SGM Journals
Research Paper

Legionella pneumophila genes that encode lipase and phospholipase C activities

Virginia Aragon, Ombeline Rossier, Nicholas P. Cianciotto

  • 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

    • Microbiology 2002; 148(7):2223–2231

    PubMed

    Abstract

    Legionella pneumophila, the agent of Legionnaires’ disease, is an intracellular parasite of aquatic protozoans and human macrophages. The type II protein secretion system of the Gram-negative Legionella organism promotes intracellular infection. A lipase activity and a p-nitrophenylphosphorylcholine (pNPPC) hydrolytic activity are two of the factors that are diminished in L. pneumophila type II secretion mutants. The Legionella lipase activity was found to include free fatty acid release from di- and triacylglycerol substrates, in addition to the previously reported cleavage of monoacylglycerol. In a number of other bacterial systems, the release of p-nitrophenol from pNPPC is due to a phospholipase C. In an attempt to identify exoproteins that potentiate intracellular infection, three genes were identified and mutated in L. pneumophila strain 130b that were predicted to encode either a secreted lipase or a phospholipase C. The first two genes, which were designated lipA and lipB, encoded proteins containing the lipase consensus sequence [LIV]-X-[LIVFY]-[LIVMST]-G-[HYWV]-S-X-G-[GSTAC]. Mutations in lipA in particular reduced supernatant activity against mono- and triacylglycerols. However, loss of lipA and/or lipB did not impair the ability of L. pneumophila to infect Hartmannella amoebae or U937 cell macrophages. The third L. pneumophila gene, which was denoted plcA, encoded a protein that was highly homologous with a phospholipase C from Pseudomonas fluorescens. Inactivation of plcA diminished secreted pNPPC hydrolase activity but did not influence Legionella infection of host cells. Taken together, these data indicate that L. pneumophila has multiple lipases and possibly several phospholipase C enzymes but that LipA, LipB and PlcA are not among those exoproteins required for optimal intracellular infection.

    • ↵a The GenBank accession numbers for the L. pneumophila lipA, lipB and plcA sequences are AF454863, AF454864 and AF454865, respectively.

    1,2-DG, 1,2-dipalmitoylglycerol • FFA, free fatty acid • 1-MG, 1-monopalmitoylglycerol • PLC, phospholipase C • PNP, p-nitrophenol • pNPPC, p-nitrophenylphosphorylcholine

    INTRODUCTION

    Legionella pneumophila is the primary aetiologic agent of Legionnaires’ disease, a potentially fatal pneumonia that especially afflicts immunocompromised individuals (Swanson & Hammer, 2000⇓ ; Winn, 1988⇓ ). An inhabitant of fresh water environments, L. pneumophila generally enters the lower respiratory tract following the inhalation of aerosols generated by air conditioners, cooling towers and other devices (Atlas, 1999⇓ ). Within its aquatic habitats, the Legionella bacterium survives as an intracellular parasite of amoebae and ciliates (Fields, 1996⇓ ; Harb et al., 2000⇓ ; Swanson & Hammer, 2000⇓ ). Within the mammalian lung, the microbe flourishes as a parasite of alveolar macrophages (Horwitz, 1992⇓ ; Roy, 1999⇓ ; Shuman et al., 1998⇓ ; Swanson & Hammer, 2000⇓ ; Vogel & Isberg, 1999⇓ ). Thus, investigations into the molecular basis of intracellular infection are critical for understanding the natural history and pathogenesis of legionellosis (Cianciotto et al., 1989a⇓ ; Cianciotto, 2001⇓ ; Hacker et al., 1993⇓ ).

    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.

    METHODS

    Bacteria and media.

