Joelle Ogier

Crystal structure of the V-region of Streptococcus mutans antigen I/II at 2.4 A resolution suggests a sugar preformed binding site.

Antigens I/II are large multifunctional adhesins from oral viridans streptococci that exert immunomodulatory effects on human cells and play important roles in inflammatory disorders. Among them, Streptococcus mutans plays a major role in the initiation of dental caries. The structure of the V-region (SrV+, residues 464-840) of the antigen I/II of S. mutans has been determined using the multiwavelength anomalous diffraction phasing technique with seleno-methionine-substituted recombinant protein and subsequently refined at 2.4 A resolution. The crystal structure of SrV+ revealed a lectin-like fold that displays a putative preformed carbohydrate-binding site stabilized by a metal ion. Inhibition of this binding site may confer to humans a protection against dental caries and dissemination of the bacteria to extra-oral sites involved in life-threatening inflammatory diseases. This crystal structure constitutes a first step in understanding the structure-function relationship of antigens I/II and may help in delineating new preventive or therapeutic strategies against colonization of the host by oral streptococci.

The A and the extended V N-terminal regions of streptococcal protein I/IIf mediate the production of tumour necrosis factor alpha in the monocyte cell line THP-1.

The induction of tumour necrosis factor (TNF)‐α from the monocytic cell line THP‐1 by the streptococcal antigen I/II from Streptococcus mutans serotype f (protein I/IIf) was studied by use of recombinant polypeptides containing discrete domains of the protein. The derivatives carrying the N‐terminal alanine‐rich region (A region) and the adjacent variable region (extended V region) of the protein bound to THP‐1 cell extracts in a saturable fashion, and one derivative lacking both the A and the extended V regions was not able to bind monocyte cell extracts, suggesting that the domains responsible for the binding of protein I/IIf to monocytes were the A and the extended V regions. Sodium metaperiodate pretreatment of THP‐1 cell extracts, tunicamycin pretreatment of monocyte cells or competition with N‐acetyl neuraminic acid (NANA) and fucose resulted in a 45–70% reduction in binding activity of the derivatives carrying the extended V region, demonstrating the lectin‐like mode of recognition of the monocytic receptor by the extended V region and the role of NANA and fucose in this recognition process. Besides, the stimulation of monocytes to release TNF‐α by the derivatives containing the A region and the extended V region was effective and was not affected by the addition of polymyxin B or vitamin D3, suggesting that CD14 does not play the role of receptor in stimulation of monocytes by protein I/IIf to release TNF‐α.

Antibacterial protection of suture material by chlorhexidine-functionalized polyelectrolyte multilayer films.

The formation of bacterial biofilms on the surface of implanted materials is a critical factor that may lead to chronic microbial infection and tissue necrosis. In the present study we analysed the stability of polyelectrolyte multilayer (ML) films on suture materials and the antibacterial effect obtained with chlorhexidine (CHX)-functionalized films built on different types of suture materials such as silk, polyester and copolymer of glycolide and l-lactide. The comparison of Escherichia coli culture on glass coverslips and glass coverslips with ML and CHX showed at 24 h an inhibition of the bacterial relative luminescence (40.68%, P < 0.5) and at 48 h (99.46%, P < 0.001). In another way, simple soaking of suture material overnight in CHX digluconate 20% without polyelectrolyte films did not at all protect sutures from bacterial colonization but CHX-functionalized polyelectrolyte films, made from poly-l-glutamic acid and poly-l-lysine, inhibited Escherichia coli proliferation.

Polyelectrolyte Multilayer Film Coating and Stability at the Surfaces of Oral Prosthesis Base Polymers: an in vitro and in vivo Study

A new type of coating involving a layer-by-layer technique has been recently reported. This coating is composed of a polyelectrolyte multilayer film that confers specific properties on surfaces to which it is applied. Here, we studied the applicability of such a technique to the coating of oral prostheses, by first testing the construction of polyelectrolyte multilayer films on several polymers used in oral prosthesis bases, and, subsequently, by studying the stability of these coatings in vitro, in human saliva, and in vivo in a rat model. We demonstrated that the multilayered films are able to coat the surfaces of all tested polymers completely, thus increasing their wettability. We also showed that saliva does not degrade the film after 7 days in vitro and after 4 days in vivo. Taken together, our results establish that the layer-by-layer technique is suitable for the coating of oral devices.

Purification and characterization of the expression product of the sr gene of Streptococcus mutans OMZ 175.

The subcloning in pBR322 of the gene of the S. mutans OMZ 175 74K SR protein, was performed after in vitro reconstitution, from two recombinant EMBL3 phages, lambda SmAD9 and lambda SmAD10. The gene is expressed in E. coli HB101 under the control of its own promoter and produces a protein with a molecular weight of 195 kDa. A strong immunological relationship between the expressed protein and the 74K SR protein was observed in ELISA, Western blotting and immunoprecipitation. The 195 kDa protein was purified by immunoaffinity chromatography to homogeneity as judged by SDS-PAGE and native PAGE. Its reactivity with monoclonal anti 74K SR antibodies indicates that it is probably a precursor form of the 74K SR protein produced in S. mutans. The adhesion properties of the two proteins, tested in solid phase adherence assays, are quite similar. This indicates that the additional peptide present in the precursor protein has little or no role in the adherence properties of protein 74K SR.

