Bacterial strain and human blood samples
The bacterial strain S. maltophilia K279a was used in this study. We chose this particular strain because the whole genome sequence of the bacterial strain is available and S. maltophilia K279a was considered as a representative genome sequence strain . The bacteria were grown in lysogeny broth (LB) or on LB agar plates. Escherichia coli (E. coli) BL21 (DE3) were used for recombinant protein production. Blood samples from healthy individuals were obtained from the healthy volunteers, Affiliated Hospital of Military Medical Science in China.
Animals and ethics
New Zealand White rabbits weighing approximately 2 kg and Female BALB/c mice (5–6 weeks old) were housed at the animal care center of the Academy of Military Medical Sciences (AMMS), China. All animals were maintained under specific pathogen-free conditions and were kept in a climate-controlled room (temperature of 22 ± 1 °C and humidity 55 ± 5%) with a 12 h light/dark cycle. Animals were sacrificed by euthanasia, and all experiments were handled according to protocols approved by Institutional Animal Care and Use Committee of AMMS. All efforts were made to minimize animal suffering.
Rabbit anti-S. maltophilia serum preparation
S. maltophilia K279a were grown in LB at 37 °C shaker to reach an optical density at 600 nm (OD600) of 1.0 (approximately 4.0 × 108 CFU/mL), and then inactivated by treatment with 0.15% (v/v) methanol at 37 °C for 24 h. The inactivated S. maltophilia was injected into two female New Zealand white rabbits (2 kg) (4.8 × 109 CFU/time) with a two-week interval between injections. The first injection was emulsified with Freund’s complete adjuvant (Sigma), and the remaining two injections were emulsified with Freund′s incomplete adjuvant (Sigma). Freund’s complete adjuvant is considered to be the gold standard adjuvant for immunization . The Freund’s complete adjuvant has been commonly used to induce antibody production in animal models . Blood samples from the rabbits were collected 7 days before the first injection (pre-immune serum) and 10 days after the last injection (post-immune serum). An indirect enzyme-linked immunosorbent assay (ELISA) was used to measure antibody levels in the serum samples. Briefly, ELISA plates were coated with 10 μg/mL S. maltophilia total protein in coating buffer [0.05 M NaHCO3 (pH 9.6)] at 4 °C overnight and then blocked with 3% BSA in phosphate-buffered saline containing 0.1% Tween-20 (PBST) buffer for 1 h at 37 °C. The pre-immune serum (1:100 dilution in PBST) and post-immune serum (1:10240 dilution in PBST) were added to the wells. Horse radish peroxidase (HRP) conjugated anti-rabbit IgG secondary antibody was added. The color was developed by using the 3,3’,5,5’-Tetramethylbenzidine (TMB) single-component substrate solution (Beijing Solarbio Science & Technology Co., Ltd. China). The color development reaction was stopped by 100 μL 1 M H2SO4. The optical density at 450 nm (OD450) was determined in an ELISA plate reader.
Preparation of outer membrane proteins (Omps)
S. maltophilia Omps were prepared according to the previous description . In brief, S. maltophilia were grown in LB at 37 °C shaker to reach OD600 = 1.0 and then collected by centrifugation. The bacterial pellets were washed and re-suspended in ice cold TS buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.4). The bacterial suspension was kept in ice, sonicated for 20 min, and centrifuged at 6000 rpm/min for 10 min at 4 °C. The supernatant was transferred to a 10 mL centrifuge tube and further centrifuged at 12800 rpm/min for 30 min at 4 °C. The supernatant was discarded and the cell membrane pellets were re-suspended in 10 mL TS buffer by repeated pipetting. An equal volume of 2% Sarkosyl was added to the cell membrane suspension to dissolve the cytoplasmic membranes. The mixture was incubated at room temperature for 30 min with intermittent mixing, and then centrifuged at 12800 rpm/min for 30 min at 4 °C to collect Omps. The supernatant was discarded. The Omp pellets were re-suspended in 5 mL PBS. Protein concentration of the Omp suspension was determined by the Plus One 2-D Quant Kit (GE Healthcare).
Two-dimensional polyacrylamide gel electrophoresis (2D PAGE) and western blot
Proteins in the Omp suspension were separated by 2D 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) isoelectric focusing (IEF) electrophoresis. Two hundred μg Omps in 350 μL rehydration buffer [7 M Urea, 2 M Thiourea, 4% (w/v) CHAPS, 50 mM DTT] were loaded in pH 4–7 IPG strips (18 cm, GE Healthcare, USA). The loaded strips were rehydrated overnight and underwent electrophoresis at room temperature at 300 V for 1 h, 600 V for 1 h, and 1000 V for 1 h, and then 8000 V for 8 h, to reach a total of 64 kVh. The proteins were separated and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, USA). The membrane was blocked with 5% skim milk in PBS overnight and then probed with the rabbit pre-immune or post-immune serum samples. The membrane was then washed and incubated with the secondary HRP-conjugated goat anti-rabbit IgG. The signals were developed by using the Super Signal West Dura Extended Duration Substrate (Pierce, USA), and the signals were detected using Image Scanner (GE Healthcare, USA).
Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) and nano liquid chromatography coupled fourier transform ion cyclotron resonance tandem mass spectrometry (nanoLC-FT ICR MS/MS)
MALDI-TOF MS was performed on a Bruker Reflex III MALDI-TOF-MS (Bruker Daltonics, Germany) operating in the reflectron mode with 20 kV accelerating voltage and 23 kV Reflecting voltage . A saturated solution of cyano-4-hydroxy-cinnamic acid in 50% acetonitrile and 0.1% trifluoroactic acid was used as the matrix. Sample preparation for the mass spectrometry followed the protocol reported by Geng and colleagues . One μL the matrix solution and one μL sample solution were added in the Score 384 target well. The mass spectrometry analysis was performed by a specialist in the Instrument Application Center of the Academy of Military Medical Sciences following the previously published protocol . In brief, mass accuracy for peptide mass finger-prints (PMF) analysis was calibrated with a 0.1–0.2 Da external standard, and internal calibration was carried out with enzyme autolysis peaks, at a resolution of 12,000. The nanoLC- FT ICR MS was performed on an APEX-Q FT-ICR tandem mass spectrometer (Bruker Daltonics, Germany) equipped with a 9.4 T superconducting magnet (Magnex Scientific, UK) and an infinity cell. The trypsin digested peptides were sequenced by auto MS mode with MS/MS boost function. The FT-ICR mass spectra were processed using Data Analysis 3.4 software (Bruker Daltonics GmbH, Germany) as a gateway to set up database searches.
Expression and purification of recombinant Omps
To express OmpA and Smlt4123 proteins in E. coli, the Omp genes were first amplified by PCR and then cloned into the pET-30a (+) vector (Takara, Japan). The PCR primers were synthesized by Sangon Biotech (Shanghai, China) Co., Ltd. The primer sequences and cloning sites in pET-30a (+) are displayed in Additional file 1: Table S1. The recombinant vector was transformed into E. coli BL21 (DE3). The gene insertion in the recombinant vector was confirmed by DNA sequencing. The 6X-His tagged proteins expressed by the transformed E. coli BL21 were purified using Ni-agarose affinity chromatography according to the manufacturer instructions (GE Healthcare, USA). Endotoxin was removed from the Omp elution by using Triton-114 . The Omp elution was analyzed by 10% SDS-PAGE.
Preparation of mouse anti-Omp serum
Omp elution was injected into female BALB/c mice (6-week old) (5 μg/time) with a 2-week interval between injections. The first injection was emulsified with Freund’s complete adjuvant (Sigma), and the remaining two injections were emulsified with Freund′s incomplete adjuvant (Sigma). Blood samples from the retro-orbital plexus of anesthetized mice were collected 7 days before the first injection (pre-immune serum) and 10 days after the last injection (post-immune serum). An indirect ELISA was used to measure the antibody levels in the serum samples. The plates were coated with 10 μg/mL of Omp elution and then blocked with 3% BSA in PBST. The pre-immune and post-immune mouse serum samples were added to the wells. The secondary antibody, HRP-conjugated goat anti-mice IgG1 and IgG2a, were then added to the wells. The color was developed by TMB single-component substrate solution (Beijing Solarbio Science & Technology Co., Ltd. China). The plate was read at 450 nm in an ELISA plate reader.
Opsonophagocytic killing assay
Opsonophagocytic killing assay (OPKA) was performed to determine the effectiveness of the anti-Omp serum in vitro. S. maltophilia were grown in LB at 37 °C shaker to reach OD600 1.0. One mL of the bacterial culture was collected and centrifuges. The bacterial pellet was washed twice and re-suspended in 100 μL PBS (4.0 × 108 CFU). The bacterial suspension was mixed with 50 μL mouse post-immune serum, for the control reaction, mouse pre-immune serum was added in place of the mouse post-immune serum. After incubation for 30 min at 37 °C, 350 μL blood samples from healthy individuals was added, and the mixture was rotated at 60 rpm at 37 °C for 1.5 h. Subsequently, 100 μL of the mixture were diluted with PBS and plated on LB agar. The plates were incubated at 37 °C overnight, and the number of colonies grown was counted on the next day.
Immunization of mice with recombinant Omp
Female BALB/c mice (6-week old) were randomized into two groups (n = 10 in each group): Smlt4123-immuned group and control group. Mice in the Smlt4123-immuned group were received a subcutaneous immunization of 5 μg Smlt4123 in PBS and was emulsified with the same volume of Freund’s complete adjuvant (CFA, total volume 100 μL/mouse) for primary immunization and Freund’s incomplete adjuvant (IFA) for boosting on days 14 and 28. Mice in the control group were immunized similarly with a mixture of PBS and adjuvant. To establish a mouse infection model, mice were challenged with intraperitoneal infection of S. maltophilia two weeks after the final immunization. In our pilot study, we found that intraperitoneal inoculation of 107 CFU S. maltophilia induced bacteremia in BALB/c mice, and inoculation of 109 CFU resulted in an overwhelming infection, which caused mouse death within 24 h. Our pilot study also showed that the lethal dose of 50% for mice (LD50) was 5.8 × 108 CFU (Additional file 1: Table S4). Thus, we used an intermediate dose, 4.0 × 108 CFU, in the current study. S. maltophilia were grown in LB at 37 °C shaker to reach OD600 = 1.0. Cells were collected, washed twice with PBS, and re-suspended in PBS at the 2.0 × 109 CFU/mL for the intraperitoneal injection. Mice in the Smlt4123-immuned group and the control group were injected intraperitoneally with 200 μL S. maltophilia suspension (4.0 × 108 CFU) . Eight hours after the injection, the mice were sacrificed by cervical dislocation. Mouse blood, liver, spleen, lung, and kidney were harvested and the tissues were homogenized in sterile PBS. Blood samples and homogenized tissue samples were cultured to determine bacterial burden.
Continuous variables are presented as mean ± standard deviation (SD). Two-group comparison was analyzed using Student’s t- test. Wilcoxon signed rank test was used for paired comparisons and Mann Whitney test was used for unpaired comparisons. P value was 2-sided and P < 0.05 was considered statistically significant.