The Type VI Secretion System Encoded in Salmonella Pathogenicity Island 19 Is Required for Salmonella enterica Serotype Gallinarum Survival within Infected Macrophages

Bacterial strains and growth conditions.The bacterial strains used in the present study are listed in Table 1. Bacteria were routinely grown in Luria-Bertani (LB) medium (10 g/liter tryptone, 5 g/liter yeast extract, 5 g/liter NaCl) at 37°C with aeration. When required, LB medium was supplemented with ampicillin (Amp; 100 mg/liter), chloramphenicol (Cam; 20 mg/liter), and kanamycin (Kan; 50 mg/liter). Media were solidified by the addition of agar (15 g/liter). For SPI-1-inducing conditions, bacteria were grown in LB medium supplemented to reach final concentrations of 0.171 M NaCl, 0.3 M NaCl, or 0.2 M sucrose, as described previously (16). LB medium (0.085 M NaCl) was used as a control condition. For SPI-2-inducing conditions, bacteria were grown in a modified N-minimal medium [5 mM KCl, 7.5 mM (NH₄)₂SO₄, 0.5 mM K₂SO₄, 1 mM KH₂PO₄, 0.1 mM Tris-HCl, pH 5.5, 38 mM glycerol, 0.1% Casamino Acids] adjusted to pH 5.5 and supplemented to reach a final concentration of 10 μM MgCl₂ (17). N-minimal medium, adjusted to pH 7.4 and supplemented to achieve/reach a final concentration of 10 mM MgCl₂, was used as a control.

Standard DNA techniques.Total genomic DNA was obtained from overnight bacterial cultures using the GenElute bacterial genomic DNA kit (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions. Plasmid DNA was obtained from overnight cultures using either the QIAprep Spin Miniprep kit or the QIAprep Spin plasmid Maxikit (Qiagen, Germantown, MD) according to the manufacturer's instructions. PCR products were purified using the QIAquick PCR purification kit (Qiagen, Germantown, MD) as recommended. The multicopy plasmid pGEM-T Easy (Promega, Madison, WI) was used for complementation studies. DNA samples were routinely analyzed by electrophoresis in 1% agarose gels (prepared in 1× Tris-acetate-EDTA buffer) and visualized under UV light after ethidium bromide staining.

Mutant constructions and analyses.Mutant strains with deletions of SPI-19 (SG1024 to SG1054), clpV (SG1034), icmF (SG1031), hcp1 (SG1029), hcp2 (SG1043), and vgrG (SG1044) were constructed using the Red-swap method (18). PCR primers 60 bases long were synthesized with 40 nucleotides (nt) on the ends corresponding to the extreme of the desired deletion (Table 2) and 20 nt that anneal to the 5′ or 3′ end of a Cam resistance cassette flanked by the FRT sites (FLP recombinase target sequence) present in plasmid pCLF2 (GenBank accession number HM047089), which was used as the template DNA. S. Gallinarum strain 287/91 (10), carrying the temperature-sensitive plasmid pKD46, which expresses the Lambda-Red recombinase system, was grown to an optical density at 600 nm (OD₆₀₀) of 0.5 at 30°C in LB medium containing Amp and l-arabinose (10 mM). Bacteria were made electrocompetent by sequential washes with ice-cold, sterile 10% glycerol and transformed with approximately 500 ng of the corresponding purified PCR product. Transformants were selected on LB Cam agar at 37°C. The presence of each mutation was confirmed by PCR amplification using primers flanking the sites of substitution. Also, each mutant was assayed for Amp sensitivity to confirm the loss of pKD46. Finally, mutant alleles were transferred to the wild-type background by generalized transduction using P22 phage.

To obtain nonpolar deletions, the Cam resistance gene was removed by transforming each mutant with pCP20, which encodes the FLP recombinase. Transformants were plated on LB Amp agar at 30°C. Individual colonies were replica plated on LB agar, LB Amp agar, and LB Cam agar at 37°C. Transformants that had lost the resistance cassette and pCP20 were selected as those colonies that were unable to grow in the presence of Amp and Cam. The absence of the antibiotic resistance gene cassette was confirmed for each mutant by PCR analysis.

