were prepared as before (Aragon et al., 2000 ). Briefly, supernatants were obtained by centrifugation followed by filtration, and then, in some cases, concentrated 100-fold by passage through a YM10 ultrafiltration cell (Millipore). For the detection of lipase activity, samples were tested for their ability to release free fatty acid (FFA) from 1-monopalmitoylglycerol (1-MG), 1,2-dipalmitoylglycerol (1,2-DG), tributyrin, tricaprylin, tripalmitin, triolein or lard oil (Aragon et al., 2000
). Thus, supernatants were incubated for up to 1 h at 37 °C in 20 mM Tris/HCl (pH 7·2) containing 3 mM sodium azide, 0·5% Triton X-100 and either 2 mg 1-MG ml-1, 1·6 mg 1,2-DG or tripalmitin ml-1, or 0·5% tributyrin, tricaprylin, triolein or lard oil. After this incubation, FFA levels were determined by the NEFA-C-Kit obtained from Wako Chemicals (Aragon et al., 2000
; Flieger et al., 2001
; Hoffmann et al., 1986
; Rossier & Cianciotto, 2001
). The release of FFA was also confirmed by TLC, as previously described (Aragon et al., 2000
). Esterase-lipase activity was assayed by monitoring the ability of supernatant samples to release p-nitrophenol (pNP) from p-nitrophenyl caprylate and p-nitrophenyl palmitate (Anguita et al., 1993
; Aragon et al., 2000
; El Khattabi et al., 1999
; Filloux et al., 1987
; Jaeger et al., 1999
; Rossier & Cianciotto, 2001
; Thorpe & Miller, 1981
). As before, 100 µl sample was added to 1 ml buffer (i.e. 100 mM Tris/HCl, pH 8, 0·2% Triton X-100) containing 1 mM substrate, and the increase in absorbance at 410 nm was measured after incubation at 37 °C. For the present study, we confirmed that the wild-type reaction was optimal at pH 7·58·0, with no activity detectable below pH 6·5, and was not inhibited by the addition of 50 mM EDTA, a chelator of divalent cations such as calcium (data not shown). One unit of enzyme activity was defined as that which yields 1 nmol pNP in 1 min. PLC activity was assayed as the ability of samples to release pNP from pNPPC (Baine, 1988
; Filloux et al., 1987
; Kurioka & Matsuda, 1976
; Strom et al., 1991
). Briefly, 100 µl supernatant was added to 1 ml 50 mM HEPES (pH 7·5) buffer containing 5 mM CaCl2, 5 mM MnCl2, 3 mM sodium azide, 0·5% Triton X-100 and 2·5 mM pNPPC, and then after overnight incubation at 37 °C, the amount of pNP was recorded as above. The PLC of Bacillus cereus served as a control in the pNPPC hydrolysis tests, with one unit of enzyme activity being defined as that which yields 1 nmol pNP in 1 min. Unless noted otherwise, all enzyme substrates and standards were obtained from Sigma. To minimize experimental variation, the supernatants, whether used for lipase or phospholipase assays, were always derived from cultures that had grown to a similar OD660
PCR and sequencing analysis.
L. pneumophila DNA was extracted as previously described (Aragon et al., 2001 ). Based on data from the L. pneumophila Philadelphia I genome (http://genome3.cpmc.columbia.edu/
legion/), three pairs of DNA primers were designed for the amplification of genes from 130b genomic DNA. The pair consisting of 5'-CAACAGGCTACCGCTAACTT-3' and 5'-CAAGCCGTGATGGTATGTCT-3' amplified a 2·8 kb fragment containing lipA. Primers 5'-GCATGAACTGGATGTGGTGT-3' and 5'-CTCTCCTGAAGAAGATGTCG-3' generated a 1·7 kb fragment containing lipB. Primers 5'-CAGGACAGCATCACCATCTT-3' and 5'-GACTTCTGTCACTGGTCTTG-3' yielded a 3·1 kb plcA-containing fragment. Sequencing reactions were performed using at least two different PCR amplicons per gene, a series of custom primers and the BigDye terminator cycle-sequencing mix from PE Applied Biosystems. Automated sequence analysis was performed on an ABI Prism 373 DNA sequencer (Applied Biosystems) at the Biotech Facility at Northwestern University Medical School, Chicago, IL. Primers were obtained from Integrated DNA Technologies. Sequence database searches were performed using programs based on the BLAST algorithm (Altschul et al., 1997
). The predicted protein was analysed with SignalP program (Nielsen et al., 1997
) and Psort (Nakai & Kanehisa, 1991
) for a signal sequence, and for protein motifs with the PROSITE database (Hofmann et al., 1999
). Protein alignments were done using the CLUSTAL method (Higgins & Sharp, 1988
).
Gene cloning and Legionella mutant constructions.
