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
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ABSTRACT |
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INTRODUCTION |
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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 SidAH 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.
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METHODS |
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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 510 kb EcoRI fragments in the vector pMMB207. The library was amplified in the E. coli host strain DH5
, 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 45 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
b were used for comparison. Within 714 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 ml1 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 ml1. 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 MannWhitney test, taking values of <0·05 as significant.
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RESULTS |
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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
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 714 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/pG4487) grow at 10100-fold higher dilutions than the corresponding icm mutant strains harbouring the empty library plasmid pMMB207
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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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 110 ORFs (Table 3
).
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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 pG34pG78) 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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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 pG34pG78 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 pG34G78). 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 45 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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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/pLcsAD 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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DISCUSSION |
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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 1718 % 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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The remote structural similarity of LcsC/LpxB and MurG corresponds to the fact that both glycosyltransferases catalyse the synthesis of a -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
-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
-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-
-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).
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ACKNOWLEDGEMENTS |
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Received 18 August 2004;
revised 13 October 2004;
accepted 14 October 2004.
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