The formation of acid end-products during fermentation of dietary sugars by acidogenic bacteria is considered the driving force to generate a cariogenic dental biofilm. However, the effect of this stressful acidic environmental condition on protein expression has been limited to in vitro studies using S. mutans
and the analyses have also focused on cellular protein change. In the present study, we investigated in vivo the protein change in the ECM of PLB since sucrose, the most cariogenic dietary carbohydrate, promotes decreases in pH and is also a substrate for EPS synthesis. The ECM has an important role in biofilm structure and function [Sutherland, 2001
To evaluate the protein composition of the ECM we used short alkali extraction at low temperature to avoid cellular membrane lysis [Fox and Dawes, 1970
]. Alkali extraction of ECM components from PLB formed in the presence of sucrose is required to dissolve the high concentration of insoluble EPS usually found in this biofilm [Cury et al., 2000
], allowing the extraction of substances that could be trapped in the matrix.
Therefore, the absence of calcium-binding proteins in the ECM of PLB formed under exposure to sucrose (table ) should not be due to their being trapped in the insoluble EPS. Thus, the presence of calcium-binding proteins, exclusively in ECM of the PLB formed in the absence of sucrose, supports the view that these proteins, in addition to plaque Ca binding [Rose et al., 1993
], may act as organic mineral reservoirs in PLB [Paes Leme et al., 2006
, for a review]. However, calcium-binding proteins may have other functions which have not yet been reported. On the other hand, prolactin-induced protein was identified in both conditions; however, 2 isoforms were uniquely found and 3 of them were found in greater abundance in the matrix of PLB formed with sucrose (tables , ).
It should be emphasized that several proteins found in the ECM of PLB formed in vivo, such as enolase, translation elongation factors, pyruvate kinase, GroEL, DnaK, and other proteins previously thought to be confined to the cytosol are associated with the cell surface or secreted into the external milieu [Joe et al., 1994
; Pancholi and Fischetti, 1998
; Len et al., 2003
; Wilkins et al., 2003
; Len et al., 2004a
; Black et al., 2004
; Nandakumar et al., 2005
; Paddick et al., 2006
]. Therefore, the presence of these proteins in the extracellular milieu may not have resulted from cellular lysis during extraction of the extracellular proteins.
Overall, our results showed that specific bacterial stress response proteins are differentially expressed in ECM of PLBs depending on the availability of sucrose (tables , ; fig. ). These proteins have been found intracellularly in in vitro studies of S. mutans
in response to environmental changes [Svensäter et al., 2000
; Quivey et al., 2000
; Wilkins et al., 2002
; Welin et al., 2003
; Len et al., 2004a
], and in the present study they were found in the ECM. The extracellular presence of these proteins may be a consequence of cell death, considering that the PBL collected was 14 days old.
Proteins linked to carbohydrate metabolism, such as pyruvate kinase and mannose-specific EIIAB, were upregulated in PBL formed under sucrose exposure (table ) in agreement with an in vitro report [Abranches et al., 2006
]. Enolase was also identified, one of the isoforms being identified only in the presence of sucrose and the other being downregulated in the presence of sucrose, but the biological functions of the differential expression of the isoforms remain to be clarified. The presence of these proteins might be an indicator of enhanced carbohydrate metabolism in the PLB formed in the presence of sucrose.
The upregulation of ATP synthase beta chain in PLB formed under sucrose exposure (table ) could reflect bacterial adaptation to acidic stress conditions imposed by eight decreases in pH per day. Even though this enzyme was found in ECM, the result agrees with the predominance of aciduric microorganisms in biofilms formed in situ [Pecharki et al., 2005
; Ribeiro et al., 2005
; Tenuta et al., 2006
]. Also, the stress conditions promoted by low pH induced upregulation of DnaK in the PLB formed under sucrose exposure (table ) in agreement with Jayaraman et al. 
, who showed increased levels of dnaK
mRNA and intracellular DnaK in S. mutans
in a continuous chemostat culture in response to acid shock. Furthermore, most proteins involved in the translation function were also upregulated, such as the translation elongation factors (EF-Tu). Thus, in addition to their regular function in translation elongation, these proteins could behave like chaperones toward protein folding and protection from stress as previously reported in Escherichia coli
[Caldas et al., 1998
], which could explain their higher levels under the low pH conditions maybe caused by sucrose fermentation (table ).
However, GroEL, another chaperone, behaved differently from DnaK, since it was downregulated in PLB formed in the presence of sucrose (table ). This protein is elevated in the intracellular compartment of S. mutans
subjected to an acidic environment [Wilkins et al., 2002
; Len et al., 2004a
]. Apparently, the different isoforms of GroEL found may be regulated by different pathways under different stress conditions as in our study; the control PLB was formed under nutrient-limited conditions, confirming a previous report [Len et al., 2003
Interestingly, NADP-specific glutamate dehydrogenase was the protein most highly expressed under sucrose exposure (table ). This enzyme is involved in the intracellular metabolism of amino acids [Wilkins et al., 2001
]. The ammonia produced could be an alternative mechanism used by aciduric bacteria to increase the pH of acidified cytoplasm. The reason for the extracellular occurrence of this protein and its role in this location remain to be investigated, but the fact that it was 30-fold more highly expressed in PLB under sucrose exposure suggests that its presence in the ECM is not an artifact of cell lysis because the control PLB was extracted under the same conditions. It can be speculated whether this protein could act as an adhesin by binding to immobilized host and bacterial proteins through the glutamate-binding domain [Joe et al., 1994
Many bacterial or salivary protein isoforms were identified in this study. However, neither the role nor the nature of different isoforms, which possibly resulted from processing by bacterial proteases or posttranslational modification, is clear yet. For instance, protein phosphatases, which are important in the phosphorylation process and signal transduction in other organisms [Mukhopadyay et al., 1999
; Vijay et al., 2000
], were previously found to be more abundant at low pH [Wilkins et al., 2003
] and they could have an effect on the PLB formed under sucrose exposure.
It should be emphasized that the proteins found in the PLB could originate from the host and different bacteria; therefore, it was necessary to include several species such as Homo sapiens, Actinomyces, Fusobacterium nucleatum, Lactobacillus, Porphyromonas gingivalis, Prevotella intermedia, Streptococcus anginosus, Streptococcus equi, Streptococcus gordonii, Streptococcus mitis,S. mutans, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus salivarius, Streptococcus sanguinis, Streptococcus sobrinus, Neisseria subflava, Veillonella parvula, and other species, such as Bacillus subtilis, Staphylococcus aureus, and also Pseudomonas aeruginosa to improve the protein identification. However, the fact that the majority of plaque bacteria are not represented in the sequence databases could explain the lack of the identification of many spots.
In summary, these results showed that sucrose induced marked changes in ECM protein composition of PLB formed in vivo. The data provide further insights into how the biochemical and microbiological changes induced by sucrose affect the ECM composition at a proteomic level. The composition of bacteria- and host-derived proteins in the ECM may be modulated by the availability of sucrose, but additional studies should be done using other dietary carbohydrates to check whether this effect is unique to this most cariogenic dietary carbohydrate. Also, in addition to the ECM composition, the intracellular or total protein composition of PLB formed in vivo should be evaluated as a control.