The amoebae plate test implicates a paralogue of lpxB in the interaction of Legionella pneumophila with Acanthamoeba castellanii

Urs Albers1, Katrin Reus1, Howard A. Shuman2 and Hubert Hilbi1

1 Institute of Microbiology, Swiss Federal Institute of Technology (ETH), Wolfgang-Pauli Str. 10, HCI G405, 8093 Zürich, Switzerland
2 Department of Microbiology, College of Physicians and Surgeons, Columbia University, 701 West 168th Street, New York, NY 10032, USA

Correspondence
Hubert Hilbi
hilbi{at}micro.biol.ethz.ch


   ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Legionella pneumophila is a bacterial parasite of freshwater amoebae which also grows in alveolar macrophages and thus causes the potentially fatal pneumonia Legionnaires' disease. Intracellular growth within amoebae and macrophages is mechanistically similar and requires the Icm/Dot type IV secretion system. This paper reports the development of an assay, the amoebae plate test (APT), to analyse growth of L. pneumophila wild-type and icm/dot mutant strains spotted on agar plates in the presence of Acanthamoeba castellanii. In the APT, wild-type L. pneumophila formed robust colonies even at high dilutions, icmT, -R, -P or dotB mutants failed to grow, and icmS or -G mutants were partially growth defective. The icmS or icmG mutant strains were used to screen an L. pneumophila chromosomal library for genes that suppress the growth defect in the presence of the amoebae. An icmS suppressor plasmid was isolated that harboured the icmS and flanking icm genes, indicating that this plasmid complements the intracellular growth defect of the mutant. In contrast, different icmG suppressor plasmids rendered the icmG mutant more cytotoxic for A. castellanii without enhancing intracellular multiplication in amoebae or RAW264.7 macrophages. Deletion of individual genes in the suppressor plasmids inserts identified lcs (Legionella cytotoxic suppressor) -A, -B, -C and -D as being required for enhanced cytotoxicity of an icmG mutant strain. The corresponding proteins show sequence similarity to hydrolases, NlpD-related metalloproteases, lipid A disaccharide synthases and ABC transporters, respectively. Overexpression of LcsC, a putative paralogue of the lipid A disaccharide synthase LpxB, increased cytotoxicity of an icmG mutant but not that of other icm/dot or rpoS mutant strains against A. castellanii. Based on sequence comparison and chromosomal location, lcsB and lcsC probably encode enzymes involved in cell wall maintenance and peptidoglycan metabolism. The APT established here may prove useful to identify other bacterial factors relevant for interactions with amoeba hosts.


Abbreviations: APT, amoebae plate test; CYE, charcoal-yeast extract; FCS, fetal calf serum; icm/dot, intracellular multiplication/defective organelle trafficking; lcs, Legionella cytotoxic suppressor; PI, propidium iodide; PYG, proteose-yeast extract-glucose


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The Gram-negative bacterium Legionella pneumophila is a natural parasite of freshwater amoebae (Rowbotham, 1980; reviewed by Steinert et al., 2002). If inhaled via contaminated aerosols, the bacteria may grow in human alveolar macrophages and cause a severe pneumonia known as Legionnaires' disease. Within phagocytic host cells, L. pneumophila establishes a pH-neutral vacuole that does not fuse with lysosomes (Horwitz & Maxfield, 1984; Horwitz, 1983a). This unique, replication-permissive vacuole associates with smooth vesicles, mitochondria and endoplasmic reticulum (ER) (Horwitz, 1983b; Swanson & Isberg, 1995; Tilney et al., 2001) and recruits early secretory vesicles at ER exit sites (Kagan & Roy, 2002; Kagan et al., 2004).

Intracellular growth in macrophages and amoebae, including Acanthamoeba castellanii and Dictyostelium discoideum, is mechanistically similar and requires the L. pneumophila Icm/Dot transporter, a type IV secretion apparatus related to conjugation systems (Hagele et al., 2000; Otto et al., 2004; Segal et al., 1998; Segal & Shuman, 1999b; Solomon et al., 2000; Vogel et al., 1998). The Icm/Dot secretion system determines the initial contact of L. pneumophila with host cells and phagosome biogenesis (Hilbi et al., 2001; Watarai et al., 2001), is required to evade immediate endocytic maturation (Roy et al., 1998; Wiater et al., 1998) and governs subsequent formation of the ER-derived, replicative vacuole (reviewed by Nagai & Roy, 2003). Once L. pneumophila resides in this nutritionally rich compartment, the vacuole may acidify (Sturgill-Koszycki & Swanson, 2000), and bacterial replication apparently proceeds without requiring a functional Icm/Dot transporter (Coers et al., 1999).

Most of the genes of the icm/dot loci are predicted to encode membrane-spanning proteins (Segal & Shuman, 1999a). Interestingly, the IcmG protein contains a t-SNARE domain, and thus this membrane protein might play a direct role in altering host cell vesicle trafficking (Morozova et al., 2004). Biochemical analysis of the soluble Icm proteins revealed that IcmS/IcmW and IcmR/IcmQ directly bind to each other (Coers et al., 2000). IcmR functions as a chaperone of IcmQ, preventing and reversing its aggregation into high-molecular-mass complexes that form pores in lipid membranes (Duménil & Isberg, 2001; Duménil et al., 2004). Intracellular multiplication of and host cell killing by L. pneumophila is inhibited by a functional plasmid mobilization system, suggesting that the nucleoprotein conjugal substrate competes with virulence effectors for transport by the Icm/Dot machinery (Segal & Shuman, 1998). Only recently, Icm/Dot-transported proteins have been identified. RalF is translocated into the host cell and acts as a guanine nucleotide exchange factor that recruits the small GTPase ARF1 to the L. pneumophila phagosome (Nagai et al., 2002). LepA and LepB share sequence similarity with SNAREs and seem to mediate the Icm/Dot-dependent release of L. pneumophila-containing vesicles from amoebae (Chen et al., 2004). Other Icm/Dot-translocated proteins are LidA, which by an unknown mechanism contributes to an efficient formation of the replication vacuole (Conover et al., 2003), and the SidA–H proteins, many of which comprise families of up to five paralogues (Luo & Isberg, 2004). Secretion into culture supernatants of the polytopic membrane protein DotA requires the Icm/Dot secretion system but neither IcmS nor IcmW, and it might lead to the formation of pores in target membranes (Nagai & Roy, 2001).