To obtain the S. Typhimurium 14028s ΔSPI-1 mutant strain, a P22 phage lysate from ΔSPI-1::Kan S. Typhimurium SL1344 (NCTC 13347) (19) was used to transduce this mutation to the 14028s background.

Construction of lacZ operon fusions.To construct lacZ operon fusions, the method developed by Ellermeier and collaborators was used (20). This method consists of the specific integration of a suicide plasmid (pCE36) that has an FRT site directly upstream of promoterless lacZY into a chromosomal FRT site from a previous Red-swap mutation. First, S. Gallinarum mutant strains Δhcp1::FRT, Δhcp2::FRT, ΔicmF::FRT, ΔclpV::FRT, and ΔvgrG::FRT were transformed with the pCP20 plasmid and selected for Amp resistance at 30°C. These strains were then grown at 30°C and transformed by electroporation with plasmid pCE36. Transformants were recovered in LB, and dilutions were plated on LB Kan 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal) agar at 37°C. Growth at 37°C allows for the optimal production of FLP recombinase and blocks the replication of the temperature-sensitive plasmid pCP20, resulting in the stable integration of pCE36 and the loss of pCP20. Fusions were checked by PCR with the appropriate set of primers (Table 2) to ensure integration of pCE36 in the correct chromosomal location and to rule out the presence of multiple plasmid integrants.

Construction of 3×FLAG gene fusions.Epitope tagging of T6SS components was performed by constructing gene fusions between the hcp1, hcp2, and vgrG open reading frames (ORFs) and the 3×FLAG epitope by a modification of the Red-swap method described by Uzzau and collaborators (21) using primers listed in Table 2. The PCR products were amplified from template plasmid pSUB11 (Table 3), and the Red-swap protocol was similar to that described above. Each fusion was checked by PCR and transduced into a clean wild-type background, and then the resistance marker was excised from the chromosome as described above.

Construction of a VgrG-GFP gene fusion.A gene fusion between the green fluorescent protein (GFP) ORF and the ORF of the VgrG T6SS component (termed VgrG-GFP) was constructed by a modification of the Red-swap method described by Gerlach and collaborators (22). The PCR product was amplified using primers listed in Table 2 and plasmid p3174 as the template (Table 3) and included the ORF encoding the GFP reporter followed by a Kan resistance cassette flanked by FRT sites. Proper integration of the reporter cassette was confirmed by PCR. Positive clones were moved into a wild-type background by generalized transduction using P22, and the resistance marker was excised from the chromosome as described above.

Protein sample preparations.For whole-cell lysates, bacteria were grown overnight in an orbital shaker at 37°C and subcultured in the appropriate growth media. When the culture reached an OD₆₀₀ of ∼0.3 to 0.4, the cells were collected by centrifugation (13,000 rpm for 2 min). The supernatant was removed and the pellet was weighted in order to normalize samples. Each mg of bacterial pellet was resuspended in 10 μl of 1× protein loading buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.01% bromophenol blue, 5% β-mercaptoethanol). Finally, the samples were boiled for 10 min and stored at −20°C.

For protein secretion analyses, bacterial cells were removed by centrifugation (13,000 rpm for 2 min). The culture supernatant was filter sterilized and mixed with trichloroacetic acid (TCA) to a final concentration of 10%. The samples were chilled on ice for 30 min and centrifuged at 13,000 rpm for 15 min. The protein pellet was washed twice with ice-cold acetone, air dried, and resuspended in 1× protein loading buffer. Finally, the samples were boiled for 5 min and stored at −20°C.

