Ethics Statement

All human studies, protocol revisions, and consent procedures were approved by the Helsinki Ethics Committee of the Soroka University Medical Center, Beer Sheva, Israel (Permit number: 10391). Written informed consent was obtained from the next of kin, carer or guardian of the minor/child participants involved in this study.

Experiments involving animals were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved either by the Institutional Animal Care and Use Committee of the Ben-Gurion University of the Negev, Beer Sheva, Israel (Permit number: 53.08.08) or by the University of Mississippi Medical Center, in accordance with the guidelines and approval of its IACUC (permit number: 00163).

Immunization and Inoculation of Mice

Seven-week-old BALB/cOlaHsd (BALB/c) female mice (Harlan Laboratories, Israel) or seven-week-old CBA/CaHN-Btk^xid (CBA/N^xid; Jackson Laboratories, Bar Harbor, ME, USA) mice were housed in sterile conditions under 12-h light/dark cycles and fed Purina Chow and tap water ad libitum.

BALB/c (highly resistant to S. pneumonia) or CBA/N^xid (highly susceptible to S. pneumoniae) mice strains were immunized subcutaneously (SC) with 25 μg of rPtsA emulsified with complete Freund’s adjuvant (CFA) and subsequently boosted (days 14 and 28) with incomplete Freund’s adjuvant (IFA). On day 42, the BALB/c and CBA/N^xid mice were challenged intranasally (IN), under deep anesthesia using Isoflurane (Piramal Critical Care Inc., PA, USA), with a sublethal dose of strain WU2 (5 × 10⁷ and 5 ×x 10⁵, respectively). Mice were humanely sacrificed 3 and 24 h later by CO₂ asphyxiation, as recommended by the AVMA Guidelines for Euthanasia in Animals: 2013 Edition (https://www.avma.org/KB/Policies/Documents/euthanasia.pdf). Strain WU2 cells were pretreated with the respective peptides and then inoculated IN into adult CBA/N^xid mice. Mice were euthanized 3, 24 or 48 h later with CO₂. The nasopharynx and right lobe lung were excised, homogenized and plated onto blood agar plates for bacterial colony counting. For survival experiments, after an immunization regime similar to that described above, BALB/c or CBA/N^xid mice were challenged with a lethal dose of strain WU2 either IN (10⁸ CFU and 10⁶ CFU, respectively) or intravenously (10⁴ CFU). In these survival experiments, the mice were humanely euthanized by CO₂ asphyxiation if they become became moribund or showed evidence of distress. The following criteria were considered sufficient evidence of distress to warrant such intervention in order to minimize pain and suffering of the animals: severe weight loss (20% body weight); reluctance or inability to move freely; appearance of bristle fur; social disengagement; refusal or inability to eat or drink. No analgesic treatment was provided, as such treatment may alter the immune response and may independently affect the outcome of the experiments [32].

Reagents

Unless otherwise stated, all chemicals and biochemical materials were of the highest purity available and were purchased from Sigma-Aldrich (St. Louis, MS, USA).

Bacterial Strains and Growth Conditions

Two genetically unrelated encapsulated S. pneumoniae strains, serotype 2 strain D39 [33] and serotype 3 strain WU2 [34], and their unencapsulated derivatives, strain R6 (ATCC, Rockville MD) and strain 3.8DW [35], respectively, were used. In addition, in this study we used also strains 1, 5, 6B, 9V, 14R. 23F (1), and 14DW (kindly provided by Prof. David Watson, Dallas USA). Pneumococci were grown in Todd-Hewitt broth supplemented with 0.5% yeast extract (THY) or on blood agar plates, as previously described [36]. Two Escherichia coli strains were used as cloning vectors, DH5α UltraMAX (DH5α; Invitrogen Corp, Carlsbad, CA, USA) and BL21 (DE3) pLysS (BL21; Promega Corp, Madison, WI, USA) and were grown in lysogenic broth (LB).

Enrichment of S. pneumoniae Total Cell-Wall and Membrane Proteins

Isolation of the cell-wall proteins was performed as previously described with minor modifications [37]. Briefly, bacteria, grown to mid log (OD₆₂₉ 0.5), were pelleted by centrifugation. The supernatant was discarded, and bacteria were resuspended in protoplast buffer (20% sucrose, 2.5 mM MgCl₂, 5 mM Tris-Cl, pH 7.4 and mixed protease inhibitors) and incubated with mutanolysin (200 U/ml) at 37°C for 1 h to release the cell-wall proteins. Following centrifugation, supernatants containing S. pneumoniae total cell-wall proteins were collected and stored at –20°C for further processing. The pellet was resuspended in the protoplast buffer and sonicated at 20,000 Hz for 15 s. Following centrifugation at 25,000 RPM for 45 min, the membrane pellet was dissolved in protoplast buffer containing 0.5% Triton X-100 and centrifuged at 40,000 RPM for 60 min. The supernatant containing the membrane proteins was collected and stored at -20°C. The cytoplasmic fraction was obtained following sonication of the bacteria at 20,000 Hz for 15 s. Following centrifugation at 25,000 RPM for 45 min, the supernatant was collected and used as the cytoplasmic fraction.

Fractionation of S. pneumoniae Cell-Wall Total Proteins into Cell-Wall Lectin and Non-Lectin Proteins

The cell-wall total proteins were fractionated into cell-wall non-lectin and cell-wall lectin proteins by using fetuin-agarose affinity chromatography, as previously described with minor modifications [38]. Briefly, cell-wall total proteins were loaded onto the column and washed with the protoplast buffer. The proteins that did not bind to the column were regarded as cell-wall non-lectin proteins and were frozen at -20°C. The proteins that bound to the column (cell-wall lectins) were eluted with 50 mM ammonium acetate, pH 3.5, dried in a vacuum centrifuge, re-suspended in PBS, and stored for further analysis at -20°C.

Identification of PtsA

Separation of the non-lectin cell-wall fraction and 2D PAGE were performed as previously described [39]. Protein spots were excised from the gel, washed with 150 μl of washing solution (50% methanol and 5% acetic acid) for 3 h and dehydrated with 200 μl of acetonitrile (5 min). Reduction and alkylation were performed with 30 μl of 10 mM DTT and 30 μl of 100 mM iodoacetamide. After rehydration with 200 μl of 100 mM ammonium bicarbonate (10 min), gel pieces were dehydrated, completely dried in a vacuum centrifuge, and subjected to enzymatic cleavage (porcine trypsin; Promega) for 16 h at 37°C in 50 mM ammonium bicarbonate buffer. Extraction of the peptides was performed by adding 30 μl of 50 mM ammonium bicarbonate and 5% formic acid. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) analysis was performed using a REFLEX III (Bruker) mass spectrometer. Peptide peak lists were searched against the database of S. pneumoniae TIGR4 (www.matrixscience.com).