The Icm/Dot-dependent establishment of the L. pneumophila vacuole eventually leads to host cell death due to intracellular replication and lysis. Additionally, the icm/dot genes mediate cytotoxic events such as (i) contact-dependent immediate cytotoxicity due to pore formation (Kirby et al., 1998; Zuckman et al., 1999), (ii) induction of apoptosis (Zink et al., 2002), and (iii) egress of the bacteria from the vacuole of the spent host cell (Molmeret et al., 2002). Other cytotoxic factors of L. pneumophila include legiolysin (Wintermeyer et al., 1991), the zinc metalloprotease Msp (Quinn & Tompkins, 1989; Szeto & Shuman, 1990), and RtxA, a member of the RTX (repeats in toxin) family of cytotoxic adhesins (Cirillo et al., 2001).

In this report, we describe the amoebae plate test (APT), a novel assay to analyse growth of L. pneumophila wild-type and icm/dot mutants on agar plates in the presence of A. castellanii. The APT was used to screen an L. pneumophila chromosomal library for multicopy suppressors of the partial growth defect of icmS or icmG mutant strains. Possible suppressors include genes that (i) enhance intracellular bacterial growth and thus kill the amoebae, (ii) are otherwise cytotoxic for the amoebae, or (iii) interfere with phagocytosis of the bacteria by the amoebae. Among the plasmids isolated, icm/dot region II and cytotoxic genes probably involved in peptidoglycan metabolism were identified.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains, cell culture and reagents.
The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli was cultured on LB agar plates or in LB broth, supplemented with chloramphenicol (Cm, 30 µg ml–1), sodium ampicillin (Ap, 100 µg ml–1) or kanamycin sulfate (Km, 50 µg ml–1) if required. Legionella pneumophila was grown on charcoal-yeast extract (CYE) agar plates (Feeley et al., 1979) or in ACES-buffered-yeast extract broth (AYE) containing 0·5 % bovine serum albumin (BSA) (Horwitz & Silverstein, 1983). Supplements for L. pneumophila were used at the following concentrations: Cm, 5 µg ml–1; Km, 50 µg ml–1; IPTG, 0·5 mM. Acanthamoeba castellanii (ATCC 30234) was grown in proteose-yeast extract-glucose medium (PYG) at 30 °C (Moffat & Tompkins, 1992; Segal & Shuman, 1999b) and subcultured once or twice a week. RAW264.7 macrophages (kindly supplied by Dr David Underhill, University of Washington, Seattle, WA, USA) were cultivated in RPMI1640 medium supplemented with 10 % fetal calf serum (FCS) and 2 mM L-glutamine at 37 °C in 5 % CO2. High-gel-strength agar was from Serva, proteose peptone from Becton Dickinson Biosciences and Bacto yeast extract from Difco. RPMI medium, glutamine and FCS were from Omnilab. All other reagents were from Sigma.


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Table 1. Bacterial strains and plasmids

 
Construction of plasmids.
Plasmids were isolated using commercially available kits from Qiagen or Macherey-Nagel, and DNA manipulations were performed according to standard protocols. In-frame deletions of single or several ORFs in the inserts of the suppressor plasmids pG34, pG54, pG65 and pG66 yielded the plasmids pUA3, pUA9, pUA11, pUA17 and pUA27 (Table 1; see also Fig. 5a) and were generated by PCR (Imai et al., 1991) after subcloning the inserts into pUC19 using the EcoRI restriction sites. To generate the deletion constructs, the oligonucleotides listed in Table 2 were used. The PCR fragments were phosphorylated, circularized, transformed into E. coli TOP10, checked by restriction digestion, and the inserts were finally cloned back into pMMB207 using the EcoRI restriction sites, yielding pUA5, pUA13, pUA15, pUA19 and pUA29. Alternatively, ORF1-3 was deleted from pG39 or pG54 by digestion with HindIII or SphI (see Fig. 5a), respectively, and religated, thus yielding pUA20 and pUA22. To overexpress lcsA, -B, -C or -D, the genes were amplified by PCR using pG34, pG54, pG65 or pG66 as templates and cloned by digestion with EcoRI and BamHI or NdeI into the expression vector pMMB207-RBS, yielding pMMB207-RBSlcsA–D. pMMB207-RBS harbours the Ptac promoter and the ribosome-binding site from the T7 gene10 taken from pGS-GFP-04 (Hilbi et al., 2001). The sequence of all PCR fragments was confirmed by sequencing.



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Fig. 5. Identification of cytotoxic suppressor genes by deletion analysis. (a) Overview of the deletions (horizontal bars) generated in suppressor plasmid pG34 (lcsA), pG54 (lcsB, ORF1-3), pG65 (lcsC, fis) and pG66 (lcsD). (b) A. castellanii was infected with icmT or icmG mutant strains harbouring the empty library plasmid (pMMB), complemented icmG (icmG/picmG), suppressor strains (icmG/pG34, /pG54, /pG65, /pG66), or with the deletion strains indicated. Cytotoxicity was determined by PI uptake 2 days post-infection. The data shown are the means and standard deviations of the percentage of PI-positive A. castellanii counted in three different wells on a 24-well plate and are representative of at least three independent experiments.

