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Laboratory for Bacteriology, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium
Correspondence
Jozef Anné
Jozef.Anne{at}rega.kuleuven.be
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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2AP, 2-antiplasmin; Lpa, Legionella plasminogen activator; MOMP, major outer-membrane protein; Omps, outer-membrane proteins; Pla, plasminogen activator; Plg, plasminogen |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Molmeret et al., 2005; Brüggemann et al., 2006; Hilbi et al., 2007), together with the identification of a great number of virulence factors, form the basis of the progress made (Albert-Weissenberger et al., 2007). In recent years the number of reported cases of legionellosis has risen dramatically, resulting in increased attention to control measurements to prevent infections by this pathogen and to the identification and mechanism of action of additional virulence factors. A variety of L. pneumophila surface components and secreted proteins have been shown to be implicated in pathogenesis. In several bacterial pathogens, functional characterization of outer-membrane proteins (Omps) has revealed diverse roles for these molecules, including iron uptake, antimicrobial peptide and multi-drug resistance, bile resistance, pore formation, adhesion, invasion, bacteriophage adsorption and serum resistance. Although significant advances have been made regarding the unravelling of the structure and function of Omps, the number of Omps that have been characterized represents only a small portion of the total number of Omps revealed by bacterial genome sequences (Lin et al., 2002). In the search for L. pneumophila genes encoding putative Omps, and more precisely Omps that might be involved in bacterial virulence, we identified a gene encoding a homologue of the plasminogen activator protein (Pla) of Yersinia pestis. The latter protein belongs to the family of the omptins, which is a class of surface proteases/adhesins found in the outer membrane of enterobacterial pathogens.
Although the omptin homologues show high sequence identity and a conserved 
-barrel, they are multifunctional and exhibit different virulence-associated functions. Pla of Y. pestis contributes to the virulence of this pathogen by several mechanisms. The protein is involved in uncontrolled plasmin activity by efficient conversion of human proenzyme plasminogen (Plg) to plasmin, inactivation of the plasmin inhibitor 2-antiplasmin (2-AP), and binding to laminin, which localizes the plasmin activity onto basement membranes. These properties are important for the bacterial invasiveness in plague (Sodeinde et al., 1992) and enhance migration of Y. pestis through tissue barriers (Kukkonen & Korhonen, 2004). PgtE, the homologue of Pla in Salmonella enterica, is significantly less efficient in plasminogen activation than Pla (Kukkonen et al., 2004), but inactivates the plasmin inhibitor 2AP more efficiently (Lähteenmäki et al., 2005). This discrepancy may reflect differences in the mechanisms of infection exerted by these two pathogens. The generation of plasmin is indispensable for the predominantly extracellular Y. pestis to proteolyse tissues in order to disseminate all over the human body. Since spread of S. enterica occurs predominantly by means of macrophages, Lähteenmäki et al. (2005) hypothesize that PgtE-mediated inactivation of 2AP serves to protect the plasmin generated by both the bacteria and the macrophages. Based on these results obtained for other bacterial pathogens, we hypothesized that the L. pneumophila omptin homologue could play an important role in the virulence of this pathogen as well.
In this study, we cloned the gene encoding the L. pneumophila plasminogen activator homologue and demonstrated, to our knowledge for the first time, that L. pneumophila possesses a surface protease that exhibits plasminogen activator activity.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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. Legionella strains were grown at 37 °C on buffered charcoal yeast extract (BCYE) agar plates or in buffered yeast extract broth (BYE) supplemented with -ketoglutarate, L-cysteine and ferric pyrophosphate (Edelstein, 1981). When necessary, kanamycin (10 µg ml–1) or chloramphenicol (5–12 µg ml–1) was added. Escherichia coli TG1 (Sambrook et al., 1989) was used as host for cloning purposes and E. coli BL21* and E. coli BL21(DE3)pLys were used for the expression of recombinant proteins (Studier & Moffatt, 1986). Cultures were grown in Luria–Bertani medium (Miller, 1972) and if necessary supplemented with ampicillin (50 µg ml–1) or chloramphenicol (50 µg ml–1). Acanthamoeba castellanii (ATCC 30234) was cultured in PYG712 medium at room temperature.
