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. 2009 Nov 30;78(2):716–725. doi: 10.1128/IAI.01056-09

Streptococcus pneumoniae Resistance to Complement-Mediated Immunity Is Dependent on the Capsular Serotype ▿

Catherine Hyams 1, Jose Yuste 1,†, Katie Bax 2, Emilie Camberlein 1, Jeffrey N Weiser 3, Jeremy S Brown 1,*
PMCID: PMC2812205  PMID: 19948838

Abstract

Streptococcus pneumoniae strains vary considerably in the ability to cause invasive disease in humans, and this is partially associated with the capsular serotype. The S. pneumoniae capsule inhibits complement- and phagocyte-mediated immunity, and differences between serotypes in these effects on host immunity may cause some of the variation in virulence between strains. However, the considerable genetic differences between S. pneumoniae strains independent of the capsular serotype prevent an unambiguous assessment of the effects of the capsular serotype on immunity using clinical isolates. We have therefore used capsular serotype-switched TIGR4 mutant strains to investigate the effects of the capsular serotype on S. pneumoniae interactions with complement. Flow cytometry assays demonstrated large differences in C3b/iC3b deposition on opaque-phase variants of TIGR4(−)+4, +6A, +7F, and +23F strains even though the thicknesses of the capsule layers were similar. There was increased C3b/iC3b deposition on TIGR4(−)+6A and +23F strains compared to +7F and +4 strains, and these differences persisted even in serum depleted of immunoglobulin G. Neutrophil phagocytosis of the TIGR4(−)+6A and +23F strains was also increased, but only in the presence of complement, showing that the effects of the capsular serotype on C3b/iC3b deposition are functionally significant. In addition, the virulence of the TIGR4(−)+6A and +23F strains was reduced in a mouse model of sepsis. These data demonstrate that resistance to complement-mediated immunity can vary with the capsular serotype independently of antibody and of other genetic differences between strains. This might be one mechanism by which the capsular serotype can affect the relative invasiveness of different S. pneumoniae strains.

The important Gram-positive pathogen Streptococcus pneumoniae has an extracellular polysaccharide capsule that inhibits complement activity, neutrophil phagocytosis, and bacterial killing by neutrophil extracellular traps (19, 23, 25, 26, 29, 31), as well as having major effects on bacterial interactions with the epithelium (8, 25, 26, 29, 31, 37). As a consequence, the capsule is essential for virulence (6, 38). Different strains of S. pneumoniae can express capsules with different structures, depending on the type of monosaccharide units and their bonds within the polysaccharide chain, the enzymes for the synthesis of which are encoded by genes within a specific locus in the genome (5, 27, 30). The different types of capsules are divided into 91 capsular serotypes. Although most S. pneumoniae strains can cause disease in humans, the ability to cause invasive infections (septicemia and meningitis) varies up to 60-fold between strains and is closely associated with the capsular serotype (4, 12). Some serotypes (e.g., 1, 4, 5, 7, and 14) are overrepresented among invasive disease isolates compared to the frequency of their isolation as nasopharyngeal commensals, while other capsular serotypes only rarely cause invasive disease despite being common nasopharyngeal commensals (4, 12, 15).

