Biofilm Formation Avoids Complement Immunity and Phagocytosis of Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae is a frequent member of the microbiota of the human nasopharynx. Colonization of the nasopharyngeal tract is a first and necessary step in the infectious process and often involves the formation of sessile microbial communities by this human pathogen. The ability to grow and persist as biofilms is an advantage for many microorganisms, because biofilm-grown bacteria show reduced susceptibility to antimicrobial agents and hinder recognition by the immune system. The extent of host protection against biofilm-related pneumococcal disease has not been determined yet. Using pneumococcal strains growing as planktonic cultures or as biofilms, we have investigated the recognition of S. pneumoniae by the complement system and its interactions with human neutrophils. Deposition of C3b, the key complement component, was impaired on S. pneumoniae biofilms. In addition, binding of C-reactive protein and the complement component C1q to the pneumococcal surface was reduced in biofilm bacteria, demonstrating that pneumococcal biofilms avoid the activation of the classical complement pathway. In addition, recruitment of factor H, the downregulator of the alternative pathway, was enhanced by S. pneumoniae growing as biofilms. Our results also show that biofilm formation diverts the alternative complement pathway activation by a PspC-mediated mechanism. Furthermore, phagocytosis of pneumococcal biofilms was also impaired. The present study confirms that biofilm formation in S. pneumoniae is an efficient means of evading both the classical and the PspC-dependent alternative complement pathways the host immune system.

INTRODUCTION

Streptococcus pneumoniae, the pneumococcus, is the leading pathogen producing acute otitis media, community-acquired pneumonia, and invasive diseases, including bacterial meningitis and sepsis (1). The growth and dispersal of microbes, whether pathogenic or environmental, commonly involve the production of biofilms, which represent the primary mode of pneumococcal growth during colonization, recurrent otitis media, and the early stages of invasive disease (2–4). This evidence supports the importance of studying pneumococcal sessile communities to understand key events in the development of disease caused by this important human pathogen.

Biofilm formation is a complex process initiated by the attachment of microorganisms to a surface or interface that is embedded in an extracellular matrix composed of various polymeric substances (5, 6). The biological and physicochemical features of the biofilm structure protect the bacterium from environmental adversities and give the microorganism an inherent resistance to antimicrobial therapies and the host immune response (6, 7). It is well known that the complement system represents one of the first lines of defense against invading pathogens such as S. pneumoniae and plays a vital role in both innate and acquired immunity (8). This unique host defense mechanism is activated by three different pathways—known as the classical, alternative, and lectin pathways—that converge at the central component C3, which is involved in essential phases of the immune response such as recognition and clearance of microorganisms, inflammatory response, and induction of phagocytosis (8, 9). The classical complement pathway is activated by the recognition of antigen-antibody complexes on the bacterial surface by the complement component C1q, and it is generally considered to be an effector of the acquired immune response. This cascade plays a vital role in complement activation against pneumococci (10, 11). However, the classical pathway also has an important role as part of the innate immune response to S. pneumoniae, since it is activated by other innate immune mediators such as natural IgM, C-reactive protein (CRP), serum amyloid P protein (SAP), and the lectin receptor SIGN-R1 (10–12). In addition, the alternative pathway is activated by the spontaneous hydrolysis of the C3 component, triggering the amplification of C3 deposition, and therefore, it contributes significantly to innate immunity (13). In addition, mannose-binding lectin-independent pathway activation has recently been demonstrated, confirming the importance of complement-mediated immunity against S. pneumoniae (14).

Although avoidance of complement immunity and phagocytosis is a clear advantage for bacterial dissemination, it may also be a common immune evasion strategy used by selected pathogens to allow long-term colonization and persistent carriage. In this sense, there are several studies reporting the importance of biofilm formation by various microorganisms in the evasion of the host immune response (15–17), although the interactions of pneumococcal biofilms with complement immunity and phagocytic cells is largely unknown.