 

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Table 2. Oligonucleotides

 
The amoebae plate test (APT).
In this test, A. castellanii is spread on CYE agar plates prior to spotting bacteria in serial dilutions onto these plates. Thus, the APT allows growth of bacteria to be determined on solid medium in the presence of amoebae. A. castellanii cultures were fed with PYG 2 days prior to an experiment. One day before the experiment, the medium was exchanged, and the amoebae were tapped off the tissue culture flask, spun down (5 min, 470 g) and resuspended in PYG (2·67x106 ml–1). Then 1·5 ml samples of suspended amoebae (or PYG only) were applied on a CYE/Cm agar plate, allowed to dry for 1–2 h in a laminar-flow hood and left at room temperature overnight. Wild-type and icm/dot mutant L. pneumophila strains used for the APT harboured the plasmid pMMB207{alpha}b, pMMB207{alpha}b-Km14 or the corresponding complementing plasmids listed in Table 1. For the APT, stationary-phase bacterial cultures (OD600 >4·5) were adjusted to an identical OD600, and series of tenfold dilutions in sterile H2O were prepared; 3 µl of stationary culture (approx. 107 bacteria) or 3 µl of each dilution were spotted onto the CYE/Cm plates and incubated for 5–7 days at 30 °C or 37 °C.

Screening of an L. pneumophila chromosomal library for icm/dot suppressors using the APT.
To perform a suppressor screen with the APT, the partially growth-defective icmS and icmG mutants were chosen as recipients for the L. pneumophila genomic library MW66 (Purcell & Shuman, 1998). MW66 harbours 5–10 kb EcoRI fragments in the vector pMMB207. The library was amplified in the E. coli host strain DH5{alpha}, isolated and electroporated into the conjugation-competent E. coli strain LW253. Mating of MW66 into the L. pneumophila icm mutant strains was done as described previously (Mintz & Shuman, 1987; Segal & Shuman, 1998). Briefly, LW253/MW66 grown on LB/Cm plates was suspended in LB, spun down and resuspended in M63 medium. Stationary-phase icmS or icmG mutants grown in liquid culture were washed and resuspended in M63 medium. Donor and recipient strains were incubated at a ratio of 1/10 on CYE plates (4 h, 37 °C), resuspended in 0·2 ml M63 medium, streaked on CYE/Cm/Km plates and incubated for 4–5 days at 37 °C. The icmS and icmG mutants harbouring the MW66 library were then suspended in AYE medium, spotted in serial dilutions onto CYE/Cm plates containing 4x106 A. castellanii and incubated at 30 °C. JR32 and the icmS and icmG mutants harbouring pMMB207{alpha}b were used for comparison. Within 7–14 days several colonies of icm mutants harbouring library plasmids appeared at dilutions where no parental icm mutant strains were detected. The APT-selected suppressor strains were grown to stationary phase for 24 h in AYE/Cm broth and spotted again onto CYE/Cm plates containing 4x106 A. castellanii. The MW66 library plasmids of suppressor strains recovered from the second round of selection by APT were analysed further. In preliminary experiments, the icmS and icmG mutants harbouring the MW66 library were spread at an m.o.i. of 1 simultaneously with 5x106 A. castellanii ml–1 in PYG on CYE agar plates and incubated at 30 °C. Colonies that appeared using this approach were subjected to a second round of selection as described above.

Analysis of suppressor plasmids.
The library plasmids of the suppressor strains were isolated, amplified in E. coli and grouped according to their EcoRI restriction pattern. A representative of each group was electroporated into the icmG or icmS mutant strain which it was isolated from and analysed again by the APT to exclude effects of chromosomal mutations. The suppressor plasmid inserts were partially sequenced using primers complementary to vector sequences at the 5' and 3' ends of the insert. The sequences obtained were mapped in the L. pneumophila genome (Chien et al., 2004; http://genome3.cpmc.columbia.edu/~legion/) and analysed for homologues of the ORFs identified.

Intracellular growth in A. castellanii and RAW264.7 macrophages.
For intracellular growth assays, A. castellanii (5x104 per well) or RAW264.7 macrophages (2x104 per well) were seeded onto a 96-well plate and allowed to adhere for 3 h or overnight, respectively. The phagocytes were infected with L. pneumophila grown to stationary phase in AYE medium for 24 h (m.o.i. 1, 880 g), and incubated at 30 °C (A. castellanii) or 37 °C (RAW264.7). Intracellular growth was quantified by plating appropriately diluted supernatant of the infected host cells on CYE agar plates at the time points indicated. RAW264.7 macrophages are derived from BALB/c mice, which are considered less susceptible to L. pneumophila than A/J mice, the established murine model of Legionnaires' disease. L. pneumophila does not replicate in peritoneal macrophages elicited from BALB/c mice (Yamamoto et al., 1988). However, bone-marrow-derived macrophages from BALB/c mice permit the intracellular replication of L. pneumophila (Wright et al., 2003), which corresponds to the observed permissiveness of RAW264.7 macrophages.

Cytotoxicity assay.
To determine cytotoxicity, 4x104 A. castellanii per well were seeded in PYG onto a 24-well plate the day prior to infection. On the day of infection, the PYG was replaced with Ac buffer (Moffat & Tompkins, 1992). Bacteria from plates 3 or 4 days old were resuspended and diluted in sterile water to infect the amoebae at an m.o.i. of 50 or 500. After spinning down the bacteria (880 g, 5 min), the plates were incubated at 30 °C. Two days post-infection, propidium iodide (PI) solution was added to the wells at a final concentration of 1 µg ml–1. After several minutes' incubation, the amoebae were viewed in brightfield or by epifluorescence with an inverse microscope (Zeiss Axiovert 200M, 20x objective). The percentage of dead (PI-positive) amoebae was determined by counting the number of total and fluorescent amoebae.