The mechanisms causing capsular serotype-dependent variation in virulence are largely unknown but could reflect differences between the abilities of strains of different serotypes to inhibit host immune responses. Potentially, strains expressing capsular serotypes that strongly inhibit immunity could be more likely to establish invasive infection than strains with capsular serotypes that weakly inhibit host immunity, and this hypothesis is partially supported by existing experimental data. The virulence of different capsular serotypes varies markedly in mouse models of infection, but as there is only a weak relationship between virulence in mice and invasive potential in humans, the clinical relevance of these findings is unclear (1, 7, 9, 33). Because of the central role of complement and phagocytosis for systemic immunity to S. pneumoniae (11, 20, 45, 46), differences in the effects of different capsular serotypes on complement activity or phagocytosis are strong candidates for explaining why the serotype can affect virulence. Indeed, existing data show that resistance to complement activity and phagocytosis varies between strains with different capsular serotypes (18, 28, 46). However, in general, these studies have not controlled for strain phase variation or for noncapsular genetic variation between strains. S. pneumoniae has two main phase variants, opaque with an increased capsule thickness and transparent with a thinner capsule but increased expression of some surface proteins, such as PspC, that can affect complement activity (24, 31). Differences in phase variation between strains could therefore affect complement susceptibility. Furthermore, there is considerable genetic variation between S. pneumoniae strains independent of the capsular serotype. Only 60% of gene clusters are common to all S. pneumoniae strains, and the genome content differs by 8 to 10%, on average, between any two strains (10, 13, 16, 17). This genetic variation is partially linked to the capsular serotype (http://www.mlst.net/), and hence, the relationship between the capsular serotype and invasiveness could be due to noncapsular genetic variation rather than direct effects of the capsule.

To overcome strain genetic variation confounding the assessment of capsular serotype interactions with the immune system, the capsular loci of one strain can be replaced with the capsular loci from another, creating otherwise isogenic strains expressing different capsular serotypes (29, 35, 43). Data obtained using capsular serotype-switched strains have shown that expression of capsular serotype 3 reduced complement deposition on a previously serotype 2 strain (2), increased the virulence in mice of an originally serotype 6B strain, and conversely decreased the virulence of a serotype 5 strain (22). Furthermore, a recent study demonstrated variations in resistance to neutrophil killing of unopsonized bacteria between capsular serotype-switched strains expressing different capsular serotypes and correlated reduced sensitivity to neutrophil killing with increased prevalence of that capsular serotype as a nasopharyngeal commensal (39). These studies have established the principle that the capsular serotype can affect the complement sensitivity, neutrophil killing, and virulence of S. pneumoniae independently of the strain background. However, as yet, there are only limited data on the effects of different capsular serotypes on complement-dependent immunity to S. pneumoniae and a more detailed assessment is required to help understand why a strain's capsular serotype is linked to its invasive potential.

We have used opaque- and transparent-phase variants of TIGR4 S. pneumoniae strains modified to express different capsular serotypes, two representative of relatively invasive capsular serotypes (4 and 7F) and two representative of less invasive serotypes (6A and 23F), to assess capsular serotype-dependent effects on immunity. We have investigated the effects of the capsular serotype on opsonization of S. pneumoniae with the complement-derived opsonins C3b and iC3b, as well as on neutrophil phagocytosis and virulence in a mouse model of septicemia.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

TIGR4(−)+ capsular serotype-switched strains were constructed by using the Janus cassette as previously described (40). Bacteria were cultured at 37°C in 5% CO2 on blood agar plates or in Todd-Hewitt broth supplemented with 0.5% yeast extract to an optical density at 580 nm of 0.4 (approximately 108 CFU/ml) and stored at −70°C in 10% glycerol as single-use aliquots. The bacterial phase was determined by using transparent medium (Tryptone Soya with catalase) under magnification and oblique, transmitted illumination as previously described (24) and stocks obtained for transparent- and opaque-phase variants for each TIGR4(−)+ capsular serotype-switched strain. There were no significant differences in growth in THY between the TIGR4(−)+ strains when assessed using optical density.

Serum sources, complement binding, and phagocytosis assays.