In this study, we investigated the recognition by the complement system and human neutrophils of S. pneumoniae growing as either biofilms or planktonic cultures by exploring how acute-phase proteins and complement downregulators interact with bacteria with these two different life styles.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

S. pneumoniae nonencapsulated strains used for this study were as follows: strain R6 (a D39 derivative) (18); strain P040 expressing the green fluorescent protein (GFP) [R6(pMV158GFP), obtained by transformation with plasmid DNA; tetracycline resistant]; and strain P064 (R6 but pspC::aad9 constructed by mariner mutagenesis, displaying anti-transcribed orientation of the antibiotic resistance cassette of the minitransposon with respect to the targeted gene; spectinomycin resistant) (19). An encapsulated pneumococcal clinical isolate of serotype 19A (strain 1041) from a patient with sepsis was also used. Bacterial strains were grown at 37°C in C medium (20) containing 33 mM potassium phosphate buffer at pH 8.0 (CpH8 medium) either unsupplemented or supplemented with 0.8 mg ml⁻¹ yeast extract (C+Y medium). Biofilm formation by pneumococcal cells was obtained using 96-well polystyrene microtiter plates (Costar 3595; Corning) as previously described (19, 21). Briefly, cells were grown in C+Y medium to an optical density at 550 nm (OD₅₅₀) of 0.5 to 0.6, sedimented by centrifugation, resuspended in an equal volume of CpH8 medium, and diluted 1/100, and then 200 μl containing 5 × 10⁶ CFU ml⁻¹ was dispensed into 96-well plates for biofilm formation or into sterile Falcon tubes for planktonic growth. Both cultures were incubated for 5 h at 34°C.

Complement factors binding to S. pneumoniae strains.

Human serum from five healthy male volunteers unvaccinated against S. pneumoniae (median age of 40 years) was obtained with informed consent according to institutional guidelines and stored as single-use aliquots at −70°C as a source of complement and serum components. C1q, C3b, factor H (FH), C4b-binding protein (C4BP), and CRP were assessed using flow cytometry assays as previously described (11). After the incubation process at 34°C, bacterial cultures growing as biofilms in microtiter plates or in the planktonic form were washed with fresh CpH8 medium and resuspended in phosphate-buffered saline (pH 7.0) (PBS). Biofilm disaggregation was performed by gentle pipetting and slow vortexing before the opsonization process to avoid possible bias by morphological differences between the two growing stages. The corresponding bacterial suspensions (20 μl) were added to tubes containing 20 μl of human serum diluted 1/5 in PBS, and samples were incubated for 20 min at 37°C to allow opsonization by the different serum components. Previously, the number of biofilm-forming CFU had been determined by viable counts of bacteria, and similar numbers of planktonic cells were used in all assays.

C1q and C3b deposition were detected by incubating the bacteria with 50 μl of a fluorescein isothiocyanate (FITC)-conjugated polyclonal sheep anti-human C1q antibody (Serotec) or a FITC-conjugated polyclonal goat anti-human C3b antibody (ICN-Cappel) diluted 1/300 or 1/500 in PBS–0.1% Tween 20, respectively. Bacterial suspensions and antibodies were incubated for 2 h at 37°C for C1q detection or for 30 min on ice for C3b analysis. The deposition of CRP, FH, and C4BP was investigated by using a polyclonal rabbit anti-human CRP antibody (Calbiochem), a polyclonal sheep anti-human FH antibody (Serotec), and a polyclonal sheep anti-human C4BP antibody (Serotec) for 1 h at 37°C, respectively, followed by a secondary staining in PBS–0.1% Tween 20 containing FITC-conjugated polyclonal goat anti-rabbit or FITC/Dylight 649 donkey anti-sheep antibodies (Serotec). After incubations, the bacteria were washed with PBS–0.1% Tween 20 to remove unbound components, fixed in 3% paraformaldehyde, and analyzed on a FACSCalibur flow cytometer (BD Biosciences) using forward and side scatter parameters to gate on at least 25,000 bacteria. The results were expressed as a relative percent fluorescence index (FI) that measures not only the proportion of fluorescent bacteria positive for the host serum component investigated but also the intensity of fluorescence that quantifies the immune component bound (11, 22).

C3b analysis by confocal laser scanning microscopy.

