The Legionella pneumophila Effector SidJ Is Required for Efficient Recruitment of Endoplasmic Reticulum Proteins to the Bacterial Phagosome

Transcription

1 INFECTION AND IMMUNITY, Feb. 2007, p Vol. 75, No /07/$ doi: /iai Copyright 2007, American Society for Microbiology. All Rights Reserved. The Legionella pneumophila Effector SidJ Is Required for Efficient Recruitment of Endoplasmic Reticulum Proteins to the Bacterial Phagosome Yancheng Liu and Zhao-Qing Luo* Department of Biological Sciences, Purdue University, 915 West State Street, West Lafayette, Indiana Received 9 August 2006/Returned for modification 29 September 2006/Accepted 2 November 2006 The virulence of Legionella pneumophila is dependent on the Dot/Icm type IV protein secretion system, which translocates effectors into infected cells. A large number of such translocated proteins have been identified, but few of these proteins are necessary for intracellular replication of the pathogen, making it difficult to correlate these genes with specific cell-biological events associated with L. pneumophila infection. We report here the identification and characterization of a family of two substrates, SidJ and SdjA, with distinctive phenotypes. In contrast to many Dot/Icm substrates, whose expression levels are elevated when bacteria are grown to postexponential phase, SidJ is produced at a constant rate during the entire bacterial growth cycle. Mutation in sidj causes a significant growth defect in both macrophage and amoeba hosts, but an sdja mutant is detectably defective only in protozoan hosts. However, in the amoeba host a mutant lacking both sidj and sdja does not display a more severe growth defect than the sidj mutant. Despite its significant intracellular growth defect, the sidj mutant is still able to effectively evade fusion with lysosomes. Importantly, recruitment of endoplasmic reticulum (ER) proteins by vacuoles containing the sidj mutant was considerably delayed in both mammalian and amoeba cells. Our results suggest that SidJ modulates host cellular pathways, contributing to the trafficking or retention of ER-derived vesicles to L. pneumophila vacuoles. Legionella pneumophila is a facultative intracellular pathogen capable of replicating within a variety of eukaryotic cells. Freshwater protozoans are believed to be the natural host of this pathogen and thus the main driving force for its evolution (15). Infection of humans by L. pneumophila occurs after inhalation of contaminated aerosols; these bacteria can survive and replicate in alveolar macrophages after being engulfed by these phagocytic cells, leading to the development of a severe form of pneumonia known as Legionnaires disease (16, 26). Upon uptake, the L. pneumophila-containing vacuole (LCV) initiates a unique trafficking route that evades fusion with lysosomes, ultimately resulting in the formation of an organelle resembling the rough endoplasmic reticulum (ER) that supports bacterial replication (18, 39). Remodeling of bacterial vacuoles into an ER-like compartment by L. pneumophila is achieved at least in part by interception of transport vesicles originating in the ER, because chemical or genetic interference with the functions of the early steps of host secretory pathways leads to depression of intracellular replication (11, 19, 20). Consistent with these observations, some ER resident proteins, such as calnexin and Bip, are actively acquired by the pathogen to modify its vacuoles and become essential components of the vacuole membrane throughout the bacterial replication cycle (11, 38). Furthermore, proteins involved in vesicle trafficking among different cellular compartments, such as the small GTPases Arf1 and Rab1 and the tsnare protein Sec22b, play important roles in the establishment of the replicative vacuoles * Corresponding author. Mailing address: Department of Biological Sciences, Purdue University, 915 West State Street, West Lafayette, IN Phone: (765) Fax: (765) Published ahead of print on 13 November (11, 20). A more recent study found that in Dictyostelium discoideum, the lipid PI(4)P is recruited to the cytoplasmic surface of LCVs for anchoring of translocated effector proteins such as SidC (44). In addition to vesicle trafficking between the ER and the Golgi apparatus, L. pneumophila also exploits the ER-associated degradation machinery to facilitate its intracellular survival and growth; perturbation of these host processes by chemical or genetic approaches leads to inhibition of L. pneumophila intracellular replication (13). Finally, modulation of host apoptosis by L. pneumophila is important for the biogenesis of the replicative phagosome (28), and recent studies showed that infection by virulent bacteria led to induction of a large number of antiapoptotic genes (1, 22). Bypass of the endocytic network, the modulation of the various host pathways, such as the interception of trafficking vesicles of ER origin, and modulation of host apoptosis by LCVs are orchestrated by the function of a specialized protein transporter called Dot/Icm (4). Dot/Icm belongs to a large family of transporters termed the type IV secretion system, which mediates the transfer of DNA or/and protein substrates into target cells (33, 42). Encoded by over 24 different dot and icm genes, the Dot/Icm system functions in the translocation of bacterial protein substrates into eukaryotic host cells. A large number of such substrates have been identified by a variety of genetic and bioinformatic tools, but the biochemical and cellbiological activities of most of these proteins are unknown (6, 7, 10, 24, 31, 34). These translocated bacterial proteins are believed to directly modulate host cellular functions for the establishment of an intracellular niche that supports bacterial growth. Consistent with the notion that L. pneumophila modulates host secretory pathways to establish the replicative phagosome, the Dot/Icm substrates RalF and SidM/DrrA are found to function as a guanine nucleotide exchange factor for 592

