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Infection and Immunity, October 2005, p. 6272-6282, Vol. 73, No. 10
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.10.6272-6282.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Characterization of Legionella pneumophila pmiA, a Gene Essential for Infectivity of Protozoa and Macrophages

Masaki Miyake,1* Takurou Watanabe,1 Hitomi Koike,1 Maëlle Molmeret,2 Yasuyuki Imai,1 and Yousef Abu Kwaik2

Department of Microbiology and COE Program in the 21st Century, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka-shi, Shizuoka 422-8526, Japan,1 Department of Microbiology and Immunology, University of Louisville College of Medicine, 319 Abraham Flexner Way 55A, Louisville, Kentucky 402022

Received 19 January 2005/ Returned for modification 15 April 2005/ Accepted 1 June 2005


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ABSTRACT
 
The ability of Legionella pneumophila to cause pneumonia is dependent on intracellular replication within alveolar macrophages. The Icm/Dot secretion apparatus is essential for the ability of L. pneumophila to evade endocytic fusion, to remodel the phagosome by the endoplasmic reticulum (ER), and to replicate intracellularly. Protozoan and macrophage infectivity (pmi) mutants of L. pneumophila, which include 11 dot/icm mutants, exhibit defects in intracellular growth and replication within both protozoa and macrophages. In this study we characterized one of the pmi loci, pmiA. In contrast to the parental strain, the pmiA mutant is defective in cytopathogenicity for protozoa and macrophages. This is a novel mutant that exhibits a partial defect in survival within U937 human macrophage-like cells but exhibits a severe growth defect within Acanthamoeba polyphaga, which results in elimination from this host. The intracellular defects of this mutant are complemented by the wild-type pmiA gene on a plasmid. In contrast to phagosomes harboring the wild-type strain, which exclude endosomal-lysosomal markers, the pmiA mutant-containing phagosomes acquire the late endosomal-lysosomal markers LAMP-1 and LAMP-2. In contrast to the parental strain-containing phagosomes that are remodeled by the ER, there was a decrease in the number of ER-remodeled phagosomes harboring the pmiA mutant. Among several Legionella species examined, the pmiA gene is specific for L. pneumophila. The predicted amino acid sequence of the PmiA protein suggests that it is a transmembrane protein with three membrane-spanning regions. PmiA is similar to several hypothetical proteins produced by bacteria with a type IV secretion apparatus. Importantly, the defect in pmiA abolishes the pore-forming activity, which has been attributed to the Icm/Dot type IV secretion system. However, the mutant is sensitive to NaCl, and this sensitivity is abrogated in the icm/dot mutants. These results suggest that PmiA is a novel virulence factor that is involved in intracellular survival and replication of L. pneumophila in macrophages and protozoan cells.


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INTRODUCTION
 
Legionella pneumophila is the causative agent of Legionnaires' disease (29, 50). The ability of L. pneumophila to grow within mammalian cells, such as monocytes and alveolar macrophages, is essential for pathogenicity in humans (1, 42). Protozoa are reservoirs of L. pneumophila in natural environments, which play a crucial role in bacterial ecology and transmission to humans (38, 54, 63). Growth of L. pneumophila within amoebae enhances invasion of epithelial cells and macrophages (20). Amoeba-grown L. pneumophila becomes resistant to various stress conditions, such as acid, oxidative, osmotic, and heat stresses (4), and becomes more resistant to antibiotics (11). These phenotypic modulations of L. pneumophila may contribute to bacterial survival in harsh environmental conditions and to invasion and replication within human phagocytic cells.

The Icm/Dot type IV secretion apparatus is a major virulence system in L. pneumophila, since it is essential for evasion of endocytic fusion and remodeling of the phagosome in a suitable replicative niche (69, 80). The Icm/Dot system is also essential for early activation of caspase-3 in the host cell, which does not result in apoptosis until late stages of the infection (6, 83). Activation of caspase-3 results in cleavage of the Rab5 effector rabaptin-5, and this cleavage may be involved in the ability of L. pneumophila-containing phagosome to evade endocytic fusion and to be remodeled by the endoplasmic reticulum (ER) (55). L. pneumophila utilizes the Icm/Dot type IV secretion apparatus for injection of bacterial effector proteins into the host cell, leading to the creation of unique vacuoles suitable for intracellular growth (22, 58). Several proteins that are substrates of the Icm/Dot secretion apparatus, such as LepAB, LidA, RalF, and SidC, have been identified (18, 23, 47, 57). Although the functions of these substrates are unclear, it is thought that they modulate host signal transductions to establish the replicative vacuoles, allowing the organism to survive and replicate within phagocytic cells. At 15 min postinfection, L. pneumophila is contained in a unique phagosome with a membrane thinner than the plasma membrane, and it is surrounded by mitochondria and the ER (1, 2, 5, 41, 75-77). The L. pneumophila-containing phagosome excludes endosomal and lysosomal markers. In contrast, phagosomes containing icm/dot mutants acquire endosomal and lysosomal markers, and the mutants are unable to replicate intracellularly (64, 67, 80). Interestingly, calnexin, Rab1, and Sec22b are acquired by phagosomes containing the wild-type strain shortly after bacterial uptake (24, 44). Upon formation of this unique replicative niche, the organism replicates in the phagosome. During late stages of the infection, the phagosomal membrane becomes disrupted, and the bacteria escape into the cytoplasm, where the last rounds of replication are completed prior to lysis of the host cell membrane and bacterial egress (7, 51-53). Interestingly, within gamma interferon-activated macrophages, the L. pneumophila-containing phagosome fuses to lysosomes, and it is not remodeled by the ER (66).