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and 2, respectively. Standard PCR reactions were carried out using SuperTaq (HT Biotechnology).
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) with an extra NdeI site introduced at the start codon of the lacZ gene and with part of the mobA gene deleted by AgeI digestion and religation. In this way, the plasmid pMMBNlpa was obtained. Using the primers PromlpaF and LpaR the lpa gene with its promoter region was amplified by PCR from chromosomal DNA (100 ng) as template. The resulting 1.2 kb PCR fragment was cloned into pGem-T Easy (Promega) and subsequently as a SacI–SacII restriction fragment cloned into pBCKS(+). In this way, the plasmid pBCpromlpa containing the gene together with its putative promoter region was obtained.
For the construction of an L. pneumophila Philadelphia mutant lacking the lpa gene, the chromosomal lpa gene was substituted by a kanamycin-resistance gene by means of double homologous recombination as described for other L. pneumophila genes (De Buck et al., 2005). To make sure that the observed effects after deletion of the lpa gene could be specifically ascribed to the deletion of the lpa gene, we constructed a complemented L. pneumophila lpa deletion strain by introducing pMMBNlpa into the mutant strain. A second complemented strain with lpa under transcriptional control of its own promoter was constructed by introducing pBCpromlpa into the mutant strain.
To construct pETlpaHis, the lpa gene was amplified by PCR using LpaFHis and LpaR. The forward primer was designed to add ten histidines at the N-terminal end of the mature protein. After cloning into pGEM-T Easy, the fragment was digested with NdeI and ligated into pET3a.
RNA isolation and reverse transcriptase (RT)-PCR.
Total RNA from exponentially growing and stationary-phase L. pneumophila cells and from L. pneumophila intracellularly grown in A. castellanii was isolated using the RNeasy mini kit (Qiagen) as described previously (De Buck et al., 2004). Samples used for RT-PCR were additionally treated with DNase I (Qiagen) (final concentration 0.5 units µl–1) to remove any residual chromosomal DNA. RT-PCR was performed with 100 ng of total RNA using the Access RT-PCR System (Promega). To investigate the presence of an operon structure encompassing the lpa gene, the primers PromlpaF and LpaR were used.
Cell fractionation.
For the preparation of protein fractions containing either extracellular proteins, total cell proteins, soluble, inner- or outer-membrane proteins, a 100 ml culture was grown overnight starting from a 1 ml overnight-grown preculture. The culture was centrifuged (5000 g, 15 min) and the supernatant obtained was filter-sterilized using a 0.2 µm filter. Proteins present in the supernatant were concentrated by TCA precipitation (20 % TCA) for 1 h on ice. After centrifugation (10 000 g, 15 min) the extracellular protein fraction was washed with acetone and dried. The remaining cell pellet was resuspended in 15 ml lysis buffer (10 mM Tris, 10 mM EDTA, pH 7.5) and subsequently lysed in a French pressure cell. To obtain the total cell protein fraction, the cell debris was removed by centrifugation (20 min, 12 000 g). Membrane proteins and the soluble protein fraction were obtained following centrifugation of the cell lysate for 2 h at 100 000 g. The soluble protein fraction is located in the supernatant, whereas the membranes with the membrane proteins are situated in the sediment. To set the inner-membrane proteins free from the membranes, the sediment was resuspended in 1.5 ml 10 mM Tris/HCl (pH 7.5) containing 1.5 % Sarkosyl and incubated for 30 min at room temperature, and subsequently recentrifuged for 2 h at 100 000 g. The supernatant, containing the inner-membrane proteins, was collected. The pellet, containing the outer-membrane proteins, was resuspended in 500 µl 10 mM Tris/HCl (pH 7.5) and 10 mM EDTA, containing 1 % Triton X-100.
