Reagents and preparation of protein samples for proteomic analysis
All reagents were obtained from Sigma-Aldrich unless otherwise specified. Salmonella enterica serovar Enteritidis (clinical isolate SE2472)  was cultured in LB broth-like normal (14N) and 15N-labeled media (Silantes GmbH, München, Germany), which are identical in chemical composition. The percentage of 15N in the labeled media is more than 98% (Silantes GmbH, München, Germany). The cultures were inoculated with a starter culture grown in normal (14N) or 15N-labeled media until mid-log phase. Two hundred fifty milliliter culture medium was inoculated with each starter culture and grown at 37°C with shaking at 225 rpm for 4 h. 15N-labeled culture was treated with 5 mM H2O2, which is well below the minimal inhibition concentration (MIC) of SE2472 (20 mM), and both cultures were grown for 2 h following the addition of H2O2. Protein extraction was performed with B-PER® bacterial protein extraction reagent (Thermo Fisher Scientific, Rockford, IL) and quantified with Dc Protein Assay Kit (Bio-Rad, Hercules, CA), which has an error rate of 2.5% in our experiments. We took this error rate into consideration by classifying any protein that had a 5% change or less as unchanged (having a 0% change).
Two-dimensional gel electrophoresis and visualization of bacterial proteins
Protein samples were further solubilized in rehydration buffer (8 M urea, 2% CHAPS, 50 mM DTT, 0.2% Bio-Lyte® 3/10 ampholytes [Bio-Rad, Hercules, CA] and trace amount of Bromophenol Blue). ReadyStrip™ IPG strips (Bio-Rad, Hercules, CA) were loaded with 200 μg of protein samples (either normal or 1:1 mixture of normal and 15N-labeled samples) for preparative 2 D gels, and allowed to rehydrate for 18-22 h. Isoelectric focusing (IEF) was performed at 20°C using PROTEAN® IEF cell (Bio-Rad, Hercules, CA). A 3-step protocol (250 V-20 min/8,000 V-2.5 h/8,000 V-10,000 V.h) was used for the IEF procedure following manufacturer's recommendations (Bio-Rad, Hercules, CA).
After the IEF procedure, the IPG strips were reduced in Equilibration Buffer I (6 M urea, 2% SDS, 0.375 M Tris-HCl [pH 8.8], 20% glycerol, 2% DTT) and alkylated in Equilibration Buffer II (6 M urea, 2% SDS, 0.375 M Tris-HCl [pH 8.8], 20% glycerol, 0.25% iodoacetamide). Strips were loaded onto 8-16% Criterion™ Tris-HCl SDS gel (Bio-Rad, Hercules, CA) and electrophoresed at 200 V for 65 min. Gels were visualized using Coomassie Brilliant Blue R-250 or silver staining (Invitrogen, Carlsbad, CA).
Mass spectrometric identification of proteins
Gels were scanned and protein spots of interest were excised using the Xcise automated gel processor (Proteome Systems, North Ryde, Australia). Gel spots were destained and washed, followed by in-gel tryptic digestion using proteomic grade trypsin (Sigma-Aldrich, St. Louis, MO). Peptide fragments were collected and purified using ZipTip™ C18 reverse-phase prepacked resin (Millipore, Billerica, MA) and mixed with an equal volume of 10 mg/ml α-cyano-4-hydroxy-trans-cinnamic acid (Sigma-Aldrich, St. Louis, MO) in 0.1% trifluoroacetic acid (TFA)/50% acetonitrile solution and directly spotted onto a stainless steel target plate for mass analysis. Axima-CFR™ Plus (Shimadzu Biotech, Columbia, MD) was used for MALDI-ToF MS analysis, and 50-100 profiles were obtained for each sample, ensuring sufficient peak data for database interrogation. Probability-based scoring method with MASCOT database search engine (Matrix Science, Boston, MA) was used to identify each protein, based on the likelihood of search results being a random match. We used the following parameters for our protein identification: Database: NCBINR, MASCOT value cut off: greater than 62 (p < 0.05), Taxonomy: Salmonella, Missed cleavage: 1, Peptide Tolerance: +/- 0.75 Da, Variable modification: none, Fixed modification: none, Enzyme: Trypsin, Mass Values: Monoisotopic.
Tryptic peak data from MASCOT database searches was tabulated and elemental composition of each peptide fragment was determined using an in-house data analysis software. The process was further automated using a custom VBScript written for Microsoft Excel, which was designed to calculate predicted 15N peak location based on the primary amino acid sequence of tryptic peptide fragments. 14N/15N mixture MS spectrum was used to obtain peak intensity ratio between labeled (15N) and unlabeled (14N) samples to give relative quantification data. An average of 10 peaks was used to calculate the mean intensity ratios and the error percentage of each protein spot. Significant outliers were manually removed from the data set to prevent them from affecting the results (less than 2%). To further increase the accuracy of our results, experiments were preformed three times, and the results were the average from the triplicate experiments. Only those proteins that were detected and identified with high confidence in all three independent experiments are listed in Table 1 and Table 2.
