The P2X(7) receptor is a ligand-gated cation channel that is highly expressed on monocyte-macrophages and that mediates the pro-inflammatory effects of extracellular ATP. Dilation of the P2X(7) channel and massive K(+) efflux follows initial channel opening, but the mechanism of secondary pore formation is unclear. The proteins associated with P2X(7) were isolated by using anti-P2X(7) monoclonal antibody-coated Dynabeads from both interferon-gamma plus LPS-stimulated monocytic THP-1 cells and P2X(7)-transfected HEK-293 cells. Two nonmuscle myosins, NMMHC-IIA and myosin Va, were found to associate with P2X(7) in THP-1 cells and HEK-293 cells, respectively. Activation of the P2X(7) receptor by ATP caused dissociation of P2X(7) from nonmuscle myosin in both cell types. The interaction of P2X(7) and NMMHC-IIA molecules was confirmed by fluorescent life time measurements and fluorescent resonance of energy transfer-based time-resolved flow cytometry assay. Reducing the expression of NMMHC-IIA or myosin Va by small interfering RNA or short hairpin RNA led to a significant increase of P2X(7) pore function without any increase in surface expression or ion channel function of P2X(7) receptors. S-l-blebbistatin, a specific inhibitor of NMMHC-IIA ATPase, inhibited both ATP-induced ethidium uptake and ATP-induced dissociation of P2X(7)-NMMHC-IIA complex. In both cell types nonmuscle myosin closely interacts with P2X(7) and is dissociated from the complex by extracellular ATP. Dissociation of this anchoring protein may be required for the transition of P2X(7) channel to a pore.

Odontoblast synthesis of dentin proceeds through discrete but overlapping phases characterized by formation of a patterned organic matrix followed by remodelling and active mineralization. Microbial invasion of dentin in caries triggers an adaptive response by odontoblasts, culminating in formation of a structurally altered reactionary dentin, marked by biochemical and architectonic modifications including diminished tubularity. Scanning electron microscopy of the collagen framework in reactionary dentin revealed a radically modified yet highly organized meshwork as indicated by fractal and lacunarity analyses. Immuno-gold labelling demonstrated increased density and regular spatial distribution of dentin sialoprotein (DSP) in reactionary dentin. DSP contributes putative hydroxyapatite nucleation sites on the collagen scaffold. To further dissect the formation of this altered dentin matrix, the associated enzymatic machinery was investigated. Analysis of extracted dentin matrix indicated increased activity of matrix metalloproteinase-2 (MMP-2) in the reactionary zone referenced to physiologic dentin. Likewise, gene expression analysis of micro-dissected odontoblast layer revealed up-regulation of MMP-2. Parallel up-regulation of tissue inhibitor of metalloproteinase-2 (TIMP-2) and membrane type 1- matrix metalloproteinase (MT1-MMP) was observed in response to caries. Next, modulation of odontoblastic dentinogenic enzyme repertoire was addressed. In the odontoblast layer expression of Toll-like receptors was markedly altered in response to bacterial invasion. In carious teeth TLR-2 and the gene encoding the corresponding adaptor protein MyD88 were down-regulated whereas genes encoding TLR-4 and adaptor proteins TRAM and Mal/TIRAP were up-regulated. TLR-4 signalling mediated by binding of bacterial products has been linked to up-regulation of MMP-2. Further, increased expression of genes encoding components of the TGF-β signalling pathway, namely SMAD-2 and SMAD-4, may explain the increased synthesis of collagen by odontoblasts in caries. These findings indicate a radical adaptive response of odontoblasts to microbial invasion of dentin with resultant synthesis of modified mineralized matrix.

Mature biofilm and planktonic cells of Streptococcus mutans cultured in a neutral pH environment were subjected to comparative proteome analysis. Of the 242 protein spots identified, 48 were significantly altered in their level of expression (P<0?050) or were unique to planktonic or biofilm-grown cells. Among these were four hypothetical proteins as well as proteins known to be associated with the maintenance of competence or found to possess a cin-box-like element upstream of their coding gene. Most notable among the non-responsive genes were those encoding the molecular chaperones DnaK, GroEL and GroES, which are considered to be up-regulated by sessile growth. Analysis of the rest of the proteome indicated that a number of cellular functions associated with carbon uptake and cell division were down-regulated. The data obtained were consistent with the hypothesis that a reduction in the general growth rate of mature biofilms of S. mutans in a neutral pH environment is associated with the maintenance of transformation without the concomitant stress response observed during the transient state of competence in bacterial batch cultures.