Cloning, Expression and Purification of Recombinant Proteins

The nucleotide sequence of the locus SP_RS05795, coding for the WP_000138135 protein was amplified from pneumococcal serotype 3 strain WU2 genomic DNA according to the published sequence of serotype 4 strain TIGR4 by PCR with the following primers: Forward: 5'-GGATCCATGACAGAAATGCTTAAAG-3' and Reverse: 5'-GAGCTCTTAATCAAAATTAACGTATTC-3' supplemented with restriction enzyme sequences of BamHI on the 3' end and Sac1 on the 5' end (Takara Biomedicals, Otsoshiga, Japan). The amplified product was cloned into the pHAT expression vector (BD Biosciences Clontech, Palo Alto, CA, USA) and transformed into E. coli DH5a UltraMAX ultracompetent E. coli cells. Insert DNA was analyzed by PCR in ampicillin-resistant transformants (data not shown). Verification of sequence identity was performed by plasmid insert sequencing (data not shown). The vector was purified using the Qiagen High-Speed Plasmid Maxi Kit and transformed into E. coli host expression strain BL21(DE3)pLysS (Invitrogen). Bacteria were grown overnight, and expression of recombinant rPtsA was induced by use of 1 mmol/L isopropyl-β-d-thiogalactopyranoside for 5 h. The cells were harvested and lysed, and the protein was purified under native conditions and then dialyzed against PBS for imidazole removal. Separation of the tagged-purified protein on SDS-PAGE showed that the 75-kDa rHAT-PtsA fusion protein was ~95% pure (S1A Fig). Immunoblotting with anti-HAT antiserum further confirmed the identity of the recombinant protein (S1B Fig). The discrepancy between the expected molecular weight of the fusion protein of 67.5 kDa (rPtsA 65.4 kDa plus 2 kDa HAT tag) may result from proteolysis. Alternatively, the altered electrophoretic mobility may stem from possible lipid moiety attachment to the protein, since TMpred software predicted the existence of hydrophobic regions in PtsA amino acids 517–535 to be a transmembrane helix (with a score of 525; http://www.ch.embnet.org/software/TMPRED_form.html) (S1B and S1C Fig). MALDI-TOF analysis of a peptide digest of this protein band identified rPtsA in 99% accordance with the expected PtsA protein (PI = 4.6, Mascot score = 92, Z score = 2.43, extent of sequence coverage = 39%).

The gene for production of the untagged rPtsA was amplified from TIGR 4 strain (using the same primers as above) and cloned into pET30a+. E. coli xl1 (Agilent Technologies, Santa Clara, CA) was transformed with pET30a+^ptsa. E. coli were grown and the plasmid was isolated and used to transform E. coli BL21. The E. coli BL21 cells were grown, centrifuged, resuspended in 10 mM Tris-HCl/10 mM EDTA (pH 8) with PMSF (final concentration 0.1 mg/ml), and then treated with lysozyme and DNase. This procedure was followed by sonication, centrifugation and precipitation of the protein using ammonium sulfate. The fraction at 70% saturation was solubilized with 10 mM Tris-HCl PMSF. The protein was purified ×3 by fractionation on Superdex 200s. Following removal of lipoprotein polysaccharide on a Detoxi-Gel^TM Endotoxin Removing Gel column (Pierce), the pooled protein was concentrated to 1 mg ml^-1, dialyzed against NaHCO_3, and lyophilized. The rPtsA was resuspended at 1 mg ml^-1 and separated on 1D SDS PAGE. The rPtsA was found to run as a ~ 70–72 kDa protein (S1D Fig). An immunoblot of the untagged protein with rabbit anti-rPtsA antiserum confirmed that under these conditions rPtsA runs as a ~72 kDa protein (S1E Fig). MALDI TOF analysis detected two peaks, one at 63,932.49 and the other at 62,492.49 Da, which are in better agreement with the hypothetical molecular weight predicted by the NCBI databases (63,165Da). Moreover, a peak at 120,090.67 Da detected by the MALDI analysis may indicate dimerization of PtsA, which is its enzymatic active form (S1E Fig). Of note, the biological experiments were performed using the tagged-rPtsA. The immunoblot analysis in Fig 1A was performed with the untagged-rPtsA as positive control.

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Fig 1. Surface expression of PtsA.

A) Immunoblot of untagged rPtsA: total S. pneumoniae extract (Total), total cell-wall proteins (CW), cytoplasmic proteins (Cyto) and membrane extract (mem) were probed with rabbit anti-rPtsA antiserum. B) Immunoblot of recombinant pneumolysin (rPly): total S. pneumoniae extract, total cell-wall proteins, cytoplasmic proteins and membrane extract were probed with rabbit anti-rPly antiserum. C) Immunoblot of tagged rFabD: total S. pneumoniae extract, total cell-wall proteins, cytoplasmic proteins and membrane extract were probed with rabbit anti-rFabD antiserum. D) FACS analysis of PtsA surface expression using anti-rPtsA monoclonal antibody (mAb) and isotype control IgG. Red line–preimmune control serum; green line–anti rPtsA mAb diluted 1:50; blue line–anti rPtsA mAb diluted 1:20. M = molecular weight markers. E) SIM orthogonal view image demonstrating the surface staining with mouse anti rPtsA antibody detected with donkey anti-mouse IgG (green). Three planes XZ, XY and YZ are shown. PtsA is visible on the perimeter of the bacterium in green, surrounding the white bacterial genome.

https://doi.org/10.1371/journal.pone.0150320.g001

Preparation of Rabbit Antiserum

Three-month-old white albino rabbits (Harlan Laboratories, Israel) were immunized intramuscularly (IM) with 200 μg HAT-rPtsA emulsified with CFA (1:1) in the first immunization or with IFA in booster immunizations. Two weeks after their final immunization, the rabbits were exsanguinated, and sera were prepared.