2 VOL. 75, 2007 LEGIONELLA EFFECTOR IMPORTANT FOR RECRUITING ER PROTEINS 593 TABLE 1. Bacterial strains and plasmids used in this study Strain or plasmid Genotype and/or relevant markers Source or reference L. pneumophila strains Lp02 Philadelphia-1 rpsl hsdr thya 3 Lp03 Lp02 dota 4 ZL25 Lp02 sidc 24 ZL101 Lp02 sidj This study ZL51 Lp02 sdja This study ZL52 Lp02 sidj sdja This study ZL102 Lp02 sidj(psidj) This study ZL103 Lp02 sdja(psdja) This study ZL104 Lp02 sidj sdja(psid-psdja) This study ZL50 Lp02 sidj(pam239) This study ZL015 Lp02 ( sdec- sdea) This study ZL015C ZL015(pSidJ) This study ZL sdea Lp02 sdea 24 ZL sdeb Lp02 sdeb 24 ZL sdec Lp02 sdec 24 E. coli strains XL1-Blue DH5 ( pir) the Arf1 and Rab1 small GTPases, respectively (25, 29, 31). In each case, the bacterial effector protein is required for the recruitment of its target small GTPase onto the surface of the L. pneumophila vacuoles (25, 29, 31). Although entry of L. pneumophila into host cells generally is considered to be a passive process that is dependent on host cell phagocytosis, in the amoeba host Dictyostelium discoideum, the bacterium appears to be actively released by the host in a process mediated by two Dot/Icm substrates, LepA and LepB (7). Unlike mutations in the dot/icm genes, which often result in L. pneumophila mutants completely defective in intracellular growth, elimination of single substrate gene or combinations of several such genes does not significantly affect intracellular growth (6, 7, 24). Host specificity and functional redundancy conferred by substrates are believed to contribute to the remarkable resilience of L. pneumophila to perturbations in genes coding for substrates of the Dot/Icm system (24). The lack of growth defects in effector mutants makes it difficult reca1 enda1 gyra96 thi-1 hsdr17 supe44 rela1 lac F proab laci q Z M15 Tn10(Tet r ) supe44 dlacu169( 80lacZ M15) hsdr17 reca1 enda1 gyra96 thi-1 rela1 pir tet::mu reca D. discoideum strains AX4 Wild-type strain for intracellular growth analysis 23 AX4::HDEL::GFP AX4 expressing a HDEL::GFP fusion, neor 21 AX3::vatM::GFP axea1 axeb1 axec1 pdxa/vatm-gfp neor 8 to identify a specific correlation between a cellular process and substrate(s). In this study, we report the identification and initial characterization of two substrates of the Dot/Icm system required for proficient intracellular growth. Our results indicate that both of these genes are important for maximal intracellular replication of L. pneumophila in an amoeba host, but only one of them is important for growth in macrophages. Furthermore, we show that loss of the activities of these substrates does not detectably affect the evasion of phagolysosomal fusion by LCVs, but deletion of one of the genes causes a delay in the acquisition of ER proteins by L. pneumophila phagosomes. MATERIALS AND METHODS Stratagene Our collection Plasmids pam239 ori RSF1010, Cm r, GFP 12 pgex-6p-1 GST fusion vector Amersham pjb908 ori RSF1010 ( orit moba), Amp r td i 2 psr47s ori R6K, orit RP4, Kan r, SacB 2 pzl199 psidc 41 pzl204 psidc C pzl sidj sidj in-frame deletion construct This study pzl sdja sdja in-frame deletion construct This study pzl223 sidj cloned into pjb908 This study pzl234 SidC C100::SidJ500 This study pzl268 sidj cloned into pgex-6p-1 This study pzl730 Construct for expression of GST-SdjA This study pzl757 SidC C100::SdjA This study pzl758 sdja cloned into pjb908 This study Media, bacterial strains, and plasmids. All bacterial strains and plasmids are listed in Table 1. Escherichia coli strains were grown on L-agar plates or in L broth. When necessary, antibiotics were added to media at the following concentrations: ampicillin, 100 g/ml; kanamycin, 30 g/ml; chloramphenicol, 30

3 594 LIU AND LUO INFECT. IMMUN. Primer TABLE 2. Primers used in this study Sequence a PL1...CTAGGTCGACTTATCACAAACGTTTATCAG PL2...CTGAGAGCTCGACACGTATTTTGGTGTTGTC PL3...CTAGGGATCCAAGTCCACCTTGGAAGAAG PL4...CTAGGGATCCTTTGGTTTCATAAAGAAAGTA PL5...CTAGGTCGACCCATAATTTTAGATCCAGAA PL6...GCCTGGATCCATCAAGTACTTTCTTTATGA PL7...CTAGGGATCCTCCGAACGTACTACTGATA PL8...CTGGGAGCTCATCACCTCGGCACAAGCCAT PL9...GCGAGGATCCATCTAATAATTTATCCAGATAAC PL10...CTAGGTCGACATAAAACACGCTTATCTCAGCC PL11...GCGGGATCCTTTGATTTTGACAGGAACAC PL12...GCGGAGCTCCATGGGTATGTACATTAAAG PL13...CTGGGATCCTTTAGTTATCTGGATAAATTA PL14...CGTGTCGACTCATAAAGGCGACTGCGATG PL15...GCGTCTAGACCAACAACGGAGAAGACTAA a Restriction enzyme sites are underlined. g/ml. All L. pneumophila strains were derivatives of the Philadelphia-1 strain Lp02 (3) and were grown on charcoal-yeast extract (CYE) plates or in ACESbuffered yeast extract (AYE) broth, as described previously (10). Thymidine was added at 200 g/ml to support growth of the thymidine autotrophical mutant Lp02 and its derivatives. For intracellular growth experiments, the Thy plasmid pjb908 (2) or its derivatives were introduced into the appropriate bacterial strains to avoid any potential variations that may arise from the use of thymidine autotrophical strains (21). Bacteria used for infection were grown in AYE broth to postexponential phase, as judged by both optical density (OD) (OD 600, 3.2 to 4.0) and motility of bacterial cells. As required, antibiotics were added to L. pneumophila media at the following concentrations: chloramphenicol, 5 g/ml; streptomycin, 100 g/ml; kanamycin, 20 g/ml. Primers used for cloning are listed in Table 2. In-frame deletion mutants were constructed as follows. To delete sidj, PCR products obtained by primers PL5/ PL6 and PL7/PL8 were digested with SalI/BamHI and BamHI/SacI, respectively, and were ligated to SalI/SacI-digested psr47s (14) to generate pzl sidj. Similarly, plasmid pzl sdja, for deletion of sdja, was constructed with primer pairs PL9/PL10 and PL11/PL12. In each case, the primers were designed such that in the mutant, the target gene is replaced with a polypeptide consisting of the first 15 and the last 15 amino acids of the original protein. Each plasmid was introduced into L. pneumophila by triparental mating, and transconjugants were selected on CYET plates containing kanamycin and streptomycin. Mutants were screened by PCR from candidates grown on medium containing 5% sucrose, as described previously (14). To make chimera proteins with SidC C100 for testing of Dot/Icm-dependent protein translocation, DNA fragments coding for the last 500 amino acids of sidj or full-length sdja were amplified by PCR with the primer pairs PL1/PL3 and PL13/PL14, respectively. Each was digested with BamHI/SalI and was inserted into similarly digested pzl204, which carries sidc C100 (41). To construct pzl223 for the complementation of sidj, the open reading frame of this gene was amplified with primer pair PL1/PL2 and was inserted into SacI/ XbaI-digested pjb908. The sdja complementation plasmid pzl758 was constructed on pjb908 with XbaI/SalI-digested PCR products generated with primers PL14 and PL15. Protein purification, preparation of SidJ-specific antibody, and immunoblotting. The predicted open reading frame of SidJ was amplified by PCR using primers PL1/PL4, digested with BamHI/SalI, and inserted into similarly digested pgex-6p-1. E. coli strain XL1-Blue, harboring the resulting plasmid, pzl268, was used to purify glutathione S-transferase (GST)-tagged SidJ with