SDS-PAGE and Western blotting.
(His)10-Lpa was isolated from the cytoplasm of E. coli BL21(DE3)pLys/pETLpaHis under denaturing conditions as described in Qia Expressionist (Qiagen). To obtain extra pure protein for immunization purposes, purified fractions were additionally separated by SDS-PAGE and subsequently the Lpa protein was electro-eluted from the gels. After dialysis of the purified fractions against PBS, 150 µg of the (His)10-Lpa protein was used for immunization of a Dutch White rabbit, in order to obtain Lpa-specific antibodies.
Expression of Lpa was monitored by Western blotting (12.5 % SDS-PAGE) and immunodetection with Lpa-specific antibodies and in a second step with alkaline-phosphatase-conjugated anti-rabbit antibodies (Sigma). Visualization of the proteins was done using the chromogenic substrate solution NBT/BCIP (Roche Diagnostics).
As a control for the protein fractionation, detection of L. pneumophila DnaK, a cytoplasmic protein, LepB (signal peptidase), an inner-membrane protein, and MOMP (major outer-membrane protein), an outer-membrane protein, was performed with antibodies against E. coli DnaK, LepB and MOMP, respectively.
Plasminogen activation tests.
Measurement of plasminogen activation was performed as described previously (Kukkonen et al., 2001), by incubating 8x107 bacteria, 4 µg human Glu-Plg (American Diagnostica) and the chromogenic plasmin substrate S-2251 (0.45 mM Val-Leu-Lys-p-nitroaniline dihydrochloride, Chromogenix) in a total volume of 200 µl at 37 °C. Plasmin activity was measured in the Infinite 200 microplate reader (Tecan) at 405 nm as the breakdown of the chromogenic substrate as a function of time. Activity of inner- and outer-membrane protein fractions was measured by the same test as described above, but instead of bacteria, 20 µl of these fractions (see preparation above) was used in the samples.
Amoebae plate test.
Growth of L. pneumophila in the presence of A. castellanii was analysed as described by Albers et al. (2005). Briefly, 1.5 ml A. castellanii (2.6x106 ml–1) was spread on BCYE agar plates, allowed to dry for 1–2 h in a laminar-flow hood and left overnight at room temperature. Stationary-phase bacterial cultures were adjusted to an identical OD600 and series of tenfold dilutions in BYE were prepared; 3 µl of stationary culture and of each dilution was spotted onto the BCYE agar plate and the plates were incubated for 5 days at 37 °C.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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The protein sequence of L. pneumophila Lpa was compared to the omptin sequences of other Gram-negative human pathogens: E. coli, Y. pestis and S. enterica serovar Typhimurium (Fig. 1). Overall identity of Lpa with these proteins is respectively 39, 49 and 47 %. The structure of the best-studied member of the omptin family, OmpT, elucidated by Vandeputte-Rutten et al. (2001), appears to consist of a -barrel with ten antiparallel -strands connected by four short periplasmic turns and five surface-exposed loops. The extracellular loops of OmpT are indicated in Fig. 1.
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; Kramer et al., 2000, 2001) are conserved (Fig. 1), they have clearly different substrates (Kukkonen & Korhonen, 2004). It is assumed that this substrate specificity is dependent on slight differences in the amino acid sequence in the regions important for substrate recognition in the extracellular loops of the protein. There are three important structural features to convert the substrate specificity of OmpT into that of Pla and as a result change it into a protein with enhanced plasminogen activator activity (Kukkonen et al., 2001). The three key elements are (i) shortening of L3 by four residues to the size of the Pla L3, (ii) deletion of two amino acids in L4, i.e. Asp213 and Pro214, and (iii) substitution of residue Lys216 by Arg in L4. These typical plasminogen activator-related structural features are also present in the L. pneumophila protein, leading to the hypothesis that this protein exhibits plasminogen activator activity.