Among the components of the Icm/Dot secretion apparatus, IcmT is essential for pore formation-mediated egress from the host cell upon termination of intracellular replication, in addition to a function required for phagosome biogenesis and evasion of lysosomal fusion (7, 51, 52). IcmQ has pore-forming capacities involving insertion into host cell membranes to form a channel for translocation of effectors from the bacterium into the host cell (25).

Loci other than the icm/dot genes are required for intracellular replication of L. pneumophila. The stationary-phase sigma factor RpoS and the stress-induced protease/chaperone HtrA are essential for multiplication within Acanthamoeba polyphaga (34, 59). The Rep helicase is required for replication within human macrophages and epithelial cells (31, 37). The type II secretion system of L. pneumophila is required for intracellular infection (33, 61, 62). The pilD gene, which encodes the prepilin leader peptidase, is required for assembly of both a type IV pilus (72) and a type II secretory apparatus, and it is also required for bacterial replication within amoebae and human macrophages (46). The LetA/LetS two-component regulatory system is required for infection of macrophages (30, 35, 48). The csrA gene is involved in regulation of the bacterial switch from the replicative form to the transmissible form and is essential for intracellular growth within both macrophages and amoebae (28, 56). Iron acquisition and assimilation are key factors for L. pneumophila virulence and intracellular growth (39, 79). The lvgA and ptsP genes of L. pneumophila affect colonization of the lungs and spleens of guinea pigs (26, 40). Therefore, numerous loci other than the icm/dot genes play major roles in the ability of L. pneumophila to replicate intracellularly and cause disease.

We previously isolated 89 insertion mutants of L. pneumophila that exhibit defects in cytotoxicity, intracellular survival, and replication within both U937 macrophage-like cells and A. polyphaga. These mutants were collectively designated pmi (protozoan and macrophage infectivity) mutants (32). Although 11 of the mutants have insertions within the icm/dot genes, most of the mutated genes of these strains have not been identified.

In this study, we characterized one of the pmi mutant strains, GB112, and identified the mutated gene, pmiA. The pmiA gene is localized outside the two genomic regions that include the icm/dot genes. The pmiA gene likely encodes an L. pneumophila-specific virulence factor required for intracellular survival and growth within macrophages and protozoan cells. The defect in intracellular survival is associated with trafficking of the pmiA mutant-containing phagosomes through the default endosomal-lysosomal degradation pathway. The predicted amino acid sequence suggests that the gene product is a transmembrane protein. Functional analyses using mutants and gene complementation indicated that the mutation does not eliminate sodium sensitivity, which is one of the phenotypes dependent on the Icm/Dot secretion apparatus, but abrogates the pore-forming activity, which is dependent on a functional Icm/Dot secretion apparatus.


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MATERIALS AND METHODS
 
Bacterial strains and media. The Legionella strains used in this study were L. pneumophila serogroup 1 strain AA100 (3), L. pneumophila Philadelphia 1 JR32 (65), L. pneumophila Philadelphia 1 Lp02 (12), L. pneumophila dotA mutant LELA3118 (65), L. pneumophila icmT mutant GS3011 (70), L. pneumophila icmT mutant AA100kmT (52), L. pneumophila serogroup 5 strain GTC297, L. pneumophila serogroup 6 strain GTC748, L. pneumophila serogroup 7 strain GTC750, Legionella bozemanii GTC298, Legionella micdadei GTC299, Legionella dumoffii GTC303, Legionella brunesis GTC515, and Legionella gratiana GTC699. The GTC strains were obtained from the Gifu Type Culture Collection at Gifu University Graduate School of Medicine, Japan. The Legionella strains used in this study were grown on buffered charcoal-yeast extract (BCYE) plates or in buffered yeast extract (BYE) broth.

Cell culture. Macrophage-like U937 cells were maintained in RPMI 1640 (Sigma, Tokyo, Japan) supplemented with 10% fetal calf serum (ICN Biomedicals, Aurora, Ohio), as described previously (32). Axenic A. polyphaga was cultured as adherent cells in PYG medium. All cells were cultured under a humidified atmosphere containing 5% CO2 and 95% air at 37°C, as described previously (32).

