CD46 Contributes to the Severity of Group A Streptococcal Infection

Bacterial strains and cell culture. S. pyogenes strain S165 of serotype T6 emm6 isolated from the blood of a patient suffering from severe invasive streptococcal disease was kindly provided by Birgitta Henriques Normark, Swedish Institute for Infectious Disease Control. The bacteria were grown in Todd-Hewitt broth (Difco Laboratories) supplemented with 1.5% yeast extract (Oxoid) at 37°C in a 5% CO₂ atmosphere. The human pharyngeal cell line FaDu (ATCC HTB-43) was maintained in Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% heat-inactivated fetal bovine serum, 2 mM l-glutamine, 0.1 mM nonessential amino acids, and 1.0 mM sodium pyruvate. Unless stated otherwise, all experiments were performed using 100% confluent cells maintained in medium supplemented with heat-inactivated serum.

Flow cytometry of FaDu cells.For analysis of CD46, HLA, or β1 integrin expression, cells were collected by trypsinization and washed with phosphate-buffered saline (PBS). The cells were incubated in 3% bovine serum albumin (BSA) on ice for 1 h and washed with PBS. Staining for CD46 was conducted by incubation with 30 μl of a primary antibody against CD46 (MAb-2, 2 μg/ml; NeoMarkers) in 0.3% BSA on ice for 1 h. Cells were washed and incubated with 50 μl of a secondary anti-mouse immunoglobulin G (IgG) antibody (R-phycoerythrin [R-PE], 4 μg/ml; Jackson ImmunoResearch) in 0.3% BSA for an additional 40 min on ice. HLA was detected by the incubation of cells with an R-PE-conjugated HLA-A, -B, and -C monoclonal antibody (diluted according to the manufacturer's recommendations; BD Pharmingen). For β1 integrin (CD29) expression, a monoclonal β1 integrin antibody (10 μg/ml; Chemicon) was used, followed by incubation with the secondary antibody anti-mouse IgG-Alexa 488 at 4 μg/ml (Molecular Probes). The cells were washed, resuspended in PBS, and analyzed for fluorescence intensity by FACScan and Cellquest Pro (Becton Dickinson).

Analysis of CD46, HLA, and β1 integrin expression after infection.FaDu cells were grown to 100% confluence, and the cell medium was changed 24 h prior to infection. Bacteria were grown overnight to the stationary phase and added at a multiplicity of infection (MOI) of 100 in cell medium (Dulbecco's modified Eagle's medium with fetal bovine serum) by replacing 100 μl cell medium with 100 μl of the concentrated bacterial suspension. As a control, cells were treated with 150 μg/ml gentamicin (Sigma) to prevent the overgrowth of extracellular bacteria, which gave results similar to those of untreated cells. At 3 and 24 h postinfection, cells were collected by trypsinization, stained for CD46 expression, and analyzed by flow cytometry. Staining for HLA and β1 integrin was performed 24 h postinfection.

Transcriptional analysis.Total RNA from FaDu cells infected with S. pyogenes was isolated at 6 and 24 h. RNA (1 μg) was reverse transcribed with 0.5 μg/μl oligo(dT) using Superscript II reverse transcriptase (Invitrogen). For real-time PCR amplification, Power Sybr green PCR master mix (ABI) was used with the ABI Prism 7300 sequence detector system (Applied Biosystems) according to the manufacturer's guidelines. The cd46 gene was amplified with primers CD46F (5′-GTGGTCAAATGTCGATTTCCAGTAGTCG-3′) and CD46R (5′-CAAGCCACATTGCAATATTAGCTAAGCCACA-3′). The β-actin gene was chosen as an internal control and amplified simultaneously with primers β-actinF (5′-GGCACCACACCTTCTACAATGAG-3′) and β-actinR (5′-CGTCATACTCCTGCTTGCTGATC-3′). cd46 gene transcription was analyzed using the standard curve method, and data were presented as an expression ratio compared to the uninfected control, which was set as 100%.