Transcriptional analysis of the L. pneumophila lpa gene
On the L. pneumophila Philadelphia chromosome the lpa gene is situated between a putative ORF with unknown function (lpg2386) and a gene encoding an amino acid permease (lpg2388) (Fig. 2a). The latter is oriented in the same direction as lpa with an intergenic distance of 130 bp. RT-PCR experiments on total L. pneumophila RNA were performed to see whether the lpa gene is effectively expressed under the experimental conditions used and to determine if it is cotranscribed with the upstream ORF. RT-PCR with lpa-specific primers clearly showed the presence of a 0.92 kb transcript in the case of L. pneumophila grown to exponential and stationary phase, confirming lpa expression in both phases (Fig. 2b). The lpa gene is also expressed in intracellularly grown bacteria (Fig. 2b). Furthermore, no transcript that encompasses the intergenic region could be obtained, indicating that transcription of the lpa gene is directed by its own promoter. RT-PCR on RNase-treated samples was negative, confirming a specific, DNA-independent amplification.
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). When the same analysis was performed with total cell proteins of L. pneumophila/pMMBNlpa, overexpressing the lpa gene upon IPTG induction, a clear band at a molecular mass of 33 kDa, corresponding to the predicted molecular mass of 32.8 kDa of the mature form of Lpa, could be observed (Fig. 3).
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). As a control for a correct cellular fractionation, detection of L. pneumophila DnaK, a cytoplasmic protein, LepB (signal peptidase), an inner-membrane protein, and MOMP, an outer-membrane protein, was performed. The control proteins were found to be present in the expected fractions (results not shown). Based on these experiments we could conclude that Lpa is an outer-membrane protein.
Functional analysis of Lpa
We studied the capacity of Lpa to convert plasminogen to plasmin with plasminogen activator tests on cells and on inner- and outer-membrane protein fractions. As stated above, the capacity to convert plasminogen into plasmin is very different for each of the characterized omptins. For example, the Pla protein of Y. pestis shows a more efficient activation of Plg than OmpT of E. coli and PgtE of S. enterica (Kukkonen et al., 2004). In order to assess the plasminogen activation ability of the Lpa protein, its activity was monitored as a function of time for L. pneumophila cells and L. pneumophila/pMMBNlpa cells with and without induction with IPTG. For a similar experiment in E. coli, the OmpT-deficient E. coli BL21* was used as host strain in order to minimize interference of residual chromosomally encoded plasminogen activator activity. Results of both tests are given in Fig. 4.
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Although L. pneumophila wild-type cells showed no detectable activation of plasminogen (Fig. 4a), plasminogen activation activity by an L. pneumophila wild-type outer-membrane preparation, as shown in Fig. 4(c), starts to increase from about 10 h of incubation, corresponding to the large amount of Lpa protein found in the outer membrane following immunoblotting and detection with Lpa-specific antibodies (Fig. 3). As expected, no activity was detected in the inner-membrane protein fraction of L. pneumophila wild-type (results not shown), which confirms the outer-membrane localization of Lpa. The outer-membrane protein fraction of L. pneumophila 
lpa has lost the ability to activate plasminogen. Providing the lpa gene in trans on a plasmid complements this defect as can be clearly seen in Fig. 4(c). Maximum plasminogen activation by the outer-membrane fraction of the complemented strain upon induction with 50 µM IPTG is reached within 5–7 h incubation. Also, the outer-membrane protein fraction of the lpa-overexpressing strain L. pneumophila/pMMBNlpa showed maximum activity after 5–7 h upon induction with 50 µM IPTG.