Intracellular bacterial growth assay. U937 cells (1 x 105 cells per well in 96-well flat-bottom plates [Falcon, Becton Dickinson, Franklin Lakes, NJ]) were allowed to differentiate into macrophages by incubation for 48 h with 50 ng ml–1 of phorbol 12-myristate 13-acetate (Sigma, Tokyo, Japan) in RPMI 1640 containing 10% fetal calf serum. A. polyphaga cells were grown in a 96-well plate at a density of 1 x 105 cells per well in PYG medium. The bacterial strains were grown in BYE medium to an optical density at 550 nm (OD550) of 2.0 to 2.2 (post-exponential phase). The differentiated U937 cells or A. polyphaga cells were infected with the bacteria at a multiplicity of infection (MOI) of 10 in triplicate cultures. The plates were spun at 250 x g for 20 min to bring the bacteria into contact with the host cell monolayer. The time at the end of this centrifugation was designated zero time. Then the monolayer was incubated for 1 h at 37°C to allow bacterial infection. At the end of the infection period, the cells were washed three times with an appropriate culture medium, and then they were incubated for 1 h at 37°C in culture medium containing 50 µg ml–1 gentamicin to kill the extracellular bacteria. The cells were washed again, and then incubation was continued in the culture medium. After several different times, cell lysis was performed by hypotonic treatment for U937 cells or by treatment with 0.04% Triton X-100 for A. polyphaga. Aliquots of the cell lysates were immediately diluted and plated on BCYE plates for enumeration of the intracellular bacteria. The number of intracellular bacteria was expressed as the number of CFU/ml (36, 52).

Cytopathogenicity of L. pneumophila for U937 cells and A. polyphaga. Infection of U937 cells with L. pneumophila strains in the post-exponential phase was performed in triplicate in 96-well tissue culture plates with 1 x 105 cells/well at an MOI of 10 for 1 h, and infection of A. polyphaga was performed in triplicate in 24-well tissue culture plates with 5 x 105 cells/well at an MOI of 10 for 1 h. After 23 h, the numbers of viable cells were determined by using the Alamar Blue (TREK Diagnostic Systems, Westlake, Ohio) assay for U937 cells and the trypan blue dye assay for A. polyphaga, and the degrees of cytopathogenicity for the two types of cells were expressed as described previously (32).

Cloning and sequencing the chromosomal junction of Kan insertions in the GB112 mutant. Genomic DNA from the GB112 mutant was digested with EcoRI, and the fragment containing the Kan cassette was ligated to EcoRI-digested pBC-SK+ (Stratagene, La Jolla, Calif.). The cloned plasmid was digested with EcoRI and probed with pUC-4K (Pharmacia, Piscataway, N.J.) by Southern blotting to confirm the fidelity of the cloning. This plasmid was designated pGB112. pGB112 was digested with XhoI to obtain chromosomal fragment GB112 flanking half-Kan cassette. This fragment was ligated to pBC-SK+, and the resulting plasmid was designated pGB112-XHOI. pGB112-XHOI was used for sequencing analysis with a T7 primer of pBC-SK+. The sequence of approximately 360 nucleotides at the junction of the insertion was determined and used in database searches of the Legionella Genome Project at the Columbia Genome Center.

Construction of a GB112 complemented strain. Amplification of lpg1728 (pmiA) was performed with primer GB112-F3, complementary to a region 136 nucleotides upstream of the lpg1728 start codon (5'-GTTGATGATATGGGGGCTG-3'), and GB112-R3, complementary to a region 138 nucleotides downstream of the lpg1728 stop codon (5'-CTCCGACAGGATAATCAGGA-3'). A 1,189-bp fragment was amplified by PCR with a Gene Amp PCR 9600 system (Perkin-Elmer, Yokohama, Japan). This fragment was cloned into the pGEM-T Easy system (Promega, Madison, Wis.). A NotI-digested fragment containing this region was subcloned into pBC-SK+. The recombinant clone was designated pGB112C. pGB112C was introduced into the GB112 strain by using a Gene Pulser II (Bio-Rad, Hercules, Calif.) as recommended by the manufacturer to generate complemented strain GB112C-5.

Sequencing of pmiA gene. Sequencing of the whole pmiA region of the AA100 strain, using pGB112C, was carried by Hokkaido System Science Co., Ltd. Sequence alignments and comparisons were performed with GENETYX-MAC 8.0 (Software Development Co., Ltd.) and National Center for Biotechnology Information (blastp) programs, respectively.

