Banga et al. 10.1073/pnas.0611030104. |
Fig. 7. Growth phase-dependent expression of sidF and Dot/Icm-mediated translocation of SidF. (A) Bacterial grown in AYE broth to either exponential (E) phase (OD600 = 2.0) or early postexponential (PE) phase (OD600 = 3.6) were harvested and analyzed by SDS/PAGE and immunoblotting with affinity-purified anti-SidF. Displayed are wild-type (Lp02:dot/icm intact), dotA-(Lp03) and DsidF(Lp02DsidF) and D sidF/pSidF(Lp02DsidF/SidF). The isocitrate dehydragenase (ICDH) protein was used as a loading control by probing with indicated density and SidF was detected with by antiserum raised against B. subtilis ICDH (a kind gift from A. L. Sonenshein, Tufts Medical School, Boston, MA). Relevant molecular markers (in Kda) were on the left of the blot. (B) Dot/Icm dependent translocation of SidF. Macrophages were infected with for indicated time points with Lp02(dot/icm intact), Lp03(dotA-) or Lp02(DsidF) and Lp02(DsidF/pSidF), respectively. Infected cells were fixed and L. pneumophila and SidF was probed with specific antibodies and secondary antibodies conjugated to distinct fluorescence dye, respectively. Stained macrophages were scored for translocation of SidF by counting phagosomes that stained positively with anti-SidF. Data shown are from two independent experiments performed in triplicate in which at least 100 phagosomes were scored per coverslip. (C) Limited diffusion of SidF from L. pneumophila phagosome. Shown are images of macrophages infected with Lp02(dot/icm intact) for 6 h. Bacteria and SidF are probed as above, with bacteria marked by red and SidF displayed in green. Note that almost all LCVs stained positively for SidF.
Fig. 8. SidF is required for proficient intracellular growth in mouse bone marrow macrophages. 4 ´ 105 macrophages were infected with indicated L. pneumophila strain at an MOI of 0.05. At each indicated time point, macrophages were lysed with 0.02% saponin and lysates or appropriate dilutions of lysates were plated onto bacteriological media. Total bacterial counts were obtained by enumerating colony-forming-units. Similar data were obtained from at least 3 independent experiments performed in triplicate.
Fig. 9. Localization of SidF to the endoplasmic reticulum required the two small C-terminal hydrophobic regions. COS1 cells were transfected to express SidF (A-C) or the DTM1/TM2 mutant (D and E) fused to the C terminus of GFP. The endoplasmic reticulum network was visualized by immunostaining using an anti-body specific for the ER protein calnexin. Note the ER/para-nuclear localization of full-size SidF (A) and the cytosolic localization of the mutant (D). Images were acquired and processed with the IPlab software package (Scanalytic, Fairfax, VA).
Fig. 10. U937 cells infected by the sidF mutant are more apoptotic. (A) The sidF is not required for intracellular growth in U937 cells. Appropriately 4 ´ 105 differentiated U937 cells were infected with indicated L. pneumophila strains and total bacterial counts at each time point were obtained by plating sample lysates or dilutions of lysates onto bacteriological medium. (B) U937 cells infected by the sidF mutant displayed higher cell death rate. 2 ´ 105 cells were infected with indicated L. pneumophila strain at an MOI of 1 for 14 h. Cells were fixed and extracellular bacteria were distinguished from intracellular bacteria by immunostaining using an anti-L.pneumophila antibody. Apoptotic status of the cells was stained by TUNEL assay and was discerned by microscopic inspection. Experiments were performed independently at least five times in triplicate and at least 150 infected cells were scored each sample. Data shown are from a representative experiment.
SI Materials and Methods
Bacterial Strains and Media and Plasmid Construction.
All L. pneumophila strains used in this study were derivatives of the Philadelphia 1 strain Lp02 (1). Bacteria were grown and maintained on CYE medium as previously described (2). E. coli stains were grown and maintained on LB agar or LB broth. When necessary antibiotics were included as described (2). Following a standard procedure (3), we generated the sidF in-frame deletion mutant ZL07 with plasmid pZLDsidF, which was constructed by ligating into SacI/SalI digested pSR47s (1) two PCR products amplified from Lp02 genomic DNA using PL08/PL09 (Table 2), PL09/PL10 (digested with BamHI/SacI). To complement the mutation, the coding region of sidF was amplified using PL012/PL07 and the PCR product was inserted into pJB908 (4) as a SacI/XbaI fragment. All infection was conducted with bacterial cultures grown to postexponential phase as judged by optical density of the cultures (OD600 ranges from 3.3-3.8) as well as bacterial motility.SidF and its derivatives were cloned into pGBKT7 and pEGFPC1 for yeast two-hybrid analysis and for expressing in mammalian cells, respectively (sequences of primers used were on Table 2S). Internal deletion of sidF was performed by ligating overlapping PCR products or specified DNA fragments (sequences of primers used for PCR are listed on Table S2). To express Flag-tagged BNIP3 and Bcl-rambo, cDNAs of these genes were amplified from a human cDNA library and pL661 (5), and were inserted into BglII/SalI digested pCMVFlag, respectively. Integrity of all inserts was verified by double strand DNA sequencing analysis.