Serum and complement were stored in single-use aliquots at −70°C. Pooled human serum was obtained from unvaccinated normal human volunteers, sera with single complement component deficiencies (C9−, C1q−, and Bf− sera) were supplied by Calbiochem (46), and baby rabbit complement was supplied by Serotec. Mouse serum was obtained from freshly culled CD1 mice. Human immunoglobulin G (IgG) was depleted in serum using purified IgG-degrading enzyme of Streptococcus pyogenes (IdeS, a kind gift from Mattias Collin and Lars Björck, Lund University), a cysteine proteinase which cleaves IgG with a unique degree of specificity for the hinge region (36, 41). One percent IdeS or bovine serum albumin (BSA) was incubated with human serum for 45 min at 37°C before use for complement and phagocytosis assays as described above. Capsule-specific antibody (Ab) titers were measured using standardized enzyme-linked immunosorbent assays (ELISAs; www.vaccine.uab.edu/ELISA%20Protocol.pdf), and total IgG and IgM binding to S. pneumoniae was measured using flow cytometry and an R-phycoerythrin goat anti-human IgG or fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgM (Jackson ImmunoResearch) as described previously (46). C3b/iC3b deposition on S. pneumoniae was measured using a previously described flow cytometry assay and FITC-conjugated polyclonal anti-human, -mouse, or -rabbit C3 Ab (ICN) (11, 44, 46). Results of complement and IgG binding assays are presented as a fluorescence index (FI; percentage of positive bacteria multiplied by the geometric mean fluorescence intensity) in arbitrary units (44, 46). To ensure consistent results, assays were repeated using two or more stock sources for each strain. Phagocytosis was investigated using an established flow cytometry assay, fresh human neutrophils (34), and S. pneumoniae labeled with 6-carboxyfluorescein succinimidyl ester (FAMSE; Molecular Probes) first incubated in baby rabbit complement or human or mouse serum (diluted in phosphate-buffered saline [PBS]) for 20 min at 37°C. Both the viability and purity (assessed by automated hemocytometer analysis) of the neutrophil preparation were routinely over 95%. Each reaction used 105 neutrophils and a multiplicity of infection of 10 to 1. A minimum of 10,000 cells were analyzed using flow cytometry to identify the proportion of neutrophils associated with bacteria (46). In a set of control reactions, neutrophils were incubated with 5 μM cytochalasin D (Sigma) for 30 min at room temperature to inhibit actin polymerization and prevent phagocytosis.

EM.

Mid-log-phase S. pneumoniae bacteria were incubated at 37°C for 20 min in serum or PBS, fixed in 1% PFA, and prepared for electron microscopy (EM) using a ruthenium red and London Resin protocol as described previously (14). Bacteria were viewed using a JEOL 1010 transmission electron microscope (100 kV), and ImageJ software was used to determine capsule thickness by measuring the cross-sectional areas of 10 or more randomly chosen bacteria, including and excluding the capsule. Assuming circularity, the areas were then used to calculate the capsule layer width.

Infection models.

Infection experiments conformed to institutional and government guidelines for working with animals. CD1 mice 6 weeks old were inoculated by intraperitoneal (i.p.) injection with 2,000 CFU of each TIGR4(−)+ strain diluted in PBS and culled at 24 h, and serial dilutions of blood and spleen homogenates were plated to calculate bacterial CFU counts (11, 44).

Statistics.

Flow cytometry data were analyzed using Student unpaired t tests (comparison of two samples) or one-way analyses of variance (ANOVAs; comparisons of multiple samples) with post-hoc tests. EM or mouse organ CFU data were compared using the Kruskal-Wallis test with Dunn's multiple-comparison test (multiple groups) and presented as medians (interquartile ranges [IQRs]). Data are representative of results obtained with repeated experiments.

RESULTS

C3b/iC3b deposition on S. pneumoniae varies with the capsular serotype.