Detection of apoptosis and cell permeability.FaDu cells were infected with bacteria at an MOI of 100 for 3 or 24 h. For chemical induction of apoptosis, FaDu cells were incubated with 10 μM camptothesin (Sigma) overnight. For microscopic analysis, cells were grown on coverslips in a 24-well plate. After infection or chemical treatment, the cells were harvested, washed, and stained by annexin V for phosphatidylserine exposure or by propidium iodide (PI) for cell permeabilization. Annexin V and PI (diluted according to the manufacturer's recommendations; BD Pharmingen) were added to the cells, followed by flow cytometry analysis. Also, FaDu cells were simultaneously stained for CD46 and phosphatidylserine using primary antibody against CD46 (MAb-2, 2 μg/ml; NeoMarkers) and annexin V. For nuclear degeneration analysis, Vectashield mounting medium containing DAPI (4′,6′-diamidino-2-phenylindole) (Vector Laboratories Inc.) was added to slides before mounting and microscopy analysis with a Leica microscope connected to a charge-coupled-device camera.

Immunoblot.Supernatants (1 ml) from infected, uninfected, or camptothesin-treated FaDu cells (2.5 × 10⁵ cells) were collected at 24 h, concentrated, subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene difluoride membranes. Identical volumes of supernatants were concentrated from infected and uninfected (control) 100% confluent cells. The membranes were blocked overnight with 5% nonfat dried milk in Tris-buffered saline (0.05 M Tris and 0.1 M NaCl [pH 7.4]) containing 0.05% Tween 20. CD46 was identified by incubation with monoclonal antibody M75 directed against complement control protein repeat 2 (46) at a 1:5,000 dilution. SpeB was detected by an SpeB-specific antiserum (48). Incubation with primary antibody was followed by horseradish peroxidase-conjugated goat anti-mouse antibody (4 μg/ml; Santa Cruz Biotechnologies). A chemiluminescence kit from Perkin-Elmer Life Sciences was used for detection.

Analysis of CD46 binding to S. pyogenes.Recombinant CD46 was purified as previously described (24, 46). Briefly, a fusion protein composed of the maltose binding protein (MBP) and the extracellular region of CD46 was purified over an amylose column according to the manual provided by the manufacturer (New England BioLabs). MBP was purchased from New England BioLabs. S. pyogenes (lag, early log, mid-log, and stationary phases) was preincubated with recombinant MBP-CD46 or MBP for 2 h at 37°C. Unbound CD46 was removed by washing, and bacteria were analyzed for CD46 binding by staining with CD46 polyclonal antibody (H294, 2 μg/ml; Santa Cruz Technologies), followed by incubation with an anti-rabbit IgG antibody (Alexa 488, 4 μg/ml; Molecular Probes) and flow cytometry analysis.

Animal infection assay.The pathogenicity of S. pyogenes was analyzed in transgenic mice expressing human CD46. These mice were previously described and express CD46 in a human-like pattern (4, 21, 22). In two independent experiments, 5- to 8-week-old CD46 transgenic mice (n = 4 to 12) and corresponding nontransgenic mice (n = 4 to 12) were infected intravenously (i.v.) with 3 × 10⁸ CFU. Blood samples were taken from the tail daily for 6 days after infection, diluted in PBS, and spread onto Todd-Hewitt agar plates to determine the bacterial load in the blood. The criteria for mice positive for arthritis were significant joint swelling in one leg. Joint swelling was graded significant joint swelling or severe joint swelling. The CD46 transgenic mice more often had severe joint swelling. Animal care and experiments were performed in accordance with institutional guidelines and have been approved by national ethical committees.

Blood survival assay.Blood from CD46 transgenic mice and appropriate nontransgenic control mice (B6/C3HeN) was collected by retro-orbital bleeding and heparin treated. Whole blood was diluted 1:1 with bacterial suspension in FaDu cell medium (10⁴ CFU). Bacterial survival was determined by serial dilutions and plating of the blood-bacterium mixture at 2, 4, and 6 h postinfection.

Statistical analysis.A Student's t test was used to assess significance in ex vivo blood survival assays. In vivo animal infection assays were assessed using Fisher's exact test, and bacteremia was monitored with a nonparametric Mann-Whitney test. All in vitro and ex vivo experiments were performed in triplicates in sets of at least two independent experiments.

