ABSTRACT
Pneumococcal surface protein A (PspA) and PspC are important virulence factors. Their absence has been shown to allow improved clearance of pneumococci from the blood of mice and to decrease pneumococcal virulence. In the presence of antibody and complement, pneumococci attach to erythrocytes in a process called immune adherence (IA), which facilitates their delivery to, and eventual phagocytosis by, macrophages. It is not known, however, if PspA and PspC affect IA. Using PspA and/or PspC isogenic mutants and complement-deficient mouse sera, we demonstrated that absence of PspA allows greater deposition of C1q and thus increased classical-pathway-mediated C3 deposition. In the absence of both PspA and PspC, there is also a major increase in C1q-independent C3 deposition through the alternative pathway. The latter was observed even though absence of PspC alone did not have a major effect on alternative-pathway-dependent complement deposition. The enhanced complement C3 deposition realized in the absence of PspA alone and in the absence of PspA and PspC resulted in both greatly increased IA to human erythrocytes and improved transfer of pneumococci from erythrocytes to phagocytes. These data provide new insight into how PspA and PspC act in synergy to protect pneumococci from complement-dependent clearance during invasive infection.
Streptococcus pneumoniae (pneumococcus) is a major pathogen causing pneumonia, bacteremia, meningitis, otitis media, and sinusitis, especially in children, the elderly, and immunocompromised patients (29). S. pneumoniae is one of the few bacteria that can cause septicemia in an immunocompetent host. As a gram-positive bacterium, the pneumococcus has a thick and rigid cell wall that protects it from lysis by the complement membrane attack complex. Largely on the basis of in vitro studies, complement-dependent opsonophagocytosis is thought to be essential to eliminate pneumococci from the circulation (5, 7, 30).
Complement can be deposited on pneumococci through activation of the classical and alternative pathways, whereas the mannose-binding lectin pathway seems to play a relatively minor role (8, 36). The classical pathway can be triggered by natural immunoglobulin M antibodies bound to the C polysaccharide of the pneumococcal cell wall or by the acute-phase-reactant C-reactive protein, which can bind the phosphocholine on the pneumococcal teichoic and lipoteichoic acids (28). The formation of immune complexes initiates the binding of C1q to Fc regions of antibodies or to C-reactive protein (16, 20). Sequential activation of C4 and C2 enables the formation of C4b2b, which is the classical-pathway-derived C3 convertase. Activation of the alternative pathway occurs spontaneously by continuous hydrolysis of C3 into C3b. On the bacterial surface, C3b binds factor B and presents factor B for cleavage by factor D. The resultant C3bBb complex is the alternative-pathway-derived C3 convertase. The C3 convertase generated from either pathway can cleave C3 into C3a and C3b. Thus, the alternative pathway is able to amplify complement activation originally triggered by C1q in the classical pathway. Once C3b binds to the gram-positive bacterium's surface, it can activate a series of downstream events leading to opsonization and phagocytosis (16).
It has been shown that C3 deposition on wild-type pneumococci is dependent on activation by the classical pathway (8). Several pneumococcal components have been shown to interfere with complement deposition. These include capsular polysaccharide, pneumococcal surface protein A (PspA), PspC, and pneumolysin (7, 11, 19, 30, 33, 40, 43). PspA is expressed by all pneumococci. It inhibits C3 activation and thus prevents subsequent C3b deposition on the pneumococcal surface (37, 40). PspC is a paralog of PspA. PspC binds complement activation regulatory protein factor H, which promotes factor I-mediated cleavage of C3b, hence abrogating C3b's opsonic activity (11, 19, 30). PspA and PspC are important virulence factors of pneumococci. PspA− strains are attenuated in virulence and are cleared from blood more rapidly than their PspA+ isogenic parents (37, 40). PspC by itself is important for the establishment of carriage and pneumonia, but not in the persistence of bacteremia, caused by at least some challenge strains. However, loss of both PspA and PspC greatly enhanced the clearance of pneumococci from blood and decreased pneumococcal virulence (4).
Pneumococci have been shown to attach to erythrocytes through immune adherence (IA), which enhances the phagocytosis of pneumococci (31). IA is mediated by complement C3b, C4b, and C1q and mannose-binding lectin on the immune complexes interacting with the CR1 receptor on erythrocytes (14, 15, 38). The immune complexes on erythrocytes can be transferred to macrophages while the erythrocytes traverse the liver and spleen. Although the complexes are thus removed from the circulation, the erythrocytes that are deprived of the immune complexes are generally not lysed or phagocytized and are returned to the circulation (10, 22).