Immunoblot Analysis

Cell-wall, membrane and cytoplasmic pneumococcal proteins, 40 μg each, were separated by SDS-PAGE under reducing conditions and transferred to nitrocellulose membranes (Bio-Rad, Carlsbad, CA, USA), as previously described [40]. The identity of PtsA was confirmed by immunoblot analysis using either rabbit or mouse anti-rPtsA antiserum. Membranes were developed using MicroChemi 4.2 (DNR, Israel), and thereafter the molecular weight markers were added to the chemiluminescent image (Markers overlay). As negative control either preimmune serum was used or the primary antibody was excluded (to reduce redundancy the negative blots are not presented).

Flow Cytometry of S. pneumoniae

Anti-rPtsA Monoclonal antibodies (mAb) were obtained using available procedures. Briefly, BALB/c mice were immunized 4 times with rPtsA, followed by harvesting of splenocytes for fusion with NSO cells by standard techniques. The hybridomas were tested for reactivity with rPtsA by ELISA. Positive clones were further subcloned, and the clone with the highest reactivity to the protein was adapted to serum-free media. The cells were expanded, and the mAb was purified from the culture supernatant by Protein G affinity chromatography. The reactivity of the purified mAb was reconfirmed by ELISA (data not shown) before sorting by FACS. The purified mAb preparations were in the range of 0.6–1.3 mg/mL^-1. Purified IgG from naive mice was used as the control. An additional control was the exclusion of the primary antibody (not shown).

Flow cytometry was performed as previously described [41]. Briefly, strain R6 bacteria were incubated with anti-rPtsA mAb or isotype control mouse serum, washed, and stained with Alexa Fluor 647^®-conjugated goat-anti-mouse-IgG (Jackson ImmunoResearch, West Grove, PA). Flow cytometry was performed using a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA), and data were acquired and analyzed using BD CellQuest^TM 3.3 software.

Cell Lines

A549 cells (lung adenocarcinoma cells; ATCC, Rockville, MD, USA), which retain the morphological, biochemical and immunological characteristics of type II lung epithelial cells [42–44], and Detroit 562 cells (pharyngeal carcinoma derived cells; ATCC, Rockville, MD, USA) are extensively used in investigations of S. pneumoniae adhesion [45]. The cells were grown in DMEM supplemented with 10% fetal calf serum.

Inhibition of Adhesion of Pneumococci to Cultured Human Cell Lines

A549 and D562 cells were cultured on fibronectin-coated 96-well plates (Fig 2 and S2 Fig) or on Ibidi 8 well chambers (Ibidi GmbH, Planegg/Martinsried, Germany; Figs 3, 4 and 5) or on 96-well plates without fibronectin coating (Figs 7 and 8 and S3 Fig), as previously described [16]. Briefly, the cells were seeded at 5 × 10⁴ cells/well in DMEM supplemented with 10% fetal calf serum (FCS) (without antibiotics). Twenty four hours later, the cells (~10⁵ cells/well) were blocked with 0.5% gelatin for 1 h. rPtsA (0–500 nM) was then added to the cultured cells for 1 h of incubation. Excess protein was removed, and S. pneumoniae bacteria at a MOI of ~10:1 were added for additional 1 h of incubation. The inoculum size was verified in each experiment. Adhesion of the bacteria to fibronectin-coated plates, under our experimental conditions, did not exceed 2000 CFU/well, and this control was performed for each experiment. Following the incubation, excess bacteria were removed, and cells were detached with trypsin and plated onto blood agar plates for counting. All the inhibitors were used at concentrations below their toxic concentrations either to the bacteria or to the cells. Keyhole limpet hemocyanin (KLH) protein was used as a negative control. Experiments were conducted in triplicates and repeated on 3 different occasions.

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Fig 2. PtsA mediates S. pneumoniae adhesion to target host cells.

A–E. A549 cells were grown to confluence and then blocked with 0,5% gelatin for 1 h. Excess gelatin was removed, and the A549 cells were incubated for 1 h with rPtsA at the denoted concentrations. Excess protein was then removed. S. pneumoniae was added for 1 h to the cells, non-adherent bacteria were removed, and cells were detached with trypsin and plated onto blood agar plates for counting. rPtsA inhibited the adhesion to A549 cells of: A) strain WU2 (p < 0.0001; r = −0.943), B) strain 3.8DW (p < 0.0001; r = −0.371), C) strain D39 (p < 0.0001; r = −1), and D) strain R6 (p < 0.0006; r = −1). E) rKLH, a protein used as a negative control, did not inhibit D39 adhesion to A549 cells (p = 0.8; r = −0.2). F–J. S. pneumoniae cells (WU2 and 3.8DW) were treated for 1 h with rabbit anti-rPtsA antiserum and added to gelatin-blocked A549 cells for 1 h; non-adherent bacteria were removed; and cells were detached with trypsin and plated onto blood agar plates for counting. Rabbit anti-rPtsA inhibited adhesion to A549 cells of: F) strain WU2 (p < 0.0001; r = −0.359); G) strain 3.8DW (p < 0.0001; r = −0.886); H) strain D39 (p < 0.0001; r = −0.943); and I) strain R6 (p < 0.0001, r = −0.406); J) Serum obtained from a rabbit prior to immunization did not inhibit the adhesion of strain WU2 to A549 (J p>0.005; r = +0.667). (p > 0.005; r = +0.667). Experiments were performed in triplicate and repeated at least 3 times. Values are means±SD. *Student's t-test p<0.05.

https://doi.org/10.1371/journal.pone.0150320.g002

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Fig 3. Putative target molecules in A549 cells.

A549 cells were fixed with 4% para-formaldehyde and stained with a combination of a mouse anti Eps 1 and one of the following: rabbit anti Int β4, PCDH19, MMRN1, or BMPER. The secondary antibodies used were either Alexa Fluor 405-conjugated AffiniPure Goat Anti-Mouse IgG (blue) or Alexa Fluor 594-conjugated AffiniPure Goat Anti-Rabbit IgG (red) in accordance with the primary antibody used. 3D SIM images were taken using the Elyra SIM imaging system.

https://doi.org/10.1371/journal.pone.0150320.g003

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Fig 4. Immunostaining of S. pneumoniae adhesion to A549 cells.

