Bacterial strains and growth conditions.
strains XL10-Gold, BL21(DE3), TOP10Fι (both Invitrogen), and JM109 (Promega) and their derivatives containing plasmids were grown at 37°C in Luria-Bertani (LB) broth with agitation or on LB agar supplemented, when appropriate, with ampicillin (100 μg/ml), spectinomycin, and streptomycin (100 μg/ml each) or kanamycin (50 μg/ml). S. pneumoniae
serotype 4 strain TIGR4; the unencapsulated TIGR4 derivative T4R; 19 clinical isolates (collection of E.I. Tuomanen); and Staphylococcus aureus
NRS193 (provided by Jon McCullers, St. Jude Children’s Research Hospital) were grown on tryptic soy agar plates supplemented with 3% defibrinated sheep blood or in defined semisynthetic casein liquid medium supplemented with 0.5% yeast extract (35
). The ΔcbpA
derivatives of unencapsulated T4R and wild-type T4 were created as described previously (28
). N. meningitidis
strains MC58, Z4181 (C: P1.5,2-1: F5-4: ST-11 [cc11]), Z4682 (B: P1.7-1,1: F5-8: ST-12 [–]), their mutant derivatives, and 68 additional clinical isolates (collection of D.A.A. Ala’Aldeen) and H. influenzae
Rd and ATCC 10211, their derivatives, and 38 clinical isolates (collection of D.A.A. Ala’Aldeen) were cultured on chocolate agar at 37°C in 5% CO2
. For selection of mutants, meningococcal cells were cultured on Mueller-Hinton agar plates supplemented with Vitox (Oxoid) and, where appropriate, streptomycin and spectinomycin (each 100 μg/ml) or kanamycin (100 μg/ml). All mutants retained wild-type growth rates. Liquid cultures of N. meningitidis
were grown in Mueller-Hinton broth supplemented with Vitox at 37°C with agitation. The ΔompP2
mutant of H. influenzae
Rd was a gift from Joachim Reidl, Biozentrum der Universität Würzburg, Am Hubland, Germany (29
Chromosomal DNA was prepared by using a DNeasy tissue kit (QIAGEN) and the protocol for bacterial cells recommended by the manufacturer. Plasmid DNA was prepared by using a QIAprep Spin Kit (QIAGEN) according to the manufacturer’s recommendations. PCR reactions contained 100 ng of chromosomal DNA or 1 ng of plasmid DNA, each primer at 200 nM, 200 μM each of dNTP, Expand polymerase buffer (including MgCl2 to a final concentration of 1.5 mM), and 0.6 U of Expand polymerase in a total volume of 25 μl (Roche). After initial incubation at 95°C for 3 minutes, reactions comprised 30 cycles of incubation at 50–55°C for 1 minute, 65–68°C for 1–6 minutes, and 94°C for 1 minute, with final incubations at 50–55°C for 1 minute and 65–68°C for 10 minutes.
Mutagenesis of N. meningitidis.