In the present study, we used isogenic strains of pneumococci that do or do not express PspA and/or PspC to investigate the ability of PspA and/or PspC to affect IA of pneumococci and the subsequent transfer of pneumococci from erythrocytes to macrophages. We also investigated the mechanisms whereby PspA and PspC, alone or in combination, affect complement deposition. Our results demonstrate that through their inhibition of C3 deposition, PspA and PspC act in synergy to greatly reduce IA and the subsequent transfer of pneumococci to phagocytes.
MATERIALS AND METHODS
Bacterial strains and fluorescein isothiocyanate (FITC) labeling.Serotype 2 pneumococcal strain D39 (PspA+ PspC+) and its isogenic mutants JY53 (PspA− PspC+), TRE108 (PspA+ PspC−), and TRE121 (PspA− PspC−) were used in this study (3, 4, 17, 42) (Table 1). The mutants were previously constructed in our laboratory by insertion duplication mutagenesis, and the absence of PspA and/or PspC was reconfirmed prior to these experiments by Western blot analysis (data not shown). These isogenic mutants have been extensively characterized in vitro and in vivo as to their infection phenotypes (4, 17, 42). A serotype 19F strain, EF3030, was also used (2).
Pneumococcal strains used in this study
Pneumococci were grown on 3% sheep blood agar in a candle jar for 16 to 18 h. Each strain was then subcultured into fresh Todd-Hewitt broth containing 0.5% yeast extract (Becton Dickinson, Sparks, MD). Appropriate antibiotics were added to the medium in accordance with the antibiotic resistance traits of the strain. When the optical density at 600 nm of pneumococci in Todd-Hewitt broth reached 0.45, the bacteria were washed three times with phosphate-buffered saline (PBS) and serially diluted prior to enumeration by plating on blood agar. A portion of the remaining bacteria was resuspended in Hanks' balanced salt solution containing 0.25% bovine serum albumin (0.25% BSA/HBSS; Invitrogen, Carlsbad, CA) and 10% glycerol, aliquoted, and frozen at −80°C. The remainder of the live bacteria were labeled with FITC (Sigma, St. Louis, MO). FITC was dissolved in PBS (pH 8.4) at a concentration of 0.2 mg/ml and filtered. Approximately 108 to 109 bacteria were mixed with 500 μl of FITC at 37°C for 1 h with shaking. The bacteria were then washed twice to remove the unbound FITC, resuspended in 0.25% BSA/HBSS with 10% glycerol, and frozen at −80°C. Before use, bacteria were adjusted to 1 × 109/ml or 5 × 108/ml.
Erythrocytes and macrophages.Erythrocytes were obtained from fresh human venous blood drawn from healthy volunteers (with informed consent and Institutional Review Board approval) and separated from other blood cells with Ficoll-Paque PLUS (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) according to the manufacturer's instructions. The purity of erythrocytes was >99%, as checked with a hemocytometer. Purified erythrocytes were preserved in Alsever's solution (MP Biomedicals Inc., Aurora, OH) and stored at 4°C. Before use, erythrocytes were washed twice with isotonic (2.37%) sodium iodide (Sigma, St. Louis, MO) to elute adsorbed serum proteins and then resuspended in 5% BSA/HBSS. Erythrocytes were enumerated and adjusted to a concentration of 2 × 108/ml.
The J774A.1 murine macrophage cell line (41) was cultured as an adherent monolayer in Dulbecco modified Eagle medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal calf serum (HyClone, Logan, UT) and 1% gentamicin (Invitrogen, Carlsbad, CA). The cells were split every 3 days to maintain a viability of no less than 90% as judged by trypan blue exclusion. The cells were resuspended in 5% BSA/HBSS at a concentration of 2 × 106/ml before use.
Complement deposition assay.Pneumococci were resuspended in 5% BSA/HBSS to a concentration of 1 × 109/ml. Ten microliters of fresh-frozen human serum from a single normal donor was added to 200 μl of bacteria, and the mixture was incubated at 37°C for 30 min. Afterwards, the bacteria were washed twice with PBS and incubated with 200 μl of biotin-labeled goat anti-human C3, C1q, or C4 antiserum (1:100 dilution in PBS; ICN Biomedicals, Aurora, OH), which was biotinylated with a biotin-labeling kit according to the manufacturer's instructions (Roche Diagnostics GmbH, Mannheim, Germany). The control bacteria were incubated with 5% BSA/HBSS and PBS instead of human serum and biotin-labeled antiserum. After 30 min of incubation at 37°C, the bacteria were washed with PBS and resuspended in 200 μl of Alexa Fluor 488-conjugated streptavidin (10 μg/ml in PBS; Molecular Probes, Eugene, OR). This incubation was performed on ice for 30 min. After being washed with PBS, the bacteria were resuspended in 300 μl of 1% paraformaldehyde and flow cytometric analysis was conducted on a FACScalibur machine with CellQuest software (Becton Dickinson, Mountain View, CA). Pneumococci were gated, and 20,000 events were collected. The amount of complement deposited on each strain was calculated by measuring the mean fluorescence (MF) of the serum-opsonized strain minus the MF of the same strain not incubated in serum. Similar results were observed at higher concentrations of serum, but the differences were a little more distinct at 5% than at the higher concentrations.