CFDA-stained S. pneumoniae strain WU2 cells were incubated with A549 cells for 1 h. Excess bacteria were removed, and the culture was fixed with 4% para-formaldehyde, stained, and viewed with a FluoView FV1000 confocal system (Olympus, Japan). The following stains were used: A) mouse anti-Eps 1 antiserum and rabbit anti-integrin β4 antiserum; C) mouse anti-epsin 1 antiserum and rabbit anti-BMPER antiserum; E) mouse anti-Eps 1 antiserum and rabbit anti-MMRN1 antiserum; and G) mouse anti-Eps 1 antiserum and anti-PCDH19 antiserum. The same cells viewed by Nomarsky microscopy overlaid with the CFDA-stained bacteria are shown, respectively, in B), D), F) and H). Secondary antibodies used were Alexa Fluor 405-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) antiserum and Alexa Fluor 594-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L (antiserum, in accordance with the primary antiserum used.

https://doi.org/10.1371/journal.pone.0150320.g004

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Fig 5. SIM images of CFDA-stained bacteria with Int β4 on A549 cells.

CFDA-stained S. pneumoniae strain WU2 cells were incubated with A549 cells for 1 h. Excess bacteria were removed, and the culture was fixed with 4% para-formaldehyde and stained with a rabbit anti Int β4. Secondary antibody used was Alexa Fluor 594 (red) conjugated AffiniPure Goat Anti-Rabbit IgG (H+L). 3D SIM images were obtained with the Elyra SIM imaging system with a 63x oil objective (NA = 1.4); actual magnification of the image is as indicated in the scale bar. A) S. pneumoniae (CFDA 488 nm—green) adhering to A549 epithelial cells are seen coated by Int β4 (red). B) Higher magnification of bacteria (green) enveloped with Int β4 (red). C) Intensity alignment profile. D) Pedestal-like structure formed at the site of adherence of S. pneumoniae (green) to A549 cells recruits Int β4 (red). E) Higher magnification demonstrates the recruitment of Int β4 (red) to the pedestal-like structure underneath the adhered S. pneumoniae (green). F) Intensity alignment profile.

https://doi.org/10.1371/journal.pone.0150320.g005

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Fig 6. MicroScale thermophoresis (MST) analysis.

In all cases, 16 serial dilutions of each putative receptor-derived peptide (~90 nM–3.175 mM) were mixed with a constant concentration of 335 nM labeled rPtsA protein. The normalized change in the fluorescence of the labeled rPtsA protein was plotted against the peptide concentration, and a fit was computed by the NTP program. The experiments were performed in triplicate. Presented here are the affinities of rPtsA binding to: the Eps1-derived peptide Kd = 43.900 ± 9.35 μM; the BMPER-derived peptide Kd = 21.00± 5.49μM; the MMRN1-derived peptide Kd = 391 ± 72.7 μM; the PCDH19-derived peptide Kd = 58.00 ± 13.4 μM; and the Int β4-derived peptide Kd = 115±20.5μM. For the control, namely, the Nox putative receptor (contactin 4)-derived peptide, a fit could not be generated due to a very low signal-to-noise ratio.

https://doi.org/10.1371/journal.pone.0150320.g006

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Fig 7. Inhibition of S. pneumoniae adhesion to A549 cells by Eps 1-derived peptide.

S. pneumoniae cells were treated for 1 h with Eps 1-derived peptide and added to A549 cells for 1 h; non-adherent bacteria were removed; and cells were detached with trypsin and plated onto blood agar plates for counting. A) Strain WU2 (p < 0.0001; r = −0.943); B) Strain 3.8DW (p < 0.0001; r = −0.056); C) Strain D39 (p < 0.0001; r = −0.943); D) Strain R6 (p <0.0001; r = −0.406; *t-test p < 0.05). Experiments were performed in 3–6 replicates and repeated at least 3 times. Values are means±SD. *Student's t-test p < 0.05.

https://doi.org/10.1371/journal.pone.0150320.g007

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Fig 8. Inhibition of adhesion of S. pneumoniae cells to A549 cells by target-derived peptides.

S. pneumoniae cells were treated for 1 h with peptide and added to A549 cells for 1 h; non-adherent bacteria were removed; and cells were detached with trypsin and plated onto blood agar plates for counting. A) BMPER, WU2 (P < 0.0001, r = −0.887); B) BMPER, 3.8DW (P < 0.0001, r = −883); C) Col VIIα WU2 (P < 0.0001, r = −1); D) Col VIIα, 3.8DW (P < 0.0001, r = −0.886); E) MMRN1, WU2 (P < 0.0001, r = −1); F) MMRN1, 3.8DW (P < 0.0001, r = −0.821); G) PCDH 19, WU2 (P < 0.0001, r = −0.829); H) PCDH 19, 3.8DW (P < 0.0001, r = −0.754); I) Int β4, WU2 (P < 0.0001, r = −0.5619); J) Int β4, 3.8DW (P < 0.0001, r = −0.967); K) No significant inhibition was observed with a scrambled peptide (K scrambled peptide, P = 0.9981). Experiments were performed in triplicates and repeated at least 3 times. Values are means±SD. *Student's t-test p<0.05.

https://doi.org/10.1371/journal.pone.0150320.g008

Inhibition of adhesion to A549 cells by anti-rPtsA antiserum, phages or peptides was performed as previously described [16]. Briefly, bacteria were incubated for 1 h with rabbit anti-rPtsA antiserum, the phages, or the selected peptides. The bacteria were then added to the cultured human cells for an additional hour. Following this incubation, excess bacteria were removed, and the procedure was continued as described above. All the inhibitors were used at concentrations below their toxic concentrations either to the bacteria or to the cells. Negative controls included pre-immune serum, phage carrying no insert or a non-inhibitory insert, and a scrambled peptide. Each experiment was performed in triplicates and repeated on 3 different occasions and a representative experiment is presented.

Immunofluorescence Microscopy

Immunostained images were obtained for live bacteria stained with polyclonal mouse anti-rPtsA antisera, detected with a Alexa-Flour 488-conjugated affinity pure donkey anti-mouse IgG (H+L) (Jackson ImmunoResearch Laboratories West Grove, PA, USA) The nuclei were stained with Hoёchst stain. Structured illumination microscopy (SIM) orthogonal view images were obtained using an Elyra SIM imaging system (Zeiss, Germany) with a 63× oil objective (NA = 1.4); actual magnification of the image is as indicated in the scale bar in the relevant figures. As negative control either preimmune serum was used or the primary antibody was excluded (the negative pictures are not presented).