Confocal laser scanning microscopy. Samples used for immunofluorescence analysis were prepared by the method described previously (36). In brief, U937 cells were cultured and then allowed to differentiate on coverslips in the wells of a six-well culture plate (Falcon, Becton Dickinson). The differentiated U937 cells were infected with L. pneumophila for 1 h. The cells were continuously incubated in culture medium containing gentamicin for 1 h to kill the extracellular bacteria. At different times after the initiation of infection (see below), the coverslips were fixed in 4% paraformaldehyde for 15 min at room temperature, and then nonspecific binding sites were blocked with 3% bovine serum albumin in phosphate-buffered saline (PBS) for 30 min. The cells were then permeabilized with 0.5% Triton X-100 in PBS for 10 min. The nuclei of both the bacteria and the U937 cells were stained with TO-PRO-3 iodide (Molecular Probes, Eugene, Oreg.) for 1 h. For colocalization experiments with endosomal markers and ER markers, 1 h of infection and 1 h of gentamicin treatment, followed by 2 h and 4 h of incubation, respectively, in culture medium without gentamicin were performed. Thus, 4-h or 6-h postinfection samples were examined. To label lysosomal and late endosomal compartments, serial 1-h incubations were performed with mouse anti-human LAMP-1 (H3B3) or LAMP-2 (H4B4) monoclonal antibodies (1:10 dilution; Developmental Studies Hybridoma Bank, University of Iowa) and Alexa Fluor 488-conjugated goat anti-mouse immunoglobulin G (IgG) (1:100 dilution; Molecular Probes). To label ER proteins, serial 1-h incubations were performed with mouse anti-KDEL monoclonal antibodies (StressGen Biotechnologies, Victoria, Canada) and Alexa Fluor 488-conjugated goat anti-mouse IgG (Molecular Probes). In this experiment, extracellular bacteria were labeled by incubation with rabbit anti-L. pneumophila serum (1:10 dilution; Denka Seiken, Tokyo, Japan) and Alexa Fluor 546-conjugated goat anti-rabbit IgG (1:100 dilution; Molecular Probes) before permeabilization. Between the incubations, the coverslips were washed three times with PBS for 5 min. The coverslips were then mounted on glass slides with Vectashield (Vector Laboratories, Burlingame, Calif.). Samples were observed with a Carl Zeiss LSM510 laser scanning confocal microscope (Carl Zeiss, Germany).

Transmission electron microscopy. For examination of infected amoebae by transmission electron microscopy, monolayers were infected by L. pneumophila strains in six-well plates at an MOI of 10 for 1 h, followed by three washes. At 6 h postinfection, the infected monolayers were washed with 0.1 M Sorenson's phosphate buffer and then incubated for 45 min in 0.1 M Sorenson's phosphate buffer containing 3.5% glutaraldehyde, pH 7.4, at 4°C. Fusion between lysosomes of the L. pneumophila phagosomes was determined by examination for the presence of the lysosomal enzyme acid phosphatase, as described previously (13, 51). The monolayers were washed three times with 0.1 M Sorenson's phosphate buffer and three times with 0.1 M acetate buffer (pH 5) and incubated with an acid phosphatase-specific substrate, ß-glycerolphosphate (0.1 M acetate buffer, 2 mM ß-glycerolphosphate as the substrate, and 1.2% lead nitrate as the capture metal), for 1 h at 37°C. After three washes in 0.1 M acetate buffer (pH 5) and Sorenson's phosphate buffer (5 min each), infected cells were postfixed with 1% OsO4 in the same buffer for 45 min. Samples were dehydrated and processed as described previously (31). Sections were stained with uranyl acetate and lead citrate and examined with an Hitachi H-7000/STEM electron microscope (Hitachi, Inc., Japan) at 80 kV, as described previously (31).

Southern blotting. Genomic DNA was isolated from L. pneumophila strains using an AquaPure genomic DNA isolation kit (Bio-Rad). Samples of genomic DNA were digested with EcoRI, separated by electrophoresis in 0.8% agarose gels, and transferred to Hybond-N nylon membranes (Amersham Biosciences, Buckinghamshire, England). pGB112C was used as a DNA probe. Labeling of DNA probes and detection of signals were performed by using the ECL direct nucleic acid and detection system (Amersham Biosciences).

Contact-dependent pore formation assay. Contact-dependent pore formation in membranes was determined by examining hemolysis of sheep red blood cells (sRBCs) by L. pneumophila at an MOI of 10 after 2 h of bacterial contact, as described previously (45). The release of hemoglobin from the lysed red blood cells was measured by spectrophotometry at 415 nm.

Sodium sensitivity assay. Bacteria were grown in BYE broth until the post-exponential phase (OD550, 2.0 to 2.2). The OD550 of bacterial cultures were adjusted to 1.0 to equalize the number of bacteria. Dilutions were plated on BCYE plates in the presence or absence of 0.6% NaCl. The ratio of the plating efficiency with 0.6% NaCl to the plating efficiency without 0.6% NaCl was calculated for each strain.

Nucleotide sequence accession number. The sequence of the whole pmiA region of the AA100 strain has been deposited in the DDBJ database under accession number AB193439.


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RESULTS
 
Characteristics of intracellular survival, replication, and cytopathogenicity of the GB112 mutant in U937 cells and A. polyphaga. We showed previously that the GB112 pmi mutant exhibits a defect in intracellular growth within both macrophages and protozoa (32). To ensure that the GB112 mutant had no other mutations, we decided to reconstruct the Kan insertion in the GB112 mutant in the wild-type strain. A DNA fragment containing the insertion and the flanking chromosomal regions was cloned from GB112 genomic DNA and introduced into wild-type strain AA100 by homologous recombination, as described previously (73). The fidelity of the allelic exchanges was confirmed by Southern hybridization (data not shown). For all the studies described in this paper, we show data only for the reconstructed mutant, which for simplicity was designated GB112.