    L. pneumophila serogroup 1 strain 130b (ATCC BAA74), a virulent clinical isolate, was described previously (Engleberg et al., 1984⇓ ). Strains NU243 and NU258, direct derivatives of 130b, contain kanamycin-resistance gene insertions in the Legionella pilD and lspDE genes, respectively (Liles et al., 1999⇓ ; Rossier & Cianciotto, 2001⇓ ). Legionellae were routinely cultured on buffered charcoal yeast extract (BCYE) agar for 3 days at 37 °C (Edelstein, 1981⇓ ). To determine the levels of secreted enzymes and compare the growth rates between strains, bacteria were cultured in buffered yeast extract (BYE) broth (Aragon et al., 2000⇓ , 2001⇓ ). Bacterial growth was assessed by measuring the OD660. Escherichia coli DH5α, the host of recombinant plasmids, was grown in Luria–Bertani broth or agar (Aragon et al., 2001⇓ ).

    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 7·5–8·0, 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⇓ ; O’Connell 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 NotI–SalI 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 ΔplcA, 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.

    RESULTS AND DISCUSSION

    Clarification of lipase secretion by L. pneumophila

    We previously demonstrated that unconcentrated supernatants from cultures of L. pneumophila strain 130b have the ability to release both pNP from p-nitrophenyl caprylate and p-nitrophenyl palmitate and FFA from 1-MG (Aragon et al., 2000⇓ ). Since these supernatants were unable to release FFA from 1,2-DG and since other supernatants that had been concentrated 40-fold did not release resorufin from 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester, we reasoned that the carboxylesterase activity of L. pneumophila supernatants includes, perhaps exclusively, a monoacylglycerol lipase (Aragon et al., 2000⇓ ; Jaeger et al., 1999⇓ ). We began the present study by determining whether Legionella culture supernatants could, if concentrated, release FFA from 1,2-DG and triacylglycerols. In addition to cleaving 1-MG, strain 130b supernatants that had been concentrated 100-fold released FFA from 1,2-DG and tricaprylin (Table 1⇓). They also cleaved tripalmitin, triolein and lard oil, but not tributyrin (data not shown). Previously, we observed that FFA release from 1-MG was diminished in L. pneumophila pilD and lsp mutants (Aragon et al., 2000⇓ ; Rossier & Cianciotto, 2001⇓ ). Mutations in pilD and lspDE also reduced the ability of strain 130b to secrete the enzyme(s) that is active against di- and triacylglycerol substrates (Table 1⇑). Taken together, these data indicate that L. pneumophila supernatants contain a variety of lipolytic activities that are dependent upon the type II protein secretion system. Given what is known about other bacteria (Jaeger et al., 1994⇓ , 1999⇓ ), these activities may result from the action of multiple lipase enzymes or a single exoenzyme that acts upon mono-, di- and triacylglycerol substrates.

    Table 1. FFA release from mono-, di- and triacylglycerol substrates

    Identification of two L. pneumophila lipase genes

    As a first step toward understanding the molecular basis of Legionella lipase activity, the L. pneumophila genome database was examined for genes that might encode lipases. Although the database is still incomplete and is reflective of the Philadelphia-1 strain of L. pneumophila, we have found it an accurate predictor of genes present in our strain 130b (Aragon et al., 2001⇓ ; Robey et al., 2001⇓ ; Rossier & Cianciotto, 2001⇓ ). The sequence [LIV]-X-[LIVFY]-[LIVMST]-G-[HYWV]-S-X-G-[GSTAC] is known to be conserved among prokaryotic and eukaryotic lipases (PROSITE accession no. PS00120). By utilizing the sequences LXLLGHSXGS and KLXFVGHSXGS to perform a blast search of the Legionella database, we identified two unlinked ORFs that were predicted to encode proteins containing both the lipase consensus sequence and a signal sequence, a situation that would be compatible with them being type II secreted lipases. Each of the ORFs was amplified by PCR from genomic 130b DNA and sequenced in its entirety. The first ORF, identified by blast searches using either variant of the consensus sequence, is predicted to encode a 282 aa protein, which has the alpha/beta hydrolase fold, a catalytic domain that is found in a variety of enzymes (Ollis et al., 1992⇓ ). We tentatively designated this ORF lipA, for lipase gene A (GenBank accession no. AF454863). The second ORF, only identified by the blast search that used KLXFVGHSXGS, encodes a 254 aa protein and was tentatively denoted lipB, for lipase gene B (GenBank accession no. AF454864). An alignment of the lipase consensus sequences in LipA, LipB and some of the known lipases is shown in Fig. 1⇓. LipA also aligns with and shares approximately 35% identity and 54% similarity with hypothetical hydrolases from P. aeruginosa and Vibrio cholerae (GenBank accession nos NP250313 and NP231620). A known protein that shares significant overall homology (31% identity and 47% similarity) with LipA was Kraken from Drosophila melanogaster (GenBank accession no. AJ000516). In contrast to the blast results for LipA, the only protein that exhibited extensive stretches of homology with LipB (i.e., 30% identity and 49% similarity) was a hypothetical membrane protein from Chlamydia trachomatis (GenBank accession no. NC_000117). Sequence analysis further suggested that lipA and lipB are monocistronic, with lipA positioned downstream of the L. pneumophila zinc-metalloprotease gene (data not shown) (Moffat et al., 1994⇓ ).