SDS-PAGE analyses and immunoblotting.Polyacrylamide gels (12%) were prepared and mounted using a Mini-Protean III (Bio-Rad, Richmond, CA) PAGE system. The samples were run at 100 V (constant) with 1× protein running buffer (1.44% glycine, 0.3% Tris, 0.1% SDS). Transfer from the polyacrylamide gel was performed in a Mini Trans-Blot system (Bio-Rad, Richmond, CA). A polyvinylidene difluoride (PVDF) membrane was activated by immersion in 100% methanol for 15 s. The transfer was performed for 65 min at 230 mA in 1× transfer buffer (1.44% glycine, 0.3% Tris, 0.01% SDS, and 20% methanol). The membrane was blocked by incubation in a solution containing 5% nonfat milk in TBS (25 mM Tris, pH 8, 125 mM NaCl) for 90 min at room temperature. Later, the membrane was incubated at 4°C for 2 h with the M2 anti-FLAG primary antibody (Sigma-Aldrich, St. Louis, MO) diluted at 1:10,000 in TBS. For sigma 70 detection, an anti-RNA polymerase sigma 70 antibody (ab12088) was used (Abcam, Cambridge, United Kingdom) at a 1:1,000 dilution in TBS. After five washes with a TBS–0.05% Tween solution, the membrane was incubated at room temperature for 2 h with a 1:20,000 dilution of the secondary antibody conjugated with horseradish peroxidase. For immunodetection, the Supersignal West-Pico solution (Pierce, Rockford, IL) was used, exposing the membrane on a CL-Xposure (Pierce, Rockford, IL) photographic film.

β-Galactosidase assays.Bacteria were grown overnight in LB broth and then subcultured and grown in 50 ml of the appropriate medium at 37°C in an orbital shaker. Every 30 min, a 1-ml sample was withdrawn to evaluate bacterial growth (OD₆₀₀ and CFU/ml), and the β-galactosidase activity was measured as described previously (20). Enzyme activities (in Miller units), normalized for cell density (OD₆₀₀), were calculated using the following equation: [A₄₂₀ − (1.75 × A₅₅₀)] × 1,000/(reaction time × culture volume × OD₆₀₀), where reaction time was measured in minutes and culture volume in ml. Each sample was analyzed in triplicate in at least three independent experiments.

Bacterial killing assays.Bacterial strains used for the interbacterial competition studies included Enterobacter cloacae (ATCC 700323), Shigella sonnei (ATCC 25931), Escherichia coli (ATCC 35218), Escherichia coli K-12 (ATCC 25922), Serratia marcescens, and Staphylococcus saprofiticus (ATCC BAA750). Each of these strains was tested for competition against antibiotic-resistant derivatives of the wild-type and ΔSPI-19 mutant strains. A Cam-resistant derivative of S. Gallinarum was generated by transformation with plasmid pWRKC (Cam^r), a derivative of plasmid pNFB9 which can integrate at the Gifsy-1 attachment site (attG1) in the chromosome (23). The chromosomal insertion of pWRKC, flanking the attG1 site, was confirmed by PCR amplification using primers LepA_F and LepA_R (Table 2). For competition assays, each strain was grown overnight in LB media and then collected and washed three times in sterile phosphate-buffered saline (PBS; 13,000 rpm for 2 min). The cell suspensions were normalized to an OD₆₀₀ of 0.5 and mixed at a 1:1 ratio with either the S. Gallinarum pWRKC (Cam^r) or the ΔSPI-19::Kan (Kan^r) strain. Fifty μl of the mixture then was spotted onto a prewarmed brain heart infusion (BHI) agar plate and incubated for 1 week at 37°C. The initial inoculum was serially diluted and plated to determine the initial CFU/ml. After 1 week, cells were recovered from the plates and resuspended in 1 ml of sterile PBS, and serial dilutions were plated out on BHI agar with or without kanamycin. The amount of prey CFU was determined by subtracting the Kan-resistant CFU counts from the total CFU counts.