Since many virulence-associated phenotypes have been shown to be induced at the postexponential phase (15), we examined the phenotype of the reconstructed GB112 mutant for intracellular growth and cytopathogenicity for host cells using bacteria grown to the postexponential phase. We first examined the cytopathogenicity for U937 human macrophage-like cells using Alamar Blue assays and the cytopathogenicity for A. polyphaga using trypan blue. The data showed that the cytopathogenicity of the GB112 mutant for both U937 macrophages and A. polyphaga was diminished compared to that of parental strain AA100 (Fig. 1A and B).



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  		FIG. 1. Cytopathogenicity and intracellular replication of L. pneumophila strains in U937 macrophage-like cells and A. polyphaga. U937 macrophages or A. polyphaga cells were infected with bacteria at an MOI of 10 for 1 h, followed by gentamicin treatment to kill extracellular bacteria, and the cells were then incubated for 24 h. The viability of host cells was determined by the Alamar Blue (for U937 cells) (A) or trypan blue (for A. polyphaga) (B) method. The following L. pneumophila strains were used: AA100 (wild type), GB112 (pmiA deficient), and GB112C-5 (pmiA-complemented GB112). The percentage of killed cells after bacterial infection was normalized to the percentage of uninfected cells, which was considered 100% viable cells. (C and D) Intracellular growth kinetics of L. pneumophila GB112 strains within U937 macrophage-like cells and A. polyphaga, respectively. Infection of the monolayers was performed exactly as described above for the cytopathogenicity assay, except that at the end of the 1-h infection period the monolayers were treated with gentamicin for 1 h to kill extracellular bacteria. The intracellular bacteria were recovered at several times postinfection, and the number of viable cells was determined by enumeration of the CFU. All experiments were done three times in triplicate; the data are the data from one representative experiment, and the error bars indicate standard deviations.

The intracellular growth of the GB112 mutant was less than that of wild-type strain AA100 in U937 cells. The number of GB112 mutant bacteria within U937 cells was approximately 1/10 the number of AA100 bacteria from 24 h to 48 h postinfection (Fig. 1C). In contrast, the GB112 mutant was severely defective in survival and replication within A. polyphaga. At 24 h postinfection, there were no detectable viable GB112 bacteria in A. polyphaga, while AA100 showed robust replication by 24 h (Fig. 1D). This result indicated that the GB112 mutant bacteria were killed in A. polyphaga.

Intracellular trafficking of the GB112 mutant within U937 macrophages and A. polyphaga. Survival and replication of L. pneumophila within mammalian and protozoan cells are totally dependent on evasion of fusion of the bacterial phagosome to lysosomes (51, 64, 74, 82). In addition, bacterial replication has also been shown to occur in a phagosome that is remodeled by the rough endoplasmic reticulum within macrophages and protozoa (1, 2, 5, 41, 75-77). Since the GB112 mutant was defective in survival in macrophages and amoebae, we hypothesized that intracellular trafficking of the mutant was likely altered in both types of host cells. To test this hypothesis, we examined colocalization of the bacterium-containing phagosomes with the late endosomal-lysosomal markers LAMP-1 and LAMP-2 in U937 cells by confocal laser scanning microscopy showing only LAMP-2 staining (Fig. 2A). Approximately 70% of the phagosomes containing the GB112 mutant colocalized with LAMP-1 and LAMP-2 (Fig. 3). In contrast, only 10 to 25% of the phagosomes containing parental strain AA100 colocalized with LAMP-1 and LAMP-2 (Fig. 2A and 3). The phagosomes harboring heat-killed L. pneumophila as a positive control colocalized with both LAMP-1 and LAMP-2, as expected (Fig. 2A and 3).



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  		FIG. 2. Representative confocal microscopy images of colocalization of the bacterial phagosomes with LAMP-2 (A) and ER proteins (B) within U937 cells infected by L. pneumophila AA100 (wild type) or GB112 (pmiA deficient). The DNA of intracellular and extracellular bacteria, as well as the U937 nucleus, were stained with TO-PRO-3 (blue). LAMP-2 and the KDEL marker were visualized with secondary antibodies conjugated to Alexa 488 (green). Extracellular bacteria were visualized with secondary antibodies conjugated to Alexa Fluor 546 (red). The arrows indicate intracellular L. pneumophila. The magnified portions are indicated by the squares in the merge panels.



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  		FIG. 3. Quantitation of colocalization of LAMP-1, LAMP-2, and ER proteins with the phagosomes containing L. pneumophila strains. Phagosomes containing AA100 (wild type), GB112 (pmiA deficient), or heat-killed AA100 (control) were examined for the presence of LAMP-1 and LAMP-2 at 4 h postinfection and for proteins with the ER retention signal (KDEL motif) at 6 h postinfection using confocal laser scanning microscopy.

