The most-studied ABP of S. gordonii
CH1, AbpA (20 kDa), is an extracellular cell wall-associated surface protein that is maximally expressed during mid-log phase of bacterial growth (32
). AbpA appears to be an essential receptor for α-amylase binding to the bacterial surface. Inactivation of AbpA entirely eliminates α-amylase binding to the bacterium (35
). Immunogold electron microscopy studies localized α-amylase binding to the cell division septum and poles on the streptococcal surface (32
). Cells in the exponential phase of growth bind more α-amylase than cells in stationary phase. Thus, it appears that AbpA is localized to the nascent cell wall, and as the cell matures, it is shed into the supernatant. Abundant amounts of AbpA protein are present in the supernatants of bacterial cultures. α-Amylase-treated bacteria do not appear to display morphological changes, as observed by electron microscopy (32
), which indicates that α-amylase binding does not cause overall perturbation to the bacterial cell surface.
Identification of S. gordonii
biofilm determinant genes was conducted by screening biofilm-deficient mutants generated following Tn916
). These studies suggested abpA
as a potential biofilm determinant gene. The mutants selected in this study were found to have similar growth characteristics. Thus, impairment of biofilm formation was not likely due to growth defects (36
). Another study from our laboratory found that an abpA
-deficient mutant showed impaired biofilm formation in a standard microtiter biofilm assay as well as in a flow model system containing 25% saliva in distilled water (35
). These findings suggest that the AbpA protein modulates adhesion and biofilm formation of oral streptococci in vitro
. Not only did mutation of abpA
reduce biofilm formation, it also reduced growth of S. gordonii
in human saliva (35
Mutation of abpA
also impaired the ability of S. gordonii
to grow with starch as the predominant carbon source (35
). S. gordonii
does not produce extracellular α-amylase (35
). Thus, the ability to bind salivary α-amylase to the bacterial surface via AbpA appears critical for bacterial growth from starch (35
). It is possible that S. gordonii
and other ABS contribute to oral microbial colonization by metabolizing dietary starch and providing nutrition for non-ABS species within the biofilm. In contrast, this interaction would make ABS better competitors against pathogenic species. This, however, has yet to be established and requires further studies using multispecies biofilm models.
studies of the AbpA mutant strain conducted on pathogen-free Osborne-Mendel rats showed quite contradictory results to those of the in vitro
studies described above. Curiously, AbpA mutant strains sometimes colonized teeth to a better extent than did the wild type, especially when the rats were fed a sucrose-rich diet and, to some degree, when they were fed a starch diet (37
). Interestingly, the activity of glucosyltransferase G (GtfG), an enzyme thought to promote biofilm formation (38
), was greater in the AbpA mutant strains, which may have been responsible for the slightly augmented cariogenicity of the mutant strain (37
). Further studies of AbpA confirmed its interaction with GtfG of S. gordonii
, in which AbpA was found to form complexes with GtfG and salivary α-amylase (34
). However, this interaction was suggested to increase GtfG enzymatic activity (34
The AbpA-α-amylase complex is also known to increase enzymatic activity of GtfB of S. mutans
). To explain this, it was suggested that a conformational change occurs in GtfG following interaction with the α-amylase-AbpA complex. This may result in a change in the structure of the synthesized glucan which has been shown to occur in the presence of starch hydrolysates (34
). These findings suggest that the α-amylase-AbpA complex represents an interaction that involves other yet-to-be-determined factors, all of which may modulate bacterial adhesion and colonization.
It is also possible that the α-amylase-streptococcus interaction functions in ways other than in nutrition or adhesion. In order to further investigate the mechanism and significance of α-amylase binding to the bacterial surface via AbpA, studies of gene expression of S. gordonii
following the binding of salivary α-amylase were conducted using microarray analysis (40
). When the bacterium was cultured in a minimal medium, a total of 33 genes were differentially expressed following exposure to purified salivary α-amylase. The greatest change in expression was observed for genes involved in fatty acid synthesis. Several of these genes were highly upregulated in response to α-amylase binding compared to the control exposure, heat-denatured salivary α-amylase. Not only was gene expression altered when the bacteria were exposed to native α-amylase, there were also changes in bacterial phenotype, as observed by increased bacterial growth, increased resistance to low pH, and increased resistance to the detergent triclosan. Mutation of abpA
, which abolishes the binding of α-amylase to the bacterial surface, eliminated the gene responses and phenotype changes, suggesting a role for the AbpA protein in this response. These findings suggest that α-amylase binding to AbpA initiates a signal resulting in differential gene expression. This may serve as an environmental sensing mechanism specific for the oral environment. Identification of other proteins that interact with AbpA will be crucial for understanding this mechanism.
Analysis of the AbpA protein sequence to predict the secondary structure identified several sites that may potentially participate in AbpA-protein interaction and signaling. Two predicted N-myristoylation sites at residues 83 to 88 and 147 to 152 may be involved in protein-protein interaction. A tyrosine phosphorylation site (residues 32 to 39) and an ATP/ADP-binding site (residues 121 to 128) may be involved in phosphorylation and signal transduction. These findings support a possible role for AbpA in environmental surveillance through α-amylase binding and interaction with other components of a putative signaling system to modulate gene expression and adaptation to the host environment.
Furthermore, multiple sequence alignment of amino acid sequences of AbpA-like proteins indicates several highly conserved areas that may mediate α-amylase binding and the interaction with other bacterial proteins (see Fig. S2 in the supplemental material). However, though there is evident homology in AbpA-like proteins, the α-amylase-binding activity of these hypothetical proteins has yet to be experimentally established. In fact, that AbpA-like proteins are conserved among some streptococcal species but not present in all the strains among the same species suggests that AbpA-like proteins likely have a common origin and perhaps were acquired by horizontal gene transfer.
Regulation of AbpA expression in S. gordonii
appears to involve at least two mechanisms: catabolite repression in response to glucose and substrate induction via maltose/maltodextrin transport (41
). The promoter region of the abpA
gene possesses the catabolite responsive element (cre
), which is the binding site for LacI/GalR transcriptional regulators. It has been determined that the RegG protein, a LacI/GalR transcriptional regulator and homolog of catabolite control protein A (CcpA), is responsible for the catabolite repressive effect on abpA
). Mutation of regG
in S. gordonii
eliminated repression of abpA
transcription in the presence of glucose, suggesting that RegG is a transcriptional regulator responsible for repression.
A recent study found that that the products of starch hydrolysis produced from the action of salivary α-amylase, particularly maltose and maltotriose, upregulate AbpA expression in S. gordonii
). While RegG represses transcription of abpA
in the presence of glucose, the identity of the transcriptional regulator that activates the expression of abpA
in the presence of maltose/maltodextrin is presently unknown. Interestingly, previous studies showed that maltotriose enhanced the binding of S. gordonii
to α-amylase-coated hydroxylapatite (43
). Thus, the products of starch hydrolysis by α-amylase on the surface of the streptococcus may increase the expression of AbpA to maximize binding of the host enzyme to the bacterial surface for better utilization of a significant dietary nutrient source or to increase adhesion of the bacterial cell to a host surface. Because starch and salivary α-amylase are abundant in the oral cavity, it seems that AbpA expression would be beneficial for bacterial colonization and proliferation. Multispecies biofilm models of oral colonization would be useful to determine the effect of increased expression of AbpA on overall bacterial survival, bacterial competence, and effects on other oral bacterial species.