Growth and survival analysis of Salmonella
Strains SipA(HF), SipC(HF) and SopB(HF) are derivatives of the wild type Salmonella enterica serovar Enteritidis strain SE2472 with a FLAG tag inserted in-frame at the C-terminus of each corresponding protein and have been described previously . Growth analysis of bacteria in LB or LB-like broth was carried out by first inoculating a single colony in 2 ml of either normal (14N) or 15N-labeled media and culturing at 37°C with shaking at 225 RPM overnight (about 16 hours) . Thirty microliters of the overnight culture were then inoculated into 3 ml fresh normal or 15N-labeled media or LB broth and cultured at 37°C with shaking at 225 RPM. At 0, 2, 4, and 6 hours after inoculation, 100 μl of bacterial culture were collected to determine their colony forming unit (CFU)/ml by plating. Salmonella grew in normal (14N) or 15N-labeled media as well as in LB broth (data not shown). To study the survival of Salmonella after exposure to H2O2, 20 μl of the overnight culture grown in normal (14N) or 15N-labeled media, or LB broth were added to 2 ml of fresh normal (14N) or 15N-labeled media, or LB broth containing 5 mM H2O2. At different time points of incubation, 100 μl of bacterial culture were collected, diluted, and plated onto LB agar plates to determine their CFU/ml [16, 36]. Each sample was analyzed in triplicates and the analysis was repeated at least three times.
In vitro studies of the expression of the tagged SPI-1 proteins
Colonies of tagged strains were inoculated in 1 ml of LB broth and cultured at 37°C with shaking at 225 RPM for 16 hours. To study the effect of H2O2 on the protein expression in vitro, 20 μl of overnight bacterial cultures were inoculated into 1 ml of antibiotic-free LB and shaken at 225 RPM at 37°C for 4 hours. The bacterial cultures were centrifuged at 5,000 × g for 5 minutes. The pelleted bacteria were re-suspended in 1 ml of fresh LB broth (control) or 1 ml of LB broth with 5 mM H2O2 and shaken at 225 RPM at 37°C for an additional 2 hours, and then collected.
To prepare protein samples from Salmonella, bacterial cultures (1 ml) were centrifuged at 5,000 × g and 4°C for 10 minutes. The pellets were re-suspended in 200 μl of bacterial lysis buffer (8 M urea, 2% CHAPS, and 10 mM Tris, pH8.0), sonicated for 15 seconds three times with an interval of 30 seconds, centrifuged at 5,000 × g and 4°C for 10 minutes, and then transferred into fresh tubes for Western blot analysis.
Infection of cultured macrophages
RAW264.7 macrophage-like cells (ATCC, Manassas, VA) were infected with stationary phase bacteria at a multiplicity of infection of 50. After incubation for 30 mins, infected cells were washed twice with phosphate-buffered saline (PBS) and incubated in DMEM medium supplemented with gentamicin (100 μg/ml) for 1 hour to eliminate extracellular bacteria. Then the cells were again washed twice with PBS, and incubated in DMEM supplemented with gentamicin (20 μg/ml). At various times postinfection, the cells were collected and resuspended in lysis buffer (120 mM NaCl, 4 mM MgCl2, 20 mM Tris-HCl [pH 7.5], 1%, Triton X-100) supplemented with protease inhibitors (complete EDTA-free cocktail, Roche Applied Science, Indianapolis, IN), incubated at 4°C for 1 hour, and centrifuged at 18,000 × g and 4°C for 10 minutes. The pellets that contained bacterial proteins were resuspended in PBS for Western blot analyses.
In vivo studies
BALB/c mice (6-8 weeks old) were obtained from Jackson Laboratory (Bar Harbor, ME). Overnight bacterial cultures were serially diluted to suitable CFU/ml in PBS before infection. To assess the virulence of the tested strains, groups of five mice were either inoculated intragastrically with 1 × 106 CFU per mouse or intraperitoneally with 1 × 102 CFU per mouse. Mice were monitored during the course of infection, and those animals that exhibited extreme stress or became moribund were euthanized. For organ colonization experiments, groups of five mice were inoculated intraperitoneally with 1 × 104 or 1 × 106 CFU per BALB/c mouse of the bacterial strains, and were euthanized at 4 days or 12 hours after inoculation, respectively. Mice were also intragastrically infected with 1 × 106 CFU per BALB/c mouse of the bacterial strains, and were euthanized at 6 days after inoculation. Organs were collected and homogenized in PBS at 4°C. An aliquot of each homogenate was used to determine its CFU/ml by serial dilution with PBS and plating onto LB agar plates. Each sample was analyzed in triplicate and the analysis was repeated at least three times. The CFU of the sample was expressed as the average of the values obtained. The concentrations of bacteria were recorded as CFU/ml of organ homogenate. The limit of bacteria detection in the organ homogenates was 10 CFU/ml. To prepare protein extracts for Western blot analyses, the homogenates of the spleen samples were centrifuged and the pellets that contained the bacteria were resuspended in PBS, following the procedures described previously . All the experimental procedures with animals were in compliance with the guidelines and policies of the Animal Care and Use Committee (ACUC) of the University of California at Berkeley, and have been approved by the ACUC.
Western blot analyses
The denatured polypeptides from bacterial lysates were separated on SDS-containing 10-12% polyacrylamide gels cross-linked with N, N''-methylenebisacrylamide (0.05%), transferred electrically to nitrocellulose membranes (Bio-Rad, Hercules, CA), and reacted in an enzyme-linked immunoassay with a monoclonal anti-FLAG antibody (Sigma, St Louis, MO) and antibodies against Salmonella FliC (BioLegend, San Diego, CA) and DnaK (StressGen, Victoria, British Columbia, Canada), followed by an anti-mouse IgG conjugated with alkaline phosphatase [16, 36]. The membranes were subsequently stained with a chemiluminescent substrate with the aid of a Western chemiluminescent substrate kit (Amersham Inc, GE Healthcare) and quantified with a STORM840 phosphorimager. Normalization of samples was also carried out by loading total proteins extracted from the same CFU (e.g. 5 × 107 CFU) of bacteria in each lane.