To investigate the effect of the capsular serotype on complement, C3b/iC3b deposition was investigated using flow cytometry on TIGR4 S. pneumoniae opaque-phase variant strains in which the serotype 4 genetic locus had been replaced with loci for two weakly invasive (6A and 23F) and two invasive (7F and 4, the latter reconstituting the original TIGR4 strain) capsular serotypes. All of the TIGR4(−)+ strains had similar capsule layer thicknesses when visualized by EM (Fig. 1). C3b/iC3b deposition was dependent on the capsular serotype, with increased C3b/iC3b deposition on the TIGR4(−)+23F strain and especially the TIGR4(−)+6A strain compared to the TIGR4(−)+4 and +7F strains (Fig. 2A and B). C3b/iC3b deposition on the TIGR4 and TIGR4(−)+4 strains in 100% human serum was similar (FIs of 5,000 [standard deviation {SD}, 1,940] and 3,910 [SD, 450], respectively), demonstrating that the process of creating the mutant strains had not significantly affected C3b/iC3b deposition. The effects of phase variation were investigated by comparing C3b/iC3b deposition on transparent- and opaque-phase variants for each TIGR4(−)+ strain. All of the transparent-phase variants of the TIGR4(−)+ strains had their capsule thickness decreased by 63.25 to 40.8 μm compared to the opaque-phase variants when visualized by EM (Fig. 1). However, there were no significant differences in capsule thickness between transparent-phase variants of the TIGR4(−)+ strains. C3b/iC3b deposition was substantially increased on all of the transparent-phase variants of the TIGR4(−)+ strains compared to the opaque-phase variants (Fig. 2C).

FIG. 1.

FIG. 1.

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Capsular thickness of opaque- and transparent-phase variants of TIGR4(−)+ capsular serotype-switched strains. (A) Representative EM images of TIGR4(−)+6A opaque- and transparent-phase variants. (B) Medians and IQRs of capsule thickness (nm) measured by EM for opaque (clear boxes)- and transparent (diagonally shaded boxes)-phase variants of TIGR4(−)+6A, +7F, +23F, and +4 strains. *, Ρ < 0.01; **, Ρ < 0.001; ***, Ρ < 0.0001 (Mann-Whitney U test).

FIG. 2.

FIG. 2.

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C3b/iC3b deposition on TIGR4(−)+ strains. (A) Mean FIs (measured in arbitrary units) of C3b/iC3b deposition measured using flow cytometry on TIGR4(−)+ strains expressing capsular serotypes 6A (▪), 7F (▴), 23F (▿), and 4 (○) in increasing concentrations of human serum. Error bars represent SDs and when not present are too small to be visible outside the symbol. **, Ρ < 0.001 (compared to the TIGR4(−)+4 strain; ANOVA with post-hoc tests). (B) Examples of flow cytometry histograms for C3b/iC3b deposition on TIGR4(−)+ capsular serotype-switched strains in 100% human serum. Gray shading indicates the results for bacteria incubated in PBS alone. (C and D) Mean FIs of C3b/iC3b deposition measured using flow cytometry on opaque- and transparent-phase variants of TIGR4(−)+ strains expressing capsular serotypes 6A (C) and on 7F, 23F, and 4 (D) in 25% human serum. Error bars represent SDs. *, Ρ < 0.01; **, Ρ < 0.001 (Student unpaired t tests).

Effect of Ab on C3b/iC3b deposition.

As Ab is a major cause of complement activity against S. pneumoniae, total IgG and IgM binding (measured using flow cytometry) and capsule-specific IgG (measured using ELISA) levels were measured for the opaque-phase TIGR4(−)+ strains (Fig. 3). Levels of both capsule-specific IgG and total IgG binding to the TIGR4(−)+6A strain were higher than to the TIGR4(−)+4 and +7F strains, although levels of IgM binding were similar. This difference in IgG recognition of the TIGR4(−)+6A strain could potentially explain the increased C3b/iC3b deposition on this strain independent of direct effects of the capsule on complement. To clarify the importance of Ab for the differences in C3b/iC3b deposition on the TIGR4(−)+ strains, C3b/iC3b assays were repeated using serum that had been treated with the Streptococcus pyogenes enzyme IdeS, which is known to cleave human IgG and to abolish IgG binding to S. pneumoniae (unpublished data) (36, 41). In IdeS-treated serum, there were still large increases in C3b/iC3b deposition on the TIGR4(−)+6A strain compared to the TIGR4(−)+7F and +4 strains, with a less impressive increase on the +23F strain (Fig. 4A). Furthermore, C3b/iC3b deposition assays performed using baby rabbit complement (Fig. 4B) and mouse serum (data not shown), both of which have no detectable IgG binding or capsule-specific Abs to S. pneumoniae (46), or in human serum depleted of C1q, in which Ab cannot activate complement activity, all gave results similar to those obtained with IdeS-treated human serum. Hence, the differences in C3b/iC3b deposition between the TIGR4(−)+ capsular serotype-switched strains probably reflect an inherent property of the capsular serotype rather than differences in Ab binding to the strains.