Cell culture conditions.The murine macrophage cell line RAW264.7 and the avian macrophage line HD11 were routinely maintained in Dulbecco's modified Eagle medium (DMEM) high glucose (high glucose is composed of 584 mg/liter l-glutamine, 4,500 mg/liter glucose, 110 mg/liter sodium pyruvate) supplemented with 10% inactivated bovine fetal serum (FBS) and antibiotic/antimycotic solution (10,000 nU/ml penicillin, 10,000 mg/liter streptomycin, 25 mg/liter amphotericin B) at 37°C in a 5% CO₂ atmosphere.

In vitro infection assays.Gentamicin protection assays were performed according to Schwan and collaborators (24). RAW264.7 and HD11 macrophages were seeded in 48-well cell culture plates at a density of ∼2.5 × 10⁵ cells per well. Bacterial strains were grown overnight at 37°C in an orbital shaker, and on the day of the assay they were subcultured in LB at a 1:30 dilution and grown until they reached an OD₆₀₀ of ∼0.6. The bacterial cells were collected by centrifugation (13,000 rpm for 2 min), washed three times with sterile PBS, and suspended in prewarmed 10% FBS-DMEM to reach a final concentration of 2.5 × 10⁷ CFU/ml. This suspension was serially diluted, and bacteria were quantified by plating on LB agar plates. Macrophages were washed once with sterile PBS, and then 100 μl of the bacterial suspension was added (multiplicity of infection [MOI] of 10). Plates were centrifuged at 250 × g for 5 min at room temperature to synchronize infection, and invasion was allowed to proceed for 45 min (time point 0). After this time, the wells were washed 3 times with warm, sterile PBS and the cells were further incubated for 1 h with 10% FBS-DMEM supplemented with gentamicin (200 μg/ml) to kill extracellular bacteria. The gentamicin concentration then was lowered to 10 μg/ml for the rest of the experiment. At 2 and 20 h postuptake, different wells were washed 3 times with warm, sterile PBS, and the macrophages were lysed with 0.5% sodium deoxycholate (DOC) in PBS. Serial dilutions were prepared and plated on LB agar plates to determine viable intracellular bacteria. For each assay, viable macrophages were determined by trypan blue exclusion. The intracellular survival of mutant strains was calculated as CFU per 10⁵ viable macrophages.

Viability and cytotoxicity assays.Salmonella-induced cytotoxicity was determined by the CytoTox 96 nonradioactive cytotoxicity assay (Promega, Madison, WI). For each assay, RAW264.7 and HD11 cells were seeded in 96-well plates at a density of ∼30,000 cells per well. The next day, the cells were infected with the bacterial strains as described for the infection assays. At 20 h postuptake, the supernatant of infected cells was collected and transferred to a new 96-well plate. The cytotoxicity reagent then was added following 30 min of incubation at room temperature protected from light. After incubation, absorbance at 562 nm was measured and the cytotoxicity levels were calculated as a percentage of lactate dehydrogenase (LDH) release normalized to the spontaneous release from noninfected cells.

For viability and sub-G₁ population analyses, RAW264.7 and HD11 macrophages were seeded at a density of ∼300,000 cells per well. Sub-G₁ analysis allows the detection of hypoploid cells, which are cells with lower DNA content due to apoptotic cell death. Gentamicin protection assays were performed as described above. At 20 h postuptake, cells from both the supernatant and the monolayer were recovered (to obtain a complete infected cell population), washed, and resuspended in 1 ml of warm 10% FBS-DMEM. For viability analysis, 5 μl of a 1 mg/ml stock of propidium iodide (PI) was added to 500 μl of the cell suspension, and the samples were analyzed by flow cytometry. For sub-G₁ analysis, a volume of 500 μl of the cell suspension was used. Cells were collected by centrifugation and then treated with 100% methanol for 1 h at −20°C. After incubation, cells were collected and resuspended in 500 μl of PBS containing RNase at a concentration of 50 μg/ml. After incubation for 1 h, 5 μl of a 1 mg/ml stock of PI was added. The levels of PI incorporation were quantified on a FACScan flow cytometer (Becton, Dickinson). Cell size was evaluated by forward-angle light scattering (FSC). PI-negative cells of normal size were considered alive. A total of 5,000 cells per sample were analyzed.