To determine C3b deposition on S. pneumoniae, the R6 strain was first grown as biofilms and planktonic cultures as described above using glass-bottom dishes (WillCo-dish; WillCo Wells B.V., The Netherlands) and sterile Falcon tubes, respectively. Pneumococcal biofilm and planktonic culture samples were incubated with human serum as a source of complement, and the samples were incubated for 20 min at 37°C to allow opsonization by the C3b component. A FITC-conjugated polyclonal goat anti-human C3b antibody (ICN-Cappel) was used to detect the C3b bound using a Leica TCS-SP5-AOBS-UV confocal laser scanning microscope (CLSM) equipped with an argon ion laser and a Leica DM4000B fluorescence microscope. Pneumococcal cells were labeled with SYTO 59. The planktonic cells were sedimented by centrifugation to improve the visualization the C3b component by fluorescence microscopy. The excitation/emission maxima were around 495/519 and 622/645 nm for anti-human C3b-FITC and SYTO 59, respectively, and the magnification was ×100. Images were analyzed using the Leica software LCS. Projections through the x-y plane (individual scans at 0.5-μm intervals) and the x-z plane (images at 3-μm intervals) were obtained by CLSM.

Quantification of IgG, phosphorylcholine (PCho) and PspC.

IgG, PCho, and PspC on the bacterial surfaces of biofilms and planktonic cultures were detected by flow cytometry. Briefly, the experimental conditions of the assay were the same as those described above except that bacterial strains were incubated for 20 min at 37°C with 50% human serum as a source of IgG or for 1 h at 37°C with the antibody TEPC-15 (monoclonal antibody to PCho, Sigma-Aldrich) diluted 1/25 or rabbit polyclonal antibody to PspC diluted 1/300 (a kind gift from Sven Hammerschmidt, University of Greifswald, Germany). The secondary antibodies used were rabbit anti-mouse FITC (Serotec) and goat anti-rabbit FITC (Serotec) for the detection of PCho and PspC, respectively. Detection of IgG by flow cytometry was measured after incubation for 20 min on ice with a phycoerythrin (PE)-conjugated donkey anti-human IgG antibody (Jackson ImmunoResearch). All secondary antibodies were diluted 1/200 in PBS-Tween 20 (0.1%) and incubated for 30 min on ice.

Phagocytosis of S. pneumoniae biofilms and planktonic cultures.

Experiments investigating human neutrophil phagocytosis were performed by a flow cytometry assay using HL-60 cells (CCL-240; ATCC) differentiated to granulocytes (11, 23). The assay included the fluorescent pneumococcal P040 strain grown as biofilm or planktonic culture in CpH8 medium containing 1% maltose and 1 μg ml⁻¹ tetracycline (19). Briefly, 96-well plates containing 1 × 10⁶ CFU per well were infected in triplicate at a ratio of 10 bacteria to 1 cell with 20 μl of a suspension of the pneumococcal P040 strain previously opsonized for 20 min with HBSS, heat-inactivated serum (HIS), or human serum, and the mixture containing cells was incubated for 30 min at 37°C with shaking (150 rpm). A minimum of 6,000 cells were analyzed using a Cytomics FC500 Beckman Coulter flow cytometer equipped with a 488 nm Ar ion laser. The presence of complement receptors on HL-60 granulocytes was previously documented, and therefore expression of CD11b (iC3b receptor and CR3 α-chain), a marker of granulocytic differentiation, was measured prior to phagocytic assays to confirm the presence of the receptor (24). Results were expressed as a FI (see above), defined as the proportion of cells positive for fluorescent bacteria multiplied by the geometric mean of fluorescence intensity, which correlates with the number of bacteria phagocytosed per cell (11, 23).

Statistical analysis.

Data are representative of results obtained from repeated independent experiments, and results are presented as means and standard deviations (SD) for 3 to 5 replicates. Statistical analysis was performed by using two-tailed Student′s t test (for two groups). GraphPad InStat version 3.0 (GraphPad Software, San Diego, CA) was used for statistical analysis.

RESULTS

C3b deposition on S. pneumoniae growing as biofilms or planktonic cultures.

