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.

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.

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).

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).

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).

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).