gene (NMB1429) and flanking DNA was amplified from the chromosomal DNA of strain MC58 using primers PorA-M1 (5′-ATCAGAAACCTAAAATCCCGTCAT-3′) and PorA-M2 (5′-TCCTCTGTTTTGAAACCCTGAC-3′). The amplicon was cloned into pGEM-T Easy (Promega) to form plasmid pPorA-4d and subjected to inverse PCR mutagenesis with the primers PorA-M3 (5′-GCGGGATCCTCGGGCAAACACCCGATAC-3′) and PorA-M4 (5′-GCGGGATCCATCGGGGCGGTGAAGC-3′). This procedure resulted in removal of the porA
coding sequence and introduction of a unique Bam
HI site, which was used to introduce an Ω cassette (encoding resistance to spectinomycin and streptomycin) (37
). The resulting plasmid was used to mutate MC58 by natural transformation and allelic exchange as described previously (38
). The deletion in the resulting mutant (MC58porA
) was confirmed by PCR analysis. The pilQ
gene (NMB1218) and its flanking sequence were amplified using the primers PilQF1 (5′-GCCGTCTGAAACAGCTGCCGACAGATGC-3′) and PilQR1 (5′-AAACCAGTACGGCGTTGCCTCGC-3′). The amplicon was cloned into pGEM-T Easy and subjected to inverse PCR mutagenesis with the primers PilQF2 (5′-CGCGGATCCCTTTCACCGTAACCTCAATCGC-3′) and PilQR2 (5′-CGCGGATCCCTGTAATGTTTCCTGCCGATGC-3′). This procedure resulted in deletion of the ORF and introduction of a unique Bam
HI site that was used to introduce the kanamycin resistance cassette digested with Bam
HI from plasmid pJMK30 (39
). The resulting plasmid was used to mutate both the wild-type MC58 and the porA
mutant, yielding strains MC58pilQ
, respectively. Successful mutagenesis of the pilQ
gene in both mutants was confirmed by PCR and immunoblot analysis. The native porA
gene of strain Z4682 was replaced with the MC58 porA
gene. Using plasmid pPorA-4d as template, we amplified a 6.2-kDa fragment by PCR using the primers M-Kan-F (5′-AGATCTGCTTTGTTTTTGACGGCTCGC-3′) and M-Kan-R (5′-AGATCTATCCGCTTCACCGCCCCG-3′). The product was gel purified, digested with Bgl
II, self ligated, and used to transform cells of JM109 (Promega). The resulting plasmid, pNAB10, was digested with Bgl
II and ligated to the Ω cassette digested with Bam
HI to form plasmid pNAM11. pNAM11 was used to transform strain MC58 and Z4682; successful transformation of strain Z4682 was confirmed by amplification of the region containing porA
and 1 kb upstream with the primers PorA-M1 (5′-ATCAGAAACCTAAAATCCCGTCAT-3′) and PorA-ER2 (5′-CCGATATTTAGAATTTGTGGCGC-3′). The sequence of the amplified product was shown to be identical to the MC58 porA
Purification of rLR.
E. coli BL21(DE3) containing plasmid pET28LR was cultured in LB containing kanamycin overnight at 37°C with agitation. Cells were harvested by centrifugation; the pellet was washed in PBS containing 0.05% v/v Tween-20 (PBS/T) and centrifuged at 6,500 g for 5 minutes. The supernatant was replaced with fresh PBS/T. This was repeated 3 times before cells were sonicated in an ice bath for 15 cycles of 10 seconds with 15 seconds of cooling. Samples were solubilized in 1% SDS sample buffer, and the suspension was incubated at 37°C for 30 minutes. The lysate was separated by SDS-PAGE and stained with SimplyBlue SafeStain (Invitrogen) for 30 minutes. A vertical section of the gel was removed for immunoblot analysis, and the band containing rLR was excised from the remaining gel, cut into small cubes, and placed in Dialyzer Midi D-Tubes (Calbiochem, EMD) in 1 ml PBS/T. The protein was eluted in SDS-PAGE running buffer at 100 V for 2 hours and dialyzed against PBS/T at 4°C for at least 24 hours.
Production of recombinant CbpA constructs.
All recombinant CbpA protein constructs were expressed and purified as previously described (24
). Constructs included the full-length N terminus (CbpA-NR12; E39-P443), domain R1 linked to R2 (CbpA-R12; E175-P443), and the full-length N-terminal domain with a point mutation ablating adhesion to the pIgR receptor (CbpA-R12/Y205G/Y358G). To create CbpA mutants, we used R12 expressed in pET15b as a template for QuikChange Multi Site-Directed Mutagenesis Kit (Stratagene). Clones were sequenced to confirm the presence of the desired mutation and subsequently used to transform BL21(DE3) cells for expression.