S. pyogenes infection leads to the loss of CD46 from epithelial cells.In order to establish whether infection with S. pyogenes could affect CD46 expression in host cells, the human pharyngeal epithelial FaDu cell line was incubated with S. pyogenes strain S165 (emm6) and analyzed by flow cytometry using CD46 antibodies. As shown in Fig. 1A, bacterial infection induced the loss of CD46 after 24 h, whereas the CD46 level remained unchanged in control cells and in cells infected for 3 h. Furthermore, the infection of FaDu cells did not affect the expression of the human major histocompatibility complex (HLA), indicating that CD46 downregulation is specific and not part of a general release of cell surface proteins (Fig. 1B). Interestingly, the expression of β1 integrin was slightly reduced at 24 h (Fig. 1C), supporting that CD46 is linked to β1 integrin, as was previously reported (38). Control experiments showed that bacteria pretreated with ethanol or heat did not mediate the loss of CD46 in FaDu cells, suggesting that the process required viable bacteria (data not shown). Furthermore, Escherichia coli did not induce the shedding of CD46 (data not shown). These data demonstrate that the infection of epithelial cells with S. pyogenes reduces cell surface-expressed CD46.

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FIG. 1.

Epithelial cell surface expression of CD46 is reduced after infection with S. pyogenes. (A) Flow cytometry analysis of CD46 expression in FaDu cells. Cells were infected with S. pyogenes at an MOI of 100 for 3 and 24 h and stained with a monoclonal CD46 antibody followed with R-PE-conjugated anti-mouse IgG. The percentage of CD46-negative cells is indicated. (B and C) Expression of HLA (B) and β1 integrin (C) in FaDu cells analyzed by flow cytometry. Cells were infected with S. pyogenes (MOI = 100) for 24 h and stained with monoclonal antibodies against HLA or β1 integrin (CD29), followed by an Alexa Fluor 633-conjugated anti-mouse IgG. The percentage of HLA- or β1 integrin-negative cells is indicated. Control cells were treated identically to the infected cells except that no bacteria were added. The x axis shows fluorescence intensity. Shown are representative pictures of three independent experiments.

CD46 is released into the extracellular environment after S. pyogenes infection.To determine if the loss of CD46 membrane staining was due to decreased transcription, we used real-time reverse transcription-PCR and showed that the CD46 mRNA level was not significantly affected after infection of cells with S. pyogenes (Fig. 2A). Since the transcription of CD46 was unaffected by bacteria, we next checked if CD46 was released from cells into the extracellular environment. Cell supernatants collected before and after infection were analyzed by Western blotting using CD46 antibodies. High levels of extracellular CD46 were detected at 24 h postinfection, suggesting that bacterial infection triggered the shedding of CD46 from the host cell surface (Fig. 2B).

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FIG. 2.

CD46 is released into the extracellular environment after S. pyogenes infection. (A) Transcription of CD46 after infection of FaDu cells with S. pyogenes. FaDu cells were infected with S. pyogenes (MOI = 100) for 6 h or 24 h. Total RNA was isolated from the infected cells or uninfected cells, which was set as a control. Transcriptional levels of CD46 were quantified by real-time reverse transcription-PCR and normalized against the housekeeping gene β-actin. Data were expressed as a percentage of the uninfected control, which was set as 100%. The results shown represent the means ± standard deviations of three independent experiments. No significant differences were identified. (B) Western blotting showing CD46 levels in supernatants (sup.) of 2.5 × 10⁵ FaDu cells. Cells were infected with S. pyogenes at an MOI of 100 (infected) or left uninfected (control). At 24 h postinfection, 1 ml of the cell supernatant was concentrated and analyzed for CD46 expression by immunoblotting using a monoclonal CD46 antibody.

CD46 is released from cells in response to infection and apoptotic cell death.Recently, Elward et al. (11) demonstrated that the chemical stimulation of apoptosis results in the loss of CD46 from various human cell lines, whereas other cell surface markers remain stable on the cell surface (11). It has also independently been shown that S. pyogenes induces apoptosis and cell death in host epithelial cells (31, 49). Therefore, we assessed apoptotic cell death in FaDu cells after S. pyogenes infection by staining with annexin V and PI at different time points. Annexin V staining to detect phosphatidylserine exposure in the outer leaflet of the cytoplasmic membrane was positive for one-third of the FaDu cells at 6 h and for all cells at 24 h, whereas uninfected cells remained annexin V negative (Fig. 3A). Annexin V did not bind bacteria by itself (data not shown). Staining with PI increased with time, and at 24 h postinfection, all cells were positive (Fig. 3B). Furthermore, nuclear staining with DAPI and microscopic analysis of cells at 24 h postinfection demonstrated nuclear shrinkage (data not shown). These data argue that the infection of epithelial cells with S. pyogenes leads to apoptotic cell death as well as the specific release of cell surface CD46 (Fig. 1).