The deposition of the complement component C3b on the surface of S. pneumoniae was investigated by a flow cytometry assay using bacteria grown either as biofilms or as planktonic cultures. Since nonencapsulated pneumococci show a higher capacity to form in vitro biofilms than encapsulated isolates (reviewed in reference 6) and to prevent any possible hindrance of complement activity by the capsular polysaccharide (12), the nonencapsulated pneumococcal R6 strain was used. In addition, to avoid possible bias in complement interaction with S. pneumoniae between the two modes of bacterial growth, biofilm disaggregation was performed before opsonization with human serum. The morphologies of S. pneumoniae cells from planktonic cultures or from disaggregated biofilms (mainly diplococci) were indistinguishable by phase-contrast microscopy confirming that disaggregation does not induce morphological changes that could affect complement interaction in further assays (data not shown). Recognition of S. pneumoniae by the key complement component C3b was explored by flow cytometry using a pneumococcal strain without capsule (Fig. 1A and B) and an encapsulated clinical isolate of serotype 19A (Fig. 1C and D). C3b deposition on pneumococcal biofilms was markedly impaired in comparison to planktonic cultures suggesting that biofilm formation in S. pneumoniae is a mechanism used by the bacterium to avoid recognition by this key complement component (Fig. 1). C3b binding was further investigated on planktonic cultures and intact biofilms of the R6 strain using fluorescence microscopy and CLSM, respectively (Fig. 2). C3b bound on the bacterial surface was detected by using FITC-conjugated polyclonal goat anti-human C3b antibody (green fluorescence), whereas the pneumococcal cells were stained with SYTO 59 (red fluorescence). The entire bacterial surface of the planktonic culture was coated with C3b (Fig. 2A and B), whereas only small patches of the pneumococcal biofilm appear to contain C3b (Fig. 2C and D). This confirmed that when S. pneumoniae cells form biofilms, a notable reduction in opsonization by C3b occurs.

Fig 1.

(A) C3b deposition on the surface of the R6 strain grown as a planktonic culture (PK) or as a biofilm (BF). Results are expressed as the FI, relative to the results for PK culture. (B) Example of a flow cytometry histogram for C3b deposition. Results for a control (CT PBS) incubated with PBS instead of human serum are also shown. (C) C3b deposition on the surface of the encapsulated clinical isolate of serotype 19A grown as planktonic culture (PK) or as a biofilm (BF) (D). Results are expressed as the FI, relative to the results for PK culture. Error bars represent standard deviations, and asterisks mark results that are statistically significant compared to those for bacteria growing as PK (two-tailed Student's t test; P < 0.001).

Fig 2.

C3b deposition on the surface of the R6 strain grown as planktonic cultures or as biofilms. A planktonic culture of R6 strain was stained with SYTO 59 (A, red), and C3b deposition on the surface of the planktonic culture of the R6 strain was visualized using a FITC-conjugated polyclonal goat anti-human C3b antibody (B, green). To enhance the quality of the picture, the culture was centrifuged and gently resuspended in PBS after labeling and prior to examination with the fluorescence microscope. (C to E) Localization by CLSM of the human C3b component on the surface of biofilm-grown R6 strain. A biofilm of the S. pneumoniae strain R6 was stained with a combination of SYTO 59 (C, red) and a FITC-conjugated polyclonal goat anti-human C3b antibody (D, green). (E) Merged image from the two channels. Scale bars = 25 μm.

Reduced activation of the classical complement pathway by pneumococcal biofilms.

As the classical pathway is essential for complement activation against S. pneumoniae (see above), deposition of its first component, C1q, on the R6 strain was investigated by flow cytometry. C1q deposition was significantly reduced on the surface of pneumococcal biofilms compared to planktonic cultures (Fig. 3A and B), indicating that pneumococcal biofilms hinder the activation of the classical pathway. Since recognition of S. pneumoniae by the pentraxin CRP (an acute-phase protein) increases the deposition of C1q on the pneumococcal surface, thus activating the classical pathway (12), we tested whether the reduced C1q level on the surface of pneumococcal biofilms was somewhat mediated by an impaired binding by human CRP on biofilms. Actually, binding to human CRP was strongly reduced on the surface of S. pneumoniae R6 biofilms in comparison to planktonic cultures (Fig. 3C and D). These results taken together demonstrate that pneumococcal biofilms enhance the resistance of S. pneumoniae to complement immunity by diminishing the classical pathway activation. Additional experiments confirmed that the impaired recognition of S. pneumoniae biofilms by C1q and CRP was not related to differences in binding to IgG (Fig. 3E and F) or variations in the amount of the PCho epitope on the bacterial surface (Fig. 3G and H).

Fig 3.

Deposition of C1q, CRP, and IgG on the surface of the R6 strain grown as planktonic culture (PK) or as biofilm (BF) and detection of PCho in PK and BF cultures. (A, C, and E) Deposition of C1q, CRP, and IgG, respectively. (B, D, and F) Examples of flow cytometry histograms for the binding of C1q, CRP, and IgG, respectively. (G) Detection of PCho on the surface of the R6 strain grown as a planktonic culture (PK) or as a biofilm (BF). (H) Example of a flow cytometry histogram for the detection of PCho. Results are expressed as the FI, relative to the results for PK culture. Results for controls incubated with PBS instead of human serum (CT PBS) are also shown. Error bars represent standard deviations, and asterisks mark results that are statistically significant compared to those for bacteria growing as PK (two-tailed Student's t test; P < 0.001).