    Figure image not available in archive

    Fig. 1. Sequence alignment of L. pneumophila LipA, LipB and known lipases. The PROSITE consensus sequence for lipases with a serine-containing active site appears at the top of the figure. Below the L. pneumophila strain 130b LipA and LipB sequences are the relevant amino acids from lipases of Bacillus subtilis, Burkholderia cepacia, Streptococcus sp. and Rattus norvegicus, which have GenBank accession numbers S23934, A39133, AAK81864 and A46696, respectively. Those residues that are completely conserved in this group of proteins are in bold. The amino acid residue immediately preceding the start of the consensus sequence has also been included, since it invariably was a lysine in the bacterial enzymes.

    To test whether LipA and LipB are associated with Legionella lipase activity, we used allelic exchange to isolate a series of 130b mutants containing an antibiotic resistance gene inserted into either lipA or lipB. For the lipA mutants, the insertion was placed 165 bp from the start of the 846 bp ORF, and for the lipB mutants, the insertion mutation was positioned 376 bp from the beginning of the 762 bp gene. Three independent lipA mutants, designated NU262, NU263 and NU264, were obtained. Two independent lipB defective strains were derived and named NU265 and NU266. All of the mutants grew in BYE broth similarly to wild-type 130b (data not shown), indicating that lipA and lipB are not generally required for extracellular replication. All further experiments described here were also performed with multiple lipA and lipB mutants with similar results. Thus, the phenotypes observed for the mutants result directly or proximately from the mutation in the lipA or lipB gene and not from spontaneous second-site mutations. For clarity, only data for NU262 and NU265 are presented in this paper.

    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.