All proteins were induced and purified as previously described (24
To create pneumococci expressing CbpA with point mutations ablating binding to LR, we cloned full-length cbpA
into pNE1 expression vector (24
). This construct was used as a template for the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Primer MUT1 (5′-GCTAAGGAAGGGGGGAACGAGGAAAAAG-3′) was used to make the P392G and R393G substitutions and cloned as previously described (24
). Clones were sequenced to confirm the presence of desired substitutions, and plasmid preparations were used to transform S. pneumoniae
by standard methods as follows. T4X ΔcbpA
was constructed by the SOEing method (40
). Briefly, an erythromycin resistance cassette (1,096 bp) was amplified from the ermB
gene using primers ermF (5′-GGAAATAAGACTTAGAAGCAAAC-3′) and ermR (5′-CCAAATTTACAAAAGCGACTC-3′). Approximately 1-kb regions flanking the cbpA
gene were amplified with 2 primer pairs: cbp1 (5′-GGCGGGAAAGAATTTGGAG-3′), CbpASOEup (5′-GTTTGCTTCTAAGTCTTATTTCCGTTTATTTCCTTCTATATTTTTTCTTTAACC-3′) and cbpA4 (5′-CGCCAACGGTGATATCCGTAC-3′), CbpASOEdn (5′-GAGTCGCTTTTGTAAATTTGGACCTAATATAACTAGTTAATACTGACTTCC-3′). Primers CbpAErmSOEup and CbpAErmSOEdn served as fusion primers and consisted of primers ermF and ermR, respectively, and the target gene–specific sequence. A mixture of gel-purified preparations of the 2 flanking products and the erm
cassette was used as a template for the SOEing PCR, using the outer CbpA1 and CbpA2 primers. The full-length PCR product consisting of the erm
cassette flanked by the CbpA-flanking sequences served as the knockout construct. The PCR fusion product was introduced into the chromosome of S. pneumoniae
T4X by transformation and homologous recombination based on standard methods. Loss of CbpA in transformants was confirmed by PCR, sequencing, and immunoblot analysis.
Affinity purification with a CbpA column.
CbpAR12 (residues E39-P443) was dialyzed against 0.1 M MOPS (pH 7.5) and concentrated to 2.5 mg/ml using Centriplus columns (Amicon, Millipore). CbpA-NR12 (10 mg) was covalently conjugated to 1 ml of Affi-Gel 15 (Bio-Rad) in 0.1 M MOPS (pH 7.5) at 4°C as instructed by the manufacturer. The column was sequentially washed with 20 ml of 100 mM glycine (pH 2.5) and 20 ml of PBS containing 1 mM MgCl2
, 0.5 mM CaCl2
, and 0.1% Triton X-100 (PBS-Ca2+
) were grown to complete confluence in 150 × 25 mm tissue culture dishes and preincubated with TNF-α at 10 ng/ml for 2 hours. Cell lysates were prepared in 1% Triton X-100 lysis buffer containing EDTA-free protease inhibitors (Roche). Cellular debris was removed by centrifugation at 135,000 g
for 1 hour at 4°C. The supernatant was recycled through the CbpA-NR12 affinity column for a period of 4 hours at 4°C. After the column was washed with 20 ml of PBS/Ca2+
/T, bound proteins were eluted in 300-μl fractions with 100 mM glycine-HCl (pH 2.5). Eluted proteins were visualized by SDS-PAGE and silver staining. Bands corresponding to eluted proteins were excised, and proteins were identified by MALDI-TOF analysis in conjunction with a database search performed with Applied Biosystems GPS Explorer software by the Hartwell Center for Bioinformatics and Biotechnology at St. Jude Children’s Research Hospital. Protein assignment was based on both mass spectrometry (MS) and MS/MS spectra and the NCBI nonredundant database.