We examined the presence of ER-associated proteins in the phagosomes at 6 h postinfection, using an antibody that recognizes the KDEL amino acid sequence, which is the signal for ER retention (Fig. 2B). Approximately 60% of the GB112 mutant-containing phagosomes did not acquire the KDEL marker. In contrast, more than 70% of the AA100-containing phagosomes acquired the KDEL marker (Fig. 2B and 3). Phagosomes harboring heat-killed AA100, as a control, did not acquire the KDEL marker, as expected. Thus, the defect in survival and replication of the GB112 mutant within U937 macrophages was associated with acquisition of the late endosomal-lysosomal markers LAMP-1 and LAMP-2 and with a reduction in the frequency of colocalization with the ER.

Since the GB112 mutant was severely defective in replication within A. polyphaga, we examined whether this defect was associated with fusion of the GB112-containing phagosomes to lysosomes. We examined the presence of the lysosomal enzyme acid phosphatase in the bacterium-containing phagosomes by electron microscopy, as described previously (51). The data showed that only 7% of the phagosomes containing AA100 contained acid phosphatase at 6 h postinfection (Fig. 4A and D). In contrast, 82% of the phagosomes harboring the GB112 mutant contained acid phosphatase (Fig. 4B and D). For the icmT null mutant, which was used as a positive control, ~60% of the phagosomes acquired acid phosphatase (Fig. 4C and D), which is consistent with previous observations (51, 52). On the basis of these findings, we concluded that the severe defect of the GB112 mutant in survival and replication in A. polyphaga was associated with the fusion of the GB112 mutant-containing phagosomes to lysosomes.



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  		FIG. 4. Fusion of phagosomes containing the GB112 mutant in A. polyphaga to lysosomes. Localization of lysosomal acid phosphatase in phagosomes within A. polyphaga was determined at 6 h postinfection. (A to C) Representative electron micrographs of L. pneumophila-infected A. polyphaga at 6 h postinfection. Signals representing acid phosphatase were detected as electron-dense lead nitrate, as indicated by the arrows. Tight phagosomes containing wild-type strain AA100 (A) were negative for acid phosphatase. Phagosomes containing GB112 (B) or AA100KmT (C) (icmT deficient) exhibited positive signals for acid phosphatase. (D) Quantitative results obtained by examining 150 Legionella-containing phagosomes for the presence of acid phosphatase. Lpn, L. pneumophila; M, mitochondrion; N, nucleus; phagoso, phagosomes. The experiment was done three times in triplicate; the data are the data from one representative experiment, and the error bars indicate standard deviations.

Identification of the mutated gene in the GB112 mutant. We cloned the DNA fragment containing the Kan insert and flanking sequences from the chromosome of the GB112 mutant. The sequence of the DNA fragments at the junction of the chromosomal DNA and the insert was analyzed. Using the BLAST program at the website of the Legionella Genome Project for the Philadelphia 1 strain (http://genome3.cpmc.columbia.edu/~legion/index.html) (19), we identified an open reading frame (ORF) that was 915 bases long (lpg1728) in the genome sequence that was interrupted by the insertion in the GB112 mutant. This ORF was also present in the complete genome sequences of two other L. pneumophila strains, Paris and Lens (17). We designated the lpg1728 ORF pmiA (accession no. AB193439) (Fig. 5A). The pmiA gene was located outside the icm/dot regions and was 1.1 x 106 bp downstream from icm/dot region I (icmVWX-dotABCD) and 1.45 x 106 bp upstream from icm/dot region II (icmTSRQPO-lphA-icmMLKEGCDJB-tphA-icmF or dotMLKJIHGFENO).



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  		FIG. 5. Genetic and structural analysis of PmiA. (A) Genetic organization of the mutated region in the GB112 mutant. Mini-Tn10::kan was found to be inserted into a putative 915-bp open reading frame (lpg1728), pmiA. In the flanking regions, there are five ORFs up- and downstream of pmiA (lpg1731, lpg1730, lpg1729, lpg1727, and lpg1726), whose direction of transcription was the same as that of pmiA. (B) Hydropathy profile of the predicted PmiA protein amino acid sequence based on a Kyte-Doolittle analysis with a default window of 21. The solid and dashed lines indicate the cutoff values for certain and putative transmembrane segments, respectively. Negative values indicate relative hydrophilicity. (C) Secondary structure of PmiA predicted by the SOSUI program.

There are five ORFs, three upstream and two downstream of lpg1728 (Fig. 5A). Since the predicted directions of transcription of all six ORFs were the same, it is possible that the Kan insertion has a polar effect on expression of the two ORFs downstream of pmiA. However, a 1.19-kb PCR-generated fragment containing only pmiA (lpg1728) was sufficient to complement the GB112 mutation (strain GB112C-5) for the defects in intracellular growth and cytopathogenicity for U937 macrophages and A. polyphaga (Fig. 1). Thus, the defect of the GB112 mutant in intracellular survival and trafficking is due to the defect in pmiA.

