Bacterial strains, culture cells and culture conditions
All bacterial strains used in the present study and their relevant characteristics are listed in Additional file 2: Table S2. All the used B. anthracis strains were derivatives of the Chinese vaccine strain A16R. Bacteria were aerobically grown at 37 °C. Escherichia coli strains were grown in Luria–Bertani (LB) broth and used as hosts for plasmid cloning and recombinant protein expression. B. anthracis strains were grown in brain heart infusion medium with 0.5 % glycerol (BHIG; BD, USA) or LB media. Antibiotics (Merck, Germany) were added to the media when appropriate to the following final concentrations: 100 μg/mL ampicillin for E. coli; 50 and 25 μg/mL kanamycin (kan) for E. coli and B. anthracis, respectively.
HeLa human cervix carcinoma or COS-7 cells were cultured in RPMI-1640 medium supplemented with 2 mM L-glutamine and 10 % FCS in a 5 % CO2 atmosphere at 37 °C.
Recombinant proteins and rabbit-anti-BslA antibodies
Total chromosomal DNA from B. anthracisA16R was used as a template to amplify bslA truncation using the primers (BslA(260-652) F: 5′-CGGGATCCGAAGAATTGAATCAAAAGTT-3′, BslA(260-652) R: 5′-CCGCTCGAGACTGTTTGGTATTCTAAGTTT-3′). To obtain the recombinant BslA(260–652) protein of B. anthracis and prepare its antibody used for the adherence activity studies, the fragment encoding BslA(260–652) was cloned into the pET28a(+) plasmid and induced to express recombinant protein in E. coli Rosetta (DE3) by IPTG. The expressed recombinant protein was purified by a column packed with ProBond™ Purification System (Life Technologies) according to the manufacturer’s instructions. Purified protein was used as the antigen to immunize rabbits three times to raise polyclonal antibodies. IgG was purified using protein G-Sepharose (GE Healthcare) affinity chromatography.
Circular dichroism (CD) spectroscopy
CD spectroscopy measurements were performed in a Chirascan CD Spectrometer (UK Applied Photophysics). The purified recombinant protein was dialyzed using 10 mM sodium phosphate buffer at a concentration of 0.2 mg/ml. Far-UV (196–260 nm) spectra were acquired using a 1-mm-path-length cell at 0.5 nm intervals. Five scans were accumulated and the mean value determined. For the analysis of the secondary structure, these data were analyzed using the online servers K2D3 [33]. Concurrently, the secondary structure prediction also was executed using the Network Protein Sequence Analysis (NPS@site at https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_seccons.html) [34].
BslA(260–652) adherence bioactive assays
Replenished B. anthracis A16R cultures, grown to an OD600 of 0.5, were inoculated into 12-well tissue culture plates containing monolayers of HeLa cells. Infected monolayers were centrifuged at 600 × g for 5 min to synchronize infection, and then incubated at 37 °C in 5 % CO2, 100 % humidity for 4 h. Cells were washed five times with PBS. Standard Gram staining was performed and bacteria were removed from the wells by adding 100 μl of 0.25 % trypsin. Serial dilutions were plated in triplicate on LB agar and grown at 37 °C overnight.
For BslA-specific antiserum inhibition experiments, cultures of B. anthracis were preincubated with 2 % BslA-specific or naive rabbit sera for 1 h at 4 °C prior to infection. For protein inhibition experiments, cells were preincubated with purified BslA(260–652) protein (25 μg of protein/1 × 106 cells) for 1 h at 37 °C in FCS free medium and washed twice before infection.