Coimmunoprecipitation of LR with rCbpA.
rBCEC6 cells were activated with TNF-α (10 ng/ml) for 1 hour, lysed with 3 ml NP-40 buffer (0.5% NP-40, 500 mM Tris, pH 7.4, 150 mM NaCl), and briefly sonicated. The lysate was combined with 4 μg CbpA-AR12 and incubated overnight with 35 μg of either anti-LR (ab711; Abcam; 1:1000) or anti-CbpA (1:2,000). Rabbit antibody against the N terminus (amino acids 39–443) of CbpA was made by Covance. Protein A beads (Invitrogen) prepared as recommended by the manufacturer were added, and the mix was incubated for 1 hour. Samples were washed 7 times with 1 ml NP-40 buffer, resuspended in 40 μl 2× sample buffer, and boiled for 10 minutes. Samples were analyzed by SDS-PAGE and immunoblotting in which samples pulled down with anti-LR were probed with anti-CbpA, and those pulled down with CbpA were probed with anti-LR.
Adhesion assays were performed in several cell culture systems. Given the technical difficulty of obtaining primary mouse brain endothelial cells, the rat brain capillary endothelial cell line rBCEC6
) was used in most in vitro tests. Human brain microvascular endothelial cells (ScienCell Research Laboratories) were also tested to confirm the relevance of all binding events studied in the rBCEC6
cells. A549 human lung epithelial cells were used to study CbpA-mediated adherence independent of the domain required for endothelial adherence. Cells were seeded to 80% confluence in a 24-well plate (Costar, Corning) and activated for 1 or 2 hours with TNF-α (10 ng/ml).
Cells were washed to remove inhibitors and incubated with 1 × 107
For adhesion assays, cells were washed 3 times with PBS after 30 minutes, trypsinized, and diluted 1:10 in PBS; 10 μl was plated on blood agar and incubated overnight. All peptides used in inhibition experiments (LR amino acids 161–180, 263–282, a scrambled version of 263–282, LMWWML, YIGSR, LGTIPG, and VGVAPG) were manufactured by Invitrogen.
To block bacterial adhesion to rBCEC6
CFU bacteria were incubated for 1 hour at room temperature with 25 μg peptide. Bacteria were pelleted by centrifugation and washed twice with Dulbecco’s PBS (DPBS).
Alternatively, monolayers were treated with antibody anti-LR 711 (Abcam) recognizing amino acids 263–282 of LR at a dilution of 1:500 or laminin from human placenta (Sigma-Aldrich; 5 μg/ml) for 1 hour and the assay continued as above.
Adhesion assays using microspheres were performed as previously described (42
). Inhibition assays were performed in a similar manner with a 1-hour preincubation with purified laminin, anti-LR 711, naive rabbit serum, LR peptides, or complex sugars dissolved in PBS prior to addition of the microspheres. For all experiments, 4 wells per sample were used, and experiments were performed in triplicate.
LR siRNA (LamR1, 5′-TTGGTTCCCATCGTAACTTAA-3′; QIAGEN) was prepared according to the manufacturer’s instructions. rBCEC6 cells (105 per well) were seeded into 24-well plates and grown to 50% confluence. For each well, 10 nM siRNA was diluted in 100 μl serum-free medium. HiPerFect Transfection Reagent (3 μl; QIAGEN) was incubated with the siRNA for 10 minutes, and the mixture was added dropwise to cells and incubated at 37°C in 5% CO2 for 48 hours. To document decreased expression of LR, 1 well per sample was lysed with NP-40 buffer and sonicated, and equal amounts of protein were loaded for SDS-PAGE and Western blot analysis with polyclonal antibody against LR (Abcam) (Supplemental Figure 3). The remaining transfected cells were used for adhesion assays with bacteria as described above.
Intravital fluorescence microscopy and mouse challenge studies.
All experiments involving animals were performed with the prior approval of and in accordance with guidelines of the St. Jude Institutional Animal Care and Use Committee. Female BALB/c (The Jackson Laboratory), C57BL/6 Pafr–/–
(provided by P. Murray, St. Jude Children’s Research Hospital), and B6N10 mice and their respective wild-type littermates were maintained in Biosafety Level 1 and 2 facilities in the St. Jude Children’s Research Hospital Animal Facility. Mice were anesthetized for all experimental procedures by inhalation of 2.5% isoflurane (Baxter).