A549 cells were cultured on Ibidi 8 well chambers (Ibidi GmbH, Planegg/Martinsried, Germany). The cells were fixed with 4% para-formaldehyde and stained with a combination of mouse anti-Eps 1 antiserum at 1:25 dilution and one of the following: rabbit anti-integrin β4 antiserum at 1:50 dilution (Santa Cruz Biotechnology, Inc. Delaware, CA, USA), anti-PCDH19 antiserum at 1:50 dilution, anti-MMRN1 antiserum at 1:200 dilution (Novus Biologicals, Littleton, CO, USA), or BMPER at 1:10 dilution (Abnova 9F, No. 108. Jhouzih, Taiwan). For bacterial adhesion studies, the combination of mouse anti- Intβ4 antiserum at 1:25 dilution and rabbit anti-Eps 1 at 1:50 dilution were used. Secondary antibodies used were either Alexa Fluor 405-conjugated AffiniPure goat anti-mouse IgG (H+L) antiserum at 1:125 dilution or Alexa Fluor 594-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) antiserum at 1:250 dilution (Molecular probes by Life Technologies, USA) in accordance with the primary antibody used. 3D images were acquired with either a FluoView FV1000 confocal system (Olympus, Japan) or an Elyra SIM imaging system (Zeiss, Germany). For SIM imaging, a 63× oil objective (NA = 1.4) was used. Five rotations and 5 phases were taken for each focal plane. Reconstructions were performed with Zen software (Zeiss, Germany). Shown here are maximum projections of 3D series. As negative control either preimmune serum was used or the primary antibody was excluded (to reduce redundancy the negative blots are not presented).

CFDA Staining of the Bacteria

Bacteria were grown in THY broth to mid-late log phase as determined by OD. Aliquots of bacteria were harvested by centrifugation (13000 rpm), re-suspended in sterile PBS, and washed twice. Bacteria (1 ml suspension, 1×10⁶ cell concentrations) were then stained with CFDA, using stock solution (0.5 mM), incubated with shaking for 30 min in 37°C, and washed twice following incubation.

Identification of rPtsA-Binding Phages

To identify putative rPtsA-binding sequences, we screened a combinatorial peptide library expressed in filamentous phage fth1, as described previously [46]. In short, the type 88 phage display library, in which a second recombinant pVIII gene has been incorporated into the fd bacteriophage genome, was used in this study. The recombinant pVIII gene also carries a tetracycline resistance gene tetA. The particular library used in this study contains a random ~12 amino acid sequence flanked by two constant cysteine residues, thus generating random 12-mer loops expressed on PVIII proteins of a mosaic fd phage. Selection and characterization of the phages were accomplished via tetracycline-resistant bacterial colonies rather than isolating phages from plaques. This library contains 10⁸−10⁹ random inserts in the original phage display library.

After incubation of the Fd phage library on HAT-rPtsA-adsorbed culture dishes, unbound phages were washed out, and bound phages were eluted at pH 2.2. Phages were then retested for their ability to bind rPtsA by using dot blot analysis. The phages were adsorbed onto filter paper, and rPtsA was added, followed by anti-rPtsA antiserum. These dot blots were repeated twice with the phages that demonstrated binding to rPtsA. As a negative control, we used a phage without an insert. The nine identified rPtsA-binding phages were tested for their ability to interfere with bacterial adhesion to cultured A549 cells. To prevent redundancy, we present only the 6 positive phages and the phage without insert. The results with the phages with insert that did not inhibit S. pneumoniae adhesion to the human cell lines were identical to the results with the phage without insert.

Bioinformatics Analysis

PtsA sequences were analyzed for homology to human or pneumococcal sequences using the NCBI database. The nucleotide sequences of the peptide insert in rPtsA-binding phages were converted into amino acid sequences and compared to human proteins in UniProt using NCBI's blastp program, with the option "Automatically adjust parameters for short input sequences". Candidate proteins from the blastp results were manually selected according to their membrane or extracellular matrix localization.

Peptide Synthesis

The following peptides were synthesized according to the sequences in the human protein homologous to the insert peptides in the phages: BMPER (VLVKNDARRTRS), Col VII (VVVSDATRVRVA), MMRN1 (TEQVSDQKNAPA), PCDH19 (DGGVPML), Intβ4 (DQVARIPVIRRV) and Eps 1 (SDGGVPV) and the negative peptide (LPADWATTLMVCSSK) (Hi-Lab laboratories, Rehovot, Israel). Contactin 4 (Q8IWV2 UniProt)-derived peptide (WECKANGRPKPTY) is a putative target for NOX, an adhesin of S. pneumoniae [17], and was used as a negative control for peptide-rPtsA binding.

MicroScale Thermophoresis (MST) Analysis

rPtsA labeled with fluorescent NT-495 dye at a constant concentration of 335 nM was mixed with sixteen serial dilutions (~90 nM–3.175 mM) of peptides derived from PCDH19, MMRN1, Eps1, Int β and BMPER. Contactin 4, was used as a negative control [17]. MST analysis was performed using Monolith NT.115 (NanoTemper Technologies GmbH, München, Germany). The normalized change in the fluorescence of bound and unbound labeled rPtsA (ΔF) is indicative of the peptide binding. Plotting ΔF vs. peptide concentration facilitated the generation the dissociation curves, computed by the NTP program. In this way the Kd, reflecting the affinity of each of the peptides for rPtsA, was obtained. The curves obtained from the interaction of rPtsA with its putative target-receptor-derived peptides exhibited a signal-to-noise ratio (SNR) of at least 2, indicating the specificity of the interaction. A curve could not be generated for the control peptide (contactin 4) due to a SNR of less than 1. A representative experiment shown is out of 3 performed. Each experiment was performed in triplicate.

Human Sera

Sera were collected at 24 and 38 months of age from healthy Bedouin children visiting maternal and child health care (MCHC) clinics for routine checkups. The children were enrolled as controls for an immunization clinical trial between November 2001 and July 2007. For each patient venous blood samples and nasopharyngeal swab were taken (S2 Table). Serum samples were stored at -70°C.

Ex Vivo Neutralization of S. pneumoniae

Strain WU2 cells were pretreated with Eps 1, BMPER and PCDH19 peptides and then inoculated IN into adult CBA/N^xid mice. Mice were euthanized 3, 24 or 48 h later. The nasopharynx and lungs were excised, homogenized and plated onto blood agar plates for counting. As a control, another group of mice was injected with bacteria treated ex vivo for 1 h with PBS alone.

In addition, CBA/N^xid mice were immunized with rPtsA as described above. Mice were euthanized, and blood was drawn from the hearts. WU2 cells were incubated at 37°C for 1 h with 1:20 diluted mice pre-immune or anti-rPtsA antiserum. A lethal dose (10⁴ CFU) of WU2 was inoculated intravenously (IV) into BALB/c or CBA/N^xid mice. Survival was monitored continuously.