    Table 2. Release of pNP from p-nitrophenyl caprylate and p-nitrophenyl palmitate

    Intracellular infection by L. pneumophila lipase mutants

    To determine whether lipA and lipB promote intracellular infection by L. pneumophila, we examined the ability of NU262 and NU265 to grow within H. vermiformis and U937 cell macrophages. Upon co-culture with the Hartmannella amoebae, the mutants grew comparably to wild-type, indicating that neither lipA nor lipB is required for intracellular infection of protozoan hosts (Fig. 2⇓). When the macrophage cell line was infected, NU262 and NU265 behaved, for the most part, similarly to wild-type (Fig. 3a⇓, b⇑). The only aberration was a modestly reduced recovery of the lipA mutant between 48 and 72 h incubation (Fig. 3a⇑). Since that mutant also exhibited reduced survivability upon incubation in the RPMI tissue culture medium (data not shown), we suspect that this reduced recovery was not due to diminished intracellular growth but rather the death of bacteria following their release from the macrophages. As an alternative way of identifying an intracellular infectivity defect (Cianciotto et al., 1989b⇓ ; Robey et al., 2001⇓ ), we examined the ability of the mutants to kill the U937 cell monolayer. However, neither NU262 nor NU265 showed an impaired cytopathic effect (data not shown). Even though the analysis of lipolytic activities in the mutants’ supernatants suggested that lipA and lipB have different substrate spectra, we explored the possibility that the two genes have overlapping function in the intracellular environment, such that the loss of lipA is compensated for by lipB and vice versa. Thus, we constructed a 130b mutant, designated strain NU267, that was lacking both lipA and lipB; i.e., a gentamicin-resistance gene cassette was inserted into the lipB gene of the kanamycin-resistant lipA mutant. Strain NU267 grew in BYE broth as well as did wild-type, but its culture supernatants had, as expected, a reduced activity against lipase substrates (data not shown). However, the double lipase mutant did not display a significant defect in intracellular infection (Figs 2b⇑ and 3c⇑). Taken together, these data indicate that the lipA and lipB genes are not required for intracellular infection by L. pneumophila. They also signal that the reduced intracellular infectivity of type II secretion mutants (see Fig. 2b⇑, for example) is not due to the loss of LipA or LipB. Our observations do not, however, rule out a possible role for other Legionella lipases in infection of macrophages and/or protozoa. It should also be acknowledged that the apparent absence of a critical role for LipA and LipB in intracellular infection does not mean that these factors have no role in L. pneumophila virulence. Indeed, like the type II secretion dependent metalloprotease (Moffat et al., 1994⇓ ), LipA and LipB may be most relevant in the extracellular spaces of the lung.

    Figure image not available in archive

    Fig. 2. Intracellular infection of H. vermiformis by wild-type and mutant L. pneumophila. (a) Infection profile for strain 130b (♦) versus lipA mutant NU262 (□). (b) Infection profile for strain 130b (♦) versus lipB mutant NU265 (▵), lipA lipB double mutant NU267 (•), plcA mutant NU268 (□) and lspDE mutant NU258 (▪). Amoebae were infected at a m.o.i. of 1, and then the numbers of bacteria in each well were quantified at 0, 24, 48 and 72 h by plating aliquots on BCYE agar. Results represent the means±SD of triplicate wells and are representative of two independent experiments.

    Figure image not available in archive

    Fig. 3. Intracellular infection of U937 cells by wild-type and mutant L. pneumophila. Infection profile of strain 130b (•) versus lipA mutant NU262 (□) (a), lipB mutant NU265 (▵) (b), lipA lipB double mutant NU267 (▽) (c), and plcA mutant NU268 (◊) (d). Macrophages were infected at a m.o.i. of 0·1. At 0, 24, 48 and 72 h post-inoculation, the monolayer was lysed and the total numbers of c.f.u. within the wells were determined. Each datum point represents the mean and standard deviation for three monolayers. Results are representative of two independent experiments. When culture supernatants obtained without the purposeful lysis of the monolayers were assayed for bacterial numbers, comparable numbers of c.f.u. of wild-type and mutant bacteria were recovered (data not shown), indicating that the lipA, lipB and plcA mutants are not defective for either intracellular growth or host cell lysis.