Recruitment of human complement regulators.

Interaction of pneumococcal cultures grown as biofilms or planktonic cultures with the major fluid-phase regulators of either the classical/lectin (C4BP) or alternative (FH) complement cascades was investigated by flow cytometry (Fig. 4). Deposition of C4BP was very similar in planktonic and biofilm cultures, indicating that interaction with the main downregulator of the classical pathway is not affected by biofilm formation (Fig. 4A and B). In contrast, recruitment of FH was significantly enhanced on pneumococcal biofilms compared to planktonic cultures (Fig. 4C and D), which suggested that the impairment of the alternative pathway in pneumococcal biofilms is mediated by increased binding to FH, the downregulator of the alternative cascade.

Fig 4.

Recruitment of downregulators of the complement system by the R6 strain grown as a planktonic culture (PK) or as a biofilm (BF). (A) Recruitment of C4BP by the R6 strain as PK (open bar) or as BF (gray bar). (B) Example of a flow cytometry histogram for the deposition of C4BP on R6 strain. (C) Recruitment of FH by the R6 strain as a PK or a BF. (D) Example of a flow cytometry histogram for the deposition of FH on R6 strain. (E and F) Recruitment of FH by the P064 (R6 pspC) strain as a PK or a BF. (G) Example of a flow cytometry histogram for the recruitment of FH on P064 strain. Results are expressed as the FI, relative to the results for PK culture, except in panel F, where results mean fluorescence intensities. Error bars represent standard deviations, and asterisks mark results that are statistically significant compared to those for bacteria growing as PK (two-tailed Student's t test; P < 0.001). Results for controls incubated with PBS instead of human serum are also shown (CT PBS).

PspC is involved in the enhanced resistance of pneumococcal biofilms to complement-mediated immunity.

It has been reported that the pneumococcal surface protein PspC (also designated CbpA) is able to bind FH (25–27). To explore the possible involvement of PspC in the increased recruitment of FH on pneumococcal biofilms, an isogenic pspC mutant of the R6 strain (P064 strain) was employed. Indeed, loss of PspC expression in P064 cells growing as biofilms caused a decrease in FH binding to levels similar to those shown by planktonic cultures of the same strain (Fig. 4E, F, and G). C3b deposition assays using biofilms of the wild-type and pspC-deficient strains demonstrated that the increased recruitment of FH mediated by PspC confers to S. pneumoniae growing as biofilms the advantage of avoiding recognition by C3b (Fig. 5A and B). However, in the absence of PspC in both biofilms and planktonic cultures, a similar C3b deposition pattern was found, confirming that this protein is clearly involved in the enhanced resistance of pneumococcal biofilms to the complement system (Fig. 5C and D). To explore the possibility that S. pneumoniae growing as biofilms might display greater levels of PspC to avoid complement-mediated immunity, detection of the PspC exposed on the bacterial surface was analyzed in pneumococcal biofilms and planktonic cultures (Fig. 5E and F). Our results showed increased levels of PspC on the surface of the biofilm, confirming that S. pneumoniae cells adopting a sessile life divert the amplification of the alternative pathway and consequently the deposition of C3b by inducing higher levels of PspC on the bacterial envelope.

Fig 5.

Effect of PspC in complement evasion and levels of PspC in planktonic cultures (PK) and biofilms (BF). (A) Deposition of C3b on the surface of the R6 strain or its isogenic pspC mutant strain, both growing as biofilms. (B) Example of a flow cytometry histogram for the deposition of C3b on R6 and P064 (R6 pspC) as a BF. (C) Deposition of C3b on the surface of the pspC strain growing as a PK or BF. (D) Example of a flow cytometry histogram for the deposition of C3b on P064 (R6 pspC). (E) Detection of PspC on the bacterial surface of R6 growing as a PK or BF. (F) Example of a flow cytometry histogram for the detection of PspC on R6 growing as a PK or BF. Error bars represent standard deviations, and asterisks mark results that are statistically significant compared to those for bacteria growing as a PK (two-tailed Student's t test; P < 0.001). Results for controls incubated with PBS instead of human serum are also shown (CT PBS).