Computational and statistical analysis.
Translated nucleic acid databases or the conserved domain database (Marchler-Bauer et al., 2003) were searched using the TBLASTX or RPSBLAST algorithms, respectively (Altschul et al., 1997). The COG database is maintained by Tatusov et al. (2001). Multiple amino acid sequence alignments were created with the CLUSTALW algorithm. Statistical analysis was done using the Mann–Whitney test, taking values of <0·05 as significant.


   RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Growth of L. pneumophila on CYE agar plates in the presence of A. castellanii – the amoebae plate test
Intracellular growth of L. pneumophila within A. castellanii requires the icm/dot genes (Segal & Shuman, 1999b). Here, we establish a novel assay, the amoebae plate test (APT) to analyse growth of wild-type and icm/dot mutant L. pneumophila on CYE agar plates in the presence of A. castellanii. Stationary-phase cultures of L. pneumophila wild-type strain JR32 and icm/dot mutants harbouring empty or complementing plasmids were spotted in tenfold serial dilutions onto agar plates in the presence or, as a control, absence of amoebae (Fig. 1). Within 5–7 days at 30 °C, wild-type L. pneumophila strain JR32 formed robust colonies even at a 10–5 dilution (m.o.i. approximately 0·02). Under these conditions, the icmT, -R, -O, -P, -C and dotB mutant strains did not grow at all. The icmS and -G mutant strains showed a partial growth defect in the presence of A. castellanii and eventually were lysed by the amoebae. An icmN mutant initially formed colonies on amoebae plates similar to wild-type L. pneumophila (data not shown). However, whereas JR32 survived on CYE agar plates in the presence of the amoebae for a prolonged time at room temperature, the icmN mutant was lysed by 14 days. Supplying the corresponding genes on a plasmid restored growth of the icm mutants almost to wild-type level (icmT, -S, -P, -O, -R, dotB) or partially (icmG, -C). In the absence of amoebae, all strains grew equally well on CYE agar plates.



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Fig. 1. Growth of wild-type L. pneumophila and representative icm/dot mutants in the APT. L. pneumophila wild-type strain JR32 and icmT, -S, -R, -P, -G and dotB mutants harbouring empty (pMMB) or corresponding complementing plasmids (picmT, -S, -R, -P, -G, dotB) were spotted in tenfold serial dilutions onto CYE agar plates in the presence or absence of A. castellanii and incubated for 5 days at 30 °C as described in Methods. The experiments were done at least three times and results similar to those shown were obtained.

 
At 37 °C, the icmS mutant grew similarly to wild-type and the icmN mutant in the APT. At this temperature, the icmT mutant did not grow at all and the icmR, -P, -O, -G, -C mutants grew only at the highest concentration. The degree of complementation of individual icm mutants was the same as at 30 °C (data not shown).

Isolation of L. pneumophila icm/dot suppressor strains using the APT
Among the icm/dot mutant strains tested, only the icmS and -G mutants showed a partial, rather than complete, growth defect in the APT at 30 °C. A partial growth defect for the icmS and -G mutant strains was shown previously using HL-60 macrophages as host cells (Purcell & Shuman, 1998; Segal & Shuman, 1997, 1999b). We reasoned that this phenotype would increase the chances to identify, in a multicopy suppressor screen, genes that increase cytotoxicity or enhance intracellular growth of the mutants. Therefore, the icmS and -G mutants were chosen as recipients for the L. pneumophila genomic library MW66 (Purcell & Shuman, 1998). The library MW66 was moved by electroporation from E. coli DH5{alpha} into the conjugation-competent E. coli strain LW253 and mated into the L. pneumophila icmS or -G mutants. The icmS and -G mutants harbouring the library MW66 were then spotted in serial dilutions onto CYE plates containing Cm and A. castellanii. The plates were incubated at 30 °C or 37 °C, respectively.

At 37 °C, the selection was not promising, since at the same dilutions similar numbers of icm mutants harbouring the library and parental mutants were obtained. At 30 °C, however, several colonies of icm mutant strains harbouring MW66 appeared within 7–14 days at dilutions where no parental icm mutants were detected. These APT-selected strains were grown to stationary phase for 24 h in AYE/Cm broth and spotted in serial dilutions onto CYE/Cm/A. castellanii plates again. In the second round of selection, 54 icmG and 2 icmS suppressor strains were recovered, representatives of which are shown in Fig. 2(a). The suppressor strains (icmS/pS37, icmG/pG44–87) grow at 10–100-fold higher dilutions than the corresponding icm mutant strains harbouring the empty library plasmid pMMB207{alpha}b (icmS/pMMB, icmG/pMMB), but not as vigorously as wild-type L. pneumophila (JR32/pMMB) or complemented icm mutants (icmS/picmS, icmG/picmG). In the absence of the amoebae, all strains grew equally well (data not shown).



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Fig. 2. Suppressor strains isolated by APT. (a) L. pneumophila icmS and icmG suppressor strains harbouring Legionella chromosomal library plasmids (pS37, pG44–pG87) were isolated by APT, grown to stationary phase and retested by APT. Representative suppressor strains are shown in comparison with wild-type, icmG and icmS mutant strains harbouring the empty library plasmid (pMMB) and corresponding complemented strains (picmG, picmS). The APT was repeated twice with strains isolated in a first round of selection, and similar results were obtained. (b) Growth defect suppression in an APT by L. pneumophila icmS or icmG mutants transformed freshly with isolated suppressor plasmids (pS37, pG34, pG54, pG65).