    Identification and mutation of a L. pneumophila PLC-like gene

    Biochemical analysis of culture supernatants has yielded mixed results concerning the existence of L. pneumophila PLC, and it has been argued that the observed hydrolysis of pNPPC might be due to a phosphatase (Baine, 1988⇓ ; Flieger et al., 2000⇓ ). Yet, a recently obtained acid phosphatase mutant is not defective for pNPPC hydrolysis (Aragon et al., 2001⇓ ). Thus, we felt that the type of genetic approach applied to lipA and lipB might be helpful in addressing the existence and relevance of a L. pneumophila PLC. Examination of the Philadelphia-I genome database suggested the presence of a L. pneumophila ORF that is related to a Pseudomonas PLC gene. Cloning of the ORF from strain 130b and subsequent complete sequence analysis confirmed that L. pneumophila contains a gene (GenBank accession no. AF454865) whose predicted 48 kDa product aligns with and is 43% identical and 62% similar to a recently described PLC that is present in P. fluorescens (GenBank accession no. AJ30443) (Preuss et al., 2001⇓ ). Since the Legionella protein was also predicted to contain a signal sequence, we viewed it as a potential type II exoenzyme. When the gene was inactivated by allelic exchange, the resulting mutant (NU268) grew normally in BYE broth (data not shown). Importantly, on five separate occasions, the mutant’s unconcentrated culture supernatants displayed a reduced ability to release pNP from pNPPC. For example, in one experiment, the wild type samples (n=3) had 2·947±0·254 U activity, whereas the mutant samples (n=3) had 1·508±0·096 (P<0·001, Student’s t-test). In that same experiment, the pilD and lspDE mutants had 0·174±0·153 and 0·021±0·019 U activity. Taken together, these data indicate that mutant NU268 has diminished PLC activity. Since L. pneumophila secretes both a phospholipase A (Flieger et al., 2000⇓ ) as well as multiple lipases (see above), we were unable to assay more specifically for PLC activity by monitoring diacylglycerol release from phospholipid substrates. On the basis of our current structural and functional data, we designated this new gene as L. pneumophila plcA, for phospholipase C gene A.

    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.

    Acknowledgments

    We acknowledge Sherry Kurtz for contributing to the sequence analysis of plcA and thank the other members of the Cianciotto lab for helpful discussions. This work was supported by NIH grant AI43987 awarded to N.P.C.