Topology analysis using the TopPred program (http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html; Institut Pasteur, France) suggested that the predicted PmiA protein has three transmembrane domains (Fig. 5B). Following the putative transmembrane domain near the N terminus, there is a predicted long hydrophilic region (SOSUI program [http://sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html]) (Fig. 5C). These results suggested that PmiA might be a transmembrane protein.

We examined the gene distribution of pmiA in several species of the genus Legionella by genomic Southern hybridization, using low-stringency conditions for hybridizations (see Materials and Methods). The pmiA gene was present in all L. pneumophila strains regardless of the serogroup, but it was not detected in any other Legionella species examined (Fig. 6). This result suggested that pmiA might be specific for L. pneumophila.



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  		FIG. 6. Southern blot analysis of the pmiA gene in L. pneumophila serogroup 1 strains (A) and in several serogroups of L. pneumophila and other Legionella species (B). pmiA was detectable in four different serogroups of L. pneumophila but was absent from other Legionella species, including L. bozemanii, L. micdadei, L. dumoffii, L. brunesis, and L. gratiana.

PmiA is essential for contact-dependent pore formation. The pore-forming activity has been shown to be dependent on a functional Icm/Dot type IV secretion system (45). Many Icm/Dot proteins that are predicted to be structural components of the Icm/Dot secretion apparatus are essential for the pore-forming activity (21, 45, 51, 52). We examined the pore-forming activity of the GB112 mutant, using contact-dependent hemolysis of sRBCs. The dotA and icmT mutants were used as controls, since both of these mutants are defective in pore-forming activity. The GB112 mutant was completely defective in pore-forming activity, similar to the dotA and icmT mutants, as well as heat-killed bacteria (Fig. 7A). Importantly, the defect of the GB112 mutant was fully complemented by the wild-type pmiA gene on a plasmid (Fig. 7A). These results indicated that the PmiA protein is essential for the contact-dependent pore-forming activity.



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  		FIG. 7. PmiA is essential for the pore-forming activity but is not required for NaCl sensitivity of L. pneumophila. (A) The pore-forming activities of L. pneumophila mutant GB112 (pmiA), LELA3118 (dotA), and GS3011 (icmT) were tested by contact-dependent hemolysis of sRBCs. GB112C-5 is a pmiA-complemented strain of GB112. Wild-type strain AA100 and heat-killed AA100 were used as positive and negative controls, respectively. (B) NaCl sensitivity of the GB112 mutant. The data are expressed as the ratio of the colony-forming activity after plating on 0.6% NaCl-BCYE agar plates to the colony-forming activity after plating on regular BCYE plates. The AA100 (wild type) and dotA strains were used as NaCl-sensitive and NaCl-resistant controls, respectively. The experiments were done three times in triplicate; the data are the data from one representative experiment, and the error bars indicate standard deviations.

pmiA mutation does not affect NaCl sensitivity. L. pneumophila virulent strains have been shown to be sensitive to sodium chloride (16, 65). It has been proposed that the sodium sensitivity is likely to be dependent on the function of an intact Icm/Dot secretion system because icm/dot mutants are resistant to high concentrations of sodium ions. It has been speculated that a functional Icm/Dot secretion apparatus is leaky to NaCl, which may explain why mutants defective in the secretion apparatus are resistant to salt (81). We examined the sodium sensitivity of the GB112 mutant and compared it to that of other icm/dot mutants. The GB112 mutant was similar to the wild-type strain in terms of sensitivity to 0.6% sodium chloride, whereas the dotA mutant was resistant to NaCl (Fig. 7B).


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DISCUSSION
 
The hallmark of Legionnaires' disease is the intracellular survival and growth of L. pneumophila within alveolar macrophages. It is thought that in natural aquatic environments, L. pneumophila is a parasite of protozoa (38, 54, 63), and the infected protozoa are important as a source of infection of humans by L. pneumophila (10, 27).

The major virulence icm/dot gene clusters of L. pneumophila, which encode components of type IV secretion systems, are required for intracellular growth and killing of human macrophages (14, 49, 60, 65, 67-69, 78, 80). The icm/dot genes of L. pneumophila are also required for intracellular growth within amoebae, as well as within human macrophages (71). However, many genes other than the icm/dot genes are also required for intracellular replication (26, 28, 30-40, 46, 48, 56, 59, 61, 62, 79). It is plausible that different sets of genes are utilized in a coordinated manner with the icm/dot genes. It is also possible that different pathways of pathogen-host interaction are host cell specific.