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FIG. 3.

S. pyogenes-induced CD46 shedding from the epithelial cell surface is associated with apoptotic cell death. (A and B) Analysis of apoptosis by staining with annexin V (A) and analysis of cell viability by staining with PI (B). FaDu cells were infected with S. pyogenes at an MOI of 100 for 3, 6, and 24 h. Uninfected cells were set as a control. The experiments were performed in triplicates and repeated at least twice, with similar results. (C) Flow cytometry analysis of CD46 expression and annexin V staining after induction of apoptosis by camptothesin. The left chart shows CD46 expression in control cells. CD46 was detected by the monoclonal antibody MAb-2, followed by an R-PE-conjugated anti-mouse IgG. The right chart shows FaDu cells treated with camptothesin overnight and stained for both CD46 expression and apoptosis using CD46 antibodies and fluorescein isothiocyanate-conjugated annexin V, respectively. (D) Western blotting showing CD46 levels in supernatants of 2.5 × 10⁵ apoptotic FaDu cells. Cells were treated with camptothesin for 24 h or left untreated (control). At 24 h postinfection, 1 ml of the cell supernatant (sup.) was concentrated and analyzed for CD46 expression. CD46 was detected with monoclonal antibody M75.

To further confirm that the apoptosis of FaDu epithelial cells caused the shedding of CD46, we induced the apoptosis of cells by treatment with camptothesin and then analyzed cell surface markers by flow cytometry. As shown in Fig. 3C, camptothesin induced both apoptosis and the loss of CD46 as detected by staining with annexin V and monoclonal antibodies against CD46. To verify that CD46 was released extracellularly after the induction of apoptosis, the cell supernatants were subjected to immunoblotting with CD46 antibodies. As shown in Fig. 3D, CD46 was clearly accumulated in the apoptotic supernatants. Strains expressing or not expressing SpeB, a cysteine protease secreted from S. pyogenes, all induced the shedding of CD46 (data not shown). Taken together, these data argue that the shedding of CD46 into the extracellular environment occurs after infection with S. pyogenes as a response to bacterium-induced apoptosis.

S. pyogenes binds soluble CD46.Since CD46 is released from human cells after S. pyogenes infection, we hypothesized that it would be beneficial for the bacteria to bind soluble CD46 as a strategy to evade early immune responses. Therefore, in a first step, we wanted to evaluate if CD46 could bind to the bacterial surface. Consequently, we generated and purified recombinant CD46 as an MBP-CD46 fusion protein. The CD46 recombinant protein contained the surface-exposed domains but not the transmembrane region and the cytoplasmic tail. Since the M protein expression of S. pyogenes may vary depending on the growth phase, we incubated bacteria from lag, early log, mid-log, or stationary phase with recombinant purified CD46 for 2 h, washed away unbound protein, and measured bacterium-bound CD46 by flow cytometry. As shown in Fig. 4, S. pyogenes in mid-log phase bound well to recombinant CD46 but not to the control MBP. S. pyogenes from lag, early log, or stationary phase showed impaired and low levels of binding to CD46, clearly demonstrating the importance of the growth phase in S. pyogenes interactions with the environment. Furthermore, CD46 from cell supernatants bound to S. pyogenes, i.e., shed native CD46, can bind bacteria (data not shown). These data argue that S. pyogenes in mid-log phase binds soluble recombinant CD46 protein, whereas S. pyogenes in other growth phases fail to bind CD46 in a strong and consistent manner.

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FIG. 4.

Soluble recombinant CD46 binds to S. pyogenes in a growth phase-dependent manner. S. pyogenes (10⁷ CFU) in lag, early log, mid-log, or stationary phase was incubated for 2 h with MBP-CD46 or MBP (control). Bacteria were washed and analyzed for CD46 binding by flow cytometry after staining with a polyclonal antibody against CD46 (H294), followed by an Alexa Fluor 488-conjugated anti-rabbit IgG. The experiments were performed in triplicates and repeated at least twice, with similar results. Representative pictures are shown.