FIG. 3.

FIG. 3.

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Ab binding to TIGR4(−)+ strains in human serum used for the C3b/iC3b and phagocytosis assays. (A) Mean FIs of total IgG binding to TIGR4(−)+6A, +7F, +23F, and +4 strains measured using flow cytometry. Treatment of the serum with IdeS prior to incubation with S. pneumoniae abolished IgG binding as detected by flow cytometry. The mean FI for total IgG binding to TIGR4 was 480 ± 40 in BSA-treated serum versus 30 ± 12 in IdeS-treated serum. (B) Mean amounts (ng/ml) of capsule-specific IgG measured using ELISA against capsular serotypes 4, 6A/6B (the assay is unable to distinguish between these subtypes), 7F, and 23F. (C) Mean FIs of total IgM binding to TIGR4(−)+6A, +7F, +23F, and +4 strains measured using flow cytometry. In all of the panels, the error bars represent SDs. *, Ρ < 0.01; **, Ρ < 0.001 [compared to the TIGR4(−)+4 strain; ANOVA with post-hoc tests]. WT, wild type.

FIG. 4.

FIG. 4.

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IgG-independent C3b/iC3b deposition on TIGR4(−)+ capsular serotype-switched strains. (A) Mean FIs of C3b/iC3b deposition measured using flow cytometry on the TIGR4(−)+ capsular serotype-switched strains in 50% human serum treated with IdeS to deplete IgG, with an example of the corresponding flow cytometry histograms. (B) Mean FIs of C3b/iC3b deposition measured using flow cytometry on the TIGR4(−)+ capsular serotype-switched strains in 25% baby rabbit complement, with an example of the corresponding flow cytometry histograms. In all of the panels, the error bars represent SDs. **, Ρ < 0.001 [compared to the TIGR4(−)+4 strain; ANOVA with post-hoc tests]. Gray shading represents flow cytometry histograms for bacteria incubated in PBS rather than serum.

Increased C3b/iC3b deposition on the specific TIGR4(−)+ strain is associated with increased neutrophil phagocytosis.

To assess the functional consequences of differences in C3b/iC3b deposition on the TIGR4(−)+ strains, neutrophil phagocytosis was investigated using flow cytometry. This assay identifies the proportion of neutrophils associated with fluorescent bacteria, which mainly represents internalization of S. pneumoniae (46). After opsonization with human serum, there were significantly greater levels of association of the TIGR4(−)+6A and +23F strains with neutrophils compared to that of the TIGR4(−)+4 strains, and these differences were abolished when phagocytosis was inhibited using cytochalasin D (Fig. 5A). The increased phagocytosis of the TIGR4(−)+6A and TIGR4(−)+23F strains persisted in additional experiments performed using human serum treated with IdeS to eliminate the effects of various levels of Ab to the opaque-phase variant TIGR4(−)+ strains (Fig. 5B). After opsonization with buffer or heat-inactivated serum, the phagocytosis of all of the strains was markedly reduced compared to that obtained with untreated serum and there were no significant differences between the TIGR4(−)+ strains (Fig. 5B). Similar data were obtained for phagocytosis assays performed after incubating bacteria in baby rabbit complement (Fig. 5C and D). These data suggest that the capsular serotype affects complement-dependent phagocytosis independently of the effects of Ab, and hence, the increase in C3b/iC3b deposition on the TIGR4(−)+6A and TIGR4(−)+23F strains results in increased phagocytosis.

FIG. 5.

FIG. 5.