Cranial windows overlying the parietal cortex on one side of the midsagittal suture and extending from the bregma to the lamboid sutures were created in 8- to 10-week-old BALB/c mice as described previously (43
). After surgery, mice were allowed to recover for 10 days before data collection was initiated. Intravital microscopy was used to visualize attachment of fluorescent latex microspheres (Polysciences Inc.) to the microvasculature of the brain. Mice with cranial windows were anesthetized, immobilized on a stereotactic frame, and placed under an industrial-scale microscope (model MM-11; Nikon) with a camera assembly and bright-field and fluorescent light sources. For some experiments, mice were injected with recombinant mouse TNF-α (12 μg/kg i.v.) 1 hour before injection of microspheres. Yellow-green fluorescent 2-μm spheres were coated with the recombinant CbpA derivatives or BSA according to the supplier’s instructions.
Mice were injected with 2.5 μl/g body weight of a 108
Video images were followed for up to 3 hours, and still pictures were captured during injection and 3 and 10 minutes after injection at an excitation wavelength of 490 nm by using MetaMorph software (Universal Imaging, Molecular Devices). Beads attached to the microvasculature were counted, and counts were confirmed by comparison of the cerebral microvasculature with baseline pictures taken prior to injection. For some experiments, mice were pretreated with 200 μl of either undiluted antibody anti–LR 711 (Abcam) or control rabbit serum i.v. 5 minutes prior to challenge with CbpA beads.
For immunohistochemical staining for LR with and without infection, mice were challenged with PBS or 107 T4 pneumococci intranasally, and pneumonia was allowed to develop over 48 hours. Lungs were harvested, and frozen sections were stained with mouse anti-LR antibody MLuC5 (ab3099; Abcam) for 1 hour, followed by detection of binding with HRP-conjugated goat anti-mouse IgM (ab5930; Abcam) and the VIP Substrate Kit (Vector Laboratories). Sections were counterstained with methyl green.
Identification of H. influenzae and N. meningitidis LR ligands by retagging.
Bacterial LR-binding proteins were purified as previously described for the H. pylori
SabA adhesion protein (44
), with some modifications. N. meningitidis
, H. influenzae,
and S. pneumoniae
were incubated with purified rLR to which the Sulfo-SBED cross-linker (Pierce, Thermo Scientific) had been conjugated according to the manufacturer’s recommendations. The photoreactive cross-linker group was activated by 2 minutes of UV irradiation, and the biotin-(re)tagged proteins were purified with streptavidin-coated magnetic beads as described previously (44
). Extracted biotin-tagged proteins were separated by SDS-PAGE, and bands were digested with sequencing grade trypsin
(Promega) and analyzed using a Micromass TofSpec E. The nanoflow liquid chromatography–MS/MS was done on a 7-Tesla linear trap quadrupole Fourier transform (LTQ-FT) mass spectrometer (Thermo Scientific) equipped with a nanospray source modified in-house. The spectrometer was operated in data-dependent mode, automatically switching to MS/MS mode. MS spectra were acquired in the FT ion cyclotron resonance analyzer (FTICR), while MS/MS-spectra were acquired in the LTQ-trap. For each scan of FTICR, the 3 most intense, doubly or triply charged, ions were sequentially fragmented in the linear trap by collision-induced dissociation. All the tandem mass spectra were searched by Mascot (Matrix Science) against the databases for the N. meningitidis
strain MC58, H. influenzae
strain Rd KW20, and S. pneumoniae
strain T4, as appropriate.
ELISA of rLR.
