Opsonophagocytic killing by neutrophils represents an important mechanism for clearance of pneumococcal infection (34
). While some pneumococcal factors are known to inhibit opsonophagocytosis, a comprehensive search to identify all factors involved in this resistance has not been performed (20
). Using a whole-genome approach, we identified that neuraminidase A (NanA) is important for resistance to opsonophagocytic killing by human neutrophils. Furthermore, we identified that NanA promotes resistance in conjunction with two other exoglycosidases from the pneumococcus, BgaA and StrH, by reducing complement deposition on the bacterial surface.
NanA is a well-characterized and ubiquitously expressed virulence factor in S. pneumoniae
strains, and its role in the pathogenesis of the pneumococcus has been studied extensively. The effect of NanA on pathogenesis in vivo
has been attributed to its various roles observed in vitro
. NanA is able to remove sialic acid to expose receptors to aid pneumococcal adherence, directly bind epithelial cells via a lectin domain, aid in formation of biofilms, desialylate the surface of its competing nasopharyngeal flora, deglycosylate human glycoconjugates, and liberate carbohydrates to aid metabolic fitness of the organism (6
). In vivo
, a role for NanA in colonization and sepsis is less clear and is dependent upon the animal model employed (26
). In contrast, during inflammatory diseases, such as pneumonia and otitis media, NanA has a more clear-cut role in the pathogenesis of this organism (33
). These infections result in a robust influx of neutrophils and the serum components necessary for opsonization (2
). Therefore, the effect of NanA in promoting resistance to opsonophagocytic killing demonstrated in this study could help explain the effect of this virulence factor observed in vivo
NanA does not, however, appear to be acting alone to promote resistance to opsonophagocytic killing. NanA has previously been shown to act together with BgaA and StrH, two other surface-anchored exoglycosidases in the pneumococcus, which remove galactose that is β1-4 linked to N
-acetylglucosamine (GlcNAc) and GlcNAc that is β1 linked to mannose, respectively (6
). All three enzymes act exclusively on terminally linked substrates, and recently it was shown that these exoglycosidases could sequentially remove sugars from complex N-linked glycans (27
). Additionally, these enzymes are surface associated and have been shown to be more effective at deglycosylating a substrate when bound to the bacterial surface (7
). Glycosylation of host proteins can affect their stability, resistance to proteolysis, and functional activity (43
). Therefore, it is possible that deglycosylation of a host glycoconjugate by the action of these three exoglycosidases is important for pneumococcal virulence in vivo
. This is supported by the fact that these exoglycosidases deglycosylate human secretory component, immunoglobulin A, and lactoferrin, three human glycoconjugates thought to be important for pneumococcal clearance (27
). The functional consequence of the deglycosylation of these host proteins or of other host proteins by these exoglycosidases, however, has not been examined.
In this study, we show that NanA, BgaA, and StrH work on the same pathway/target to promote resistance to opsonophagocytic killing by reducing complement deposition on the pneumococcus. As these enzymes function as exoglycosidases, we concluded that deglycosylation of a host glycoconjugate(s) promotes this resistance.
Using antibody binding assays and blocking antibodies to complement receptor 3, we showed that NHS is a source of both complement and antipneumococcal antibodies. Glycosylation of antibodies is known to be critical for their ability to fix complement (1
); therefore, we wanted to test the hypothesis that deglycosylation of antibodies could help promote resistance to complement deposition. Using serum depleted of IgG, the predominant antibody isotype in NHS, we still saw a significant effect of NanA in opsonophagocytic killing assays. This suggests that IgG is not necessary for the effect of NanA in NHS. Additionally, using heat-inactivated NHS as a source of antibodies, we did not see an increase in phenotype for a nanA
mutant when added to BRS. This suggests that a heat-labile component in NHS is responsible for promoting the greater effect of nanA
in this serum source. Therefore, the difference between NHS and BRS could be due to increased exoglycosidase substrate specificity, since S. pneumoniae
is a pathogen adapted to humans and glycosylation patterns can vary between species (17
). Therefore, we believe that antibodies in NHS contribute to the deposition of complement on the pneumococcal surface but are not being acted upon by pneumococcal exoglycosidases. This is further supported by the fact that we do not see deglycosylation of human IgG using lectins that bind specifically to terminal mannose (data not shown). Thus, we conclude that deglycosylation of a serum component in NHS downstream of antibody binding and subsequent complement activation is important for resistance to complement deposition on the pneumococcus.
There has been a recent interest in studying glycosylation of complement components, and in some instances glycosylation is important for the function of these proteins (11
). Most complement components are synthesized predominantly in the liver and contain complex biantennary glycans, as displayed in Fig. (44
). Therefore, deglycosylation of a complement component by NanA, BgaA, and StrH could provide a direct mechanism to promote resistance to complement deposition. Reducing the function of complement components, however, is only one mechanism whereby deglycosylation can directly affect complement deposition. Glycosylation can also be important for resistance to proteolysis; therefore, deglycosylation of a complement component(s) could increase its turnover by increasing its susceptibility to serum proteases (47
). Opsonophagocytic killing assays looking at the effect of NanA on the alternative pathway suggest that deglycosylation affects at least this pathway of complement activation (Fig. ). This does not, however, rule out a role for pneumococcal exoglycosidases in the classical pathway. In fact, since the effect of NanA on the alternative pathway was not as dramatic as that seen in complete NHS (containing both classical and alternative pathways intact) (Fig. ), this suggests that NanA may have an effect on the classical pathway as well. Both the classical and alternative pathways of complement activation converge at the formation of the C3 convertase and deposition of C3 onto the bacterial surface. As C3 is shared by both complement pathways, it would seem to be a likely target for these exoglycosidases; however, this complement component does not have complex-type N-linked glycans (44
). Therefore, the pneumococcal exoglycosidases could act upstream on a regulatory component of complement that affects C3 deposition. Alternatively, this resistance could require deglycosylation of multiple complement components or have an indirect role in promoting resistance to opsonophagocytic killing. With over 30 serum proteins involved in the complement cascade, however, pinpointing the factor reducing complement deposition is complex.
In summary, we have shown that NanA, BgaA, and StrH promote resistance to opsonophagocytic killing by increasing the ability of the pneumococcus to evade complement deposition and subsequent phagocytic killing. As complement contributes to both antibody-dependent and -independent clearance, this may be a mechanism to limit the effectiveness of both innate and adaptive immunity. Since glycoside hydrolases are not a unique feature of S. pneumoniae, deglycosylation may be a conserved strategy to evade the immune response.