Phagocytosis by neutrophils is impaired in S. pneumoniae biofilms.

Activation of complement immunity is a very efficient mechanism of the host immune response involved in phagocytosis of pneumococci and other encapsulated bacteria. The susceptibility of pneumococcal biofilms to opsonophagocytosis mediated by human neutrophils was investigated by flow cytometry using strain P040. Uptake of S. pneumoniae grown as a biofilm was markedly impaired in comparison to that of the planktonic culture, demonstrating that the sessile growth of S. pneumoniae represents a benefit to the microorganism by avoiding very efficiently the phagocytosis mediated by human neutrophils (Fig. 6). However, in the absence of complement (HBSS or heat-inactivated serum), phagocytosis levels were drastically reduced in comparison to those seen with bacteria opsonized with serum, confirming that the increased resistance to phagocytosis by pneumococcal biofilms is complement dependent (Fig. 6). Overall, these findings mirror the results described above regarding the interaction with different components of the complement immune response and strongly suggest that the reduced complement activation on the surface of pneumococcal biofilms confers to the bacterium enhanced resistance to phagocytosis by professional phagocytes.

Fig 6.

Opsonophagocytosis of the R6-GFP strain grown as a planktonic culture (PK) or as a biofilm (BF). (A) Opsonophagocytosis of the P040 strain as PK or as BF. Negative controls of bacteria incubated with HBSS or heat-inactivated serum (HIS) instead of human serum are also shown. (B) Example of a flow cytometry histogram for the opsonophagocytosis. Results are expressed as the FI relative to the results for PK culture. Error bars represent standard deviations, and asterisks mark results that are statistically significant compared to those for bacteria growing as a PK (two-tailed Student's t test; P < 0.001).

DISCUSSION

Bacterial biofilms are widely accepted as a frequent cause of chronic persistent infections (5). The ability of respiratory pathogens to persist in the nasopharynx and disseminate throughout the host under certain favorable circumstances is associated with their capacity to form biofilms on the mucosal epithelium (7). Nasopharyngeal colonization provides a stable environment for S. pneumoniae from which it can spread to other hosts and/or give rise to an infection (28). Compared to their planktonic counterparts, bacteria living as biofilms appear to have developed an evolutionary advantage because they are less sensitive to antibiotics, which complicates the effectiveness of the antimicrobial therapy (7, 29). Two main questions that remain unanswered to date are those of how the host defense immune system reacts to S. pneumoniae biofilms and whether pneumococcal biofilms can be efficiently recognized by professional phagocytes. One of the major immunological mechanisms against microbial pathogens is complement-mediated immunity, which consists of a complex network of circulating and cell surface-bound proteins that play an essential role in host defense (8, 9). In this regard, it has been shown that biofilm formation by Mycoplasma pulmonis protects against the lytic effects of complement immunity (16), whereas Staphylococcus epidermidis growing as biofilms has developed the ability to avoid neutrophil killing by preventing C3b opsonization (30). In this study, we investigated the interaction of S. pneumoniae with the complement system, exploring the activation and regulation of complement immunity by biofilm and planktonic bacteria. Our results indicate that pneumococcal cells within biofilms are much more effective in diverting C3b deposition on the bacterial surface than planktonic bacteria. This is relevant from the immunological perspective because C3b is essential for both innate and adaptive immunity against pyogenic bacteria such as S. pneumoniae (8, 10, 11).

To unravel the mechanism behind the impaired C3b deposition on pneumococcal biofilms, the classical pathway activation was investigated, as this cascade is essential for complement immunity against pneumococcus (10, 11). Pneumococcal biofilms have been identified in children with acute otitis media (2), and therefore, the impaired classical pathway activation on S. pneumoniae biofilms may have functional consequences, as C1q protects not only against pneumococcal pneumonia and sepsis (10) but also against acute otitis media and invasive disease by preventing the dissemination of S. pneumoniae from the middle ear to the systemic circulation (31). Our results demonstrate that biofilm formation confers to S. pneumoniae an enhanced ability to circumvent the early activation of this pathway by a C1q-dependent mechanism, which is consistent with previous results obtained with Acinetobacter baumannii (17).