Purified rLR or BSA (2–50 μg/ml) diluted in carbonate buffer (150 mM; 142 mM NaHCO3, 8 mM Na2SO3, pH 9.0) was used to coat amino-reactive 96-well microtiter plates (Immoblizer Amino; Nunc) for 2 hours at room temperature. Bacterial strains were grown in liquid culture and washed 3 times in PBS/T before being resuspended in carbonate buffer to an OD of 0.1 at 600 nm. Bacteria were added to ELISA plates unlabeled (N. meningitidis) or labeled by adding 10 μg of digoxigenin (Roche) per 1 ml bacterial suspensions for 2-4 hours at room temperature (H. influenzae and S. pneumoniae). Liquid was removed from the plates, plates were washed 3 times with PBS/T, 100 μl of bacterial suspensions were added to each well, and plates were incubated for 2–4 hours at room temperature. Plates were washed several times with PBS/T and incubated with polyclonal anti-digoxigenin Fab fragment–conjugated antibody (1:5,000; Roche) in 1% BSA in PBS/T (blocking buffer) at 100 μl per well (digoxigenin-labeled cells) or with rabbit anti–whole N. meningitidis followed by goat anti-rabbit HRP. Plates were incubated at room temperature for an additional 1 hour and washed several times as described above. One hundred microliters of ABTS tablets (5 mg/ml; Roche) were added to each well, and the absorbance was measured at 405 nm after 30 minutes using an ELISA plate reader. Inhibition assays were performed as described above, except that bacteria were preincubated with 20 μg/ml rLR for 2 hours at room temperature and washed twice before being added to the ELISA plates. In the reverse inhibition assay, wells were coated with either N. meningitidis or H. influenzae (OD600, 0.1), and then incubated with digoxigenin-labeled rLR. In this experiment, rLP was preincubated with rPorA, rPilQ, rOmpP2, or nothing. Wells coated with BSA or ethanolamine were used as negative controls.
For studies on Factor H binding to CbpAs, plates were coated with 106 bacteria expressing CbpAWT or CbpAP392G-R393G and blocked with 10% bovine serum. Wells were exposed to 1 μg Factor H (Sigma-Aldrich), washed, and treated with 1:100 or 1:1,000 anti–Factor H antibody (Sigma-Aldrich). Binding was quantitated as the OD405 developed by anti-goat IgG alkaline phosphatase (AP) and yellow AP substrate (Sigma-Aldrich).
Bacterial suspensions were adjusted to an OD of 0.1 at 600 nm, and 1 ml of suspension was labeled with 10 μg CFSE (Molecular Probes, Invitrogen) for 10 minutes at room temperature, after which the cells were harvested by centrifugation and washed in 1 ml PBS/T. Purified rLR was labeled with Cy5 (Amersham, GE Healthcare) according to the manufacturer’s recommendations. CFSE-labeled bacterial cells were incubated with Cy5-labeled rLR for 2–4 hours. Cells were washed 2 times in PBS/T and transferred to the confocal plates. Images were captured using a Zeiss LSM 510uv META combi confocal system on a Zeiss Axiovert 100 microscope with a Zeiss C-Apochromat 63×/1.2 W objective lens. Sequential scanning with an argon 488-nm laser was used to excite the CFSE with a 505- to 550-nm band pass filter and an HeNe 633-nm laser with a 650-nm-long pass filter for the Cy5. The image size was 1,024 × 1,024 pixels, with 4 averages taken per line and a zoom of 3. All images were captured with the same laser, gains, and zoom settings. Colocalization analysis was performed using Zeiss LSM software and its Pixel Spreadgram function.
In instances where values from 2 experimental conditions were compared with each other, we used a 2-tailed Student’s t test to test for statistical significance. In instances where multiple experimental conditions were compared with a single control group, we tested for statistical significance using 1-way ANOVA. In regard to the latter, a Holm-Sidak ANOVA test was used for experiments with values passing normality and equal variance tests; otherwise, a Kruskal-Wallis ANOVA by ranks test was used. A P value less than 0.01 was considered significant.