Epifluorescence microscopy.For epifluorescence analysis of VgrG-GFP production upon internalization of S. Gallinarum into RAW264.7 and HD11 macrophages, cells were seeded on 25-mm coverslips in 6-well plates at a density of ∼375,000 cells per well. After 20 h, the cells were infected with the bacterial strains as described for the infection assays. At either 45 min or 2 h postuptake, the cells were washed 3 times with warm, sterile PBS and fixed with a 4% formaldehyde solution for 20 min at room temperature. After incubation, the coverslips were washed with sterile PBS and mounted in a 4′,6-diamidino-2-phenylindole (DAPI)-containing solution from VectaShield (Vector Laboratories, Burlington, CA). For Cytochalasin D analysis, cells were incubated for 1 h with 10 μg/ml Cytochalasin D before infection and maintained with this concentration throughout the experiment. The samples were stored at 4°C and visualized under an epifluorescence microscope. Fluorescence microscopy analysis was performed using a Spinning Disk Olympus IX 81 fluorescence microscope, and fluorescence micrographs were captured using a Zeiss Axiocam MRC5 and CellR software.

VgrG translocation assays.The vgrG ORF (SG1044), without the corresponding stop codon, was amplified by PCR using as the template a preparation of S. Gallinarum 287/91 genomic DNA and cloned into plasmid pFlagTEM-1 (25, 26). The production of the VgrG-BlaTEM recombinant protein was assessed by SDS-PAGE and Western blot analysis using anti-3×FLAG monoclonal antibodies. To demonstrate T6SS-dependent VgrG translocation, a modified gentamicin protection assay was performed as described previously (8). RAW264.7 and HD11 macrophages were seeded in 96-well optical plates (black walls and clear, flat bottoms) and infected with each of the bacterial strains expressing the corresponding BlaTEM fusions.

Bacteria were grown to an OD₆₀₀ of 0.3 and induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) until they reached an OD₆₀₀ of 0.6. The cells were collected and washed three times with sterile PBS (13,000 rpm for 2 min) and resuspended in the appropriate volume of prewarmed 10% FBS-DMEM. Infections were performed for 45 min at an MOI of 50. Noninternalized bacteria were removed by three washes with sterile PBS, and cells were further incubated with 200 μl 10% FBS-DMEM supplemented with 1 mM IPTG and 200 μg/ml gentamicin. After 1 h of incubation, the cell supernatant was replaced with 100 μl Hanks' buffered salt solution (HBSS; Gibco) supplemented with 20 mM HEPES and 3 mM probenecid (pH 7.4; Sigma-Aldrich, St. Louis, MO) (designated HBSS-HP), and 20 μl freshly prepared CCF4-AM β-lactamase substrate (LiveBLAzer FRET-B/G loading kit; Invitrogen) was added. After 90 min of incubation at room temperature in the dark, the cells were washed five times with HBSS-HP. Fluorescence emission at 450 and 520 nm was measured from the bottom using a plate reader fluorometer (Synergy H1; BioTek) (410-nm excitation wavelength, 10-nm band pass). The translocation level was calculated as recommended in the LiveBLAzer FRET-B/G loading kit manual. Briefly, emission values were first corrected by subtraction of the average background signals recorded for empty wells, and the mean 450-nm/520-nm emission ratio of a triplicate of wells was calculated for each sample. The translocation level is expressed as fold increase of the mean 450-nm/520-nm emission ratio of each infected sample relative to the mean emission ratio of uninfected cells. Epifluorescence microscopy analysis was performed using a Spinning Disk Olympus IX 81 fluorescence microscope. Fluorescence micrographs were captured using a Zeiss Axiocam MRC5 and CellR software. Images were imported into the ImageJ software (v.1.44).

Statistical analysis.The statistical significance of differences in the data was determined using one-way analysis of variance (ANOVA) with Dunnett's posttest (GraphPad Prism v5.0).