Three surface exoglycosidases from Streptococcus pneumoniae, NanA, BgaA, and StrH, promote resistance to opsonophagocytic killing by human neutrophils

Abstract

Streptococcus pneumoniae (the pneumococcus) is a major human pathogen and a leading cause of inflammatory infections such as pneumonia and otitis media. An important mechanism for host defense against S. pneumoniae is opsonophagocytic killing by neutrophils. To persist in the human host, the pneumococcus has developed strategies to evade opsonization and subsequent neutrophil-mediated killing. Utilizing a genomic approach, we identified NanA, the major pneumococcal neuraminidase, as a factor important for resistance to opsonophagocytic killing in ex vivo killing assays using human neutrophils. The effect of NanA was shown using both type 4 (TIGR4) and type 6A clinical isolates. NanA promotes this resistance by acting in conjunction with two other surface-associated exoglycosidases, BgaA, a beta-galactosidase, and StrH, an N-acetylglucosaminidase. Experiments using human serum showed that these exoglycosidases reduced deposition of complement component C3 on the pneumococcal surface, providing a mechanism for this resistance. Additionally, we have shown that antibodies in human serum do not contribute to this phenotype. These results demonstrate that deglycosylation of a human serum glycoconjugate(s) by the combined effects of NanA, BgaA, and StrH, is important for resistance to complement deposition and subsequent phagocytic killing of S. pneumoniae.

FIG. 1.

Survival of S. pneumoniae in neutrophil killing assays, showing comparisons of WT and nanA mutant strains in two independent backgrounds (TIGR4 and 6Atr). (A) Opsonophagocytic killing of S. pneumoniae by human neutrophils after bacteria were preopsonized in 66% BRS. Biochemical complementation (+Nan) was performed by addition of 0.008 U of C. perfringens neuraminidase per reaction. All results are relative to killing of the WT strain (T4 [black bars] or 6Atr [white bars]). (B) Opsonophagocytic killing of S. pneumoniae by human neutrophils after bacteria were preopsonized in 10% NHS. Data are the means from at least three independent experiments performed in duplicate ± standard errors of the means (SEM). *, P < 0.05; ***, P < 0.001; NS, not significant (compared to WT or as indicated).

FIG. 2.

NanA acts upstream of phagocytosis, and complement is the predominant opsonin responsible for opsonophagocytic killing of S. pneumoniae. (A) Uptake of S. pneumoniae strains by human neutrophils. FITC-labeled bacteria were preopsonized in 10% NHS, followed by incubation with human neutrophils. The proportion of adherent and ingested pneumococci was assessed by flow cytometry. (B and C) The contribution of complement in the killing of S. pneumoniae was shown by performing opsonophagocytic killing assays using heat-inactivated NHS (HI NHS) to opsonize bacteria (B) and by performing assays in the presence of a blocking antibody to CR3 (CBRM1/5) or a control antibody (Ct Ab) (C). Gray boxes in panels B and C represent reactions where neutrophils were added, while white boxes represent control reactions where no neutrophils were added. Box-and-whiskers plots indicate high and low values, median, and interquartile ranges. Data are the results of at least two independent experiments performed in duplicate. *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, not significant.

FIG. 3.

C3 deposition on S. pneumoniae in NHS and BRS. Complement deposition was analyzed by flow cytometry. (A and B) Bacteria were preopsonized in 10% NHS, and C3 deposition was assessed using MAb 130.1, a monoclonal antibody specific for C3 breakdown products. All results are relative to bacteria preopsonized in 10% heat-inactivated NHS. (C and D) Bacteria were preopsonized in 20% BRS, and C3 deposition was assessed using a polyclonal antibody to C3. All results are relative to bacteria preopsonized in 20% heat-inactivated BRS. (A and C) Means from three independent experiments ± SEM. **, P < 0.01 compared to T4. (B and D) Representative histograms of the data comparing complement deposition on T4 (black lines), T4 nanA (gray lines), and T4 opsonized in heat-inactivated serum (gray fill).

FIG. 4.

NanA has an effect on the alternative pathway of complement activation. To assess killing by the alternative pathway, opsonization was carried out with 30% IgG-depleted NHS in GVB-MgEGTA buffer. Data are the means from at least three independent experiments performed in duplicate ± SEM. ***, P < 0.001 compared to WT.

FIG. 5.

Contribution of antibodies on the effect of NanA in NHS. (A to C) Levels of IgG (A), IgA (B), and IgM (C) in NHS were assessed by antibody binding assays on both T4 (black lines) and T4 nanA (gray lines) cells relative to unopsonized cells (gray fill). (D) Western blot detecting IgG heavy chain presence in NHS (1) and IgG-depleted NHS (2). (E) IgG-depleted serum was used to opsonize bacteria in opsonophagocytic killing assays. (F) Opsonophagocytic killing assays where bacteria were opsonized in either 10% NHS, 66% BRS, or a mixture of 10% BRS and 10% HI NHS (as a source of antibodies) to determine the role of antibodies in mediating the difference in phenotype observed in NHS versus BRS. Data are the means from at least two independent experiments performed in duplicate ± SEM. ***, P < 0.001 compared to WT.

FIG. 6.

Role of two other surface-exposed exoglycosidases of S. pneumoniae, BgaA and StrH, in resistance to opsonophagocytic killing by neutrophils. (A) Prototypic structure of complex N-linked biantennary glycans present on human glycoconjugates. Cleavage sites for the three pneumococcal exoglycosidases are indicated by arrows. (B) Neutrophil killing assays of S. pneumoniae, using 10% NHS to preopsonize bacteria. (C) C3 deposition assays of S. pneumoniae preopsonized in 10% NHS. Data are the means from three independent experiments ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (compared to T4).