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Effect of the capsular serotype on human neutrophil phagocytosis measured using flow cytometry. (A) Mean (SD) proportions of neutrophils associated with fluorescent (labeled with FAMSE) TIGR4(−)+6A (white columns), +7F (stippled columns), +23F (gray columns), and +4 (black columns) strains after incubation in 20% human serum or 20% human serum treated with 5 μM cytochalasin D (Cyto D). (B) Mean (SD) proportions of neutrophils associated with fluorescent (labeled with FAMSE) TIGR4(−)+6A (white columns), +7F (stippled columns), +23F (gray columns), and +4 (black columns) strains after incubation in Hanks balanced salt solution (HBSS) buffer, heat-treated (HT) 20% human serum, or 20% human IgG-depleted serum (using treatment with IdeS). (C) Mean (SD) proportions of neutrophils associated with fluorescent TIGR4(−)+6A (white columns), +7F (stippled columns), +23F (gray columns), and +4 (black columns) strains after incubation in HBSS buffer, heat-treated 20% baby rabbit complement, or 20% baby rabbit complement. For panels A, B, and C, a single asterisk indicates a Ρ value of <0.01 and double asterisks indicate a Ρ value of <0.001 (compared to the TIGR4(−)+4 strain; ANOVAs with post-hoc tests). (D) Examples of flow cytometry histograms for the association of TIGR4(−)+4, +6A, and +23F capsular serotype-switched strains with neutrophils after opsonization with 20% baby rabbit complement. Gray shading indicates the results for bacteria incubated in HBSS alone. The histogram for the TIGR4(−)+7F strain is not included for ease of interpretation and because this strain had no significant differences from the TIGR4(−)+4 strain.

Increased C3b/iC3b deposition on TIGR4(−)+ strains is associated with increased virulence in a mouse.

Whether the effects of the capsular serotype on C3b/iC3b deposition and phagocytosis were associated with differences in virulence was investigated using a mouse model of septicemia. Initially, we confirmed that the differences in sensitivity to neutrophil phagocytosis between the TIGR4(−)+ strains persisted for bacteria incubated in mouse serum (Fig. 6A). After incubation in mouse serum, there were increased associations of fluorescent TIGR4(−)+6A and TIGR4(−)+23F bacteria with human neutrophils, and these differences were abolished by heat treatment of the serum to denature complement. Groups of five mice were then infected by i.p. inoculation with 2,000 CFU of the TIGR4(−)+ strains, and the numbers of bacterial CFU recovered from blood and spleens after 24 h were calculated by plating serial dilutions (Fig. 6). In this model, no unencapsulated TIGR4 strain bacteria are recovered from either spleens or blood at 24 h. Large numbers of all of the TIGR4(−)+ strain bacteria were recovered from the blood and spleens after 24 h, compatible with the known virulence of the TIGR4 strain in mice (Fig. 6) (29). The median number of CFU in the spleens depended on the capsular serotype, with significantly lower numbers of CFU of the TIGR4(−)+6A and +23F strains recovered (median numbers of CFU, 3.46 × 106 and 5.88 × 106, respectively) than of the TIGR4(−)+7F and +4 strains (median numbers of CFU, 8.4 × 106 and 13.2 × 106, respectively) (Fig. 6A). Similar results were obtained for the median number of CFU in blood (Fig. 6B). Hence, the TIGR4(−)+ strains with increased levels of C3b/iC3b deposition and neutrophil phagocytosis were less virulent in a mouse model of septicemia.

FIG. 6.

FIG. 6.

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Virulence of capsular serotype-switched TIGR4(−)+ strains in mice. (A) Mean (SD) proportions of human neutrophils associated with fluorescent (labeled with FAMSE) TIGR4(−)+6A (white columns), +7F (stippled columns), +23F (gray columns), and +4 (black columns) strains after incubation in various concentrations of mouse serum and in heat-treated (HT) mouse serum. Error bars represent SDs. *, Ρ < 0.01; **, Ρ < 0.001 (compared to the TIGR4(−)+4 strain; ANOVAs with post-hoc tests). (B and C) Virulence in a mouse model of septicemia. Each symbol represents the median number of bacterial CFU recovered from an individual mouse spleen (B) or blood (C) 24 h after i.p. inoculation with 2,000 CFU of the TIGR4(−)+6A, +7F, +23F, or +4 strain. Bars represent median numbers of CFU of each bacterial strains, and very similar data were obtained in a repeat experiment. For both blood and spleen, a P value of 0.0005 (Kruskal-Wallis test) was obtained for the overall comparison between strains. P values for individual strains compared to the TIGR4(−)+4 strain are given above the symbols for the strains (Dunn's multiple-comparison test).