 
The library plasmids of the suppressor strains were isolated, amplified in E. coli and grouped according to their EcoRI restriction pattern into 18 groups (1 icmS, 17 icmG background; data not shown). A representative plasmid of each group was electroporated into the icmG or -S mutant strain which it was isolated from, and analysed by APT. The strains thus obtained grew better in the APT than the parental icm mutant harbouring an empty library plasmid, indicating that suppression of the growth defect in the presence of amoebae was caused by the library plasmid and not due to a second-site mutation in the bacterial chromosome. Representative strains belonging to different EcoRI restriction groups are shown in Fig. 2(b). Retransformation of an icmS mutant strain with plasmid pS37 led to growth in the APT similar to that of wild-type L. pneumophila, consistent with the finding that pS37 complements the icmS mutant strain (see below).

Analysis of suppressor plasmid inserts
The inserts from representative suppressor plasmids were partially sequenced and mapped in the L. pneumophila strain Philadelphia genome (http://genome3.cpmc.columbia.edu/~legion/). Some of the inserts were identical (pS36/pS37; pG54/pG58/pG63; pG65/pG72; pG71/pG79/pG83), and the inserts of the suppressor plasmids pG47 and pG61 were independently selected twice, since they covered the same loci in the L. pneumophila genome but were of different length (1·8 kb and 2·5 kb, respectively). The inserts of the suppressor plasmids were analysed by BLASTX searches for homologues to ORFs, and 12 different inserts were identified (pS37, pG34, pG39, pG41, pG47, pG53, pG54, pG65, pG66, pG67, pG71 and pG78), each harbouring 1–10 ORFs (Table 3).


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Table 3. Suppressor plasmids identified in this study

 
The insert of the icmS suppressor plasmid pS37 contains a 9·0 kb fragment of icm/dot region II. The fragment spans a region between 1·7 kb upstream of the icmT and the icmL gene and includes the icmS gene. This result indicates that pS37 complements the icmS mutant, and thus increased growth of the icmS/pS37 strain in the APT is due to intracellular growth within and killing of the amoebae. In contrast, the ORFs identified in the inserts of the 11 different icmG suppressor plasmids did not immediately suggest a mechanism accounting for their growth defect suppression. The suppressor strains might grow better on agar plates in the presence of amoebae because of more efficient intracellular replication and host cell killing or as a result of a cytotoxic activity not related to intracellular replication. To test these possibilities, intracellular growth of the icmG suppressor strains within A. castellanii or murine RAW264.7 macrophages and cytotoxicity towards amoebae was determined.

Intracellular growth of icmG suppressor strains in A. castellanii and macrophages
Intracellular growth of the icmG suppressor strains was assayed by quantifying bacteria released into the supernatant from A. castellanii infected with bacteria at an m.o.i. of 1. To match the conditions used for the APT, the infected amoebae were incubated for 4 days at 30 °C, rather than at 37 °C as described previously (Segal & Shuman, 1999b). At 30 °C, the number of wild-type L. pneumophila or icmG mutant bacteria harbouring the empty library plasmid increased within 3 days, by about 4 or 1 orders of magnitude, respectively (Fig. 3a). Supplying the icmG gene on a plasmid only partially complemented the intracellular growth defect, as was observed previously (Segal & Shuman, 1999b). None of the 11 icmG suppressor strains (harbouring pG34–pG78) grew better than the icmG mutant, ruling out that the growth defect suppression observed in the APT was due to enhanced intracellular growth. Rather, we noted that the suppressor strains tended to grow even worse than the icmG mutant strain. We also performed an intracellular growth assay with a high m.o.i. of 500. Under these conditions, wild-type L. pneumophila grew 2 orders of magnitude within 3 days (data not shown). In this experiment, the relative growth of the wild-type and icmG mutant strains was the same as above, and the 11 icmG suppressor strains tested also grew worse than the icmG mutant. At 37 °C, the suppressor strains icmG/pG54 or icmG/pG65 did also not grow better in amoebae (m.o.i. of 1) than an icmG mutant strain harbouring the empty library plasmid (data not shown).



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Fig. 3. Intracellular multiplication of icmG suppressor strains within A. castellanii and macrophages. Intracellular growth of the icmG suppressor strains was assayed using (a) A. castellanii or (b) murine RAW264.7 macrophages as host cells. The phagocytes were infected at an m.o.i. of 1, and bacteria released into the supernatant were quantified at the time indicated. Growth of suppressor strains icmG/pG34–pG78 ({circ}), an icmG mutant harbouring the empty library plasmid ({triangleup}), or plasmid-encoded icmG ({blacktriangleup}) and wild-type strain JR32 ({blacksquare}) is shown. The experiments were repeated at least twice at two different m.o.i.'s each, and similar results were obtained.

 
To analyse intracellular multiplication of the 11 icmG suppressor strains in macrophages, the murine macrophage cell line RAW264.7 was used. Within 4 days, the number of wild-type or icmG mutant bacteria released into the supernatant increased 4 or 3 orders of magnitude, respectively (Fig. 3b). Supplying the icmG gene on a plasmid complemented growth of the icmG mutant almost to wild-type level. In RAW264.7 macrophages, the icmG suppressor strains (icmG/pG34–pG78) grew worse than the icmG mutant strain, similar to what was observed for growth of these strains in A. castellanii. Taken together, the results indicate that growth defect suppression of the icmG suppressor strains is not due to enhanced intracellular growth within phagocytes.