    References

    1. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389-3402.
    2. Anguita, J., Rodriguez Aparicio, L. B. & Naharro, G. (1993). Purification, gene cloning, amino acid sequence analysis, and expression of an extracellular lipase from an Aeromonas hydrophila human isolate. Appl Environ Microbiol 59, 2411-2417.
    3. Aragon, V., Kurtz, S., Flieger, A., Neumeister, B. & Cianciotto, N. P. (2000). Secreted enzymatic activities of wild-type and pilD-deficient Legionella pneumophila. Infect Immun 68, 1855-1863.
    4. Aragon, V., Kurtz, S. & Cianciotto, N. P. (2001). Legionella pneumophila major acid phosphatase and its role in intracellular infection. Infect Immun 69, 177-185.
    5. Atlas, R. M. (1999). Legionella: from environmental habitats to disease pathology, detection and control. Environ Microbiol 1, 283-293.
    6. Baine, W. B. (1985). Cytolytic and phospholipase C activity in Legionella species. J Gen Microbiol 131, 1383-1391.
    7. Baine, W. B. (1988). A phospholipase C from the Dallas 1E strain of Legionella pneumophila serogroup 5: purification and characterization of conditions for optimal activity with an artificial substrate. J Gen Microbiol 134, 489-498.
    8. Cianciotto, N. P. (2001). Pathogenicity of Legionella pneumophila. Int J Med Microbiol 291, 331-343.
    9. Cianciotto, N. P. & Fields, B. S. (1992). Legionella pneumophila mip gene potentiates intracellular infection of protozoa and human macrophages. Proc Natl Acad Sci USA 89, 5188-5191.
    10. Cianciotto, N. P., Long, R., Eisenstein, B. I. & Engleberg, N. C. (1988). Site-specific mutagenesis in Legionella pneumophila by allelic exchange using counterselectable ColE1 vectors. FEMS Microbiol Lett 56, 203-208.
    11. Cianciotto, N., Eisenstein, B. I., Engleberg, N. C. & Shuman, H. (1989a). Genetics and molecular pathogenesis of Legionella pneumophila, an intracellular parasite of macrophages. Mol Biol Med 6, 409-424.
    12. Cianciotto, N. P., Eisenstein, B. I., Mody, C. H., Toews, G. B. & Engleberg, N. C. (1989b). A Legionella pneumophila gene encoding a species-specific surface protein potentiates initiation of intracellular infection. Infect Immun 57, 1255-1262.
    13. Dowling, J. N., Saha, A. K. & Glew, R. H. (1992). Virulence factors of the family Legionellaceae. Microbiol Rev 56, 32-60.
    14. Edelstein, P. H. (1981). Improved semiselective medium for isolation of Legionella pneumophila from contaminated clinical and environmental specimens. J Clin Microbiol 14, 298-303.
    15. El Khattabi, M., Ockhuijsen, C., Bitter, W., Jaeger, K. E. & Tommassen, J. (1999). Specificity of the lipase-specific foldases of gram-negative bacteria and the role of the membrane anchor. Mol Gen Genet 261, 770-776.
    16. Engleberg, N. C., Drutz, D. J. & Eisenstein, B. I. (1984). Cloning and expression of Legionella pneumophila antigens in Escherichia coli. Infect Immun 44, 222-227.
    17. Fields, B. S. (1996). The molecular ecology of legionellae. Trends Microbiol 4, 286-290.
    18. Filloux, A., Murgier, M., Wretlind, B. & Lazdunski, A. (1987). Characterization of two Pseudomonas aeruginosa mutants with defective secretion of extracellular proteins and comparison with other mutants. FEMS Microbiol Lett 40, 159-163.
    19. Flieger, A., Gong, S., Faigle, M., Deeg, P., Bartmann, P. & Neumeister, B. (2000). Characterization of a novel phospholipase A (PLA) activity secreted by Legionella species. J Bacteriol 182, 1321-1327.
    20. Flieger, A., Gong, S., Faigle, M., Stevanovic, S., Cianciotto, N. P. & Neumeister, B. (2001). Novel lysophospholipase A secreted by Legionella pneumophila. J Bacteriol 183, 2121-2124.
    21. Gribbon, E. M., Cunliffe, W. J. & Holland, K. T. (1993). Interaction of Propionibacterium acnes with skin lipids in vitro. J Gen Microbiol 139, 1745-1751.
    22. Hacker, J., Ott, M., Wintermeyer, E., Ludwig, B. & Fischer, G. (1993). Analysis of virulence factors of Legionella pneumophila. Zentbl Backteriol 278, 348-358.
    23. Hales, L. M. & Shuman, H. A. (1999). Legionella pneumophila contains a type II general secretion pathway required for growth in amoebae as well as for secretion of the Msp protease. Infect Immun 67, 3662-3666.
