Bacterial strains, plasmids, media and experimental animals
The bacterial strains and plasmids used in this study are listed in Table 3. Bacterial strains were maintained in LB broth plus 20% glycerol at -80°C. S. Typhimurium cultures were grown at 37°C in LB broth or on LB agar. Diaminopimelic acid (DAP) (50 μg/ml) was added to grow the Δasd strains [43]. LB agar containing 5% sucrose was used for sacB gene-based counter-selection in the allelic exchange experiments [44]. Chloramphenicol (Cm, 25 μg/ml) was added to the media when needed. The seven-week-old female BALB/c mice used in this experiment were purchased from Dashuo Experimental Animal Ltd. (Chengdu, China). Mice were acclimated for 7 days after arrival before starting the experiments. All animal procedures were approved by the Southwest University Animal Care and Use Committee.
Plasmids and mutant strain construction
The primers used in this study are listed in Table 4. DNA manipulations were carried out as described [47]. Transformation of E. coli and S. Typhimurium was achieved by electroporation. Transformants were selected on LB agar plates containing the appropriate antibiotics. For the ECA operon deletion experiments, Doperon-1F/Doperon-1R and Doperon-2F/Doperon-2R primers were used to amplify approximately 400-bp fragments upstream and downstream of the ECA operon from the χ3761 genome, respectively. The two fragments were then joined by PCR using Doperon-1F and Doperon-2R primers. Terminal A was simultaneously added to both ends of the resulting PCR product by LA-Taq enzyme (Takara, Japan) and ligated to pYA4278 to generate plasmid pYA5493. The mutation was introduced into S. Typhimurium χ3761 by allelic exchange using suicide vectors pYA5493 to generate χ12357. The mutation was also introduced into S. Typhimurium vaccine strain χ9241 to yield strain χ12358.
ECA western blot and LPS analyses
ECA was prepared as previously described [48]. ECA phenotypes were characterized by immunoblotting. Bacteria grown overnight on LB plates were pelleted and washed twice with physiological saline, re-suspended in 100 μl of lysis buffer (0.065 M pH 6.8 Tris-HCl, 5% β-mercaptoethanol, 2% SDS, 10% glycerol, 0.05% bromophenol blue) and heated at 100°C for 10 min, and then incubated overnight at 60°C with proteinase K to a final concentration of 1.6 μg/ml. Samples were boiled again for 10 min. ECA samples were separated on 12% Tricine-SDS-PAGE gels, and transferred to nitrocellulose membranes. Membranes were probed with a 1:1,000 dilution of murine anti-ECA monoclonal antibody mAb898 [48]. Blots were developed using a BCIP/NBT chromogenic substrate kit and imaged using a Gel Doc TM XR+ with Image LabTM Software (BIO-RAD, USA). The LPS and silver staining analyses were based on previously described protocols [49]. Briefly, cells from overnight cultures were lysed and boiled for 10 min, and then incubated with proteinase K for 1 h at 37°C. Subsequently, 10 μl of each sample was separated by SDS-PAGE (12%), and the gels were stained with silver nitrate. For the immunoblotting experiments, the isolated ECA samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the resulting bands were transferred to nitrocellulose membranes, and the nitrocellulose membranes were probed with the murine anti-ECA monoclonal antibody mAb898 at a 1:1000 dilution [48].
Motility assays
LB plates containing 0.3% agar were inoculated with each strain and dried at room temperature for 2 h. For the motility assays, three microliters of fresh bacterial liquid cultures were spotted onto the middle of plates and incubated at 37°C for 6 h. The diameter of the colonies was measured. The experiments were repeated three times.
DOC sensitivity assay
Bacteria were grown until they reached an OD600 of 0.8-0.9. Dilutions from exponential cultures of each strain were spread on LB plates supplemented with 1% DOC [11]. Three-microliter portions of the appropriate dilutions of the exponential cultures of the wild-type and mutant strains were incubated overnight at 37°C in LB plates with or without 1% DOC. To precisely calculate the survival rate in DOC, 100 μl of a 100-fold dilution of the bacterial cultures with an OD600 of 0.8 were transferred to a new tube, DOC was added to final concentration of 10 mg/ml, and then incubated for 1 h at 37°C. The samples were then diluted to the appropriate concentration, and then 100 μl of the dilutions were spread onto LB plates, followed by overnight incubation at 37°C. The assays were repeated three times.
