Protein Expression by Streptococcus mutans during Initial Stage of Biofilm Formation

Cells growing on surfaces in biofilms exhibit properties distinct from those of planktonic cells, such as increased resistance to biocides and antimicrobial agents. In spite of increased interest in biofilms, very little is known about alterations in cell physiology that occur upon attachment of cells to a surface. In this study we have investigated the changes induced in the protein synthesis by contact of Streptococcus mutans with a surface. Log-phase planktonic cells of S. mutans were allowed to adhere to a glass slide for 2 h in the presence of a (14)C-amino acid mixture. Nonadhered cells were washed away, and the adhered cells were removed by sonication. The proteins were extracted from the nonadhered planktonic and the adhered biofilm cells and separated by two-dimensional gel electrophoresis followed by autoradiography and image analysis. Image analysis revealed that the relative rate of synthesis of 25 proteins was enhanced and that of 8 proteins was diminished ≥1.3-fold in the biofilm cells. Proteins of interest were identified by mass spectrometry and computer-assisted protein sequence analysis. Of the 33 proteins associated with the adhesion response, all but 10 were identified by mass spectrometry and peptide mass fingerprinting. The most prominent change in adhered cells was the increase in relative synthesis of enzymes involved in carbohydrate catabolism indicating that a redirection in protein synthesis towards energy generation is an early response to contact with and adhesion to a surface

Parvimonas micra stimulates expression of gingipains from Porphyromonas gingivalis in multi-species communities

Dental biofilms are complex ecosystems containing many bacterial species that live in mutualistic relationships. These interactions can profoundly affect the virulence properties of the community. In this study we investigated how the production of gingipains, virulence factors from Porphyromonas gingivalis important in periodontal disease, was affected by other commonly found members of the sub-gingival microbiome. To mimic the subgingival microbiome, multispecies consortia (P. gingivalis, Fusobacterium nucleatum, Actinomyces naeslundii, Streptococus oralis, Streptococcus mitis, Streptococcus gordonii and Streptococcus cristatus, with or without Parvimonas micra) as well as dual species consortia (P. gingivalis with P. micra, S. oralis or F. nucleatum) were constructed and maintained anaerobically in 10% serum for up to seven days. The number of P. gingivalis was determined by plating on Brucella agar and the gingipain specific fluorogenic substrate BikKam-10 was used to investigate gingipain activity. The effect of secreted products from P. micra on gingipain activity was investigated by adding supernatants from P. micra to P. gingivalis cultures. The most prominent secreted proteins in the supernatant were identified using mass spectrometry. P. gingivalis was unable to grow in serum, either alone or in the presence of S. oralis or F. nucleatum. In contrast, with P. micra growth was significantly enhanced and this was associated with an increase in gingipain activity. In the multi-species consortia, the presence of P. micra caused a 13-fold increase in gingipain activity. Exposure of P. gingivalis to supernatants from P. micra for 24 h caused a 3-fold increase in gingipain activity. This effect was reduced by 43% after heat-treatment of the supernatant. Two dimensional gel electrophoresis revealed that several of the most prominent proteins in the P. micra supernatant were glycolytic enzymes. The results from this study suggests that gingipains are produced in response to a P. micra derived signalling molecule that is most likely a protein. This is the first time it has been shown that P. micra can affect P. gingivalis virulence properties. This is likely to be of significance for the development of be of periodontitis since these two microorganisms are often found together in the subgingival biofilm

Acid tolerant streptococci in dentine caries using pH-selective agars

Objectives: To investigate the acid tolerant microflora in dentine caries, with special respect to oral streptococci cultivable on pH-selective agar media, as acid stress might be a major selective force in dentine caries. Methods: Five patients with primary dentine caries lesions (vital teeth, no symptoms) participated in the study. Caries lesions were excavated aseptically under rubber-dam using rose-burs. Dentine sampling was performed using sterile rose-burs at 3 levels: superficially, in the centre and in the cavity floor. Samples were incubated on neutral (blood agar) and pH-selective agars (Todd-Hewitt agar with citrate-phosphate buffer; pH 4.5, 5.0 and 5.5). Numbers of colony-forming units (CFUs) were determined, characterized morphologically, isolated to blood agar and then identified (Gram-reaction, cell- and colony morphology). Each colony type was frozen in skim milk (158 isolates). Isolates described as streptococci were revived, re-incubated (65 isolates) and further characterized by enzymatic- and sugar fermentation tests. Results: The same bacterial counts were recovered from superficial, center and cavity floor, respectively. Each dentine sample exhibited a unique microflora. There was no significant difference in proportions of aciduric microorganisms in different levels in the lesion. Approximately 38% (superficial), 5% (centre) and 38% (cavity floor) of the total cultivable microbiota was able to grow at pH 5.5, a pH value critical for demineralisation of dentine. The acid-tolerant streptococcal isolates included mutans streptococci, S. anginosus, S. constellatus and a group of unidentified streptococci. Lactate consuming taxa were found in one case, but only at pH 5.5. Conclusion: There was no significant difference in numbers of microorganisms on different levels in the lesion. Composition of the dentine caries microflora differs from lesion to lesion. The dentine caries microflora consortia differ from lesion to lesion. A common property of these bacteria was acid tolerance. Approved by the ethical committee Lund University. Funded by Faculty grants