expresses endogenous intracellular amylase, which is not secreted, and is thus unable to metabolize starch as a primary nutritional source unless it binds host salivary amylase (13
). α-Amylase hydrolyzes starch at α-1,4 glucosidic linkages to form various maltooligosaccharides and eventually maltose. Once bound to the streptococcal surface, host amylase retains its enzymatic activity, allowing the organism to metabolize dietary starch efficiently (13
). The presence of both starch and amylase in the oral cavity likely allows S. gordonii
to persist and proliferate. Previous studies identified genes in S. gordonii
that were differentially expressed in response to exposure to human whole saliva (16
). Considering that amylase is one of the most abundant components of human saliva, this interaction could mediate differential gene expression and could serve as an important factor for bacterial host adaptation.
The results of this study provide evidence that the binding of amylase to the surface of S. gordonii elicits differential gene expression in the bacterial cell. This appears to result in an alteration of the bacterial phenotype that may be advantageous for bacterial survival in the oral environment. Here we show evidence that after exposure to salivary amylase, S. gordonii is better able to tolerate an acidic environment. The bacterium also appears to grow better in the presence of bound amylase. Salivary amylase upregulates genes involved in the synthesis of fatty acids, which are key components of bacterial membranes. Considering the fact that in all the experiments we performed, the presence of complex carbohydrate as a source of nutrition in the medium was avoided through the use of a chemically defined medium, it is clear that S. gordonii not only binds host amylase but also responds to its binding by differential regulation of gene expression and phenotype adjustment.
The amylase-induced upregulation of FAS genes could be a mechanism for the “fine tuning” of bacterial metabolism to redirect acetyl-CoA from the citric acid cycle to fatty acid synthesis for membrane building and bacterial proliferation. In the first step of fatty acid synthesis, acetyl-CoA from the citric acid cycle receives a CO2
group and forms malonyl-CoA; this involves the action of acetyl-CoA carboxylase (accD
). Next, 3-oxoacyl-(acyl carrier protein) synthase III (fabH
) initiates the condensation of acetyl-CoA with malonyl-CoA and attaches it to acyl carrier protein (acpP
), creating acetoacetyl-ACP (9
). Then 3-ketoacyl-(acyl-carrier-protein) reductase (fabG
) reduces the 3-carbon ketone of acetoacetyl-ACP to hydroxyl and converts it to hydroxybutanoyl-ACP. These steps are repeated to elongate the fatty acid chain. The acyl carrier protein is essential for the cycle, since it carries the synthesizing fatty acid chain. In fact, acpP
is an essential gene in E. coli
that possesses several promoters, ensuring that mutation of one of the promoters does not prevent the transcription of fully functional protein (51
). Another essential gene, accD
, previously reported in E. coli
temperature-sensitive mutations, blocked fatty acid synthesis (9
). KAS III-3-oxoacyl-(acyl carrier protein) synthase III, encoded by fabH
, produces only C4
compounds and cannot make long-chain fatty acids (47
), suggesting that this enzyme initiates fatty acid synthesis (9
, encoding 3-ketoacyl-(acyl-carrier-protein) reductase, is also an essential gene; its transcriptional termination in Salmonella enterica
serovar Typhimurium blocked cell growth (51
). Considering the fact that several amylase-induced genes affect bacterial growth and proliferation, the findings of the present study suggest that upregulated fatty acid synthesis genes in amylase-exposed S. gordonii
modulate the initiation of fatty acid synthesis and stimulate proliferation. Thus, the effect of upregulation of FAS genes may be to increase the overall amount of fatty acids, but it does not change the proportion of fatty acids in the membrane (see Table S1 in the supplemental material).
We established that the cell wall-attached protein AbpA plays a role in the amylase-induced gene response. Insertional inactivation of abpA drastically reduced the binding of salivary amylase to the bacterial surface and eliminated amylase-induced gene expression and phenotypic response.
Considering the fact that AbpA is a cell wall-associated protein localized at the cell surface (41
), the question of its ability to convey a signal upon binding to salivary amylase arises. AbpA is maximally expressed during the mid-log phase of S. gordonii
). Recent sequence analysis indicates that AbpA shares homology with several AbpA-like proteins produced by other amylase-binding oral streptococci. To date, no conserved domains have been identified (see Fig. S2 in the supplemental material). The AbpA protein is translated with a leader peptide directing its secretion outside the bacterial cell, where it attaches to the cell surface to serve as the receptor for amylase (H. Wu, personal communication). It has been suggested recently (28
) that sortase B (SrtB), encoded by srtB
and located immediately downstream of abpA
, plays a role in the attachment of AbpA to the cell wall. AbpA does not have a classic sortase B recognition domain, such as NXZTN, as described for Listeria monocytogenes
, or NPQTN, for Staphylococcus aureus
). It apparently does possess a novel, yet to be identified sortase recognition motif at the C terminus (28
), which is likely responsible for the covalent binding of AbpA to the cell wall.