Determination of the LD50 and colonization in mice
Following our standard procedures, the oral 50% lethal dose (LD50) was determined [35]. Bacteria were grown statically overnight at 37°C in LB broth, diluted to 1:50 in fresh media, and grown with aeration at 37°C. When the cultures reached an OD600 of 0.8-0.9, they were harvested at room temperature by centrifugation at 4000 rpm, washed once, and normalized to the required inoculum density in buffered saline gelatin (BSG) by adjusting the suspension to the appropriate OD600 value. Groups of five mice each were infected orally with 20 μl containing various doses of S. Typhimurium χ3761 or the mutant χ12357, ranging from 1.0×103 CFU to 1.0×109 CFU. Mice were observed for 4 weeks post-infection, and deaths were recorded daily. To evaluate colonization, mice were inoculated orally with 20 μl of BSG containing 1×109 CFU of the wild type and mutant strains. Peyer’s patches (PP), spleen and liver tissues were harvested at days 3, 6, 9, 12, 15 and 20 post-infection. The standard procedure for determining the bacteria colonization in PP, spleen and liver in the mouse was described in our previous publications [23, 35]. Briefly, we collected all the PP from the small intestinal surface, spleen from each mouse, or cut a slice of mouse liver into 1.5 ml tube. 1 ml BSG was added to the tube containing PP, and spleen or a slice of liver were measured the weight and extra BSG were added to a total volume of 1 ml. The samples were homogenized and plated onto LB plates to determine the number of viable bacteria after appropriate dilutions. The means of pooled PP were calculated in each mouse as bacterial colonization number in PP, and the bacterial number of liver and spleen were calculated as bacterial colonization number in spleen and liver.
Western blot analyses
To test the expression level of the heterologous antigen (PspA) delivered by the S. Typhimurium vaccine carrier χ9241 and its ECA operon mutant strain, western blotting analyses were performed. Two proteins, LacI and GroEL, were used as the controls. The standard western blotting procedure were followed to visualize protein expression in bacteria [35], Briefly, the proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked with 3% skim milk in 10 mM Tris and 0.9% NaCl (pH 7.4) and incubated with rabbit polyclonal antibodies specific for PspA, LacI or GroEL (Sigma, St. Louis, USA) [46]. Later, the membranes were washed three time using 10 mM Tris (pH 7.4) with 0.05% Tween 20, and inoculated in the AP-conjugated goat anti-rabbit immunoglobulin G (IgG) (Sigma) for 1 hr at room temperature. Last, the membranes were rinsed three time with 10 mM Tris (pH 7.4) with 0.05% Tween 20 and were visualized by addition of reactive substrate of BCIP-NBT solution (Sigma), the reaction was stopped by washing the blots with large volumes of pure water when the bands developed clearly.
Immunogenicity of the vaccine strains in mice
The attenuated vaccine strains χ9241 (pYA4088), χ12358 (pYA4088) and χ9241 (pYA 3493) were cultured in LB with and without 0.1% arabinose and diluted with BSG for immunization following previous described procedures [35]. Briefly, BALB/c mice were orally inoculated with 20 μl of BSG containing approximately 1.0 × 109 colony forming units (CFU) of each strain suspension prepared as described above, and then boosted on day 28 with the same dose of the same strain. The vaccinated mice were monitored for symptoms and mobility during the immunization period. Blood were obtained by mandibular vein puncture at biweekly intervals. Serum samples were obtained and stored at -80°C for ELISA analysis.
ELISA analysis of serum antibodies
To measure the levels of antibodies triggered by the vaccine strains in the serum, ELISA assays were performed with serum antibodies against E. coli recombinant PspA (rPspA) and outer membrane proteins (SOMP) as previously described [35]. Briefly, rPspA and SOMP proteins of S. Typhimurium were prepared and purified as described previously [46]. Polystyrene 96-well flat-bottom microtiter plates were coated with 100 ng/well of purified rPspA or SOMPs in the coating buffer (50 mM Na2CO3, 50 mM NaHCO3, 0.1% sodium azide, pH 9.6), and 100 ng/well of goat anti-mouse IgG (H+L) in PBS was applied with 100 μl volumes to coat the two lines in 96-well plates for standard curve drawing. The coated plates were incubated overnight at 4°C, followed by blocking with PBS containing 10% FBS (Sigma, USA) for 1 h at room temperature. A 100 μl volume of serum diluted sample or mouse IgG (BD Pharmingen) for the standard curve were added to individual wells in triplicate and incubated for 1 h at 37°C. Plates were treated with biotinylated goat anti-mouse IgG, IgG1 or IgG2a (Southern Biotechnology). Wells were developed with a streptavidin-alkaline phosphatase conjugate (Southern Biotechnology), followed by p-nitrophenylphosphate substrate (Sigma) in diethanolamine buffer (pH 9.8). Color development (absorbance) was recorded at 405 nm using a microplate reader. The standard curve was drawn using Curve Expert, and the serum antibody concentration was calculated using the standard curve.
Pneumococcal challenge
The protective efficacy of immunization with the attenuated Salmonella strains expressing PspA was assessed. Vaccinated mice at day 56 were challenged with 2×104 CFU of S.pneumoniae WU2 in 200 μl of BSG by intraperitoneal (i.p.) injection and monitored and recorded daily for 15 days[45].
Statistical analysis
The antibody titer data are expressed as the geometric means, and the relative immunoreactivity is expressed as an arithmetic mean. The means were evaluated by two-way analysis of variance and chi-square test for multiple comparisons among the groups. A P value of <0.05 was considered statistically significant.