For some experiments, sera from wild-type (C57BL/6J) mice and mice with a genetic deficiency of C1q (C1q−/−) or factor B (Bf−/−) were used. Except that the sera were diluted 1:1 prior to opsonization and goat-anti-mouse C3 was used, the rest of the complement deposition assay was conducted as described above. The complement C3 deposition on the serum-opsonized bacteria and the bacteria incubated in PBS were illustrated in the graphs without correction for the background.
Erythrocyte adherence assay.FITC-labeled pneumococci were resuspended in 5% BSA/HBSS at a concentration of 5 × 108/ml. A volume of 150 μl of bacteria was mixed with 20 μl of human serum and incubated at 37°C for 30 min while shaking. Next, 150 μl of autologous erythrocytes was added and the incubation was repeated. Erythrocytes were then washed thoroughly with 0.1% BSA/HBSS to remove unbound bacteria. Erythrocytes and adherent bacteria were fixed with 1% paraformaldehyde and transferred to microtubes. Fluorescent bacteria attached to erythrocytes were detected by flow cytometry on a FACScalibur instrument (Becton Dickinson, Mountain View, CA). Erythrocytes were gated, and 20,000 events were counted. The MF and the percentage of erythrocytes with associated pneumococci were calculated for each sample. In some experiments, the pneumococci were opsonized in heat-inactivated serum (56°C water bath for 30 min) or C3-depleted serum replenished with pure C3 protein (both from Calbiochem, La Jolla, CA) at final concentrations of 1.15 μg/ml, 2.3 μg/ml, and 4.6 μg/ml. Controls run with nonlabeled pneumococci showed that they have no effect on the detected sizes of the much larger erythrocytes.
Transfer reaction.After the erythrocyte adherence assay, instead of fixing the samples for flow cytometry, 200 μl of J774A.1 macrophages was added and the mixture was incubated at 37°C for 30 min while shaking. The samples were then loaded onto Ficoll-Paque PLUS and centrifuged at 400 × g for 30 min to separate erythrocytes from macrophages. After washing with 0.1% BSA/HBSS, erythrocytes and macrophages were fixed separately with 1% paraformaldehyde and analyzed by flow cytometry. Erythrocytes and macrophages were gated separately, and 15,000 events were collected for each. The transfer reaction was indicated by a decrease in the MF of erythrocytes and a concomitant increase in the MF of macrophages. The decrease in the MF of erythrocytes was calculated by subtracting the MF of erythrocytes in the presence of macrophages from the MF of erythrocytes in the absence of macrophages. Each transfer experiment included a control for each pneumococcal strain where no erythrocytes were added to the opsonized bacteria. These tubes were carried through the entire procedure to control for the number of bacteria that the macrophages might acquire from residual bacteria that survived the previous washing steps. The increase in the MF of the macrophages was calculated by subtracting the MF of the controls from the MF of the corresponding samples. To study the influence of complement C3 on the transfer reaction, the pneumococci were opsonized in C3-depleted serum alone or with serum replenished with pure C3 protein to a final concentration of 2.3 μg/ml.
Fluorescence microscopy.The same samples that were used to measure erythrocyte adherence were concentrated and applied to microscope slides for viewing under a fluorescence microscope. The number of attached pneumococci per erythrocyte was quantified by viewing 10 microscopic fields for each sample. Differential interference contrast and epifluorescence images were captured separately with a monochrome digital camera and then merged and colored with Zeiss AxioVision 3.1 software.