a glutathione-sepharose matrix according to protocols suggested by the manufacturer (Amersham Biosciences, Carlsbad, CA). After removal of the GST tag with Precision protease (Amersham Biosciences), the SidJ protein was used to raise antibodies in rabbits, according to standard protocols (Pocono Rabbit Farm and Laboratory, Canadensis, PA). SidJ-specific antibody was affinity purified against the same protein coupled to Affigel15 (Bio-Rad) according to a standard procedure (17). To detect SidJ in L. pneumophila lysates, cells grown in AYE to the indicated OD 600 were isolated by centrifugation and lysed by boiling in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. SDS-PAGE-resolved proteins were transferred to nitrocellulose membranes and were incubated with anti-sidj antibody at a 1:5,000 ratio. After incubation with a secondary antibody conjugated to horseradish peroxidase, signals were detected by the Enhanced Bioluminescence method (Pierce, Rockford, IL). Preparation of host cells and L. pneumophila infection. Bone marrow-derived macrophages were prepared from 6- to 10-week-old female A/J mice with L-cell supernatant-conditioned medium as described previously (10). Macrophages were seeded into 24-well plates 1 day before infection. Cells at /well and /well were used for intracellular growth and immunofluorescence assays, respectively. U937 cells were grown in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 10 mm glutamate. Cells were differentiated into macrophages in fresh medium by phorbol myristate acetate at a final concentration of 10 ng/ml for 36 h as described previously (39). D. discoideum strains were maintained and grown at 21.5 C and prepared for infection as described previously (21, 36). Strains AX4 (21), AX4-HDEL::GFP (21), and AX3-vatM::GFP (8) were grown axenically at 21.5 C in HL-5 liquid medium supplemented with penicillin and streptomycin (100 U/ml) (Invitrogen, Carlsbad, CA), as described previously (21). G418 was added at a concentration of 10 g/ml to maintain the integrated plasmids expressing green fluorescent protein (GFP) fusions. Prior to being infected with L. pneumophila, D. discoideum cells were plated in MB medium at a concentration of cells per well for intracellular growth study. For immunostaining, cells were plated onto each poly-l-lysine-coated coverslip. For assays of L. pneumophila growth within bone marrow-derived macrophages or D. discoideum, we infected host cells plated on 24-well plates with L. pneumophila at a multiplicity of infection (MOI) of Two hours after adding the bacteria, we synchronized the infection by removing extracellular bacteria by washing the monolayers three times with fresh medium. Infected macrophages were incubated at 37 C in the presence of 5% CO 2, whereas infected D. discoideum was incubated at 25.5 C. At each time point, cells were lysed with 0.02% saponin, dilutions of the lysate were plated onto bacteriological media, and CFU were determined from triplicate wells of each strain. Isolation of L. pneumophila vacuoles. Postnuclear supernatant (PNS) of infected cells was prepared as described previously (11). Briefly, appropriately differentiated U937 cells or murine macrophages seeded in a 15-cm petri dish were infected with postexponential L. pneumophila expressing GFP at an MOI of 5. At the indicated time, the infected cells were suspended in 0.5 ml of homogenization buffer (20 mm HEPES-KOH, ph mm sucrose 0.5 mm EGTA) and were disrupted in a 7-ml Dounce homogenizer (Wheaton). Unbroken cells and nuclei were pelleted by centrifugation at 4 C (3 min, 200 g). The PNS containing the L. pneumophila vacuoles was allowed to adhere to poly-l-lysine-coated coverslips prior to fixation and probing with the indicated antibodies. The integrity of the vacuolar membrane was assessed by immunostaining of the bacteria by using a polyclonal anti-l. pneumophila antibody (11). Only batches of samples containing more than 85% intact vacuoles are used for further experiments. Saponin extraction of translocated proteins. Detergent extraction of translocated proteins was carried out as described previously (41) with minor modification. Briefly, approximately differentiated U937 cells were plated in a 15-cm petri dish and allowed to adhere for one hour. We then infected the cells at an MOI of 5.0 with L. pneumophila grown to postexponential phase. Infected cells were incubated at 37 C in a 5% CO 2 incubator. At the indicated time points, infected cells were collected into a 15-ml centrifuge tube and were lysed by the addition of 2 ml of phosphate-buffered saline containing 0.2% saponin. Lysis was allowed to proceed for 30 min on ice with intermittent vortexing. After centrifugation for 15 min at 8,000 g, the top 1.6 ml of the supernatant was subjected to precipitation with by the methanol-chloroform method (45). The detergentsoluble and -insoluble materials were suspended in SDS-PAGE sample buffer containing 5.5 M urea. Equivalent-amount samples were analyzed by SDS- PAGE, resolved proteins were transferred to nitrocellulose membranes, and proteins of interest were detected by immunoblotting with the appropriate antibodies. Antibodies, immunostaining, and fluorescence microscopy. Anti-L. pneumophila antibodies and antiserum specific for Bacillus subtilis isocitrate dehydrogenase (ICDH) were generous gifts from R. R. Isberg and A. L. Sonenshein, respectively. For immunofluorescent analyses, cells were fixed and stained by standard procedures (27, 43). Anti-L. pneumophila antibodies were used at a dilution of 1:10,000. Extracellular and internalized bacteria were distinguished by differential staining with anti-l. pneumophila antibody and secondary antibodies conjugated to distinct fluorescent dyes (10). Extracellular bacteria were decorated with a secondary antibody conjugated to the blue fluorescent dye 7-amino- 4-methylcoumarin-3-acetic acid; total bacteria were labeled with a secondary antibody linked to Texas red fluorescent dye (Jackson ImmunoResearch, West Grove, PA). Antibodies against calnexin and Rab1 were purchased from Stressgen (San Diego, CA) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Both antibodies were used at 1:100 for immunostaining. LAMP-1 was labeled with antibody clone ID4B (Development Studies Hybridoma Bank, Iowa