Biochemical treatment of cells
HeLa cells were grown to 80–90 % confluency and washed with PBS, then detached from the plate with 0.04 % EDTA (10 mM in PBS). Cells were washed and resuspended in approximately 1 × 107 cells/ml in ice cold PBS. For protease treatment, the cells were incubated with 40 μg/ml trypsin or 6 μg/ml proteinase K for 30 min at 4 °C. To degrade the carbohydrate structures on cell surfaces, the cells were incubated with peptide-N-glycosidase F (PNGase F) for 1 h at 4 °C or incubated with 30 mM sodium periodate in 50 mM sodium acetate buffer (pH 4.5) for 1 h at room temperature in the dark. After washing three times with PBS and blocking with 3 % FCS in medium, cells were used for flow cytometry analysis experiments as described further.
Flow cytometry analysis
BslA(260–652) protein was added to the pretreated cells (25 μg of protein/1 × 106 cells) and incubated for 1 h at 4 °C. Cells were washed three times with PBS, and suspended in PBS containing 3 % FCS and BslA(260–652)-specific antiserum (1:100, v/v) and incubated on ice for 1.5 h. After washing 3X with PBS, the cells were incubated with FITC-conjugated goat anti-rabbit IgG (1:1000, v/v; Abcam) on ice for 1 h. Then the cells were stained with 0.5 mg/ml of 7-AAD (KeyGen Biotech) for 10 min and the FITC-labeled cells (7-AAD negative, live) were examined with FACSCalibur (BD Biosciences, USA).
Blot overlay/Far western blotting assay
For blot overlays, the commercial extracellular matrix (ECM, Corning Life Sciences) were blotted onto nitrocellulose membranes and air dried. For the far Western blot, ECM proteins (collagenase-digested or non-pretreated) were separated on 4–12 % SDS-PAGE and transferred to nitrocellulose membranes. All the membranes were blocked in 5 % fat-free milk in PBST for 1 h. Following several washes with PBST, BslA(260–652) protein in PBST/milk (1 μg/ml) was added to the nitrocellulose membranes, incubated for 1 h at 37 °C, and washed several times. Detection was performed using BslA(260–652)-specific antiserum and HRP-conjugated goat anti-rabbit IgG. For blot overlay test, the sonicated E. coli BL21(DE3) was used as negative control.
Mass spectrometry analysis
Further identification of the ligand of BslA was conducted using a combination of the Far Western blotting studies and mass spectrometry (MS). To separate the proteins well and fit mass spectrometry analysis, the ECM protein were digested by collagenase and separated on 4–12 % SDS-PAGE. Separated proteins were electrotransferred to nitrocellulose membranes or stained with Coomassie Blue. The far western blotting was used to determine signals of corresponding protein spots located on the colloidal coomassie gel.
Polypeptides cut out from the coomassie gel were analyzed by ESI-QUAD-TOF. The proteins were identified against the NCBI database using the MASCOT program (www.matrixscience.com), where protein scoring of >82 was significant (p < 0.05).
Blocking assays
For ligand antiserum inhibition experiments, COS-7 cells were preincubated with laminin-specific polyclonal antibodies (Merck Millipore, 1:50 dilution) or naive rabbit sera for 1 h prior to incubation with BslA(260–652) at 4 °C. After washing three times with PBS and blocking with 3 % FCS in medium, cells were labeled with BslA(260–652), followed by mice anti-His tag antibodies and FITC-conjugated rabbit anti-mice IgG for flow cytometry analysis experiments.
Solid-phase binding assays
To determine binding of BslA(260–652) to immobilized laminin, wells of Costar 96-well plates (Corning) were coated overnight with 20 μg/ml EHS laminin in 50 mM Na2CO3 (pH 9.6) at 4 °C. Plates washed three times with PBS plus 0.5 % Tween-20 (PBST). Wells were blocked for 2 h at 37 °C with Superblock (Thermo Fisher), and then washed three times with PBST. Wells were incubated for 2 h at 37 °C with 100 μl of various concentrations of recombinant BslA(260–652) in PBST (plus 0.1 % BSA) and developed with BslA(260–652)-specific rabbit polyclonal antiserum, followed by goat-anti rabbit HRP-IgG. Plates were washed three times with PBST, incubated for 1 h at 37 °C with HRP-conjugated goat anti-rabbit IgG (Jackson), and diluted 1:5000 in PBST-2 % fat-free milk. After washing, 100 μl of TMB substrate solution (TianGen) was added to the wells in the microtiter plate. After 10 min at room temperature, the reaction was interrupted by adding 50 μl of 1 M H2SO4. Absorbance was read at 450 nm in a Spectra Max 190 ELISA microplate reader (Molecular Devices). A binding curve was generated using Graphpad Prism, version 5.