Statistical analysis.All flow cytometry studies were run until 20,000 gated events were observed. The 95% confidence interval for 20,000 events is less than 1.4% of the number of counts according to the Poisson distribution. Each experiment was run in triplicate tubes and was repeated three times. Only studies giving the same general result in all three runs are reported. The means and standard errors (SEs) reported are from one experiment that is representative of three experiments. Comparisons of MFs were based on triplicate experiments where the mean MF and the SE of the measurements were expressed. Statistically significant differences were calculated by Student two-tailed t tests. In the case of calculations of percentages of erythrocytes that have associated pneumococci, we again used triplicate data sets and calculated statistically significant differences in two ways. One method was to treat the percent positive as values from which we determined averages, SEs, and statistically significant differences with Student's two-tailed t test. The other method was to use the actual numbers of positive and negative cells from the three fluorescence-activated cell sorter (FACS) runs and calculate the P values by chi-square analysis. In most cases, comparisons showed statistically significant differences by either method. Where visual counts of positive versus negative cells were made by microscopic analysis, comparisons were made by Fisher's exact test. P values of <0.05 were considered to be statistically significant.
RESULTS
Effects of PspA and PspC on complement deposition.Since prior studies have shown that in nonimmune sera complement is deposited onto pneumococci through the classical pathway (8) and that PspA can inhibit this deposition (37), we investigated the effects of PspA and PspC on complement C3, C4, and C1q deposition before examining their effects on IA. Complement protein deposition onto wild-type bacteria and PspA- and/or PspC-deficient mutants (Table 1) was measured by flow cytometry (Fig. 1). As measured by MF of the gated bacteria, the greatest human complement C3 deposition was observed on PspA− PspC− strain TRE121 and PspA− strain JY53 (P = 0.0076 and 0.0132, respectively [Student's two-tailed t test], compared to D39) (Fig. 1A). In contrast, the amount of human C3 deposited onto PspC− strain TRE108 was not significantly different from that on D39. Compared to that on D39, the amount of C3 deposited on pneumococci in the absence of PspA (JY53) increased by about 170 MF units, whereas when both PspA and PspC were absent (TRE121) the increase was about 460 MF units. This finding suggests that PspA and PspC have a synergistic inhibitory effect on C3 deposition. The high level of C3 deposition on wild-type strain EF3030, compared to that on D39, is consistent with its low virulence in the mouse models of bacteremia and sepsis (6).
Complement C3, C1q, and C4 deposition onto pneumococci. Pneumococci were opsonized in human serum at 37°C for 30 min, and the C3 (A), C1q (B), and C4 (C) deposited onto the pneumococcal surface were detected by flow cytometry. C3, C1q, and C4 deposition on JY53 and TRE121 was significantly greater than that on D39; C3 and C4 deposition on EF3030 was significantly greater than that on D39 (*, **, and *** indicate statistically significant differences from D39 at P values of <0.05, <0.01, and <0.001, respectively). The expression of PspA and PspC on D39 and its isogenic mutants is indicated in parentheses beside each strain, where PspA and PspC are abbreviated as A and C, respectively.
We also observed a significant increase in the deposition of human C1q and C4 in the absence of PspA and in the absence of both PspA and PspC (Fig. 1B and C). C1q deposition on JY53 and TRE121 was significantly greater than that on D39 (P = 0.0007 and P = 0.0003, respectively). However, while there was almost twice as much C3 deposition on PspA− PspC− strain TRE121 as on PspA− strain JY53, we observed virtually identical C1q deposition on both JY53 and TRE121. Thus, while the C1q deposition might explain the increased complement deposition on strain JY53, the even greater increase in C3 deposition on TRE121 requires additional explanation.
To investigate more directly the relative abilities of PspA and PspC to blunt C3 deposition via the classical and/or alternative pathways, we assessed C3 deposition on pneumococci opsonized with sera from wild-type, C1q-deficient (C1q−/−), and factor B-deficient (Bf−/−) mice (Fig. 2A and B). Using serum from wild-type mice, we observed the same relative C3 deposition on the various pneumococcal strains that was observed with human serum (Fig. 1A). In serum from C1q−/− mice, C3 deposition is entirely dependent on the alternative pathway. In the C1q−/− serum, C3 deposition on both D39 and JY53 decreased to the level of the PBS control, indicating that the bulk of the basal C3 deposition on wild-type pneumococci and the additional C3 deposition on PspA− pneumococci were driven by C1q and, as expected (8, 36), dependent on the classical pathway. However, for TRE121 a high level of C3 deposition was realized, indicating that most of the complement deposition in the absence of both PspA and PspC was independent of C1q (Fig. 2A). Thus, it appeared that only in the absence of PspA and PspC was significant complement deposition on pneumococci achieved through the alternative pathway, without the need for classical-pathway complement activation.