DISCUSSION

Clinical and epidemiological data suggest that different S. pneumoniae strains vary in the ability to cause severe disease and that this variation is linked to the capsular serotype, with nasopharyngeal colonization with strains expressing some capsular serotypes associated with a greatly increased incidence of septicemia and meningitis (4, 12, 15). However, the recent genome sequence data showing considerable genetic variation between S. pneumoniae strains unrelated but linked to the capsular serotype (13, 16) suggest that phenotypic differences between strains cannot be assumed to be due to the effects of the capsular serotype alone. We have therefore used otherwise isogenic bacterial strains expressing four different capsular serotypes to investigate the influence of the capsular serotype independently of other genetic variation on S. pneumoniae interactions with the host immune response.

Using flow cytometry, we have shown increased C3b/iC3b deposition on otherwise isogenic strains expressing serotype 23F and 6A capsules compared to strains expressing the serotype 4 and 7F capsules. Capsule thickness, and therefore phase variation, probably influences complement-mediated immunity to S. pneumoniae, but the strains investigated were opaque-phase variants and had similar capsule thicknesses when measured by EM. Transparent-phase variants of the same strains had increased C3b/iC3b deposition, probably relating to their reduced capsule thickness and compatible with previous data and the known association of opaque-phase variants with invasive disease rather than colonization (23, 24). Abs to capsular polysaccharide increase complement deposition on S. pneumoniae, and we found that capsule serotype-specific IgG levels did correlate with C3b/iC3b deposition on the TIGR4(−)+ capsular serotype-switched strains in our human test serum. However, C3b/iC3b deposition was also increased on the TIGR4(−)+6A and +23F strains in human serum depleted of IgG and in complement sources (rabbit and mouse) that do not contain S. pneumoniae-specific IgG, suggesting that there are effects of the capsular serotype independent of Ab. Overall, our data provide good evidence that different capsular serotypes can have different effects on the resistance of S. pneumoniae to complement and support existing data showing that expression of capsular serotype 3 in a serotype 2 strain affected complement deposition (2). Intriguingly, the TIGR(−)+ strains expressing serotypes associated with invasive S. pneumoniae infection (serotypes 4 and 7F) had the lowest level of C3b/iC3b deposition, suggesting perhaps that resistance to complement-mediated immunity may influence invasiveness. Precisely how different capsular serotypes can have various effects on C3b/iC3b deposition is not clear; the structure of the serotype 6A and 23F capsules may allow greater access of complement to the bacterial surface, but differences in capsular structure could also influence complement deposition indirectly by modifying the interactions of surface proteins such as PspA and CbpA with complement factors (31, 45).

Phagocytosis of S. pneumoniae is markedly increased in the presence of complement (23, 28, 46), and any differences between capsular serotypes in interactions with complement would be expected to also influence phagocytosis. Using IdeS-treated human serum to opsonize the bacteria to prevent Ab from confounding the results through Ab-Fcγ receptor interactions, we found that the TIGR4(−)+6A and +23F strains were the most susceptible to neutrophil phagocytosis. These results largely match the C3b/iC3b deposition results, although in human serum (but not rabbit or mouse) there was no difference in phagocytosis between the TIGR4(−)+6A and +23F strains despite there being more C3b/iC3b deposition on the +6A strain. The reason for this discrepancy is not clear. Recently, Weinberger et al. demonstrated differences between strains based on the TIGR4 strain expressing different capsular serotypes in neutrophil-mediated killing, suggesting direct capsular serotype effects on interactions with neutrophils (39). In contrast, we have found that differences in phagocytosis between strains were abolished when the TIGR4(−)+ strains were not opsonized with C3b/iC3b, confirming that they were due to complement activity rather than other potential differences between capsular serotypes in their interactions with noncomplement phagocytic receptors. The reasons for the discrepancies between our results and those of Weinberger et al. are not clear but probably relate to technical differences, as different assays with very different ratios of bacteria to neutrophils were used.