Yeast Manipulation and Two-Hybrid Assay.
Yeast strains were grown in YPD medium or in appropriate amino acid dropout minimal media. The sidF gene was fused to the activation domain of the Gal4 activator on pGBKT7 (BD Biosciences) as an EcoRI/SalI fragment and the resulting plasmid pZL513 was introduced into yeast strain PJ-64A (6) by standard transformation protocols. Expression of the fusion protein in yeast was assessed by Western blot using anti-SidF antibody. The human kidney cDNA two-hybrid library was purchased from BD Bioscience; library amplification and screening were performed according to instructions of the manufacturer.Protein Purification.
The ORF of sidF was cloned into pGEX-6-P-1 as a BamHI/SalI fragment to generate pZL264, GST-SidF was purified from E. coli and the GST tag was subsequently removed using the Precision protease (GE Biosciences). To purify histidine(6)-tagged proteins, genes or fragments of genes were inserted into pQE30 (Qiagen) and proteins produced in strain XL1Blue were purified using Ni-NTA agarose following manufacturer's instructions (Qiagen). Protein concentrations were determined by the Bradford assay; the purity of all proteins was more than 95% as assessed by SDS-PAGE followed by Coomassie bright blue staining.Cell Culture, Transfection, and Infection.
U937 cells were cultured in RPMI medium supplemented with 10% fetal bovine/calf serum (FBS) and 5 mM glutamate. Other cell lines, including 293T, HeLa, MCF-7 and COS1 were cultured in DMEM supplemented with 10% FBS. When necessary, U937 were differentiated into macrophages by incubating at 37°C with 5% CO2 for 36-48 h with 50 ng/ml phorbol myristic acid (PMA). Cells grown to about 80% confluence were transfected with Lipofectamine 2000 (Invitrogen) following manufacturer's instructions. For experiments designed to assess apoptosis inhibitory activity of SidF, to ensure that cells transfected by SidF also received the Bcl-rambo plasmid, a molar ration of 1:3 (SidF:Bacl-rambo) ratio of DNA was used. Mouse macrophages were prepared from marrow of female mice of 6-10 weeks of age following published protocols (7). For infection, macrophages were plated into 24-well plates at 2 ´ 105 cell per well. Infection was carried out at indicated MOIs as required by the objectives of the experiments. In all cases, one hour after adding bacteria to cultured cells, infection was synchronized by washing infected cells three times with warm PBS buffer.Immunoprecipitation.
We seeded 293T cells on 100-mm plates at 5 ´ 105 cells per plate one day before transfection, and transiently transfected cells with combinations of plasmids. In all experiments, the total amounts of plasmids were kept constant by adding appropriate amounts of vector DNA. Thirty hours posttransfection, we washed cells with ice-cold PBS and lysed cells with a solution containing Tris (20 mM, pH 8.0), NaCl (150 mM), 0.2% Nonidet P-40 (wt/vol), 10% glycerol (wt/vol), NaF (100 mM), PMSF (1 mM), sodium orthovanadate (Na3VO4)(1 mM), aprotinin (20 mg/ml), and leupeptin (40 mg/ml). After incubation on ice for 30 min, cell lysates were centrifuged at12,000g for 15 min at 4°C. To prepare immunoprecipitates, we incubated cell lysates with an appropriate antibody and protein G agarose (Roche) for 7-12 h at 4°C. We then washed the agarose beads five times with lysis buffer, and solubilized adsorbed proteins by boiling the washed beads for 5 min in SDS-PAGE loading buffer. Proteins of interest were detected by Western blot after SDS-PAGE. To detect the second protein, membranes were treated with a striping buffer (2% SDS, 100 mM b-mercaptoethanol, 62.5 mM Tris-Cl, pH 6.8) for 30 min at 55°C and the proteins were detected by appropriate antibodies. For co-immunoprecipitation using lysates from infected cells, 5 ´ 107 U937 macrophage cells seeded in 100 mm Petri dish were infected at an MOI of 5 for 6 h with indicated L. pneumophila strains, respectively. Cell lysates and precipitates were prepared as described above (we predetermined that up to 1% Nonidet P-40 did not lyse L. pneumophila under the extraction condition).Antibodies, Immunostaining, and Western Blot.