4 VOL. 75, 2007 LEGIONELLA EFFECTOR IMPORTANT FOR RECRUITING ER PROTEINS 595 FIG. 1. sidj and sdja are closely linked to members of the side protein family. sidj is situated between sdeb and sdec and is separated from sdec by an open reading frame (orf2) predicted to code for a protein of 301 amino acids (A), and sdja is directly downstream of sded (B). A plasmid expressing SidJ almost completely complements the intracellular growth defect caused by the deletion of a chromosomal region spanning sdec to sdea (C). After challenge of murine macrophages with appropriate L. pneumophila strains, growth of bacteria was monitored by plating cell lysates at the indicated time points on bacteriological media. Similar results were obtained from more than three independent experiments performed in triplicate, and data shown were from one representative experiment. L. pneumophila strains used: Lp02, wild type (dot/icm ); Lp03, Lp02 (dota); ZL015, Lp02 ( sdec- sdea); ZL015C, ZL015 (psidj). City, IA) at a 1:50 dilution. The anti-sidc antibody (24) was used at a dilution of 1:500 for immunostaining. An anti-sdec antibody (a generous gift from J. Vogel, Washington University) was used to detect members of the SidE protein family at a 1:5,000 dilution. Processed coverslips were mounted on glass slides with an antifade reagent (Bio-Rad). Protein translocation, recruitment of the GFP:: HDEL, and the formation of replicative vacuoles were scored by visual inspection with an Olympus IX-81 fluorescence microscope. Images acquired with an Orca camera were processed with the IPlab software package (Scanalytic, Inc. Fairfax, VA). RESULTS Identification of the SidJ protein family as substrates of the Dot/Icm transporter. Our previous study of the SidE family of substrates revealed that three members of this protein family, sdea, sdeb, and sdec, are organized in an operon-like structure (Fig. 1A) (24). To simplify our initial analysis of the roles of these three genes in L. pneumophila intracellular replication, we constructed a mutant lacking the entire region spanning from sdec to sdea; this mutant exhibited about a 20-fold intracellular growth defect in bone marrow-derived macrophages (Fig. 1C). Unexpectedly, such a defect cannot be complemented by either sdea, sdeb, or sdec (data not shown). However, when open reading frame 3 (lpg2155), situated between sdec and sdeb, was used to complement the mutant, the growth defect was almost completely restored, indicating that this gene is responsible for the observed growth defect (Fig. 1C). Orf3 is separated from the upstream sdec by an open reading frame (orf2 in Fig. 1A) predicted to encode a 301- amino-acid protein, and the intergenic region between this smaller gene and orf3 is only 53 bp (Fig. 1A). These genes, along with the upstream sdec and the further downstream sdeb and sdea, are organized into a gene cluster separated by very short intergenic spaces (Fig. 1A). Orf3 is encoded by all three sequenced L. pneumophila genomes, but the predicted protein encoded by this gene does not share detectable homology with any protein from other species. However, this protein has a closely related paralog (lpg2508) (orf1 in Fig. 1B) present in the L. pneumophila genome. Interestingly, the paralog of orf3 is 71 bp downstream of sded, a member of the SidE family (2, 24). orf3 and orf1 code for two hypothetical proteins of 873 and 807 residues, respectively. These proteins have 52% identity and 60% similarity. Since many substrates of the Dot/Icm system form families with multiple members (24), we considered the possibility that these two proteins are substrates of the Dot/Icm transporter; they were tentatively designated SidJ and SdjA. To test whether these proteins are indeed substrates of Dot/ Icm, we first examined whether SidJ and SdjA can promote Dot/Icm-dependent transfer of the Cre recombinase in our interbacterial protein transfer assay (24). Thus, we fused regions coding for the last 500 amino acids of sidj and full-length sdja to cre on pzl180 (24), respectively. Stable proteins were made for both fusions in L. pneumophila, but inconsistent data were obtained in the interbacterial transfer assay and we cannot confidently conclude from these experiments that these two proteins are transferred by the Dot/Icm transporter (data not shown). To determine the substrate status of these two proteins, we employed a protein fusion assay that utilized the SidC protein known to localize to the cytoplasmic face of the L. pneumophila vacuole after being translocated (24). Similar to that of RalF (30), signals sufficient for Dot/Icm-mediated SidC translocation have been localized to the carboxyl terminus of the protein (Z.-Q. Luo et al., unpublished data). Removal of the last 100 amino acids of SidC (SidC C100) completely abolished Dot/Icm-mediated translocation of the protein (41). Fusion of candidate signal recognizable by Dot/Icm to the carboxyl terminus of SidC C100 can lead to translocation of the fusion protein into host cells, which can be easily detected by immunostaining with anti-sidc antibody. This method is highly useful in determining the translocation of proteins that do not associate with LCVs after transfer (41). To test for SidJ and SdjA translocation, we made chimera proteins by fusing the last 500 amino acids of SidJ and full-length SdjA, respectively, to SidC C100 and expressed these fusions in L. pneumophila strain ZL25, a sidc deletion mutant of wild-type strain Lp02 (24). We infected murine macrophages with these strains; one hour after incubation, infected monolayers were fixed, stained with anti-sidc antibody, and analyzed by immunofluorescence microscopy to score for the presence of SidC staining signal around the L. pneumophila vacuole (24, 41). In infections with a strain expressing full-length SidC, greater than 90% of L. pneumophila-containing vacuoles stain positively with anti-sidc antibody (Fig. 2A and E), a rate slightly higher than that of infection with the wild-type strain (24). On