Complement is an essential component of the host immune response to systemic infection with S. pneumoniae (11, 20, 42, 45, 46), and the differences in C3b/iC3b deposition between the TIGR4(−)+ strains would therefore be predicted to have significant consequences for virulence. Indeed, the TIGR4(−)+6A and +23F strains had reduced virulence in a mouse model of sepsis, with a nearly fourfold difference in the number of recovered CFU of the TIGR4(−)+6A strain compared to the TIGR4(−)+4 strain at 24 h after infection. Although these differences in CFU counts are relatively small and probably will not lead to major differences in survival in this model, these data support previously published data showing that expression of capsular serotype 3 can increase, decrease, or have no effect on virulence in a mouse model of sepsis, depending on the strain background (22). Hence, the capsular serotype can affect virulence independently of other genetic variation between strains. Given the importance of complement for systemic immunity to S. pneumoniae and that the TIGR4(−)+6A and +23F strains were the two strains with increased C3b/iC3b deposition and with reduced virulence, it is plausible that the effects of the serotype on virulence are due to the effects on complement. Alternatively, the differences between capsular serotypes in C3b/iC3b deposition could correlate with effects on a wider range of host protein-bacterium interactions, for example, recognition by macrophages via the lectin SIGN-R1 (which specifically recognizes the S. pneumoniae capsule) or other noncomplement cell surface receptors, and these may influence virulence in the mouse model (3, 21, 42). Pleiotropic effects of the capsular serotype on different aspects of host immunity may explain why the TIGR4(−)+7F strain, although it had a lower (albeit not statistically significantly lower) median number of CFU in blood and spleen, was more resistant to phagocytosis than the TIGR4(−)+4 strain.

Previous data have shown that clinical serotype 7F and 23F isolates are not virulent in mice (9, 33), but we have shown that the TIGR4(−)+7F and +23F strains caused a significant septicemia that would almost certainly result in fatal infection. This suggests that differences between S. pneumoniae strains in virulence in mouse models are strongly influenced by noncapsular genetic variation, so that the TIGR4 genetic background of the TIGR4(−)+7F and +23F strains allows these strains to be virulent in mice whereas clinical isolates that express the same serotype but have a different genetic background are not. This is supported by other data showing marked differences in virulence in mice between different S. pneumoniae strains with the same capsular serotype (9, 32). Hence, although the capsular serotype can modulate virulence in mice, there is also a considerable, perhaps dominant, effect of noncapsular genetic variation.

To conclude, opsonization with C3b/iC3b of S. pneumoniae TIGR4(−)+ strains can vary markedly with the capsular serotype, with a higher level on two capsular serotypes associated with weakly invasive strains. Differences between capsular serotypes in opsonization with C3b/iC3b were not due to differences in capsular thickness or Ab binding and were associated with effects on neutrophil phagocytosis and in virulence in mice. Overall, our results show that the serotype can influence S. pneumoniae resistance to host immune responses, and they suggest that this maybe one cause of the variation in invasive potential associated with the capsular serotype.

Acknowledgments

This work was undertaken at UCLH/UCL, which received a portion of the funding from the Department of Health NIHR Biomedical Research Centre funding scheme. C.J.H. is supported by the Astor Foundation and GlaxoSmithKline through the University College London MB Ph.D. program. J.Y. was supported by the British Lung Foundation (P05/3). E.C. is supported by the Medical Research Council. J.N.W. is supported by grants from the U.S. Public Health Service (AI44231 and AI38446).

Editor: A. Camilli

Footnotes

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Published ahead of print on 30 November 2009.

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