Statistical Analysis

Non-parametric Pearson correlation analysis (r value) was used to evaluate the dose-dependent inhibition of pneumococci binding to A549 or Detroit 562 cells. To determine the significance of inhibition among all tested groups and relative to the positive control ANOVA analysis and a two-tailed unpaired Student's t-test were performed, respectively. Inhibition of bacterial colonization was determined using Student's t-test. Analysis of survival was done using the log-rank (Mantel Cox) test Or Gehan-Breslow_Wilcoxon tests using GraphPad Prism V6 software.

Cell-Wall Localization and Immunogenicity of PtsA

Our previous 2D PAGE immunoblot analysis studies of the different cell-wall fractions with sera obtained from mice immunized with the total cell-wall lectin or cell-wall non-lectin fractions revealed a highly immunogenic spot in the total and non-lectin fractions (Spot 1) [39]. The spot was found to contain DnaK, which has low antigenicity in humans and is incapable of eliciting a protective immune response in mice [47]. We therefore continued to explore the non-lectin fraction of strain WU2 by MALDI-TOF analysis and found that co-localized with DnaK in Spot 1 was an additional protein, PtsA (PI 4.6, molecular weight 63.4, Mascot score 134, SP_RS05795, WP_000138135). We thus assumed that the immunogenic protein in this spot was PtsA (and not DnaK), an assumption that we confirmed in this study. MALDI-TOF analysis detected PtsA in the cell-wall of seven of the nine different S. pneumoniae strains tested (S1 Table). Pneumolysin, enolase, and glyceraldehyde 3-phosphate dehydrogenase were identified in the cell-wall preparations of all strains analyzed. These results encouraged us to further explore the function of PtsA in the bacterial cell-wall and its potential as a vaccine.

Surface Expression of PtsA in Different Pneumococcal Strains

The ptsA gene was cloned, expressed, and purified, and antibodies were produced as described in the Materials and Methods section (S1 Fig). The surface expression of PtsA was analyzed using rabbit anti-rPtsA antiserum. PtsA was found in the cell-wall, membrane and cytoplasmic cellular fractions of strain WU2 (Fig 1A). Pneumolysin, which is known to be present in the S. pneumoniae cell wall [26], was used as a positive control for cell-wall localization, and FabD, a protein involved in lipid synthesis, was used as a negative control [27]. Pneumolysin (Fig 1B) was detected in the cell-wall, membrane and cytoplasm of strain WU2, while there was no detection of FabD protein in the bacterial cell-wall (Fig 1C).

To validate the presence of PtsA on the bacterial surface, flow cytometry analysis of live strain R6 probed with a mouse monoclonal anti-rPtsA antibody was undertaken. PtsA was indeed found to be expressed on the bacterial surface (Fig 1D). Purified mouse IgG served as a negative control. Consistently, SIM orthogonal view imaging of live bacteria stained with mouse anti-rPtsA antisera (green) indicated that PtsA (green) was located on the perimeter, surrounding the bacterial genome (white). This configuration was most prominently observed in the XZ plane and further confirmed in the XY and YZ planes (Fig 1E). No such staining was observed with preimmune serum, used as the control.

PtsA Mediates S. pneumoniae Adhesion to Target Host Cells

To further establish the extent to which PtsA is involved in pneumococcal interaction with the host, the ability of rPtsA to inhibit pneumococcal adhesion to human lung adenocarcinoma A549 cells was tested using the previously described assay for inhibition of S. pneumoniae adhesion to A549 cells [16, 17, 41, 48]. rPtsA significantly inhibited the adhesion of the four strains tested: S. pneumoniae strain WU2 (Fig 2A; p < 0.0001; r = −0.943), the unencapsulated derivative of strain WU2, namely, strain 3.8DW (Fig 2B; p < 0.0001; r = −0.371); serotype 2 strain D39 (Fig 2C; p < 0.0001; r = −1); and the unencapsulated derivative of strain D39, namely, strain R6 (Fig 2D; p < 0.0001; r = −1). All groups demonstrated dose dependency except strain 3.8DW. Nonetheless, a significant reduction, by an order of magnitude, in the adhesion of strain 3.8DW was observed in the presence of rPtsA. KLH was used as a negative control. A representative experiment demonstrating the inability of KLH to inhibit D39 adhesion to A549 cells is presented in Fig 2E (p = 0.8; r = −0.2). Of note, in a previous study [16] no inhibition of adhesion could be observed using either rPsipD, a surface S. pneumoniae protein not involved in adhesion, or KLH. In the current study we used only KLH as a negative control protein.

We then tested the ability of anti-rPtsA antiserum to inhibit S. pneumoniae adhesion to A549 cells. S. pneumoniae cells were incubated with rabbit anti-rPtsA antiserum and then added to the gelatin-blocked A549 cells. Rabbit anti-rPtsA antiserum significantly inhibited the adhesion to A549 cells of strains: WU2 (Fig 2F; p < 0.0001; r = −0.359), 3.8DW (Fig 2G; p < 0.0001; r = −0.886), D39 (Fig 2H; p < 0.0001; r = −0.943) and R6 (Fig 2I; p < 0.0001; r = −0.406). Serum obtained from a rabbit prior to immunization did not inhibit the adhesion of strain WU2 to A549 (Fig 2J; p > 0.005; r = +0.667).

Identification of rPtsA-Binding Sequences

To identify PtsA target molecules in the host, we screened a combinatorial peptide library expressed in a filamentous phage with rPtsA. Of the nine phages that bound to rPtsA, six inhibited adhesion of strain WU2 to the A549 cells: phage D3 (S2A Fig; p < 0.0001, r = −1); phage E6 (S2B Fig; p < 0.0001, r = −0.6); phage D8 (S2C Fig; p<0.0001, r = −0.8); phage D10 (S2D Fig; p < 0.0001, r = −0.8); phage H9 (S2E Fig; p<0.0001, r = −0.7); and phage H10 (S2F Fig; p < 0.0001 r = −0.164). The phages significantly inhibited the adhesion by more than 75% in comparison to adhesion of bacteria without inhibitors. Moreover, for phage D3, phage D8 and phage D10, the inhibition of adhesion was dose dependent. Inhibition of bacteria below 25% was considered as negative. To prevent redundancy, the controls include the phage without an insert and only one phage with a non-inhibitory insert. The phage without an insert inhibited pneumococcal adhesion to A549 cells only by 20% (S2G Fig p<0.001, r = − 0.7). In addition an inactive page with insert demonstrated only about 15% reduction in bacterial adhesion (S2H Fig; p<0.0001, r = −0.7)."