5 596 LIU AND LUO INFECT. IMMUN. FIG. 2. SidJ is translocated into host cells by the Dot/Icm system. Macrophages were infected at an MOI of 1 for 1 h with sidc strains harboring plasmids expressing wild-type sidc (psidc/pzl199) (A) or sidc lacking the region coding for the last 100 residues (psidc C100/ pzl204) (B); fusions of the last 500 amino acids of SidJ to SidC C100 (psidc 100::sidJ500/pZL234) (C); or full-length sdja (psidc C100::sdjA/ pzl757) (D). L. pneumophila was labeled with an anti-l. pneumophila antibody and a secondary antibody conjugated to Texas red; SidC was labeled with an anti-sidc antibody and a secondary antibody linked to fluorescein isothiocyanate. Bar, 5 m. Data were obtained by inspecting at least 100 vacuoles in each sample (E). Experiments were performed three times in triplicate, and similar results were obtained. Data shown are the means and standard deviations from one representative experiment. (F) Dot/Icm-dependent translocation of SidJ into host cells. U937 cells were infected with the indicated L. pneumophila strains at an MOI of 5.0 for 14 h, and infected cells were collected and extracted with 0.2% saponin as described in Materials and Methods. After being resolved by SDS-PAGE, proteins transferred to nitrocellulose membranes were detected with antibodies against SidJ and ICDH. WT (wild type), U937 cells infected with strain Lp02; dota, U937 cells infected with Lp03; psidj, U937 cells infected with strain ZL101 ( sidj) expressing sidj from a high-copy-number plasmid (pzl223);, uninfected U937 cells; WT-Bacteria, Lp02 grown to postexponential phase and lysed with SDS sample buffer. Experiments were repeated three times, and similar results were obtained. Statistical analyses were performed with the Student t test. ***, P the other hand, less than 5% of vacuoles stained positively for SidC in infections with a strain expressing SidC C100 (Fig. 2B and E). Fusion of SidJ or SdjA to SidC C100 yielded 82% and 75% SidC-positive vacuoles, respectively (Fig. 2C, D, and E). These data strongly suggest that these two proteins are translocated by the Dot/Icm transporter. To further confirm the Dot/Icm-mediated translocation of SidJ, we prepared polyclonal anti-sidj antibody using recombinant SidJ protein generated from E. coli. However, though affinity-purified anti-sidj antibody specifically recognizes SidJ expressed from L. pneumophila, in immunofluorescence staining analyses, we were unable to detect clear association of SidJ with bacterial vacuoles at several different infection times (data not shown). Since some Dot/Icm substrates are not detectably associated with bacterial vacuoles during infection (6, 41), we employed a biochemical fractionation technique that has been successfully used to determine the translocation of LidA and VipD into host cells by the Dot/Icm transporter (12, 41). We infected differentiated U937 cells with L. pneumophila strains for 14 h and extracted the infected cells with saponin, a detergent that does not lyse the bacteria or release cytosolic bacterial proteins (41). SidJ was found in supernatants of saponin extracts when wild-type bacteria or the complementation strain was used to infect mammalian cells (Fig. 2F). No SidJ was detected in the supernatant of infections with the dota mutant L. pneumophila strain, indicating that translocation was dependent on an intact Dot/Icm transporter (Fig. 2F). Furthermore, SidJ was absent from the supernatant fraction in mock infections (Fig. 2F). As a control for bacterial lysis, we examined the presence of the bacterial cytoplasm protein ICDH with a specific antibody in all extraction fractions; this protein was found only in the pellet fraction and was absent from the supernatant, further indicating that the detected SidJ was released via the Dot/Icm transporter (Fig. 2F). The above two sets of data clearly demonstrate that SidJ is a substrate of the Dot/Icm system that is translocated from L. pneumophila to mammalian cells. We were unable to examine the translocation of SdjA by this fractionation method, because the anti-sidj antibody does not detectably react with even purified SdjA protein (data not shown). However, given the high-level similarity of SdjA to SidJ and the observation that fusion of SdjA to SidC C100 resulted in a high frequency of translocation of the hybrid protein, we propose that SdjA is also a substrate of the Dot/ Icm transporter. SidJ is constantly produced by L. pneumophila during its entire growth cycle. Upon entering the postexponential growth phase, expression of many L. pneumophila virulence factors is significantly induced to ensure maximal survival of the bacterium in host cells (5). Consistent with this observation, expression of almost all characterized substrates of the Dot/Icm system, such as RalF, SidC and members of the SidE family, is elevated when bacterial cultures reach postexponential phase (10, 24, 31). We analyzed whether the expression of sidj is similarly regulated by probing the SidJ protein level in cell lysates of L. pneumophila grown at different phases. As predicted, a protein of 100 kda was detected in both the wild type and the dota mutant of L. pneumophila (Fig. 3A). This protein, which matches well with the predicted molecular weight of SidJ, was absent from strain ZL101, the sidj deletion mutant (Table 1 and Fig. 3A). These data indicate that the

6 VOL. 75, 2007 LEGIONELLA EFFECTOR IMPORTANT FOR RECRUITING ER PROTEINS 597 FIG. 3. Growth-phase-independent expression of sidj and the expression of members of the side protein family in the sidj mutant. (A) SidJ is produced by L. pneumophila constitutively during its growth cycle. Tested L. pneumophila strains were grown in AYE broth to the indicated OD 600, identical amounts of bacterial cells were lysed in SDS sample buffer, and cleared protein samples were separated by SDS- PAGE. After being transferred to membranes, proteins were probed with antibodies specific for SidJ and ICDH. WT (wild type), Lp02; dota, Lp03; ZL101, Lp02 ( sidj); ZL102, Lp02 sidj/psidj. (B) Expression of sdea, sdeb, and sdec is not affected by the deletion of sidj. Total proteins from equivalent amount of bacteria grown to postexponential phase were resolved by SDS-PAGE and probed with an anti-sdec antibody (2). Bands representing individual proteins were indicated by arrows, and the cytosolic protein ICDH was used as a loading control. Relevant molecular mass standards are shown on the left (in kilodaltons). Dot/Icm transporter does not influence the expression of sidj. Furthermore, introduction of a plasmid harboring the open reading frame of sidj into the deletion mutant resulted in the production of a protein with the identical molecular weight (Fig. 3A). Intriguingly, in contrast to most characterized Dot/ Icm substrates, such as RalF, SidC, and members of the SidE family, which are significantly induced when L. pneumophila enters postexponential phase (2, 24, 31), very similar levels of SidJ protein were detected in all growth phases examined (Fig. 3A). These results, along with the observation that high levels of SidJ were detected in the cytosol of infected cells at 14 h postinfection, suggest that SidJ is continuously translocated into infected cells during the entire cycle of intracellular growth or that SidJ translocated in the early phase of infection remains stable during the first 14 h of infection. These results also suggest that this protein continues to exert its activity in late-phase intracellular replication of the bacterium. Mutants of sidj and sdja exhibit distinct intracellular growth phenotypes. To further