CD46 Contributes to the severity of group A streptococcal infection

Abstract

Streptococcus pyogenes (group A Streptococcus) is a human pathogen that causes a wide variety of diseases ranging from uncomplicated superficial infections to severe infections such as streptococcal toxic shock syndrome and necrotizing fasciitis. These bacteria interact with several host cell receptors, one of which is the cell surface complement regulator CD46. In this study, we demonstrate that infection of epithelial cells with S. pyogenes leads to the shedding of CD46 at the same time as the bacteria induce apoptosis and cell death. Soluble CD46 attached to the streptococcal surface, suggesting that bacteria might bind available extracellular CD46 as a strategy to survive and avoid host defenses. The protective role of human CD46 was demonstrated in ex vivo whole-blood assays showing that the growth of S. pyogenes was enhanced in blood from mice expressing human CD46. Finally, in vivo experimental infection showed that bacteremia levels, arthritis frequency, and mortality were higher in CD46 transgenic mice than in nontransgenic mice. Taken together, these results argue that bacterial exploitation of human CD46 enhances bacterial survival and represents a novel pathogenic mechanism that contributes to the severity of group A streptococcal disease.

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.

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.

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.

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.

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).

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.