icmG suppressor strains are cytotoxic for A. castellanii
Since compared to the parental icmG mutant strain, the icmG suppressor strains did not grow better in amoebae or macrophages, we tested whether the suppressor plasmids pG34–pG78 confer increased cytotoxicity to icmG mutants. A. castellanii was infected with wild-type L. pneumophila, an icmG mutant harbouring the empty library plasmid, a complemented icmG mutant, or the icmG suppressor strains (harbouring pG34–G78). Cytotoxicity was determined by PI uptake 2 days post-infection. Under the conditions described, the amoebae infected with either wild-type L. pneumophila or the complemented icmG mutant rounded up and were all dead as judged by their complete disintegration or by PI uptake (Fig. 4a). In contrast, amoebae infected with an icmT mutant harbouring the vector control all survived and remained spread out and firmly attached. Amoebae infected with an icmG mutant harbouring the vector control rounded up, but only about 15 % stained with PI. Most of the suppressor strains were found to be significantly more cytotoxic than the icmG mutant strain, thus providing a rationale for their isolation in the suppressor screen (Fig. 4b). Notably, icmG mutants harbouring pG34, pG39, pG41, pG54 or pG66 showed an increased cytotoxicity in 4–5 out of 5 independent experiments. The suppressor strains icmG/pG47, /pG53, /pG65 and /pG71 were more cytotoxic than the icmG mutant in some experiments, and icmG/pG67 and /pG78 did not show increased cytotoxicity in any of the experiments performed. At 37 °C, the suppressor strains icmG/pG54 or icmG/pG65 were cytotoxic for amoebae to the same extent as at 30 °C (data not shown).



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Fig. 4. Suppressor strains are cytotoxic for A. castellanii. (a) A. castellanii was infected with wild-type L. pneumophila, icmT or icmG mutant strains harbouring the empty library plasmid (pMMB) or with suppressor strains (icmG/pG34–pG78). Cytotoxicity was determined by PI uptake 2 days post-infection as described in Methods. (b) The data shown are the means and standard deviations of the percentage of PI-positive A. castellanii counted in three different wells on a 24-well plate and are representative of at least five independent experiments.

 
Identification of cytotoxic suppressor genes by deletion analysis
To identify cytotoxic suppressor genes, individual or several ORFs were deleted in the suppressor plasmid inserts, and cytotoxicity was assessed by the PI uptake assay. For the deletion analysis, we focused on the cytotoxic suppressor plasmids pG34, pG39, pG41, pG47, pG54, pG65 and pG66, and only complete ORFs were considered. The suppressor plasmid pG41 was not studied further since the encoded DNA-interacting proteins (integration host factor, histone methyltransferase) presumably increase cytotoxicity unspecifically. Deletion of the LuxR-type transcription factor in pG47 did not decrease cytotoxicity (data not shown), and therefore this plasmid was not analysed further. Deletion of all the complete ORFs in the insert of pG39 also did not decrease cytotoxicity, but left the fragment of an ORF encoding the C-terminus of a putative lytic murein transglycosylase (data not shown; see Discussion). These findings and considerations left the cytotoxic suppressor plasmids pG34, pG54, pG65 and pG66 to be analysed in detail.

The inserts of the plasmids pG34, pG54, pG65 and pG66 harbour homologues of hydrolases, NlpD-like metalloproteases, lipid A disaccharide synthases and ABC transporters, respectively (Table 3, Fig. 5a). Deletion of the genes encoding the hydrolase, NlpD-like metalloprotease, lipid A disaccharide synthase or ABC transporter by a PCR-based method or by restriction enzyme digestion reproducibly reduced cytotoxicity, indicating that the corresponding proteins are required for the enhanced cytotoxicity of the suppressor plasmids (Fig. 5b). The genes were termed lcs (Legionella cytotoxic suppressors) -A (hydrolase), -B (NlpD-like metalloprotease), -C (lipid A disaccharide synthase homologue) and -D (ABC transporter). Deletion of ORF1-3 in the insert of pG54 also decreased cytotoxicity, presumably because lcsB forms an operon with ORF3 and consequently will not be expressed in the pG54-ORF1-3 deletion mutant. Deletion of the DNA-binding regulator protein Fis (factor of inversion stimulation) substantially increased cytotoxicity of plasmid pG65-fis carrying lcsC as the only remaining complete ORF (Fig. 5b) and, as expected, deletion of lcsC and fis in the insert of suppressor plasmid pG65 abolished cytotoxicity (data not shown).

Overexpression of suppressor genes
Prompted by the finding that strain icmG/pG65-fis showed enhanced cytotoxicity compared to icmG/pG65, we cloned lcsA, -B, -C, and -D into the expression vector pMMB207-RBS, overexpressed the proteins under the control of the Ptac promoter, and quantified cytotoxicity of icmG mutant strains harbouring these plasmids by PI uptake. Interestingly, the strain icmG/pLcsC, overexpressing the LpxB homologue, was 9 or 4 times more cytotoxic than icmG/pMMB or icmG/pG65, respectively (Fig. 6a, b), suggesting that LcsC-induced cytotoxicity might be dose-dependent. The icmG/pLcsC strain did not grow more efficiently in A. castellanii (30 °C, 37 °C) or in RAW264.7 macrophages compared to icmG/pMMB (data not shown). In contrast, icmG mutants expressing the putative hydrolase LcsA, NlpD-like metalloprotease LcsB or the ABC transporter LcsD were less cytotoxic than the icmG mutant strain. We noted, however, that induction of LcsB prevented growth of the bacteria, and induction of LcsC and LcsD resulted in fewer and smaller bacterial colonies, indicating that overexpression of these proteins is toxic for L. pneumophila (data not shown). In agreement with this assumption, the strains icmG/pLcsA–D grew well on agar plates, if IPTG was omitted, and therefore the genes were expressed only at low levels from the Ptac promoter. Under these conditions, strain icmG/pLcsC was consistently more cytotoxic than the parental suppressor strain icmG/pG65 in three independent experiments (data not shown). Strain icmG/pLcsB was more cytotoxic than the negative control icmG/pMMB207 but not as cytotoxic as the parental suppressor strain icmG/pG54. Finally, the strains icmG/pLcsA and icmG/pLcsD were not cytotoxic for the amoebae (data not shown).