C3 deposition onto wild-type and mutant pneumococci in the presence of normal mouse serum and mouse serum from mice with complement deficiencies. (A) C3 deposition onto pneumococci opsonized with normal mouse serum (NMS) and C1q−/− mouse serum. C3 deposition onto all four strains in NMS and C1q−/− mouse serum was compared with the corresponding PBS controls (*, P < 0.05, Student's two-tailed t test). (B) C3 deposition onto pneumococci opsonized with NMS and Bf−/− mouse serum. C3 deposition onto all four strains in NMS and Bf−/− mouse serum was compared with the corresponding PBS controls (*, P < 0.05, Student's two-tailed t test). The expression of PspA and PspC on D39 and its isogenic mutants is indicated in parentheses below each strain, where PspA and PspC are abbreviated as A and C, respectively.
To test this possibility, we performed experiments with Bf−/− mouse serum, in which the alternative pathway is not functioning. The absence of factor B nearly eliminated complement deposition on the PspA− PspC− mutant (Fig. 2B) and showed only the classical-pathway deposition achieved without alternate-pathway amplification of C3 cleavage.
These findings are consistent with the notion that C3 deposition onto D39 pneumococci is initiated by C1q but propagated via recruitment of the alternative pathway. PspA interfered with the C1q-dependent initiation step, and PspC interfered with the factor B-dependent amplification step, but PspC's effect was not very apparent unless PspA was also absent.
Effects of PspA and PspC on the adherence of pneumococci to erythrocytes.The adherence of pneumococci to erythrocytes and the impact of surface expression of PspA and PspC on this phenomenon were measured by two methods. First, FACS analysis was used to measure the increase in fluorescence gained by erythrocytes upon incubation with opsonized, FITC-labeled pneumococci. Second, the samples were viewed under a fluorescence microscope and the number of adherent pneumococci per erythrocyte was determined. In the FACS analysis, the erythrocytes were gated by forward and side scatter to exclude debris and aggregates (Fig. 3A) and adherence was gauged by the MF of the erythrocytes and by the percentage of erythrocytes that had associated pneumococci. A low level of natural fluorescence was detected on erythrocytes without adding FITC-labeled pneumococci (Fig. 3B), and all four serotype 2 strains plus one serotype 19F strain of pneumococci adhered to erythrocytes after exposure to human serum (Fig. 3C). The results mirrored our complement deposition data in Fig. 1. PspA− strain JY53 showed more adherence to erythrocytes than wild-type strain D39 but did not show nearly as much adherence as PspA− PspC− strain TRE121 (Fig. 3C), and absence of PspC by itself had only a small, sometimes undetectable, effect on adherence to erythrocytes, as shown by PspC− strain TRE108. EF3030 adhered to erythrocytes more than highly virulent strain D39 (Fig. 3C). Table 2 summarizes these results and provides the P values calculated for differences between the fractions of erythrocytes with bound pneumococci.
Adherence of pneumococci to erythrocytes. FITC-labeled pneumococci were opsonized in human serum and then incubated with autologous erythrocytes. After washing, the samples were fixed and analyzed by flow cytometry. The MF of erythrocytes and percentage of erythrocytes decorated with pneumococci were calculated. (A) Erythrocytes gated by side scatter and forward scatter. (B) Autofluorescence of erythrocytes. (C) Adherence of different pneumococcal strains to erythrocytes. The expression of PspA and PspC on D39 and its isogenic mutants is indicated in parentheses beside each strain, where PspA and PspC are abbreviated as A and C, respectively.
Summary of the adherence of pneumococci to erythrocytes determined by FACS analysis
The binding of FITC-labeled bacteria per erythrocyte was evaluated directly with a fluorescence microscope with between 98 and 154 erythrocytes visualized in each sample. Although the numbers of erythrocytes examined were 200-fold less than the 20,000 examined by FACS analysis, the results were supportive of the FACS data (Table 3), indicating that the expression of PspA alone or together with PspC inhibits the erythrocyte adherence of pneumococci.