characterize the roles sidj plays in L. pneumophila pathogenicity, we constructed an in-frame deletion mutant of this gene and examined its intracellular growth in three different host systems. Compared to wild-type strains, the sidj deletion mutant did not display any detectable growth defect in AYE broth (data not shown). Furthermore, when the mutant was used to challenge murine macrophages, internalization of the bacteria by host cells was indistinguishable from that of the wild-type strain (data not shown), indicating that this gene is not involved in bacterial uptake. Importantly, when intracellular multiplication was examined in murine macrophages, the sidj mutant displayed a significant growth defect in each time point analyzed, with the most pronounced defect being observed at 24 to 48 h after uptake, showing about a 20-fold growth defect (Fig. 4A). A significantly lower bacterial yield was also detected in the late phase of infection (72 h postinfection), exhibiting about a 15-fold growth defect (Fig. 4A). Introduction of a plasmid carrying sidj expressed from a constitutive promoter fully complemented the growth defect in murine macrophages, indicating that the observed growth phenotype was caused by the loss of sidj (Fig. 4A). We also tested the growth of this mutant in the macrophage-like host U937 cells, and a very marginal growth defect was detected for the SidJ mutant (data not shown). The lack of a significant growth defect of the mutant in U937 cells is not surprising because this cell line is considerably more permissive for L. pneumophila replication, and an earlier study showed that this cell line is even able to support multiplication of several dot/icm mutants missing accessory components of the transporter (9). We then examined the growth of the sidj mutant in D. discoideum, which is closer to the natural host of L. pneumophila (46). In this host, the sidj mutant failed to multiply in the first 24 h of infection, whereas the wild-type strain grew about threefold (Fig. 4C). The sidj mutant exhibited a sixfold growth defect at 48 h postinfection; such a defect increased to about 20-fold at 72 h postinfection (Fig. 4B). Thus, the defect was apparent throughout the 72-h experiment duration (Fig. 4B). In the protozoan host, introduction of the complementation plasmid fully restored the growth defect of the sidj mutant exhibited at the 48-h time point (Fig. 4B). However, at the 24- and 72-h time points, the SidJ-expressing plasmid only partially complemented the growth of the mutant, restoring its growth rate to about 80% of that of the wild-type strain (Fig. 4B). We have constructed several additional complementation plasmids derived from different vectors, but none of these constructs was able to fully complement the mutation at 72 h postinfection (data not shown). Given that members of the side family proteins are required for proficient intracellular growth in the amoeba host (2), we considered the possibility that deletion of sidj caused some polar effect on the expression of downstream genes, such as sdea and sdeb, and such effect only becomes apparent in the amoeba host, which is known to be more restrictive to L. pneumophila replication (35). Thus, we examined the expression of sdea and sdeb in the sidj mutant by probing the protein levels of these two genes. An antibody against the SdeC protein that also recognizes both SdeA and SdeB (2) was used to immunoblot SDS-PAGE-resolved bacterial lysates of the sidj mutant and the wild-type bacteria. Our results indicate that expression of either sdea or sdeb was not detectably affected in the sidj deletion mutant (Fig. 3B). Thus, the incomplete complementation of the mutant in the protozoan host most likely results from some other factors, such as the elevated expression level of sidj, which may interfere with the translocation of other Dot/Icm substrates. To analyze the role of sdja in intracellular growth, we con-

7 598 LIU AND LUO INFECT. IMMUN. FIG. 4. sidj and sdja mutants display different growth defect phenotypes in host cells. L. pneumophila strains grown to postexponential phase were used to infect murine bone marrow macrophages (A) or D. discoideum (B and C) at an MOI of Two hours after addition of bacteria, infection was synchronized by washing of the cells with medium. At the indicated time points, infected monolayers were lysed and the bacterial yield was determined by plating appropriate dilutions on CYE plates to obtain CFU. (A) Bone marrow macrophages were infected with Lp02 (wild type), Lp03 (dota), ZL101 (Lp02 sidj), ZL102 (Lp02 sidj/psidj), or ZL51 (Lp02 sdja). (B) Growth of the wild type (Lp02), the sidj mutant (ZL101), and the sidj complementation strain (ZL102) in D. discoideum. (C) Growth of the sdja mutant (ZL51) and complementation strain ZL103 (Lp02 sdja/psdja) in D. discoideum. The dota mutant Lp03 was included in all experiments, and no growth was observed for this strain; instead, it was digested by the host efficiently, as described earlier (35). For clarity, the data on this mutant are not shown. Growth was determined by dividing CFU at a given time point by the input bacterial cell numbers. All experiments were performed in triplicate at least three times, and data shown are from one representative experiment. Statistical analyses were performed with the Student t test. *, P 0.05; **, P ; ***, P structed an in-frame deletion mutant of this gene and tested its multiplication in all three host systems. Despite the high-level similarity to SidJ, in murine macrophages the sdja mutant did not display any detectable growth defect throughout the entire 72-h experiment cycle (Fig. 4A, strain ZL51). As expected, this mutant also grows proficiently in the differentiated U937 cells, indistinguishable from its parent strain, Lp02 (data not shown). However, this mutant exhibited detectable intracellular growth attenuation in the amoeba host D. discoideum (Fig. 4C). Intriguingly, the growth defect was almost not detectable in the first 48 h of incubation and became detectable after the incubation proceeded to 72 h, showing a five- to sevenfold defect (Fig. 4C). The growth defect exhibited by the sdja mutant in the protozoan host can be complemented by a plasmid expressing SdjA, but like SidJ, the complementation restores only about 70% of the defect (Fig. 4C). To test whether sidj and sdja exhibit functional redundancy to each other, we constructed a mutant lacking both genes and tested its intracellular growth phenotypes. In murine macrophages, the double mutant displays a growth defect indistinguishable from that of the sidj single mutant (data not