Although foods and beverages and oral hygiene practices are still currently considered major contributors to oral infectious diseases such as caries and gingivitis/periodontitis, specific foods and beverages have been demonstrated to exert antimicrobial, antiadhesive and biofilm disgregating activities [5, 6]. Among the numerous potentially active foods, fractions of them and identified bioactive compound(s), we have previously identified a LMM fraction of shiitake mushroom aqueous extract with promising properties [7, 8]. This LMM fraction has been further fractionated and the best in vitro biological activities demonstrated to be associated with sub-fraction #5. [Papetti et al., manuscript in preparation].
S. mutans and P. intermedia, were chosen as appropriate representatives of disease; being an etiological agent of dental caries and a representative of the bacterial complex involved in gingivitis and later in progression to periodontitis, respectively. These were coupled with relevant assays which included inhibition of adhesion, inhibition of biofilm formation and biofilm disruption .
Because a main target of the bioactive compound(s) is the adhesion of bacteria to abiotic or biotic surfaces and with cell-cell interaction as in the case of biofilm formation, the aim of this work was to evaluate which bacterial surface molecules were targeted by the bioactive compounds. To do this, candidate bacterial surface compounds CWPs, TA and LTA in the case of S. mutans and OMPs and LPS of P. intermedia were prepared and used to characterise sub-fraction #5.
As far as S. mutans was concerned, 15 out 16 CWPs were bound by sub-fraction #5; this implies that the sub-fraction may play an important, but complex, role in inhibiting bacterial cell adhesion and biofilm formation. SpaP, the surface protein antigen A also known as protein I1, antigen B, Pac, SR and antigen I/II is a well characterised adhesin of S. mutans. Gene cloning and sequencing  has revealed a block of alanine-rich repeats and another of proline-rich repeats which are implicated in binding to salivary agglutinin glycoprotein gp340, a protein involved in saliva-mediated aggregation and adherence . Importantly, antibody raised against SpaP blocked attachment of S. mutans to saliva-coated hydroxyapatite . Sub-fraction #5 could act similarly. Wall associated protein WapA or Antigen III is released in the growth medium in a 29 KDa form although its gene encodes for a protein of 48,769 KDa. Knockout of wapA has an effect on other surface components, surface ultrastructure and biofilm formation . WapE appears to alter cell surface and biofilm formation [18, 24]. Glucan binding protein (Gbp) A, B, C and D are mediators of the sucrose-dependent adherence of S. mutans to polymers formed from sucrose. GbpA and GbpD contain a series of repeats (glucan binding domains) similar to those found in glucosyl transferases (GTFs) and, in addition to this, GbpD contains a lipase activity . In vitro testing of knockouts of either GbpA or GbpD results in altered biofilm architecture suggesting a fundamental role in dental plaque structure . GbpB and GbpC lack the repeats characteristic, sharing GbpB sequence homology with putative peptidoglycan hydrolases of other streptococci, thus hypothesizing a crucial role in cell wall turnover and stress response . GbpC binds dextran tightly and, for this reason, is considered the major receptor involved in dextran-dependent aggregation, a mechanism involved again in biofilm architecture . AtlA is a surface-associated protein that plays a critical role in surface biogenesis, biofilm formation, genetic competence and autolysis . SloC, a cell wall associated component of the complex SloABC, is a solute-binding lipoprotein and a metal-dependent regulator which is involved in manganese and iron transport, thus regulating virulence gene expression of S. mutans[29, 30]. SrtA or sortase is a transpeptidase that covalently links LPXTGX-containing surface proteins to the Gram-positive bacterial cell wall, included S. mutans. The S. mutans SrtA mutant is markedly less hydrophobic than wild-type, non adherent to hydroxyapatite, non aggregating in the presence of saliva and salivary agglutinin. Thus, sortase plays a crucial role in the surface-related properties by modulating the bacterial cell surface . RgpG protein is involved in the synthesis of S. mutans rhamnose-glucose polysaccharide . Although the mechanisms of cell surface polysaccharide synthesis are poorly characterized in Gram-positive bacteria, it is however clear that polysaccharides play crucial role in cell wall architecture and bacterial virulence . DexA is a dextranase which can partially degrade glucan and, thus may affect S. mutans virulence . GapC, an extracellular glyceraldehyde-3-phosphate, is involved in acid production from glucose . Interestingly, inhibition of glycolysis by chlorexidine was demonstrated at GapC level .
It is known that polyphenols are capable of protein binding with high affinity and denaturation [37, 38]. In this study we have shown for the first time that these polyphenol properties are expressed against proteins fundamentally involved in cell adhesion, biofilm formation and architecture, thus, justifying the previous in vitro observations.
In addition to the inhibition of biological functions of several CWPs involved in S. mutans adhesion and biofilm formation, TA has been shown to be bound by sub-fraction #5. This takes into account the biological role played by TA in cell wall structure of Gram-positive bacteria, an effect of disorganizing this fundamental bacterial structure may be suggested. On the contrary, purified LTA, at least in the experiments we performed, was unable to bind sub-fraction #5 and this may caused by the lipid moiety rather than the polysaccharide chain. It may be that in growing bacteria, since the lipid moiety is included in the cytoplasmic membrane and the polysaccharide component is included in the width of the cell wall, sub-fraction #5 may interact with this macromolecule.
If this were true, since LTA is involved in bacterial adhesion to both biotic and abiotic surfaces, further inhibition of this fundamental virulence property may occur.
As far as P. intermedia is concerned, we have evaluated the binding capability of the OMPs, showing that a few proteins interact with sub-fraction #5. OMPs play fundamental roles in Gram-negative bacteria functioning as a dynamic interface between the cell and the surrounding environment. Functions of these proteins include maintaining of cell structure, passive and active transport, adhesion to other cells, and binding a variety of substances . Although studied to a lesser extent than those of Gram-negative facultative bacteria such as E. coli, similar functions can be attributed to the OMPs of Gram-negative anaerobes [19, 40]. As in the case of S. mutans, binding sub-fraction #5 to surface proteins of P. intermedia may result in denaturation and inactivation of their physiological functions. This is compatible with the observed intererence of P. intermedia adhesion to gingival cells [8, 41], biofilm formation and disgregation .
Capacity of binding sub-fraction #5 to P. intermedia LPS was also studied but in this case negative results were obtained. This event, however, may be justified by the fact that P. intermedia strain ATCC 25611 is a rough strain, i.e. containing a LPS composed only by lipid A and core but without a polysaccharide chain. Consequently, lipid alone is incapable of binding. Support of this statement comes from the observation that a commercially purchased LPS purified from an E. coli smooth strain, containing the polysaccharide O chain, is capable of binding sub-fraction #5. It is concluded, thus, that LPS may represent a target bacterial structure only in smooth strains. It is worthy of note to recall, however, that other major bacterial receptors may not be excluded and have been not tested.
Experiments performed more recently have shown that sub-fraction #5 contains 11 sub-sub-fractions mainly composed of quinic acid, uridine, adenosine, inosine, aconitic acid, oxalic acid and succinic acid [Papetti et al, manuscript in preparation] and biological activity relies mainly in quinic acid (QA). Very recently Papetti et al.  have shown that QA present in Cychorium intybus is one of the most active compounds capable of inhibiting virulence-related properties of oral pathogenic bacteria.