Sequence determination of the six inhibitory peptide inserts was achieved following sequencing the oligonucleotide inserts in the envelop gene of the phages and translating them into amino acids. The peptide sequences were compared to the UniProt human proteins database by using NCBI's blastp program. Candidate proteins from the blastp results were selected manually according to their localization in the cell membrane or the ECM (Table 1). The following proteins were selected for further study: epsin 1 (Eps 1), bone morphogenetic protein binding endothelial regulator (BMPER), multimerin 1 (MMRN1), protocadherin 19 (PCDH 19), integrin β4 (Int β4) and collagen VII α1 (Col VIIα).

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Table 1. Inhibitory peptide sequences.

https://doi.org/10.1371/journal.pone.0150320.t001

Existence of the Putative Target Molecules in A549 Cells

Immunofluorescence microscopy revealed the expression in A549 cells of Eps 1, Int β4, BMPER, MMRN1, and PCDH 19, but not of Col VIIα (Fig 3A–3D, respectively). Similar results were obtained for the pharyngeal carcinoma cell line, Detroit 562 (data not shown). Experiments with bacteria stained green with carboxyfluorescein diacetate (CFDA) and A549 cells confirmed the attachment of S. pneumoniae to the A549 cells (Fig 4A, 4C, 4E and 4G). The interaction of the bacteria with the A549 cells was more clearly visible in Nomarsky microscopy overlaid with the CFDA-stained bacteria (Fig 4B, 4D, 4F and 4H). Furthermore, Fig 4A and 4C imply the association of Int β4 and BMPER with the adhered bacterium. SIM imaging further processed with a maximum intensity projection method revealed that S. pneumoniae cells (stained with CFDA) adhering to A549 epithelial cells are surrounded by Int β4 (Fig 5A). The higher magnification better demonstrates the bacteria enveloped with Int β4 (Fig 5B), as was further confirmed by the intensity alignment profile (Fig 5C). In addition, S. pneumoniae cells adhering to A549 cells are shown to be associated with a pedestal like structure. The bacterium 'sits' on a cellular protrusion that contains Int β4 (Fig 5D). The higher magnification provides a better visualization of this association of S. pneumoniae with the A549 cell via Int β4 in the pedestal-like structure (Fig 5E), as was also confirmed by the intensity alignment profile in Fig 5F. Of note, the interaction of S. pneumoniae with its target cells consists of sequential stages initiated by pilus binding, in pili-carrying strains, followed by intimate adhesion with different adhesins and culminating in cell invasion with invasins. Two different stages of S. pneumoniae interaction with its host cell are depicted in Fig 5. The first is attachment to a pedestal-like host-cell structure, and the second is engulfment by the host cell.

Affinity of rPtsA Interactions with Target-Molecule-Derived Peptides

Following the verification of the existence of the putative receptors for PtsA in the A549 (Figs 3, 4 and 5) and Detroit 562 (data not shown) cells, peptides spanning the regions in the putative target molecules that were homologous to the insert peptides in the inhibitory phages were synthesized (Table 1). MicroScale thermophoresis (MST) was used to test the affinity of rPtsA to the target-derived peptides. Normalized change in the fluorescence of labeled rPtsA following peptide binding facilitated the determination of the affinity of rPtsA for each of the five peptides but not for a control peptide (contactin 4, a peptide derived from the NOX putative receptor [17]), as reflected by their Kd values (Fig 6). Represented here are the affinities for rPtsA binding to: the Eps1-derived peptide Kd = 43.900 ± 9.35 μM; the BMPER-derived peptide—Kd = 21.00± 5.49μM; the MMRN1-derived peptide Kd = 391 ± 72.7 μM; the PCDH19-derived peptide Kd = 58.00 ± 13.4 μM; and the Int β4-derived peptide Kd = 115±20.5μM.

Target-Derived Peptides Inhibit S. pneumoniae Adhesion to Epithelial Cells

Peptides spanning the homologous regions in the target molecules were synthesized (Table 1) and tested for their ability to inhibit bacterial adhesion to A549 cells. Peptides derived from Eps 1 significantly inhibited the adhesion to A549 cells of strains WU2 (7A; p < 0.0001, r = −0.943), 3.8DW (7B; p < 0.0001, r = −0.5), D39 (Fig 7C; p < 0.0001, r = −0.943) and R6 (Fig 7D; p < 0.0001, r = −0.406). The dose dependency was more pronounced for the encapsulated strains than for the unencapsulated strains. Similarly, there was a significant inhibition of WU2 and 3.8DW adhesion to lung-derived A549 cells in the presence of peptides derived from BMPER (Fig 8A; WU2 p < 0.0001, r = −0.887; Fig 8B; 3.8 DW p < 0.0001, r = −0.883), Col VIIα (Fig 8C; WU2 p < 0.0001, r = −1; Fig 8D; 3.8DW p < 0.0001, r = −0.886), MMRN1 (Fig 8E; WU2 p < 0.0001, r = −1; Fig 8F. 3.8DW p < 0.0001, r = −0.821), PCDH 19 (Fig 8G; WU2 p < 0.0001, r = −0.829; Fig 8H; 3.8DW p < 0.0001, r = −0.754) and Int β4 (Fig 8I; WU2 p < 0.0001 r = 0.058; Fig 8J; 3.8DW p < 0.0001, r = −0.9). Dose dependency was observed in all cases except for inhibition of strain 3.8DW with PCDH 19 and of WU2 strain with Int β4. No significant inhibition was observed for a scrambled peptide (Fig 8K). Similar results were obtained for the nasopharynx-derived cell line, Detroit 562. There was significant inhibition of the adhesion of S. pneumoniae strain WU2 to Detroit 562 cells in the presence of peptides derived from: BMPER (S3A Fig; p < 0.0001, r = −0.9), PCDH19 (S3B Fig; p < 0.0001 r = −0.829), Int β4 (S3C Fig; p < 0.001 r = −0.562) and Eps 1 (S3D Fig; p < 0.0001, r = −0.771). Moreover, for BMPER and PCDH19, this inhibition was dose dependent.