Distribution of erythrocytes according to pneumococcal adherencea
Effects of PspA and PspC on IA.The adherence of pneumococci to erythrocytes could be mediated by complement and other serum components. We have referred to the complement-dependent adherence of pneumococci to erythrocytes as IA. To study the effects of PspA and PspC on the IA of pneumococci, we heat inactivated normal human serum to prevent complement activation and then measured the erythrocyte adherence of pneumococci in heat-inactivated serum and in the same serum prior to heat inactivation (fresh serum) (Fig. 4A). The IA of pneumococci attributable to complement (Fig. 4A) was calculated by subtracting the adherence exhibited in heat-inactivated serum from the amount of adherence exhibited in fresh serum. As in the above studies, the greatest adherence in fresh serum was observed for TRE121(PspA− PspC−), which was significantly greater than that of D39 (wild type), JY53 (PspA−), or TRE108 (PspC−) (P < 0.01, Student's two-tailed t test). In each case, the bacteria opsonized in heat-inactivated serum exhibited less adherence to erythrocytes than those opsonized in fresh serum. Heat inactivation caused the adherence of TRE121 to decrease more than that of other strains. The IA of JY53, TRE108, and TRE121 was greater than that of D39. More importantly, the IA of TRE121 was greater than the total IA of JY53 and TRE108. These data suggest that PspA, and to a lesser extent PspC, may inhibit the IA of pneumococci; together, PspA and PspC had a synergistic effect on inhibition of the IA of pneumococci, similar to their synergistic effect on C3 deposition on pneumococci.
Relationship between IA of pneumococci and complement. (A) Adherence of pneumococci to erythrocytes in the presence of fresh versus heat-inactivated human serum. The adherence of TRE121 to erythrocytes was significantly greater than that of D39, JY53, or TRE108 (*, P < 0.01, Student's two-tailed t test). Complement-specific IA was calculated by subtracting the adherence exhibited in heat-inactivated serum from the adherence exhibited in the fresh serum. (B) Association of IA with deposition of complement C3, C1q, and C4 on pneumococci. The association of IA with C3, C1q, and C4 exhibited R2 values of 0.8809, 0.3764, and 0.8331, respectively. The associations of IA with C3 and C4 was significant at P = 0.0181 and 0.0305, respectively. (C) Adherence of pneumococci to erythrocytes in the presence of C3-depleted serum alone or replenished with C3 at a final concentration of 1.15, 2.3, or 4.6 μg/ml. The expression of PspA and PspC on D39 and its isogenic mutants is indicated in parentheses below each strain, where PspA and PspC are abbreviated as A and C, respectively.
To obtain further evidence that the effects PspA and PspC had on IA were related to their effects on complement deposition, we did a correlation analysis (Pearson's correlation) with data from the experiments depicted in Fig. 1 and 4A. We found that the IA of pneumococci correlated significantly with the deposition of complement C3 (R2 = 0.8809, P = 0.0181) and C4 (R2 = 0.8331, P = 0.0305) (Fig. 4B). The association between C1q deposition and IA showed the same trend but was not a statistically significant correlation. These data suggested that PspA and PspC inhibit IA of pneumococci through their inhibition of complement deposition on pneumococci.
To confirm that the effects of PspA and PspC on IA are dependent on complement C3, we did the erythrocyte adherence assay with C3-depleted serum with or without replenishment with purified C3 protein. In C3-depleted serum, the adherence of the PspA−, PspC−, and PspA− PspC− strains to erythrocytes was the same as that of the wild-type strain (Fig. 4C), in contrast to their elevated adherence to erythrocytes in normal human serum (Fig. 4A). The elimination of IA of PspA− and/or PspC− strains by blocking C3 deposition demonstrated that PspA and PspC inhibit IA through their influence on complement C3. In general, the adherence of pneumococci to erythrocytes increased in relation to the amount of C3 added and was saturated by a C3 concentration of 2.3 μg/ml. TRE121 exhibited a more dramatic C3-dependent increase in adherence than was observed for D39, JY53, or TRE108 (Fig. 4C). These data are in agreement with our finding (Fig. 1A) that there was greater C3 deposition on TRE121 than on the other two mutant forms of D39.
Effects of PspA and PspC on the transfer of erythrocyte-bound pneumococci to macrophages.One of the outcomes of IA is the transfer of immune complexes on erythrocytes to phagocytic cells (10, 22). Therefore, we determined whether the effects of PspA and PspC on IA could subsequently influence the transfer of erythrocyte-bound pneumococci to macrophages. After macrophages were added to erythrocytes decorated with FITC-labeled pneumococci, the MF of erythrocytes decreased (Fig. 5A), which suggests that pneumococci detached from erythrocytes. Consistent with its great IA, TRE121 (PspA− PspC−) was detached from erythrocytes more readily than the D39 (wild type), JY53 (PspA−), and TRE108 (PspC−) strains (P < 0.05, Student's two-tailed t test). Concomitantly, macrophages obtained pneumococci, as indicated by the increased MF (Fig. 5B). TRE121 was obtained by macrophages in greater numbers than were D39, JY53, and TRE108 (P < 0.05, Student's two-tailed t test).