In this study, we characterized the GB112 mutant. This mutant has been identified as one of the pmi mutants (32). Our confocal laser scanning and transmission electron microscopic studies showed that phagosomes containing the GB112 mutant are defective in evasion of acquisition of late endosomal and lysosomal markers at early stages of infection of both U937 macrophages and A. polyphaga. Thus, pmiA is involved in inhibition of fusion of the bacterium-containing phagosomes to lysosomes in both types of host cells. Interestingly, the pmiA mutant is severely defective in protozoa but exhibits a less severe defect in intracellular growth in the U937 human macrophage cell line. Although the intracellular growth of the pmiA mutant within U937 cells shows a partially defective phenotype, LAMP-1 and LAMP-2 colocalize with phagosomes containing the pmiA mutant. The acquisition of these late endosomal and lysosomal markers by the pmiA mutant-containing phagosomes is correlated with the defect in intracellular survival and replication. It is interesting that despite the colocalization of LAMP-1 and LAMP-2 with phagosomes containing the pmiA mutant within U937 macrophages, the bacteria that were able to establish a replicative niche replicated in these cells. It has been reported that the dotA and dotB mutants reside in a nonlysosomal LAMP-1-positive compartment within mouse-derived bone marrow macrophages (43). The pmiA mutant might reside in the same type of phagosome as dotA and dotB mutants within macrophages.

The pmiA gene encodes a putative transmembrane protein that has three membrane-spanning domains based on hydropathy and membrane topology analysis. Interestingly, PmiA has a distinct long hydrophilic region between the transmembrane domains. This region has high similarity to hypothetical proteins of other bacteria possessing type IV secretion systems, such as Rickettsia species and Helicobacter pylori. A comparison using the protein-protein BLAST (blastp) program revealed that the region between amino acids 36 and 269 of PmiA, which covers the entire long hydrophilic region and the neighboring two transmembrane regions, exhibits similarity (20% identity and 40% similarity) to the corresponding part of hypothetical protein RP489 (accession no. Q9ZD57) of Rickettsia prowazekii (9). Interestingly, the corresponding hypothetical protein of Rickettsia species is predicted to be a transmembrane protein, and the secondary structure of the whole protein is also similar to that of PmiA (data not shown). The region between amino acids 68 and 155, which covers most of the long hydrophilic region, exhibits similarity (29% identity and 48% similarity) to hypothetical protein jhp0336 (accession no. C71944) of H. pylori (8). At least five ORFs both up- and downstream of pmiA, which have the same transcriptional direction as pmiA, exhibit no similarity with any genes of these bacteria. The genomic positions of the genes encoding each of the corresponding hypothetical proteins of these pathogens are far from the genetic loci that encode the type IV secretion apparatus (data not shown). The functions of these proteins in intracellular survival and replication and whether there is any relationship to the type IV secretion apparatus have not been reported.

We demonstrate here that PmiA is involved in the pore-forming activity which is attributed to the Icm/Dot type IV secretion system (45). The loss of the pore-forming activity in many icm/dot mutants, including dotA, icmT, icmQ, and icmR mutants, has been demonstrated previously (45, 51, 52). Recently, it has been shown that the IcmQ protein is localized on the bacterial surface shortly after contact with a macrophage (25). It has been suggested that pore formation in the mammalian cell membrane is caused by membrane insertion of IcmQ after its translocation to the bacterial surface (25). It is possible that PmiA is a component of the Icm/Dot secretion apparatus involved in export of IcmQ.

In summary, we show here that a defect in pmiA results in a defect in survival and replication of L. pneumophila in U937 macrophages and protozoa. Interestingly, the pmiA mutant exhibits a severe defect in intracellular growth within protozoa, in contrast to the partial defect in intracellular growth within macrophages. This defect is associated with trafficking of the pmiA mutant-containing phagosome through the endosomal-lysosomal pathway in macrophages and protozoa. The severe defect in the pore-forming activity of the pmiA mutant, in addition to a defect in its intracellular trafficking, suggests a potential contribution of the PmiA protein to export of Icm/Dot substrates. However, this potential role of PmiA in export of Icm/Dot substrates must be demonstrated directly.


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ACKNOWLEDGMENTS
 
We thank Howard A. Shuman (Columbia University) and Masahisa Watarai (Obihiro University of Agriculture and Veterinary Medicine) for kind gifts of the L. pneumophila strains. We are also grateful to Takashi Fukui, Toshihiko Harada, Maki Kato, and Asano Ikegaya for technical assistance.

This work was supported in part by grant-in-aid 16790258 and the COE Program in the 21st Century from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by Grant for Research on Cancer Prevention and Health Services H15-gan-yobou-095 from the Ministry of Health, Labor and Welfare. Y.A.K. was supported by Public Health Service awards RO1AI43965 and R21AI038410-06A1 and by the Commonwealth of Kentucky Research Challenge Trust Fund.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka-shi, Shizuoka 422-8526, Japan. Phone: 81-54-264-5711. Fax: 81-54-264-5715. E-mail: miyakem{at}ys7.ushizuoka-ken.ac.jp. Back

Editor: J. N. Weiser


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Infection and Immunity, October 2005, p. 6272-6282, Vol. 73, No. 10
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.10.6272-6282.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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