Target-Derived Peptides Inhibit Colonization

The ability of Eps 1 to interfere with bacterial colonization was tested in the intranasal (IN) mouse inoculation model. Strain WU2 bacteria were pretreated with Eps 1 (14.7 μM) and then inoculated IN into CBA/N^xid mice. The mice were euthanized 3 and 24 h following inoculation, and the nasopharynx and the lungs were excised, homogenized and plated for bacterial colony counting. Mice inoculated with peptide-treated bacteria demonstrated a significant reduction in bacterial load in the nasopharynx and the lungs in comparison to mice inoculated with sham-treated bacteria at 3 h (Fig 9A; P < 0.0001; Fig 9B; P < 0.0001, respectively) and at 48 h (Fig 9C; P < 0.0001) and Fig 9D; P < 0.0001) post inoculation. In a different experiment, bacteria were pretreated with BMPER-derived peptide (7 μM and 14 μM) and then inoculated IN into CBA/N^xid mice. Forty eight hours following inoculation the mice were euthanized, and the nasopharynx and lungs were excised, homogenized and plated onto blood agar plates for bacterial colony counting. A significantly reduced bacterial load was observed in the mice inoculated with BMPER-derived peptide in comparison with mice inoculated with sham-treated bacteria (Fig 9E; P < 0.0001 and Fig 9F; P < 0.0001, respectively). Similar results were obtained for mice inoculated with WU2 bacteria pretreated with PCDH19-derived peptide (14.7 and 27.4 μM) in comparison with mice inoculated with sham-treated bacteria (Fig 9G; P < 0.0001; Fig 9H; P < 0.0001).

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Fig 9. Effects of target-derived peptides on nasopharyngeal and lung colonization and survival in mice.

Strain WU2 cells were pretreated with peptide and then inoculated IN into adult CBA/N^xid mice. As a control, another group of mice was injected with bacteria treated ex vivo for 1 h with PBS alone. Mice were euthanized 3, 24 or 48 h later. The nasopharynx and lungs were excised, homogenized and plated onto blood agar plates for counting. A) Eps 1 (14.7 μM), nasopharynx, 3 h (P < 0.001); B) Eps 1 (14.7 μm), lung, 3 h (P < 0.0001); C) Eps 1 (14.7 μm), nasopharynx, 24 h (P < 0.0001); D) Eps 1 (14.7 μm) lung, 24 h (P < 0.0001); E) BMPER, nasopharynx, 48 h at 7 and 14 μM (P < 0.0001); F) BMPER, lung, 48 h at 7 and 14 μM (P < 0.0001); G) PCDH19, nasopharynx, 48 h at 14.7 and 17.4 μM (P < 0.001); H) PCDH19, lung, 48 h (P < 0.0001).

https://doi.org/10.1371/journal.pone.0150320.g009

Antigenicity of PtsA in Children

Previous studies have revealed that conserved surface proteins demonstrating antigenicity in children constitute immunogens that elicit a protective immune response in mice [47–50]. To test whether PtsA is antigenic in children, rPtsA was immunoblotted with sera obtained from healthy children (S2 Table). PtsA antigenicity was observed in 7 out of 8 children at 24 and 38 months of age (S4 Fig). These results suggest that PtsA is likely to be antigenic in children.

Vaccine Potential of PtsA

The observations that PtsA resides in the cell-wall, is immunogenic, and functions as an adhesin suggest that it could be considered as a candidate for a vaccine antigen. BALB/c mice immunized with rPtsA demonstrated a significant reduction in colonization at 3 h (Fig 10A; p < 0.01) and 24 h (Fig 10B; P < 0.05) after inoculation with a sublethal dose of strain WU2. Immunization with rPtsA reduced the mortality of BALB/c mice following an IN inoculation with a lethal dose (10⁸ CFU) of strain WU2, although it did not reach significance (Fig 10C). However, immunization of CBA/N^xid mice significantly protected the mice against an intravenous (IV) lethal challenge (Fig 10D; P < 0.02). In addition, serum obtained from rPtsA-immunized CBA/N^xid mice neutralized S. pneumoniae WU2 virulence in BALB/c and CBA/N^xid mice following IV inoculation with10⁴ CFU of the WU2 bacteria both in BALB/c and CBA/N^xid (Fig 10E; P < 0.002; Fig 10F; P > 0.05, respectively). In contrast, serum obtained from CBA/N^xid mice immunized with adjuvant alone was not able to neutralize bacterial virulence (Fig 10G, Fig 10H, respectively).

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Fig 10. Vaccine potential of rPtsA.

BALB/c mice were immunized subcutaneously (SC) with 25 μg of rPtsA emulsified with CFA and boosted (days 14 and 28) with IFA. Mice were challenged IN on day 42 with a sublethal dose (5 × 10⁷) of strain WU2. Mice were sacrificed 3 and 24 h (strain WU2) following inoculation, and the nasopharynx and right lobe lung were excised, homogenized and plated onto blood agar plates for bacterial colony counting. Control mice were immunized with the adjuvant alone. A) Bacterial load in the nasopharynx and the lungs 3 h post inoculation of BALB/c mice with strain WU2 (Student's t-test; P < 0.01 and P < 0.05, respectively). B. Bacterial load in the nasopharynx (P < 0.05) and lung of BALB/c mice 24 h post inoculation with WU2 strain. C) After a similar immunization regimen, BALB/c mice were challenged IN with a lethal dose (10⁸ CFU) of strain WU2; control mice were immunized with the adjuvant alone. Mortality was monitored daily. Each group contained 10 mice (p = ns). D) After a similar immunization regimen, CBA/N^xid mice were challenged intravenously (IV) with a lethal dose (10⁴ CFU) of strain WU2. Control mice were immunized with the adjuvant alone. Mortality was monitored continuously (P<0.02). Each group contained 15 mice. E) S. pneumoniae serotype 3 (10⁴ CFU) was incubated with 1:10 dilution of serum obtained from adjuvant or 1:50 dilution of serum obtained from rPtsA + adjuvant immunized BALB/c mice and inoculated IV into BALB/c mice. Survival was monitored continuously (P < 0.002). Each group contained 10 mice. F) S. pneumoniae serotype 3 strain WU2 was incubated with 1:10 dilution of serum obtained from adjuvant or rPtsA + adjuvant (1:50) immunized CBA/N^xid mice. Then, 10⁴ CFU of WU2 were inoculated IV into CBA/N^xid mice, and survival was monitored continuously (P < 0.006). Each group contained 10 mice.*Log-rank (Mantel-Cox) test.

https://doi.org/10.1371/journal.pone.0150320.g010