S. pyogenes shows enhanced survival in the presence of human CD46.We hypothesized that the presence of CD46 might be beneficial for bacterial survival. To evaluate the importance of CD46 during early steps of streptococcal systemic infection, we investigated bacterial survival and growth in blood collected from transgenic mice expressing human CD46 and nontransgenic control mice lacking CD46. Bacteria were mixed with blood for 2, 4, or 6 h; incubated at 37°C; diluted; and spread onto plates for viable counts. In the ex vivo blood assay, S. pyogenes showed significantly better growth in blood from CD46 transgenic mice than in blood from nontransgenic mice after 6 h of incubation (Fig. 5), whereas there was no growth difference in serum from these mice (data not shown), indicating that human CD46 facilitates bacterial survival and suggesting that the binding of CD46 might help the bacteria to survive host defenses. It was shown that soluble CD46 is present in normal human plasma (18) and that CD46 levels are elevated under autoimmune conditions (12, 25). To investigate the importance of CD46 in human blood, we preincubated S. pyogenes with recombinant CD46 (MBP-CD46) before adding the bacteria to human blood. Bacteria preincubated with MBP-CD46 survived significantly threefold better in human blood than did bacteria incubated with buffer or control protein (data not shown).

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FIG. 5.

Enhanced growth of S. pyogenes in blood containing human CD46. Blood from CD46 transgenic mice and nontransgenic control mice were collected and incubated with S. pyogenes (10⁴ CFU) for 2 h, 4 h, or 6 h. Bacterial counts were determined by plating onto plates of Todd-Hewitt broth supplemented with 1.5% yeast extract after serial dilutions. Data are shown as percentages compared to the inoculum that was set to 100%. The experiments were performed in triplicate and repeated twice. An asterisk marks significant values compared to bacterial growth in control blood (P < 0.05).

Human CD46 increases disease severity in mice.The finding that S. pyogenes survived better in blood from CD46^+/+ mice than in blood from CD46^−/− mice prompted us to evaluate the in vivo infection of mice. The capacity of S. pyogenes to cause arthritis as well as lethal disease has been studied in mouse model systems using i.v. inoculation doses of 2 × 10⁸ to 4 × 10⁸ cells per mouse (40). To study the in vivo impact of CD46, we challenged CD46 transgenic mice and nontransgenic mice with 3 × 10⁸S. pyogenes cells i.v. The majority (80%) of the nontransgenic mice survived, but only 40% of the CD46 transgenic mice survived (Fig. 6A), indicating that CD46 augments disease severity. To analyze bacteremia, blood samples from the tail vein were collected at day 3 postinfection, serially diluted, and spread onto bacterial growth plates for viable count. The bacterial blood counts in CD46 transgenic mice were significantly higher than those in nontransgenic mice (Fig. 6B). Furthermore, survival correlated with bacteremia levels (data not shown). As a control of the murine model system, we previously showed that CD46 transgenic mice are not more sensitive than nontransgenic mice to infections with bacteria unable to interact with CD46 (21).

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FIG. 6.

S. pyogenes induced severe disease more often in CD46 transgenic mice than in nontransgenic mice. CD46 transgenic mice (n = 12) and nontransgenic control mice (n = 12) were infected i.v. with 3 × 10⁸ bacteria, and disease development was monitored for 7 days. (A) Survival of mice after infection with S. pyogenes. Differences in survival were significant (P < 0.05). (B) Bacteremia levels in mice infected with S. pyogenes at day 3 postinfection. The blood samples were serially diluted and spread onto agar plates for viable count. The horizontal lines mark the logarithmic median value. The counts were analyzed by the nonparametric Mann-Whitney test. ND, nondetectable. (C) Development of arthritis after S. pyogenes infection. Shown are the results of two independent experiments. Differences between CD46 transgenic mice and control mice were significant (P < 0.05). The counts were analyzed by the nonparametric Mann-Whitney test. (D) Representative pictures of arthritis.

S. pyogenes is frequently isolated from patients with arthritis and accounts for 8% to 16% of cases of septic arthritis (17, 26). Arthritic joint swelling of the S. pyogenes-infected mice was visually examined three times a day during 6 days. About 70% of the CD46 transgenic mice developed arthritis, whereas only 15% of the nontransgenic mice showed arthritic joint swelling (Fig. 6C and D). There was no correlation between bacteremia and arthritis. Taken together, these data support that human CD46 may play an important role in protecting bacteria during invasive S. pyogenes disease.