    24. Harb, O. S., Gao, L.-Y. & Abu Kwaik, Y. (2000). From protozoa to mammalian cells: a new paradigm in the life cycle of intracellular bacterial pathogens. Environ Microbiol 2, 251-265.
    25. Higgins, D. G. & Sharp, P. M. (1988). CLUSTAL: a package for performing multiple sequence alignment on a microcomputer. Gene 73, 237-244.
    26. Hoffmann, G. E., Schmidt, D., Bastian, B. & Guder, W. G. (1986). Photometric determination of phospholipase A. J Clin Chem Clin Biochem 24, 871-875.
    27. Hofmann, K., Bucher, P., Falquet, L. & Bairoch, A. (1999). The PROSITE database, its status in 1999. Nucleic Acids Res 27, 215-219.
    28. Horwitz, M. A. (1992). Interactions between macrophages and Legionella pneumophila. Curr Topics Microbiol Immunol 181, 265-282.
    29. Jaeger, K. E., Ransac, S., Dijkstra, B. W., Colson, C., van Heuvel, M. & Misset, O. (1994). Bacterial lipases. FEMS Microbiol Rev 15, 29-63.
    30. Jaeger, K. E., Dijkstra, B. W. & Reetz, M. T. (1999). Bacterial biocatalysts: molecular biology, three-dimensional structures, and biotechnological applications of lipases. Annu Rev Microbiol 53, 315-351.
    31. Jepson, M., Howells, A., Bullifent, H. L., Bolgiano, B., Crane, D., Miller, J., Holley, J., Jayasekera, P. & Titball, R. W. (1999). Differences in the carboxy-terminal (putative phospholipid binding) domains of Clostridium perfringens and Clostridium bifermentans phospholipases C influence the hemolytic and lethal properties of these enzymes. Infect Immun 67, 3297-3301.
    32. Konig, B., Jaeger, K. E., Sage, A. E., Vasil, M. L. & Konig, W. (1996). Role of Pseudomonas aeruginosa lipase in inflammatory mediator release from human inflammatory effector cells (platelets, granulocytes, and monocytes). Infect Immun 64, 3252-3258.
    33. Koster, M., Bitter, W. & Tommassen, J. (2000). Protein secretion mechanisms in Gram-negative bacteria. Int J Med Microbiol 290, 325-331.
    34. Kovach, M. E., Elzer, P. H., Hill, D. S., Robertson, G. T., Farris, M. A., Roop, R. M. & Peterson, K. M. (1995). Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166, 175-176.
    35. Kurioka, S. & Matsuda, M. (1976). Phospholipase C assay using p-nitrophenylphosphorylcholine together with sorbitol and its application to studying the metal and detergent requirement of the enzyme. Anal Biochem 75, 281-289.
    36. Lee, V. T. & Schneewind, O. (2001). Protein secretion and the pathogenesis of bacterial infections. Genes Dev 15, 1725-1752.
    37. Liles, M. R., Viswanathan, V. K. & Cianciotto, N. P. (1998). Identification and temperature regulation of Legionella pneumophila genes involved in type IV pilus biogenesis and type II protein secretion. Infect Immun 66, 1776-1782.
    38. Liles, M. R., Edelstein, P. H. & Cianciotto, N. P. (1999). The prepilin peptidase is required for protein secretion by and the virulence of the intracellular pathogen Legionella pneumophila. Mol Microbiol 31, 959-970.
    39. Merino, S., Aguilar, A., Nogueras, M. M., Regue, M., Swift, S. & Tomas, J. M. (1999). Cloning, sequencing, and role in virulence of two phospholipases (A1 and C) from mesophilic Aeromonas sp. serogroup O:34. Infect Immun 67, 4008-4013.
    40. Moffat, J. F., Edelstein, P. H., Regula, D. P.Jr., Cirillo, J. D. & Tompkins, L. S. (1994). Effects of an isogenic Zn-metalloprotease-deficient mutant of Legionella pneumophila in a guinea-pig pneumonia model. Mol Microbiol 12, 693-705.
    41. Muller, H. E. (1981). Enzymatic profile of Legionella pneumophila. J Clin Microbiol 13, 423-426.
    42. Nakai, K. & Kanehisa, M. (1991). Expert system for predicting protein localization sites in gram-negative bacteria. Proteins 11, 95-110.
    43. Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. (1997). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10, 1-6.
    44. Nolte, F. S., Hollick, G. E. & Robertson, R. G. (1982). Enzymatic activities of Legionella pneumophila and Legionella-like organisms. J Clin Microbiol 15, 175-177.
    45. Nunn, D. (1999). Bacterial type II protein export and pilus biogenesis: more than just homologies? Trends Cell Biol 9, 402-408.
    46. O’Connell, W. A., Bangsborg, J. M. & Cianciotto, N. P. (1995). Characterization of a Legionella micdadei mip mutant. Infect Immun 63, 2840-2845.