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Fig. 6. Overexpression of LcsC is cytotoxic for A. castellanii in an icmG but not in other icm or rpoS mutants. A. castellanii was infected with the icm (a–c) or rpoS (c) mutant strains indicated harbouring the empty library plasmid (pMMB), complementing plasmid (picmG), suppressor plasmids (pG34, pG54, pG65, pG66), or expression plasmids (pLcsA–D). Cytotoxicity was determined by PI uptake 2 days post-infection. The data shown are the means and standard deviations of the percentage of PI-positive A. castellanii counted in three different wells on a 24-well plate and are representative of at least three independent experiments.

 
In the presence of IPTG LcsC was cytotoxic for A. castellanii only if overexpressed in an icmG but not in an icmS, -F or rpoS mutant background (Fig. 6c), even though the icmF as well as the rpoS mutant strains harbouring the empty plasmid pMMB207 were cytotoxic for the amoebae to a similar extent as the icmG mutant. Like the icmS mutant strain, the icmC, -E, -O, -R, -T, -W, or dotB mutants overexpressing LcsC were not cytotoxic for amoebae (data not shown). Moreover, about 30 % of A. castellanii infected with wild-type L. pneumophila (m.o.i. of 5) harbouring either an empty plasmid or pLcsC were killed within 2 days, and thus LcsC apparently did not increase cytotoxicity of wild-type L. pneumophila against the amoebae (data not shown).


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Screen for icm suppressors using the APT
In this paper we describe a novel assay, the amoebae plate test (APT), to determine growth of bacteria on CYE agar plates in the presence of amoebae. Wild-type L. pneumophila replicates within and kills amoebae, and thus grows on solid medium. In contrast, icm/dot mutant strains are defective for intracellular growth, are killed by the amoebae and do not form colonies on agar plates (Fig. 1). The APT was routinely done at 30 °C, allowing the amoebae to feed on the icm/dot mutant strains, as judged from the observation that most mutants did not form colonies under these conditions. At 37 °C, the icmS and other icm/dot mutants grew better than at 30 °C in the presence of amoebae. Possible explanations for this finding include (i) the bacteria grow faster on agar plates at 37 °C and thus gain an advantage over the amoebae in the putative competition between growth on solid medium and being phagocytosed, or (ii) the amoebae are stressed at a temperature far from environmental conditions, and therefore are less phagocytic or less bactericidal.

In screens using the partially growth-defective icmS or icmG mutants, we identified icm/dot region II complementing the icmS mutant, and isolated several plasmids that conferred increased cytotoxicity to an icmG mutant, without alleviating its intracellular growth defect (Figs 3 and 4). Some cytotoxic icmG suppressor genes were identified by deletion analysis (lcsAD, Legionella cytotoxic suppressor); these encode a putative hydrolase, an NlpD-like metalloprotease, a lipid A disaccharide synthase homologue and an ABC transporter, respectively (Fig. 5, Table 3). To obtain clues about the cellular pathways which these genes participate in, we inspected their vicinity in the L. pneumophila genome and searched the conserved domain and translated nucleic acid databases. The lcsB and lcsC genes were found to be of particular interest, since both genes may be involved in peptidoglycan metabolism. Another gene possibly involved in cell wall degradation is a lytic murein transglycosylase (COG2821), a fragment of which is encoded by the cytotoxic suppressor plasmid pG39 (Table 3). Deletion of ORF1-3 of plasmid pG39 left only the truncated lytic murein transglycosylase downstream of the Ptac promoter. This construct was still cytotoxic in an icmG mutant background, possibly due to expression of a cytotoxic 242 amino acid C-terminal enzyme fragment by using the Ptac promoter and the first internal ATG as a start codon.

LcsB, a homologue of NlpD-like membrane-bound metalloproteases
LcsB is 26 % identical to the E. coli YibP protease and 17–18 % identical to the NlpD orthologues from L. pneumophila and E. coli (Fig. 7). The C-terminal domains of the four proteins belong to COG4942 (membrane-bound metallopeptidases, involved in cell division and chromosome partitioning) and are characteristic for members of the M23/M37 family (Pfam01551) of putative zinc metallo-endopeptidases from Gram-negative and Gram-positive bacteria (http://www.sanger.ac.uk/Software/Pfam/). The M37 protease family includes staphylococcal glycylglycine endopeptidases that hydrolyse the polyglycine interpeptide bridges of peptidoglycan of Gram-positive bacteria (Ramadurai & Jayaswal, 1997; Ramadurai et al., 1999; Recsei et al., 1987; Sugai et al., 1997) and the E. coli lipoprotein NlpD that probably functions in cell wall formation and maintenance of Gram-negative bacteria (Ichikawa et al., 1994; Lange & Hengge-Aronis, 1994). In E. coli as well as in L. pneumophila, the nlpD gene is located immediately upstream of the rpoS gene encoding the stationary growth phase transcription factor RpoS (Hales & Shuman, 1999). Among the proteins found in the databases, L. pneumophila LcsB is most closely related to E. coli YibP. Purified YibP has endoprotease activity which is inhibited by EDTA, and a fraction of the protein is membrane-bound (Ichimura et al., 2002). A chromosomal yibP deletion mutant is defective for cell division, FtsZ ring formation and growth at 42 °C but not at 37 °C. At the non-permissive temperature, the yibP mutant forms filamentous, multi-nucleoided cells that tend to lyse, indicating that YibP is required for cell wall degradation and proper cell division.



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Fig. 7. Alignment of L. pneumophila LcsB, E. coli YibP and the membrane-bound metalloproteases NlpD from L. pneumophila and E. coli.