Influence of O-antigen on Lpa activity
The fact that Lpa activity of the L. pneumophila cells did not exceed background levels is remarkable, as transcriptional analysis revealed lpa expression during both the exponential and stationary growth phase (Fig. 2b). A Western blot experiment of total L. pneumophila using Lpa-specific antibodies showed expression as well, albeit at a very low level (Fig. 3). L. pneumophila might need specific conditions for an increased production of Lpa such as intracellular growth in protozoa or macrophages, as has been shown for the production of PgtE in S. enterica (Lähteenmäki et al., 2005). As well as the possibility that plasminogen activator activity did not exceed background levels due to a low in vitro expression level, it is possible that the protein is not fully active under the laboratory conditions used and that it has specific requirements with respect to, for example, the lipopolysaccharide (LPS) structure or composition. Kramer et al. (2002) showed that reconstitution of an enzymically active, purified OmpT was only achieved after addition of LPS. LPS was shown to be required for obtaining a native conformation of the protein by inducing subtle conformational changes in the surface-exposed loops. Otherwise, the L. pneumophila Lpa protein might be sterically hindered by long O-polysaccharides present in smooth-type LPS, as was shown for PgtE of S. enterica (Kukkonen et al., 2004). To get an idea about the influence of the length of the O-polysaccharides on the activity of Lpa, plasminogen activation was studied in a modified LPS environment. To simulate a rough Legionella LPS background, an LPS mutant from L. pneumophila serogroup 1 strain Corby (Lück et al., 2001) was used. This LPS mutant has lost reactivity with monoclonal antibody 3/1 and fails to produce high-molecular-mass long-chain O-polysaccharides. Whereas the wild-type L. pneumophila Corby strain produces long-chain O-polysaccharides (40–75 repeating units), the LPS mutant strain produces O-polysaccharides of highly reduced chain length of 7–15 repeating units (Lück et al., 2001). Plasminogen activation by these two strains was monitored as a function of time (Fig. 5). Although the plasminogen activation is lower compared to that of the induced L. pneumophila and E. coli Lpa-overexpressing strain, a clear difference in activity can be seen between the wild-type Corby strain and the isogenic LPS mutant strain. The LPS mutant strain shows a more than threefold increase in the plasminogen activation capacity. This experiment indicates that, like OmpT of E. coli, Pla of Y. pestis and PgtE of S. enterica, Lpa of L. pneumophila might also be sterically hindered by O-antigen repeats in smooth LPS.
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). As can be seen in Fig. 6, wild-type L. pneumophila was able to grow in the presence of amoebae up to a dilution of 10–6. The lpa mutant strain formed robust colonies until a dilution of 10–2, but failed to grow from dilution 10–6, showing a partial growth defect in the presence of A. castellanii. Supplying lpa on a plasmid restored growth to the wild-type level. In the absence of A. castellanii all strains grew equally well.
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With respect to the pathogenesis of L. pneumophila, research has focused almost exclusively on the interaction of the bacteria with the macrophage host. Nevertheless, besides a possible role for Lpa in the adhesion to the host cell and in intracellular replication, one might hypothesize a role in later stages of infection as well. These include penetration of the alveolar epithelial barrier and basement membrane and dissemination of the bacteria in the lung tissue, causing alveolar damage and bacterial spread. Very recently a new peptidyl prolyl cis/trans isomerase (PPIase)-dependent mechanism for bacterial tissue invasion as well as a model for alveolar damage and bacterial spread was proposed (Wagner et al., 2007). The virulence protein Mip binds collagen and renders the extracellular matrix protease sensitive in a PPIase-dependent way. An as yet not further defined serine protease would contribute to the perforation of the basal membrane allowing bacterial transmigration. We hypothesize that Lpa might fulfil the role of this additional protease. We report here, to our knowledge for the first time, that L. pneumophila produces a protein with the capacity to convert plasminogen to plasmin. Whether this protein is important for the virulence of L. pneumophila will be the issue of further research.
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ACKNOWLEDGEMENTS |
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Edited by: J. Tommassen
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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Received 29 May 2007;
revised 13 August 2007;
accepted 14 August 2007.
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); L. pneumophila/pMMBNlpa both non-induced (
) and induced with 50 µM IPTG (
); (b) E. coli BL21* (
), L. pneumophila 
) and L. pneumophila/pMMBNlpa (50 µM IPTG) (