    47. Ollis, D. L., Cheah, E., Cygler, M. & 10 other authors (1992). The alpha/beta hydrolase fold. Protein Eng 5, 197–211.
    48. Polesky, A. H., Ross, J. T., Falkow, S. & Tompkins, L. S. (2001). Identification of Legionella pneumophila genes important for infection of amoebas by signature-tagged mutagenesis. Infect Immun 69, 977-987.
    49. Pratt, J., Cooley, J. D., Purdy, C. W. & Straus, D. C. (2000). Lipase activity from strains of Pasteurella multocida. Curr Microbiol 40, 306-309.
    50. Preuss, I., Kaiser, I. & Gehring, U. (2001). Molecular characterization of a phosphatidylcholine-hydrolyzing phospholipase C. Eur J Biochem 268, 5081-5091.
    51. Robey, M., O’Connell, W. & Cianciotto, N. P. (2001). Identification of Legionella pneumophila rcp, a pagP-like gene that confers resistance to cationic antimicrobial peptides and promotes intracellular infection. Infect Immun 69, 4276-4286.
    52. Rollof, J., Braconier, J. H., Soderstrom, C. & Nilsson-Ehle, P. (1988). Interference of Staphylococcus aureus lipase with human granulocyte function. Eur J Clin Microbiol Infect Dis 7, 505-510.
    53. Rossier, O. & Cianciotto, N. P. (2001). Type II protein secretion is a subset of the PilD-dependent processes that facilitate intracellular infection by Legionella pneumophila. Infect Immun 69, 2092-2098.
    54. Roy, C. R. (1999). Trafficking of the Legionella pneumophila phagosome. ASM News 65, 416-421.
    55. Russel, M. (1998). Macromolecular assembly and secretion across the bacterial cell envelope: type II protein secretion systems. J Mol Biol 279, 485-499.
    56. Sandkvist, M. (2001a). Biology of type II secretion. Mol Microbiol 40, 271-283.
    57. Sandkvist, M. (2001b). Type II secretion and pathogenesis. Infect Immun 69, 3523-3535.
    58. Schmiel, D. H. & Miller, V. L. (1999). Bacterial phospholipases and pathogenesis. Microbes Infect 1, 1103-1112.
    59. Shuman, H. A., Purcell, M., Segal, G., Hales, L. & Wiater, L. A. (1998). Intracellular multiplication of Legionella pneumophila: human pathogen or accidental tourist? Curr Topics Microbiol Immunol 225, 99-112.
    60. Songer, J. G. (1997). Bacterial phospholipases and their role in virulence. Trends Microbiol 5, 156-161.
    61. Stone, B. J. & Abu Kwaik, Y. (1999). Natural competence for DNA transformation by Legionella pneumophila and its association with expression of type IV pili. J Bacteriol 181, 1395-1402.
    62. Strom, M. S., Nunn, D. & Lory, S. (1991). Multiple roles of the pilus biogenesis protein pilD: involvement of pilD in excretion of enzymes from Pseudomonas aeruginosa. J Bacteriol 173, 1175-1180.
    63. Swanson, M. S. & Hammer, B. K. (2000). Legionella pneumophila pathogenesis: a fateful journey from amoebae to macrophages. Annu Rev Microbiol 54, 567-613.
    64. Szeto, L. & Shuman, H. A. (1990). The Legionella pneumophila major secretory protein, a protease, is not required for intracellular growth or cell killing. Infect Immun 58, 2585-2592.
    65. Terada, L. S., Johansen, K. A., Nowbar, S., Vasil, A. I. & Vasil, M. L. (1999). Pseudomonas aeruginosa hemolytic phospholipase C suppresses neutrophil respiratory burst activity. Infect Immun 67, 2371-2376.
    66. Thanassi, D. G. & Hultgren, S. J. (2000). Multiple pathways allow protein secretion across the bacterial outer membrane. Curr Opin Cell Biol 12, 420-430.
    67. Thorpe, T. C. & Miller, R. D. (1981). Extracellular enzymes of Legionella pneumophila. Infect Immun 33, 632-635.
    68. Titball, R. W. (1998). Bacterial phospholipases. Soc Appl Bacteriol Symp Ser 27, 127S-137S.
    69. Viswanathan, V. K., Edelstein, P. H., Pope, C. D. & Cianciotto, N. P. (2000). The Legionella pneumophila iraAB locus is required for iron assimilation, intracellular infection, and virulence. Infect Immun 68, 1069-1079.
    70. Vogel, J. P. & Isberg, R. R. (1999). Cell biology of Legionella pneumophila. Curr Opin Microbiol 2, 30-34.
    71. Weingart, C. L. & Hooke, A. M. (1999). Regulation of expression of the nonhemolytic phospholipase C of Burkholderia cepacia. Curr Microbiol 39, 336-341.
    72. Winn, W. C.Jr. (1988). Legionnaires disease: historical perspective. Clin Microbiol Rev 1, 60-81.