 
LcsC, a paralogue of the lipid A disaccharide synthase LpxB
LcsC is homologous to lipid A disaccharide synthases (LpxB) and, as determined by searching the conserved domain database, distantly to MurG glycosyltransferases involved in peptidoglycan synthesis (COG0707). Interestingly, the genome of L. pneumophila Philadelphia-1 harbours two lipid A disaccharide synthase paralogues which are 42 % or 30 % identical to E. coli LpxB, respectively (Fig. 8). The gene more closely related to E. coli lpxB is most likely the L. pneumophila lpxB orthologue since it is located within a cluster encoding components of the lipopolysaccharide (LPS) biosynthesis pathway, including a neuraminidase, lauroyl/myristoyl acyltransferase, LpxA, LpxD, LpxB and the ABC transporter WlaB. The arrangement of the lpx gene cluster is similar in E. coli, where lpxB is located downstream of lpxA, fabZ and lpxD (Raetz, 1996).



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Fig. 8. Alignment of L. pneumophila LcsC with lipid A disaccharide synthase (LpxB) homologues from L. pneumophila and E. coli.

 
In the L. pneumophila genome, the lcsC gene appears to form an operon with an oxidoreductase/dehydrogenase gene and is separated from the putative rodAftsI operon by only one ORF. The latter genes encode the rod-shape-determining protein RodA and the peptidoglycan transglycosylase/peptidase FtsI. RodA and FtsI are membrane-bound, interact with each other and are required for cell envelope biogenesis and cell division, respectively (Begg et al., 1986). Analogously, in the E. coli genome, the murG glycosyltransferase gene is flanked by genes involved in peptidoglycan biosynthesis and cell division (Heijenoort, 1996). At present, we can not exclude the possibility that LcsC participates in lipid A biosynthesis. However, the vicinity of lcsC to rodAftsI, its lower homology to lpxB and its similarity to murG suggest that LcsC catalyses a disaccharide synthase/glycosyltransferase reaction in peptidoglycan biosynthesis.

The remote structural similarity of LcsC/LpxB and MurG corresponds to the fact that both glycosyltransferases catalyse the synthesis of a {beta}-linked lipodisaccharide moiety from an acylated uridine 5'-diphosphate (UDP)-D-glucosamine (GlcN) and a lipid, and both enzymes liberate UDP during the reaction. LpxB catalyses the {beta}-1'-6 condensation of UDP-2,3-diacyl-GlcN with lipid X (2,2-diacyl-GlcN-1-phosphate), and MurG couples N-acetyl-GlcN via a {beta}-1'-4 linkage to lipid I (N-acetyl-muramyl-pentapeptide-phosphoryl-undecaprenol) to form the lipodisaccharide lipid II that is the minimal subunit of peptidoglycan (Heijenoort, 1996).

Mechanism of LcsC cytotoxicity
Not only was cytotoxicity of an icmG mutant strain harbouring the suppressor plasmid pG65 decreased upon deletion of lcsC (Fig. 5), but overexpression of the gene in an icmG background enhanced cell death of A. castellanii (Fig. 6), suggesting that the product(s) of the LcsC enzyme are cytotoxic. While the structure of the toxic product of LcsC remains to be identified, it might either associate with bacteria or be released into the supernatant. Soluble cytotoxic peptidoglycan derivatives have been described for Bordetella pertussis, Neisseria gonorrhoeae, Haemophilus influenzae and other bacteria (Burroughs et al., 1993; Cloud & Dillard, 2002; Cookson et al., 1989; Luker et al., 1993, 1995). For example, the murein-derived tracheal cytotoxin (TCT; N-acetyl-GlcN-1,6-anhydro-N-acetyl-muramyl-L-ala-{gamma}-D-glu-meso-diaminopimelyl-D-Ala) from B. pertussis is released by growing bacteria and is sufficient to reproduce the cytopathology observed during whooping cough. The murein-derived toxins from N. gonorrhoeae or H. influenzae are also disaccharide muramyl tetrapeptides, i.e. they consist of the monomeric subunit of Gram-negative bacterial peptidoglycan.

Overexpression of LcsC apparently is cytotoxic for the amoebae specifically in an icmG mutant but not in other icm/dot mutants or an rpoS mutant background (Fig. 6c). One possibility to account for this observation would be that LcsC requires a functional Icm/Dot secretion system to exert cytotoxicity. Among the strains tested, the icmG, icmS, icmF, icmW, icmR and rpoS mutants are expected to form at least partially functional Icm/Dot secretion systems, since these mutants can grow intracellularly in macrophage cell lines (Coers et al., 2000; Hales & Shuman, 1999; Segal & Shuman, 1999b). Moreover, the icmG mutant is only partially defective for intracellular growth within A. castellanii at 30 °C (Fig. 4), the icmG and icmF mutants persist in D. discoideum (Otto et al., 2004), and the icmS and icmW mutants are not impaired in Icm/Dot-dependent immediate cytotoxicity (Coers et al., 2000; Zuckman et al., 1999). However, LcsC was cytotoxic specifically in an icmG mutant but neither in other mutant strains only partially defective for intracellular growth, nor in wild-type L. pneumophila (data not shown). Therefore, a (limited) functional Icm/Dot transporter is apparently not sufficient for LcsC cytotoxicity. In contrast to other icm/dot mutants, the icmG mutant grows to some extent in A. castellanii and persists in D. discoideum, suggesting that entry and/or intracellular trafficking of the icmG mutant differs from that in other icm/dot mutants as well as from that in wild-type L. pneumophila. It is feasible that overexpression of LcsC exerts cytotoxic effects only in certain cellular compartments. We are currently investigating entry and intracellular trafficking of the icmG mutant and effects of LcsC on these processes to establish a link between loss of IcmG (possibly involved in intracellular trafficking) and cytotoxicity of LcsC (a putative enzyme of peptidoglycan metabolism).


   ACKNOWLEDGEMENTS
 
We wish to thank Natalie Schlegel und Judith Zaugg for help with cloning. This work was supported by grants from the Swiss National Science Foundation (631-065952) and the Swiss Federal Institute of Technology (17/02-3).


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RESULTS
DISCUSSION
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Received 18 August 2004; revised 13 October 2004; accepted 14 October 2004.



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