Exoglycosidases in pathogenic bacteria are important enzymes for infection and colonization of host cells. The essential role of BgaA, a surface-associated β1,4-galactosidase of S. pneumoniae
, was demonstrated in degalactosylation of human glycoconjugates for colonization and/or pathogenesis (19
). In contrast to typical β-galactosidases that comprise approximately 1,000 amino acids and are cytoplasmic proteins, S. pneumoniae bgaA
encodes a 2,235-amino-acid polypeptide with a putative signal sequence at the N terminus (41
). In S. pneumoniae
BgaA, 365 residues located in the N-terminal half of the protein show homology to the E. coli
and Streptococcus thermophilus
β-galactosidases, but the remainder of the protein displays no homology to the other proteins. Interestingly, the complete genome sequencing of S. pneumoniae
) reveals that S. pneumoniae
has another β-galactosidase of 595 amino acids, BgaC, which shows relatively high homology to eukaryotic β-galactosidases as well as to pathogenic microbial enzymes.
In the present study, we showed that the β-galactosidases encoded by bgaA
of S. pneumoniae
R6 differ in their substrate specificities. While S. pneumoniae
BgaA releases a terminal galactose β1,4 linked to GlcNAc in sugar chains of glycoproteins, BgaC shows high substrate specificity only for Galβ1-3GlcNAc (Table ). It was previously reported that BgaC proteins of B. circulans
and X. axonopodis
pv. manihotis could cleave terminal galactoses of Galβ1-3GlcNAc or Galβ1-4GlcNAc, but the specific hydrolysis of Galβ1-3GlcNAc was 1,000-fold more efficient than that of Galβ1-4GlcNAc (11
). Moreover, BgaC proteins from C. piscicola
and B. circulans
had specificity for galactose linked to both GalNAc and GlcNAc with a β1,3-glycosidic bond (15
). In addition, BgaC of B. circulans
also has activity for galactose linked to GlcNAc modified with Neu5Ac or Fuc (11
). In contrast, BgaC of S. pneumoniae
cleaved only the terminal galactose linked to GlcNAc that was not modified with Fuc or Neu5Ac (Table ). The analysis of BgaC activity with various sugar chains indicates that S. pneumoniae
BgaC is a β-galactosidase with high oligosaccharide specificity for the β1,3-glycosidic bond rather than the β1,4-glycosidic bond with GlcNAc.
Localization analysis of BgaC indicated that it is expressed on the cell surface, even though BgaC does not have a typical signal peptide and LPXTG motif or choline-binding repeats on its amino or carboxyl terminus, which are required for anchorage of proteins on the cell surface. Recently, it was reported that there was a new class of virulence factors that did not have anchors but rather underwent surface-located adhesion and invasions (7
). Expression of BgaC on the cell surface may belong to this new class of virulence factors and play a role in adherence to the host cell by exposing GlcNAc in glycolipid. Amino acid sequence alignment and motif scanning of BgaC revealed an interesting motif, homologous to the NHL repeat sequence (28
), located at amino acids 562 to 573. NHL is defined by amino acid sequence homologies among Ncl-1, HT2A, and Lin-41 proteins (28
) and is a conserved structural motif present in a large family of growth regulators. According to structural model analysis, the NHL domain is expected to be involved in protein-protein interaction (28
). Bacterial NHL repeat domains are homologues of the YWTD repeat family of proposed β-propeller domains, which are widespread in eukaryotic extracellular proteins (29
Adherence is an initial stage of the pathogen invasion into the host cells and involves a number of ligands, such as oligosaccharides and protein adhesins. In S. pneumoniae
, NanA, BgaA, and StrH act sequentially to remove sialic acid, galactose, and N
-acetylglucosamine and expose mannose on human glycoproteins for binding by pneumococci, suggesting that S. pneumoniae
deglycosylates host airway defense glycoproteins, thereby enhancing adherence of S. pneumoniae
to airway components (18
). Interestingly, although adherence of nonencapsulated nanA
mutants to epithelial cells was decreased in vitro, in vitro results revealed no decrease in the colonization of bgaA nanA strH
triple exoglycosidase mutants (19
). The surface-anchored pullulanases of S. pneumoniae
recognize and bind multivalently to host glycogen, thus increasing interaction with alveolar type II cells in mouse lung tissue (36
). Moreover, sugar moieties such as lacto-N
-neotetraose and asialoganglioside GM1 can contribute to the adherence of pneumococci to host cells (33
). These results suggest that exoglycosidases can unmask receptors upon hydrolysis of targets.
It was reported that the disaccharide unit of a glycoconjugate receptor for pneumococci attaching to human pharyngeal epithelial cells was Galβ1-3GlcNAc (1
). Adherence inhibition tests with neolactotetraose and lactotetraose indicated that pneumococci prefer binding to the lactotetraose structure (1
). This lactotetraose structure is one of the major core structures of vertebrate glycosphingolipids, suggesting that BgaC can remove galactose from β1,3-linked GlcNAc in lacto-N
-tetraose of the glycolipid that could serve as a possible binding site for S. pneumoniae
infection. If that is the case, absence of BgaC protein caused by gene deletion or by antibody treatment would increase adherence rather than decrease adherence, as observed in Fig. and .
Notably, we report for the first time that surface-associated exoglycosidase BgaC can hydrolyze and remove the host cell ligand so that binding of pneumococci to the host is decreased in vivo and in vitro. This appears to be analogous to the function of influenza virus sialidase, which causes release from host cells by cleaving sialic acid, the sugar residue important for binding to host receptor (8
). On the other hand, the bgaC
deletion mutant could adhere more efficiently than the wild-type by some other factors. For example, galactoside hydrolysis by BgaC might trigger mucin synthesis; this would inhibit binding of pneumococci to the epithelial cells since infection with bacteria induces mucin synthesis in epithelial cells as an antimicrobial response of the innate immune system to protect the host (10
). More work is required for investigation of key factors that might play a role in BgaC-mediated adherence.
During the progression from colonization to invasive disease, adaptation to different environmental niches in the host is mediated by changes in the expression of key virulence factors. Modulation of gene expression for a few virulence factors in S. pneumoniae
has been reported, although the exact mechanisms involved in S. pneumoniae
are not well characterized (17
). Through microarray analysis, we also observed that the bgaC
gene was induced 2.54-fold and slightly decreased to 0.91-fold in A549 human lung cells infected with wild-type D39 after 10 min and 2 h of infection, respectively (data not shown). This suggests that BgaC is immediately induced upon contact with the host cells, indicating the involvement of BgaC during host cell invasion.
The present study showed that BgaC could hydrolyze the host galactoside moiety and thus affect adherence to the host cells and viability in phagocytes. The results indicate that BgaC is localized on the outer surface to hydrolyze β-galactosides on the surfaces of host cells and subsequently to remove ligands responsible for pneumococcal binding to the host cells. The underlying mechanism of BgaC expression and its role in pathogenesis should be investigated. This study will provide further insight into one of the diverse microbial strategies employed during pathogenesis.