SidF and Bcl-rambo specific antibodies were prepared by injecting rabbits with purified proteins following standard protocols (Pocono Rabbit Farm and Laboratory, Canadensis, PA). Antibodies were affinity-purified against the same proteins covalently coupled to an Affigel matrix (Bio-Rad) using standard protocols (8). Monoclonal and polyclonal antibodies against FLAG, calnexin, actin, BNIP3 and cytochrome c were purchased from Sigma, Stressgen and Santa Cruz, respectively. For immunostaining, anti-Legionella antisera: 1:10,000; purified anti-SidF was used at 1:200; anti-Bcl-rambo: 1:200; anti-cytochrome C: 1:50. Cell fixation, permeabilization and immunostaining were performed as described (2). Intracellular bacteria were distinguished from extracellular bacteria by differential immunostaining with anti-L. pneumophila antibodies.For Western blot, samples resolved by SDS-PAAGE were transferred onto nitrocellulose membranes. After blocking with 4% milk, membranes were incubated with the appropriate primary antibody: anti-SidF, 1:10,000; anti-Bcl-rambo, 1; 10,000; anti-FLAG, 1:20,000; anti-actin, 1:1000; anti-BNIP3, 1:1000. Serum specific for Bacillus subtilis isocitrate dehydrogenase (ICDH) was generously provided by A.L. Sonenshein, Tufts University Medical School (Boston, MA) and was used at 1:10,000. Horseradish peroxidase-conjugated secondary antibodies and the enhanced bioluminescence reagents were used to detect the signals (Pierce, Rockford, IL).
Recombinant Adenoviral Particle Preparation and Transduction.
sidF was cloned into pShuttle CMV-YFP (Stratagene) as a BglII/SalI PCR fragments using primers PL05/PL07. This clone was used to prepare a recombinant adenoviral vector containing sidF through homologous recombination in bacterial strains using Adeasy system (Stratagene). The recombinant adenoviral plasmid was transfected into AD-293 cells using Lipofectamine 2000 to prepare packagable primary adenovirus stock. The virus was harvested from AD-293 cells using four rounds of freeze/thaw cycle and collected by microcentrifugation at 12,000 ´ g for 20 min. The primary stock was propagated (by passage) four times in AD-293 cells to achieve titers of 1010-1012 infectious particles/ml. This viral suspension was used to transduce HeLa cells and U937 cells at an MOI of 10. The control viral particles were prepared in a similar manner using the empty vector.Cell Death Assays.
Apoptotic status of mammalian cells was determined by using the terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay (9) with the in situ cell death detection kit TMR red (Roche) or by staining of nuclei with Hoechst 33342. Cell death was determined by scoring cells with positively labeled fragmented chromatin (TUNEL assay) or condensed nuclei (Hoechst staining). We determined that the percentages of apoptotic cells obtained by TUNEL staining or Hoechst staining are highly similar.Data Collection and Statistic Analyses.
All experiments involved in quantitative data were performed in triplicate for at least three independent times; statistic analysis was carried out with Student's t test.1. Berger KH, Isberg RR (1993) Mol Microbiol 7:7-19.
2. Conover GM, Derre I, Vogel JP, Isberg RR (2003) Mol Microbiol 48:305-321.
3. Dumenil G, Isberg RR (2001) Mol Microbiol 40:1113-1127.
4. Bardill JP, Miller JL, Vogel JP (2005) Mol Microbiol 56:90-103.
5. Kataoka T, Holler N, Micheau O, Martinon F, Tinel A, Hofmann K, Tschopp J (2001) J Biol Chem 276:19548-19554.
6. James P, Halladay J, Craig EA (1996) Genetics 144:1425-1436.
7. Swanson MS, Isberg RR (1995) Infect Immun 63:3609-3620.
8. Harlow E, Lane D (1999) in Using Antibodies, a Laboratory Manual (Cold Spring Harbor Lab Press, Cold Spring Harbor, NY), pp 311-343.
9. Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) J Cell Biol 119:493-501.