Macrophage transfer reaction of erythrocyte-bound pneumococci. FITC-labeled pneumococci were opsonized in human serum and then incubated with erythrocytes. After washing, J774A.1 macrophages were added and the mixture was incubated for 30 min. The erythrocytes and macrophages were separated through centrifugation with Ficoll, after which the erythrocytes and macrophages were fixed and analyzed by flow cytometry. Panels A and B illustrate the transfer reaction of pneumococci opsonized in normal human serum. (A) Detachment of pneumococci from erythrocytes as measured by the decrease in the MF of erythrocytes. (B) Gain of pneumococci by macrophages as measured by the increase in the MF of macrophages. TRE121 was detached from erythrocytes, as well as obtained by macrophages, more significantly than D39, JY53, and TRE108 (* in panels A and B, both P values of <0.05, Student's two-tailed t test). Panels C and D illustrate the transfer reaction of pneumococci opsonized in C3-depleted serum alone or replenished with pure C3 protein. (C) Detachment of pneumococci from erythrocytes was measured as for panel A. (D) The gain of pneumococci by macrophages was measured as for panel B. The expression of PspA and PspC on D39 and its isogenic mutants is indicated in parentheses below each strain, where PspA and PspC are abbreviated as A and C, respectively.
Statistical analyses with Pearson's correlation among the five strains of pneumococci showed that the detachment of pneumococci from erythrocytes correlated strongly with the gain of pneumococci by the macrophages (R2 = 0. 93, P = 0.0070), and both of them correlated strongly with complement C3 deposition of the pneumococci (R2 = 0.99, P = 0.0002, and R2 = 0.93, P = 0.0084, respectively). These results suggest that the transfer reaction of pneumococci is dependent on complement C3 deposition and that PspA and PspC in combination result in reduction of the transfer reaction through inhibition of C3 deposition.
To confirm that the transfer reaction depends on complement C3 deposition, the transfer reaction of pneumococci was conducted with C3-depleted serum alone or replenished with pure C3 protein at a final concentration of 2.3 μg/ml (Fig. 5C and D). The pattern of the decrease in the MF of erythrocytes for the pneumococcal strains (Fig. 5C) was similar to that of the increase in the MF of macrophages for these strains (Fig. 5D). The pneumococci were transferred from erythrocytes to macrophages more efficiently in the presence of C3 than in the absence of C3. Moreover, the greatest transfer of pneumococci to macrophages in the presence of C3 was with TRE121. This observation was anticipated since more C3 was deposited on strain TRE121 than on any of the others (Fig. 1A). These results verify that the transfer of pneumococci from erythrocytes to macrophages is dependent on complement C3 deposition, a process which is inhibited on pneumococci by their expression of PspA and PspC.
DISCUSSION
S. pneumoniae expresses a number of proteins on its surface that are able to protect the pathogen from host defenses. In particular, PspA and PspC contribute to pneumococcal virulence by interfering with complement deposition on the pneumococcal surface (34, 36, 40). Studies of capsular serotype 3 pneumococci have shown that the additional complement deposited in the absence of PspA is dependent on both the classical and alternative pathways (35, 36, 40).
PspC and its alternative form Hic have been shown to bind to factor H (11, 19), a regulatory protein that inhibits the formation of the alternative-pathway C3 convertase C3bBb and thereby the amount of C3b generated. Furthermore, the binding of factor H to PspC may facilitate factor I-mediated cleavage of C3b and hence also reduce C3b production (19). pspA and pspC mutations have been shown to act synergistically in decreasing virulence (4), and in a very recent paper by Quin et al., they have been shown to also have a synergistic effect on complement deposition (34).
The present study confirms the previous finding that PspA inhibits complement activation and goes on to demonstrate that PspA inhibits C1q deposition, thereby providing a mechanism by which PspA blocks classical-pathway activation and the subsequent amplification by recruitment of the alternative pathway. This study also demonstrates for the first time that the synergistic effect that PspA and PspC have on complement deposition is dependent in part on their synergistic effects on the activation of complement through the alternative pathway.
We observed that the deposition of C3 on a PspA− PspC− strain was almost completely blocked in Bf−/− mouse serum but not in C1q−/− mouse serum. Thus, the absence of both PspA and PspC results in C3 deposition that is only partially dependent on the classical pathway and almost completely dependent on the alternative pathway. Acting together, PspA and PspC are able to prevent the extensive complement deposition that would otherwise occur on pneumococci if the alternative and classical pathways were unchecked.