To determine the binding of laminin to immobilized BslA(260–652), recombinant BslA(260–652) was diluted in carbonate buffer, pH 9.6, to 1 μg/ml, and 50 μl/well was coated on Costar 96-well plates (Corning) overnight. The wells were washed, blocked, probed with 100 μl of soluble EHS laminin (0–100 mg/ml), and developed with rabbit anti-mouse laminin antibody (Merck Millipore), followed by goat-anti rabbit HRP-IgG.
Surface Plasmon Resonance (SPR)
All surface plasmon resonance (SPR) experiments were performed using a Biacore T200 instrument at 25 °C (GE Healthcare) using the single-cycle kinetics (SCK) experiments. Briefly, The CM-5 chip was activated with a 1:1 mixture of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 0.1 M N-hydroxysuccinimide for 7 min. Laminin (10 μg/mL in 10 mM sodium acetate buffer, pH 4.0) was immobilized on the surface of individual CM5 chips a flow rate of 5 ml/min. Approximately 2000RU of laminin were immobilized. After immobilization, CM5 chips were inactivated with 1 M ethanolamine-HCl. Increasing concentrations of BslA(260–652) in HBS-EP+ buffer(10 mM HEPES,150 mM NaCl,3 mM EDTA,0.05 % P20,pH 7.4) were injected at 30 μl/min over both the ligand and reference surfaces for 3 min then allowed them to dissociate for 15 min at 25 °C. Data processing and analysis were performed using BiacoreT200 evaluation software in a 1:1 binding model (GE Healthcare).
B.anthracis AP422 Expressing BslA
PCR reactions were performed with Pfu DNA polymerase (Transgene) using the primers (BslAF: 5′-GAAGCTTAAGGAGGAAGCAGGTATGAAAAAAAGAAAGATAAAAG-3′, BslAR: 5′-CATGCATGCTTAACTGTTTGGTATTCT-3′). The fragment coding BslA was cloned into the shuttle vector pDG148and the ligation products were transformed into E. coli DH5α, and plasmid DNA into E. coli SCS100 (dam-, dcm-) and purified (nonmethylated) plasmid DNA was transformed into B. anthracis following a previously developed protocol [35].
Bacterial adherence laminin assays
Replenished B. anthracis AP422, AP422(pDG148) and AP422(bslA) cells, induced to express BslA protein by IPTG 4 h before harvest, were washed and resuspended in filtered PBS supplemented with 2 % BSA. Laminin (2 μl of a 1 mg/ml solution) was added to microtubes (1.5 ml), and incubated at 37 °C in the dark for 1 h. Following three washes with PBST, bacteria were incubated for 2 h at 37 °C with a 1:100-diluted rabbit anti-mouse laminin antibody in PBS containing 2 % BSA and incubated on ice for 1.5 h. After washing three times with PBS, the cells were incubated with Alexa Fluor 488 -labeled goat anti-rabbit IgG (1:100, v/v) (Life Technologies) on ice for 1 h. The Alexa Fluor 488-labeled cells were examined using FACSCalibur (BD Biosciences). Unlabeled bacteria AP422 were used as negative controls.
Statistical methods
All the statistical analyses were performed with the GraphPad Prism 5 software. Data are presented as mean value with the standard deviation (±1 S.D.). Statistical significance was tested using Student’s paired t-test or One-way ANOVA. Differences were considered significant at p < 0.05. The correlation coefficient (R) and p-value (two-tailed) were calculated at 95 % confidence interval.