AbpA is present on the surface of the nascent cell wall in the annular and polar zones but is later shed into the environment (42
). Its function, other than mediating the binding of amylase to streptococci, remains unknown. The fact that AbpA is present only at cell division sites and is later shed into the environment leads us to postulate that AbpA may play a role in conveying information about the environment to new daughter cells during cell division. Although AbpA is an extracellular cell wall-attached protein and lacks a membrane-spanning domain, it could potentially function as a transient coreceptor or signal transducer. Consequently, it is possible that AbpA is part of a putative signal transduction system. Analysis of the AbpA protein sequence suggests several sites for protein-protein interaction, phosphorylation, and ATP binding.
AbpA may interact with another component(s) of a putative transduction system, such as an integral membrane protein or a transport system. The latter, activated by AbpA, could carry the signal to the transcription machinery. In fact, our microarray analysis revealed two candidates for such an interaction. Two genes downregulated upon exposure to salivary amylase encoded an IMP (SGO_0091) and a PTS (SGO_1890). One or both could be involved in transmitting the signal. The IMP is a predicted functional partner of a TetR repressor family regulator (SGO_0090) and could be a member of the putative t
omponent signaling s
ystem (TCS) (34
). Interestingly, the downregulation of this predicted TetR repressor system could be linked to upregulation of the FAS operon through the release of the suppression effect (31
). However, the function of the IMP as part of an amylase-triggered transduction system, as well as its role in the regulation of the FAS operon, has yet to be determined experimentally. The second candidate for such a possible regulatory role is a PTS (SGO_1890), which was downregulated upon exposure to salivary amylase. Multiple phosphotransferase systems responsible for carbohydrate transport have been shown previously to be involved in signal transduction in Gram-positive bacteria (25
) and E. coli
). Thus, it is possible that AbpA could interact with the PTS on the cell surface level and initiate signaling through this carbohydrate transport system. The actual roles of these candidates in amylase-induced signal transduction have yet to be determined.
While wild-type S. gordonii showed increased resistance to triclosan after exposure to native versus denatured amylase, peculiarly, the AbpA mutant, when exposed to amylase, showed less resistance to triclosan than did the control. Since triclosan binds to and inhibits the enzymes of fatty acid synthesis, it is logical that increased expression of FAS genes in the WT after amylase exposure would cause increased levels of FAS enzymes that overcome the effect of triclosan. We can only speculate on the possible explanations for why the AbpA mutant showed the opposite effect. Perhaps the absence of AbpA protein on the surface of the bacterium results in an as yet unknown perturbation that affects the bacterial response to the presence of unbound amylase in the environment. AbpA may not be the only protein that is involved in signaling following the binding of amylase. Perhaps the deletion of AbpA from the signaling system creates a countersignal in the presence of amylase that actually reverses triclosan resistance. Studies to identify other components that may be involved in amylase-dependent signaling are in progress. We anticipate that information on the regulatory pathway will be valuable in the interpretation of this phenomenon.
Identification of the mechanisms of amylase-induced signaling in differential gene expression has implications for understanding and controlling bacterium-host interactions and also for the detection of new paradigms for the regulation of bacterial fatty acid synthesis that may stimulate the development of novel antimicrobial agents.
Now that we have demonstrated that amylase may serve as an environmental signal for the modification by S. gordonii of its gene expression and phenotype in order to adapt to a changing host environment, other questions arise. Does amylase binding provide an advantage for S. gordonii in competition with other bacterial species within the niche? Does it aid in colonization by other species? We observed that exposure to salivary amylase stimulates S. gordonii growth, as well as its survival under acidic conditions, which are often found in the oral environment. Does the interaction of S. gordonii with amylase decrease colonization by pathogenic bacteria, such as Streptococcus mutans, or does it benefit S. mutans by delivering an available source of utilizable carbohydrates? These results suggest further avenues for research to determine the fundamental role of S. gordonii in bacterium-host interaction and oral colonization.