To facilitate mutant construction, the PCR fragments were ligated into the pGem-T Easy vector (Promega), yielding pVA14 for the cloned lipA, pVA15 for the cloned lipB, and pVA16 for the cloned plcA. The lipA mutants were generated by allelic exchange, a process that proceeded in three steps. First, a kanamycin-resistance gene cassette, obtained from pVK3 (Viswanathan et al., 2000 ), was cloned into the BglII site of pVA14, yielding pVA14-1. Then, a BstZI fragment containing the insertionally inactivated lipA was released from pVA14-1 and ligated into NotI-digested pBOC20, producing pVA14-2. The pBOC20 vector facilitates allelic exchange in Legionella by virtue of its counterselectable sacB gene (Cianciotto et al., 1988
; OConnell et al., 1995
). Following electroporation of pVA14-2 into competent 130b cells (Cianciotto & Fields, 1992
), mutants were selected based upon their kanamycin and sucrose resistance. The construction of the lipB mutant followed a similar progression. After a gentamicin-resistance gene cassette, obtained from pBBR1MCS-5 (Kovach et al., 1995
), was inserted into the XcmI site within pVA15, the insertionally inactivated lipB was transferred, on a NotISalI fragment into NotI/SalI-digested pBOC20. Following electroporation or transformation of the resulting pVA15-2 into strain 130b (Cianciotto & Fields, 1992
; Stone & Abu Kwaik, 1999
), mutants were obtained based upon their resistance to gentamicin and sucrose. To obtain a single strain deficient in both lipA and lipB, pVA15-2 was also introduced into one of the lipA mutants, and the resulting allelic exchange event yielded a kanamycin-, gentamicin-, sucrose-resistant double mutant. The plcA mutant was obtained as follows. pVA16 was digested with EcoRV and then religated, producing pVA16-1, which contains a 310 bp deletion in the cloned plcA. Next, the kanamycin resistance cassette from pVK3 was inserted into
plcA, and then the mutated, tagged gene was transferred, on a BstZI fragment, into NotI-digested pBOC20. Following electroporation of the resulting pVA16-3 into strain 130b, mutants were obtained based upon their resistance to kanamycin and sucrose. Verification of all of the mutant genotypes was carried out by PCR and Southern hybridization (Robey et al., 2001
).
Intracellular infection of U937 cells and Hartmannella amoebae.
U937, a human cell line that differentiates into macrophage-like cells after treatment with phorbol esters, served as a host for in vitro infection by L. pneumophila (Cianciotto et al., 1989b ). The cell line was infected as previously described (Aragon et al., 2000
, 2001
; Liles et al., 1999
; Rossier & Cianciotto, 2001
). To quantitate intracellular growth, monolayers containing 106 macrophages were inoculated with approximately 105 c.f.u., incubated for 0, 24, 48 or 72 h and then lysed. Serial dilutions of the lysates were plated on BCYE agar, supplemented with kanamycin or gentamicin for the mutants, to determine the numbers of bacteria per monolayer. To establish the cytopathic effect of L. pneumophila on U937 cells, the viability of infected monolayers was tested by their ability to reduce alamar blue (Aragon et al., 2001
). To examine the ability of legionellae to grow within a protozoan host, Hartmannella vermiformis was infected as previously indicated (Aragon et al., 2000
, 2001
; Cianciotto & Fields, 1992
; Liles et al., 1999
). Thus, approximately 105 c.f.u. were added to wells containing 105 amoebae and then, at 0, 24, 48 or 72 h post-inoculation, the numbers of bacteria within the co-culture were determined by plating serial dilutions on BCYE agar.
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RESULTS AND DISCUSSION |
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Concentrated culture supernatants of NU262 had a reduced ability to release FFA from 1-MG and tricaprylin (Table 1), as well as tripalmitin, triolein and lard oil (data not shown), further indicating that lipA encodes a secreted lipolytic enzyme. Although not defective for the cleavage of the monoacylglycerol substrate, NU265 was modestly impaired for FFA release from the triacylglycerol substrate (Table 1
), suggesting that lipB also encodes a Legionella lipase-like enzyme. Neither the lipA nor the lipB mutant was significantly defective for FFA release from 1,2-DG (Table 1
), suggesting that lipase(s) other than LipA and LipB are largely responsible for the cleavage of diacylglycerol substrates. The ability to release pNP from p-nitrophenyl caprylate and p-nitrophenyl palmitate has often been associated with lipase activities, including those of L. pneumophila (Aragon et al., 2000
; Thorpe & Miller, 1981
). NU262 culture supernatants were lacking in their ability to cleave p-nitrophenyl caprylate and p-nitrophenyl palmitate, whereas the lipB mutant appeared to be unaltered for these activities (Table 2
). Taken together, the relationship between LipA and LipB sequences and those of known lipases and the altered lipolytic activity of the mutants indicate that lipA and lipB are lipase genes of L. pneumophila. Since the lipA mutant was defective in assays that utilized unconcentrated supernatants, we strongly suspect that LipA is a bona fide type II exoenzyme. In contrast, because the lipB mutant was only impaired in an assay that utilized concentrated supernatants, LipB is either an exoenzyme that is exported in low amounts or is a periplasmic enzyme that is released into supernatants upon cell lysis.
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To determine the relevance of plcA for Legionella intracellular growth, we examined the relative ability of NU268 to multiply within Hartmannella co-cultures and U937 cell monolayers. The plcA mutant did not show evidence of a replication defect in either amoebae (Fig. 2b) or macrophages (Fig. 3d
). Furthermore, it exhibited a cytopathic effect in U937 cells that was comparable to that of its wild-type parent (data not shown). Taken together, these data indicate that plcA, though associated with a PLC-like activity, is not required for L. pneumophila intracellular infection. This observation does not, however, rule out a possible role for Legionella phospholipase C enzymes in infection of macrophages and/or protozoa. Indeed, recent examination of the L. pneumophila genomic database suggests the presence of additional PLC genes (data not shown); a situation that is compatible with the residual pNPPC hydrolysis exhibited by the plcA mutant. Thus, our further exploration into the question of Legionella PLC enzymes will initially be targeted toward the characterization of other candidate phospholipase C genes.
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ACKNOWLEDGEMENTS |
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Received 5 December 2001;
revised 20 March 2002;
accepted 21 March 2002.