shown); this result is expected given the fact that sdja is not important for growth in this host (Fig. 4A). Unexpectedly, in the amoeba host the double mutant displayed a defect highly similar to the sidj single mutant (data not shown), exhibiting about a 6-fold defect in the first 48 h of incubation and about a 20-fold defect after 72 h of infection (data not shown). The lack of a more severe growth defect in the double mutant strongly suggests that SidJ and SdjA do not function synergistically. The Dot/Icm transporter is intact in the sidj mutant. Previous studies indicated that deletion of genes coding for some Dot/Icm substrates could affect the integrity of the Dot/Icm system and thus cause growth defects (10). To examine whether activity of the Dot/Icm system is still intact in the sidj mutant, we analyzed two functions of the transporter. First, we examined the translocation of SidC by Dot/Icm during infection, a process known to be sensitive to subtle dysfunction of the transporter (40). In murine macrophages, translocation of this substrate by the sidj mutant was indistinguishable from that of the wild-type bacterium; in each case, about 85% of the bacterial vacuoles stained positively for this protein by 1 h after infection (data not shown). Second, we probed the ability of vacuoles containing the mutant to recruit the small GTPase Rab1, a process known to be completely dependent on an intact Dot/Icm system via the function of its substrate, SidM/ DrrA (11, 19, 25, 29). Immunostaining of Rab1 in infected cells revealed that vacuoles containing the sidj mutant acquired this host protein as efficiently as the wild-type strain, with about 80% of the vacuoles stained positively for this host protein (data not shown). These results indicate that function of the Dot/Icm remains intact in the sidj mutant and that the growth defect exhibited by the mutant was solely due to the loss of this gene. The sidj mutant forms more small vacuoles in murine macrophages. At least two possibilities can lead to the intracellular growth defect exhibited by the sidj mutant: the initiation of multiplication by the mutant was delayed in the host cells, or the doubling time of the mutant was longer than that of the wild-type strain. Similarly, lower total bacterial counts could result from two possibilities: first, the mutant forms uniformly

8 VOL. 75, 2007 LEGIONELLA EFFECTOR IMPORTANT FOR RECRUITING ER PROTEINS 599 smaller vacuoles containing fewer bacteria than those of wildtype; second, unlike the wild-type strain, which forms predominantly big vacuoles containing large numbers of bacteria, vacuoles formed by the mutant might consist of a high percentage of small vacuoles and some large vacuoles comparable to those formed by the wild-type strain. To distinguish these possibilities, we conducted an infection center assay (10) to determine the initiation of multiplication of the mutant as well as the distribution of vacuole sizes. We infected murine macrophages with the appropriate bacterial strains at an MOI of 0.5; the use of a low MOI was to ensure that few cells were infected by multiple bacteria. We then scored the distribution of vacuoles categorized by the number of bacteria they contained at different time points in the first 14 h of infection. Fourteen hours was chosen because normally, within this time frame, no infected cells have been lysed and there is thus no secondary infection (39). As expected, bacterial numbers in vacuoles containing the growth defect dota mutant remained at one bacterium per vacuole throughout the infection cycle (data not shown). At 4 h after infection, vacuoles formed by all testing strains contained only one bacterium (data not shown); at 6 h postinfection, we began to detect vacuoles containing more than one bacterium; the percentage of such vacuoles formed by the sidj mutant (about 5%) was similar to that of the wild-type strain (about 8%) (data not shown), suggesting that initiation of replication of the mutant is not affected. A similar pattern was observed at the 8-h time point. As infection proceeded to 14 h, about 35% of the sidj mutant vacuoles are small phagosomes containing 1 to 2 bacteria, about 58% of the vacuoles are medium sized with 3 to 10 bacteria, and only about 6% are large vacuoles containing more than 10 bacteria (Fig. 5). This pattern is sharply different from that of the wild-type strain, which has a 12%, 32%, and 57% distribution of the three categories of vacuoles (Fig. 5). The distribution of vacuole categories in infections using the sidj complementation strain was almost identical to that with the wild-type strain, further indicating full complementation of the intracellular growth defect by the plasmid-expressed protein in murine macrophages (Fig. 5). These results suggest that the doubling time of the bacterium was affected by the deletion of sidj. Actively avoiding fusion with the lysosomal compartment is one of the essential steps in successful biogenesis of the L. pneumophila replicative vacuole; this is particularly true in the early phase of infection (32, 37). Since the sidj mutant exhibits a significant growth defect in murine macrophages, we analyzed whether SidJ plays a role in inhibition of phagolysosomal fusion by examining the acquisition of lysosomal markers by vacuoles containing this mutant. We found that in primary macrophages, the sidj mutant is still able to efficiently block the acquisition of the lysosomal marker, because less than 14% of mutant phagosomes stained positively for LAMP-1, a rate indistinguishable from the 13% exhibited by the wild-type strain (data not shown). In contrast, about 85% of dota mutant vacuoles have incorporated this marker. Similar data were obtained in experiments using the D. discoideum strain AX3, expressing the major subunit gene vatm of the vacuolar ATPase fused to GFP (VatM::GFP) (7, 23). Four hours after infection, whereas more than 90% of the vacuoles containing the dota mutant have acquired the vatm::gfp signal, about 52% of the vacuoles harboring the sidj mutant are positive for FIG. 5. The sidj mutant forms more small vacuoles in bone marrow macrophages. Murine bone marrow macrophages attached to glass coverslips were infected with the indicated bacterial strains at an MOI of 0.5. After removal of extracellular bacteria by washing at 2 h after infection, samples were withdrawn and fixed at the indicated time points. Extracellular and intracellular bacteria were labeled with an anti-l. pneumophila antibody and different fluorescent dyes. The number of bacteria per vacuole in each strain was scored and categorized into small (1 to 2 