Carbohydrate modification plays an essential role in pneumococcal pathogenesis; carbohydrates are required for growth of the bacteria and compose the pneumococcal capsule, which is essential for disease. The finding that BgaA probably acts as an adhesin supports the hypothesis that pneumococcal glycosidases contribute to colonization independently of their enzymic activity. This hypothesis is also supported by the recent finding that the lectin-like domain of NanA promotes pneumococcal adherence and invasion of human brain microvascular endothelial cells (Uchiyama et al., 2009
). Our current study indicates that NanA reveals a receptor on airway epithelial cells. Together, these findings suggest distinct roles for NanA in adherence to different cell types, probably due to the differential distribution of receptors.
Pneumococcal adherence appears to be complex. Several mechanisms of pneumococcal adherence have been identified previously (Cundell et al., 1995b
; Hammerschmidt, 2006
; Zhang et al., 2000
). Given the importance of bacterial adherence to colonization and different stages of disease, it is not surprising that S. pneumoniae
possesses multiple adherence mechanisms. Adding another layer of complexity is the fact that the expression of some pneumococcal adhesins differs between strains (Bagnoli et al., 2008
; Moschioni et al., 2008
; Paterson & Mitchell, 2006
). Furthermore, it is likely that different host cells vary in their expression of receptors for adherence. Thus, we can predict that the relative contribution of different mechanisms to adherence is probably dependent on both the host-cell type and the pneumococcal strain. We demonstrated that bgaA
contributed significantly to adherence of R6, D39 and C06_18. The residual adherence observed is probably due to expression of other adherence mechanisms. We were unable to demonstrate a phenotype for two further strains, TIGR4 and 384. It is unknown whether these strains express other adherence mechanisms that compensate completely for the absence of BgaA or whether this protein does not contribute to adherence of these strains. Our data allow us to conclude that BgaA contributes to adherence of at least some pneumococcal strains to all airway epithelial cells tested, including primary NHBE cells.
Our experiments demonstrated that rBgaA inhibited pneumococcal adherence and bound directly to epithelial cells, both key characteristics of adhesins. Analysis of the BgaA sequence reveals features supporting the hypothesis that this protein may have functions besides that of a β-galactosidase (). BgaA is approximately twice the size of the majority of other predicted β-galactosidases. The N-terminal region of the protein (aa 110–1136) shares significant sequence similarity with other β-galactosidases, but the C-terminal half of the protein, with the exception of the surface-localization marker, has no known function and lacks sequence similarity to most predicted β-galactosidases. The uncharacterized, predicted β-galactosidases from some other streptococcal species, including Streptococcus oralis
; AEDW01000000), Streptococcus mitis
(ATCC 6249; AEEN01000000), Streptococcus sanguinis
(ATCC 49296; AEPO01000000) and Streptococcus gordonii
(Vickerman et al., 2007
), are approximately the same length as BgaA and share high levels of amino acid identity over 2166 aa. Whether BgaA is a member of a novel family of β-galactosidase adhesins is unknown.
The region of BgaA that mediates adherence is currently unknown. The C-terminal region may confer the Galβ1,4GlcNAc enzyme specificity or perform a second independent function, for example mediating adherence. Homology searches using the C terminus of BgaA did not reveal a function for this region; however, it does contain an 80 aa partial G5 domain (aa 2083–2162). G5 domains are present in many enzymes that bind GlcNAc and have been proposed to contribute to this binding (Bateman et al., 2005
). This G5 domain may contribute to BgaA-mediated adherence, either directly by enabling binding to surface-associated GlcNAc or indirectly by contributing to the restricted cleavage specificity of BgaA. This may in turn enable BgaA to adhere to glycan substrates that it is unable to cleave. G5 domains present in staphylococcal surface proteins have also been proposed to mediate intercellular adhesion, although a single repeat was insufficient for this activity (Conrady et al., 2008
). Also, within the C-terminal region are several bacterial Ig folds (pfam07532), which are often identified in both glycosidases and other surface proteins (Marchler-Bauer et al., 2007
). The role of the G5 domain and Ig folds will be the focus of further research.
Generation of the active-site mutant R6BgaAE564Q demonstrated that residue 564 is essential for enzymic activity and confirmed that β-galactosidase activity is not required for BgaA to mediate pneumococcal adherence. Mutation of the corresponding residue in E. coli
was reported to reduce substrate binding (Cupples et al., 1990
); however, it is unknown whether this is true in S. pneumoniae
. R6BgaAE564Q adhered to epithelial cells at a significantly higher level than the parental strain, raising several possibilities for the increased adherence. If the active site is responsible for adherence and the glutamine substitution at aa 564 does not reduce substrate binding, Galβ1,4GlcNAc could act as an additional receptor on the epithelial-cell surface. Alternatively, if the region mediating adherence is distinct from the active site, it is possible that the substrate is still bound efficiently and that the inability to cleave this linkage may provide more time for increased interaction between the adhesin and receptor. A third possibility is that substrate binding is reduced, and that BgaA which is not interacting with the substrate has increased ability to bind to the receptor.
The receptor for BgaA on the epithelial surface is currently unknown. Previous studies have reported that S. pneumoniae
adheres to a number of different host-cell glycoconjugates, including GalNAcβ1,4Gal found in ganglioside GM1 (Krivan et al., 1988
). However, our studies suggest that BgaA is binding to the glycan portion of a glycosphingolipid, but not GM1. It has previously been proposed that the initial adherence of S. pneumoniae
to resting epithelial cells is mediated by a glycoconjugate receptor (Cundell et al., 1995a
; Hammerschmidt, 2006
); therefore, NanA and BgaA may contribute to the initial adherence of S. pneumoniae
to human epithelia. Mutation of bgaA
was shown previously to have a small but significant effect in a model of pneumonia, but the role of this protein in colonization was not examined (Robertson et al., 2002
). We observed no reduction in the ability of a bgaA
mutant to colonize the oro-nasopharynx or to bind to mouse airway epithelial cells, suggesting that the BgaA receptor is human-specific.
Collectively, these data support the hypothesis that pneumococcal glycosidases have functions independent of their enzymic activity. Glycosidases are probably upregulated during colonization, as the concentration of free carbohydrates in the nasopharynx is normally low and the bacteria require carbon for growth. Additional functions for these proteins may efficiently co-ordinate growth and other processes, including adherence, that are required to establish and maintain colonization.