No abstract

The ff gene, coding for the cell-bound 13-D-fructosyltransferase (YIUF) of Streptococcus salivarius ATCC 25975, has been analyzed, and its deduced amino acid sequence has been compared with that of the secreted FM of Streptococcus mutans and the levansucrases (SacBs) of Bacilus species. A unique proline-rich region detected at the C terminus of the FTF of S. salivarius preceded a hydrophobic terminal domain. This proline-rich region was shown to possess strong homology to the product of the prgC gene from pCF10 in Enterococcus faecalis, which encodes a pheromone-responsive protein of unknown function, as well as homology to the human proline-rich salivary protein PRP-4. A series of 3'-OH deletions of the S. salivariusftf gene expressed in Streptococcus gordonii Challis LGR2 showed that the C terminus was required for cell surface attachment in this heterologous organism, as only the complete gene product was cell bound. This cell-bound activity was released in the presence of sucrose, suggesting that the mode of attachment and release of the S. salivarius FTF in S. gordonii was similar to that in its native host.The P-D-fructosyltransferases (FTFs) of oral streptococci and the levansucrases (SacBs) of bacilli form a family of enzymes that polymerize the fructose moiety of sucrose into fructans which may possess either levan or inulin structures (7,10,11). Unlike the bacilli and mutans streptococci, which secrete their FTFs directly into the culture fluid (5, 6), the higher-molecular-weight Ca2"-dependent FTF of Streptococcus salivarius is initially cell associated (15,22). This cell association is lost in the presence of the enzyme's substrate, sucrose, for under these conditions, the FTF is released from the cell and secreted into the culture medium (25).The sucrose-dependent secretion of the cell-associated FTF of S. salivarius raises not only the question as to the mechanism of release of the enzyme from the cell surface but also the question as to how the enzyme is bound to the cell in the first place. In this communication, we report the sequencing of the ftf gene from S. salivarius ATCC 25975 and show that the C-terminal region is obligatory for anchoring the FTF to Streptococcus gordonii when the latter is used as the host organism. The intact enzyme was also shown to be released from S. gordonii in the presence of sucrose, suggesting that the same release mechanism may occur in both species of streptococci. (22), and S. gordonii Challis LGR2 (38) were used throughout these studies. E. coli strains were grown in Luria-Bertani medium at 370C (35), while cultures (10 ml) of both streptococcal species were grown at 370C without aeration in either Todd-Hewitt broth containing serum (THBS) (Oxoid, Basingstoke, England) supplemented with 1% inactivated horse serum and 0.2% glucose or semidefined medium (SDM) supplemented with 1 mM CaCl2 and containing 25 mM glucose (20). Erythromycin (40 pg/ml) was added where appropriate. For growth on solid medium, the streptococci were plated on brain heart infusion medium...

Compared with traditional two-dimensional (2D) proteome analysis of Streptococcus mutans grown as a biofilm from a planktonic culture at steady state (Rathsam et al., Microbiol. 2005, 151, 1823-1837), the use of 2D fluorescence difference gel electrophoresis (DIGE) led to a 3-fold increase in the number of identified protein spots that were significantly altered in their level of expression (P < 0.050). Of the 73 identified proteins, only nine were up-regulated in biofilm grown cells. The results supported the previously surmised hypothesis that general metabolic functions were down-regulated in response to a reduction in growth rate in mature S. mutans biofilms. Up-regulation of competence proteins without any concomitant increase in stress-responsive proteins was confirmed, while the levels of glucosyltransferase C (GtfC), involved in glucan formation, O-acetylserine sulfhyrylase (cysteine synthetase A; CsyK), implicated in the formation of [Fe-S] clusters, and a hypothetical protein encoded by the open reading frame, SMu0188, were also up-regulated.

No abstract

Analysis of the region downstream of the ftf gene of Streptococcus salivarius led to the detection of two open reading frames (ORFs). The deduced amino acid sequences of these ORFs were homologous to proteins encoded by genes not previously described and/or sequenced in Gram-positive bacteria. The deduced amino acid sequence of the first of these ( o r -) showed strong homology to the product of the FUR1 gene of Saccharomyces cereuisiae, which codes for a uracil phosphoribosyltransferase. The over-expression of the product of this gene appeared to be the source of the detrimental effect observed with phagemids carrying or$? in Escherichia coli hosts. The deduced amino acid sequence of the second ORF (orJJ) was homologous to the ClpP family of proteases. Examination of the upstream region of theftfgene led to the discovery of a new insertion sequence-like element which has been designated IS1161.