bacteria), medium (3 to 10 bacteria), and large (more than 10 bacteria) vacuoles. Similar results were obtained in at least three independent experiments; data shown are averages of triplicate infections in which more than 100 vacuoles were scored per sample. Statistical analyses were performed with the Student t test. **, P ; ***, P this marker, a rate similar to that observed for the wild-type strain (48%) (data not shown). Thus, SidJ is not important for blocking fusion of the L. pneumophila phagosome with the lysosomal compartment in the protozoan host. SidJ is required for efficient recruitment of proteins of ER origin. In addition to bypassing the endocytic pathways, LCVs actively incorporate membrane components originating in the ER and eventually are transformed into compartments characteristic of the rough ER (20). We then examined whether deletion of sidj affects the acquisition of calnexin, an ER resident protein commonly used to assess interception of ER membrane materials by L. pneumophila phagosomes (11, 20). Early studies indicated that, compared to whole-cell staining, the use of isolated L. pneumophila vacuoles for assessing calnexin recruitment is a more sensitive method because positive signals were detected on a high percentage of vacuoles within 10 min of infection (11). Thus, we infected murine macrophages with GFP-labeled bacterial strains and prepared PNS from infected cells by a described procedure (11). Isolated vacuoles adhering to coverslips were stained with an anti-calnexin antibody, and association of this protein with the vacuoles was scored by fluorescence microscopy inspection. In vacuoles prepared from murine macrophages of the A/J mouse strain, 10 min after infection, about 82% of the vacuoles containing the wild-type bacteria stained positively for this protein, whereas about 60% of sidj mutant vacuoles were positive (Fig. 6A). After the infection was allowed to proceed for one hour, more than 88% of the wild-type vacuoles had acquired this protein, and the calnexin-positive vacuoles in the mutant infection increased to 78%, which still is significantly (P 0.003) lower than that of the wild-type strain (Fig. 6A). Few vacuoles

9 600 LIU AND LUO INFECT. IMMUN. FIG. 6. SidJ is required for efficient acquisition of ER protein markers. For the recruitment of calnexin, macrophages were infected with the indicated bacterial strains at an MOI of 5 for 10 min or 1 h, and postnuclear supernatant containing LCVs was prepared and stained for calnexin (see Materials and Methods). Data shown are from infections using bone marrow macrophages (A) or differentiated U937 cells (B). For kinetic analysis of the recruitment of HDEL-GFP (C), D. discoideum strain AX4::HDEL::GFP was infected with the appropriate bacterial strains and samples withdrawn at the indicated times were processed for fluorescence microscopy analysis. Similar results were obtained in three independent experiments, and data shown are from one representative experiment performed in triplicate. At least 100 vacuoles were scored from each sample. Statistical analyses were performed with the Student t test. **, P 0.001; ***, P containing the dota mutant were positively stained for this marker (Fig. 6A). A very similar pattern was observed in the more-permissive host U937 cell, in which more than 86% of wild-type vacuoles stained positively for calnexin, compared to 64% of the mutant in samples prepared from 10-min infections (Fig. 6B). However, at one hour postinfection, no difference was detected between these two strains; in both cases, about 90% percent of the vacuoles stained positively for this protein (Fig. 6B). These results suggest that mutation in sidj affects the efficiency of ER recruitment by the bacterial phagosome and that the efficiency of ER protein recruitment may contribute to the permissiveness of the host cells. Because the sidj gene also is required for proficient replication in D. discoideum, we assessed whether the mutant is defective in recruiting ER markers in this host. To this end, we utilized a strain of D. discoideum stably transfected to express GFP-HDEL, a marker that has been successfully used to study Dot/Icm-dependent recruitment of ER proteins by LCVs (21). After incubation of the D. discoideum transfectants with bacteria, samples were withdrawn at time points ranging from 30 min to 8hand processed for immunofluorescence analysis. At 30 min, about 70% of the LCVs containing replication-competent wild-type L. pneumophila were surrounded by the GFP-HDEL signal (Fig. 6C); the ratio of HDEL-GFP positive vacuoles increased to about 85% at 4 h after infection and was maintained at this level throughout the extra 4 h of experiment duration (Fig. 6C). No such association was observed in infections using the growth-defective dota mutant (Fig. 6C). In sidj mutant infections, at 30 min after uptake, only 35% of the vacuoles have recruited the HDEL-GFP signal; this ratio was maintained for 2 h (Fig. 6C). As the infection proceeded to 4 h, more HDEL- GFP-positive sidj vacuoles (62%) were detected, and the ratio continued to increase until it became indistinguishable from that of the wild-type strain at 8 h after uptake (Fig. 6C). The kinetics of HDEL-GFP recruitment exhibited by phagosomes containing the complementation strain is highly similar to that of the wild-type bacteria (Fig. 6C). These data further indicate that sidj is solely responsible for this phenotype. Thus, SidJ is important for efficient acquisition of ER materials, i.e., interception of ER-derived vesicles to bacterial phagosomes. DISCUSSION In this study we describe the identification and characterization of two proteins important for the biogenesis of L. pneumophila replicative vacuoles. Our immunostaining analysis using the SidC C100 fusion and biochemical fractionation data clearly show that SidJ is a substrate of the Dot/Icm system and that it is delivered into host cells during infection. Given the high similarity between SidJ and SdjA and the fact that SdjA promotes translocation of SidC C100, we propose that SdjA is also translocated into host cells by Dot/Icm during infection. In addition to its important role in L. pneumophila intracellular replication, the SidJ protein distinguishes itself from many other Dot/Icm substrates in its expression level in response to bacterial growth phase transition. The SidJ protein is maintained at a constant level during the entire growth cycle of the bacterium (Fig. 3A). Consistent with this observation, a large amount of SidJ was injected into host cells even when the infection proceeded to 14 h after bacterial uptake (Fig. 2F),