Polyelectrolyte multilayer-mediated gene delivery for semaphorin signaling pathway control.

The capability of multilayered polyelectrolyte films (MPFs) to control the sequential expression of two genes encoding cell receptors involved in a common cell signalling activity is shown, while achieving a fully functional signal transduction. As a functional model system representative of a cell signalling process that proceeds in a top-down manner, cell collapse induced by semaphorin 3A (Sema3A) was chosen as the target. Polyelectrolyte multilayers were sequentially functionalized with two plasmids encoding Neuropilin-1 (NRP-1) and Plexin-A1 (Plx-A1), respectively, acting as co-receptors for Sema3A. By using hyaluronan and chitosan as structural components for the incorporation of plasmid DNA layers onto precursor films made of poly-allylamine hydrochloride and poly-sodium-4-styrenesulfonate, the polyelectrolyte system is established; this systems is capable of delivering both plasmids to Cos-1 cells in a manner that permits control over the timing and the respective order in which the two plasmid DNA constructs are expressed. Importantly, it was observed that, following Sema3A stimulation, COS-1 cells co-expressing Plx-A1 and NRP-1 display a collapse phenotype, which is determined by the multilayer build-up scheme, and that the expression products of both transgenes embedded in MPFs are temporally functional over several days while acting their role of co-receptors for Sema3A.

TNF-alpha release by monocytic THP-1 cells through cross-linking of the extended V-region of the oral streptococcal protein I/II.

We tested the hypothesis of a conserved activation mode of monocytic THP‐1 cells by proteins I/II expressed by several species of oral streptococci through the specific role of the extended V‐region. We studied the binding and modulating activities of six proteins I/II purified from strains representing four different species of oral streptococci, and of expression products of polymerase chain reaction‐amplified sequences encoding corresponding extended V‐regions. We found that the different proteins I/II bound to THP‐1 cells in a sugar‐dependent mode involving the extended V‐region. Furthermore, all the proteins I/II stimulated THP‐1 cells to produce tumor necrosis factor α, indicating that these properties are not strain‐ or species‐specific. Despite the weak stimulation of THP‐1 cells by the extended V‐region alone, we obtained evidence that cross‐linking of this region can be one of the mechanisms involved in monocytic cell activation by proteins I/II. J. Leukoc. Biol. 67: 81–89; 2000.

Streptococcal antigen I/II binds to extracellular proteins through intermolecular β‐sheets

One of the functions associated with the oral streptococcal surface protein I/II is to bind to human extracellular matrix molecules or blood components, which could act as opportunistic ligands in pathological circumstances. In order to understand the relative specificity of the binding repertoire of this bacterial adhesin, we examined by infrared measurements the mode of binding of the protein I/II from Streptococcus mutans OMZ175 (I/IIf) to fibronectin and fibrinogen. This approach revealed the β‐structure forming capacity of I/IIf upon interaction with both proteins. The forming of intermolecular β‐structures may provide a non‐selective way of interaction between I/IIf and its possible targets.

Molecular characterization of the gene sr of the saliva interacting protein from Streptococcus mutans OMZ175

The saliva interacting protein (74KSR) from Streptococcus mutans serotype f, which is immunologically related to antigen I/II from serotype c, also termed B, P1, PAc, is probably involved in the adherence process of Strep. mutans to the tooth surface. A solid-phase adherence assay showed that 38% of the binding of salivary glycoproteins to Strep. mutans OMZ175 was due to this protein. We have cloned and sequenced the 74KSR gene (sr), which produces a recombinant protein (rec195K) with a relative molecular mass of 195,000, as estimated by SDS-PAGE. The strong immunological relationship and functional identity of the 74KSR and rec195K indicate that the Mr 195K protein is probably a precursor form, post-translationally processed, of the 74KSR produced in Strep. mutans. The gene sr consists of 4667 bp and codes for a 171,177 Mr protein. Biochemical features of the protein (density in proline residues and hydrophobicity) may explain the difference observed between the SDS-PAGE estimated molecular mass of the immature protein and the one deduced from the nucleotide sequence. Intra-species hybridization experiments using three contiguous restriction fragments isolated from gene sr as probes showed that the sequence is highly similar in strains from serotypes c and e. We have also shown that a fraction of the heart specific antibodies induced in rabbits during immunization with the 74KSR or rec195K reacts predominantly with human IgG and suggest the hypothesis of antigen mimicry as an explanation for the production of anti-IgG autoantibodies. It will be of great importance to identify the cross-reactive epitopes within the molecule before considering its use in protective immunization against oral streptococci.

Studies of Streptococcus mutans interactions with saliva glycoproteins by an enzyme linked immunosorbent assay

Abstract An enzyme linked immunosorbent assay was developed for the measurement of saliva binding to Streptococcus mutans cells adsorbed on polystyrene microtitre plates. Bound saliva was quantified by means of specific rabbit anti-saliva immunoglobulin G and alkaline phosphatase conjugated goat anti-rabbit immunoglobulin. The amount of specific saliva binding to S. mutans OMZ 175 was evaluated by competition with homologous bacterial cells and bacterial receptors were detected in fractions of wall associated antigens from various S. mutans strains. The saliva binding assay was shown to be applicable to a variety of oral streptococcal strains.