The classical pathway can also be activated on S. pneumoniae by acute-phase proteins, such as CRP (22). CRP is the main acute-phase reactant in humans, and indeed, CRP levels markedly increase after pneumococcal infection, which confirms the importance of this molecule for S. pneumoniae recognition (32). Our findings show that biofilm formation in S. pneumoniae is associated with reduced recognition by human CRP. This is in agreement with results previously reported for coagulase-negative staphylococcal biofilms (33) but contrasts with the claim that an enhanced production of choline phosphate, which is known to bind CRP, occurs during biofilm development (34). Our results suggest that the impaired activation of the classical pathway on the surface of pneumococcal biofilms is not due to differences in phosphorylcholine or variation in the binding to IgG. There is evidence confirming that CRP binds the complement component C1q through its globular head region, thus activating the classical pathway (35, 36). In this regard, our results indicate that S. pneumoniae growing as a biofilm has the ability to avoid the direct interaction of C1q with the pneumococcal surface, as a recent study has demonstrated that C1q can directly recognize S. pneumoniae in the absence of any mediator (37).

Recruitment of regulators for complement activation is a common strategy used by different microorganisms for complement evasion (38). The PspC protein of S. pneumoniae binds FH, reducing the activation of the alternative complement pathway (27). Our results demonstrated that recruitment of FH was markedly enhanced by pneumococcal biofilms compared to their planktonic counterparts in a PspC-dependent manner and are in accordance with a recent report showing that pneumococci increase production of PspC when grown under biofilm-forming conditions (34). Our findings demonstrate that the increased presence of PspC on the surface of pneumococcal biofilms have functional consequences in terms of subversion of complement mediated immunity by reducing the activation of the alternative pathway through an FH-dependent mechanism. In contrast, a significant difference in the deposition of C4BP onto biofilms compared to planktonic pneumococcal cultures was not apparent. A variety of bacteria interact with C4BP to facilitate immune evasion (reviewed in reference 39). It has been reported that the binding of C4BP to S. pneumoniae is PspC allele dependent, with the R6/D39 allele being a weak binder (40). More recently, however, it was reported that the pneumococcal glycolytic enzyme enolase, a moonlighting surface protein (41), recruited C4BP but not FH (39). Previous proteomic analyses using procedures different from those employed in this study to grow pneumococcal biofilms have shown either a transient increase (42) or a marked inhibition in enolase biosynthesis (43). This discrepancy has been attributed to the different strains used, the different ages of the biofilms examined, and/or differences in the criteria used for protein identification in both studies. Assuming that significant changes in the binding of C4BP to biofilm-grown and planktonic cells have not been found, we propose that no major alterations on enolase production take place under our experimental conditions. Overall, our study shows for the first time that S. pneumoniae biofilms avoid complement immunity by targeting both the classical and alternative pathways using a complex mechanism of impaired activation and increased downregulation, respectively.

Clearance of S. pneumoniae by professional phagocytes requires efficient opsonization of the bacterium by the complement system (8). Biofilm formation has been suggested to be a pivotal event in the pathogenesis of numerous infectious diseases (5) and is consistent with our findings as long as the reduced complement activation on S. pneumoniae biofilms confers a significant benefit to the virulence of the microorganism. Reduced phagocytosis has been documented previously for other bacterial species growing as biofilms, confirming the idea that sessile communities of certain microbial pathogens are more resistant to opsonic killing by host phagocytes than planktonic cells (15, 44, 45). In the case of S. pneumoniae, biofilm matrices consist of a mixture of extracellular polymeric substances composed of extracellular DNA, proteins, and polysaccharides that are synthesized in large part by the pneumococcal strains producing the biofilm. The relevance of these matrices lies in the fact that they are responsible for the cohesion and three-dimensional architecture of biofilms (46). In terms of host-pathogen interaction, the presence of an extracellular matrix during biofilm formation improves the virulence of S. pneumoniae (47). The results of our study fully confirm that opsonophagocytosis of pneumococcal biofilms is significantly impaired in comparison to that in planktonic cultures and demonstrate that pneumococcal biofilms have developed an increased resistance to the phagocytosis process mediated by human neutrophils. Taken together, our findings suggest that biofilm formation may constitute an evolutionary advantage in certain phases of the pneumococcal pathogenic process, such as nasopharyngeal colonization or during the early steps of microbial attachment for invasion, by avoiding the host immune system.