The ability of PspA and PspC in combination to markedly reduce complement C3 deposition also results in a striking inhibition of IA and the transfer of pneumococci to macrophages. Moreover, in C3-depleted serum, where full complement activation could not occur, the erythrocyte adherence and transfer reaction of all of the strains studied decreased to the same low level (Fig. 5C and D), thus confirming that the IA and the transfer reaction visualized in our studies are indeed dependent on complement. When two pneumococcal strains of different capsular serotypes were compared, such as D39 and EF3030, the strain that was easily cleared from blood (EF3030) exhibited greater complement deposition, greater IA, and a more efficient macrophage transfer reaction than the D39 strain, which is much more resistant to clearance from blood.
Two types of adherence of pneumococci to erythrocytes were observed in our study. In the case of wild-type strain D39, which is highly resistant to complement deposition in vitro, most of the adherence to erythrocytes in fresh serum was not dependent on complement. It is possible that the low level of binding of D39 to erythrocytes is dependent on lipoteichoic acid (25). In strains such as TRE121, on which complement was readily deposited in the presence of fresh serum, there was significantly more adherence to erythrocytes compared to D39. Virtually all of this additional adherence was dependent on complement (IA). As in the case of soluble immune complexes (9, 10, 12), the complement-mediated IA of pneumococci could facilitate the transfer of pneumococci from erythrocytes to macrophages. In mice, IA is not supported by CR1, which is not expressed by rodent erythrocytes (23). Rather, in mice IA appears to utilize complement factor H associated with platelets (1). Thus, it is possible that in mice the clearance of pneumococci lacking PspA and PspC might also involve IA.
The exact mechanisms that support the transfer reaction following IA to human erythrocytes are controversial (26, 27, 32, 39). The requirement of factors H and I to cleave C3b into iC3b and hence unload the immune complexes from erythrocytes has been questioned by previous studies (12, 13, 24). In our experiments, transfer reactions were conducted with erythrocyte-bound pneumococci and J774A.1 macrophages in the absence of serum. All of the pneumococci studied were transferred to macrophages whether measured by flow cytometry or observed by fluorescence microscopy. When the transfer reaction were conducted with the erythrocyte-bound pneumococci that were reconstituted in fresh human serum after washing off the nonadherent bacteria, more bacteria were transferred from erythrocytes to macrophages for each strain, but the overall pattern was the same (data not shown).
It has been shown that CR1 and Fc receptors on phagocytic cells are involved in the transfer reaction of immune complexes (13, 39) and that CR3 and CR4 might participate in the clearance of immune complexes. The newly described macrophage receptors CRIg and SIGN-R1 are thought to be critical in the clearance of blood-borne pathogens. CRIg is a dominant C3 receptor on Kupffer cells in the liver. It mediates the clearance of C3-opsonized particles from the circulation (18). SIGN-R1 is expressed on marginal-zone macrophages in the spleen. SIGN-R1 binds C1q, as well as pneumococcal polysaccharides, leading to activation of the classical pathway without requiring antibodies (21). It is possible that the interaction of both CRIg and SIGN-R1 with pneumococci could facilitate the transfer reaction.
In summary, we have demonstrated that PspA and PspC, in combination, markedly reduce complement deposition by inhibiting both the classical and alternative pathways and, as a result, inhibit IA and subsequent transfer of pneumococci from erythrocytes to macrophages. The absence of PspA by itself has a smaller effect than the absence of PspA and PspC together. The additional C3 deposited in the absence of PspA was dependent on both the classical pathway and amplification through the alternative pathway. Although the absence of PspC alone had almost no effect on complement deposition, it synergized with the absence of PspA to lead to complement deposition, which was largely, but not completely, independent of C1q and the classical pathway. The direct relationship between complement C3 deposition and IA provided a mechanism by which PspA and PspC act to inhibit IA, as well as the transfer of pneumococci from erythrocytes to phagocytes. It has been shown that antibodies to PspA can increase complement activation and C3 deposition on pneumococci (36). It therefore seems likely that antibodies to PspA will further enhance the removal of pneumococci through IA.
ACKNOWLEDGMENTS
We thank Bernard P. Arulanandam for providing mouse macrophage cell line J774A.1 and Varija N. Budhavarapu for suggestions regarding cell culture.
This work was supported by National Institutes of Health grant AI21548 to D.E.B.
FOOTNOTES
- Received 18 June 2007.
- Returned for modification 30 August 2007.
- Accepted 27 September 2007.
↵▿ Published ahead of print on 8 October 2007.
Editor: A. Camilli
- American Society for Microbiology
