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Formation of salivary-mucosal pellicle: the role of transglutaminase.

ABSTRACT: The present investigation was carried out to identify salivary components of mucosal pellicles in vivo and explore further the mechanism of interaction between salivary molecules and buccal epithelial cells. By using specific antisera and immunoprotein blotting, high-(MG1) and low-(MG2) molecular-mass salivary mucins, amylase, salivary cystatins and proline-rich proteins were detected within mucosal pellicle in vivo. In addition, the data indicated that the mucins and proline-rich proteins could be cleaved into lower-molecular-mass products, whereas the proline-rich proteins could also be cross-linked into higher-molecular-mass complexes. The role of buccal epithelial cell transglutaminase in these interactions was further studied by utilizing purified iodinated amylase, neutral cystatin SN and acidic proline-rich proteins 1 and 3 (APRP1 and 3). After incubation with buccal epithelial cells in vitro 125I-labelled APRPs appeared to undergo a greater degree of cross-linking than 125I-labelled cystatin SN, as determined by SDS/PAGE/autoradiography. Amylase did not appear to be cross-linked at all. Recovery of 125I-labelled APRPs and 125I-labelled cystatin SN with epithelial cell envelopes after repeated extraction suggested that both molecules were cross-linked to envelope proteins, but that 125I-labelled APRPs were cross-linked to a greater degree than 125I-labelled cystatin SN. Cross-linking in buccal epithelial cell preparations was inhibited by an excess of methylamine hydrochloride, a transglutaminase substrate. In a further assessment of amylase, cystatin and APRPs as transglutaminase substrates, only APRP3 and a partially purified preparation of APRPs acted as an amine acceptor for the cross-linking of [14C]methylamine by purified transglutaminase, as determined by SDS/PAGE/fluorography. This reaction was completely inhibited by excess EDTA. The combined data from this study suggest that during mucosal pellicle formation multiple components of saliva adsorb to buccal epithelial cell surfaces, and that, within this group, selected components are enzymically cross-linked by an epithelial transglutaminase and/or proteolytically cleaved into smaller fragments.

SUBMITTER: Bradway SD

PROVIDER: S-EPMC1132674 | biostudies-other | 1992 Jun

REPOSITORIES: biostudies-other

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Project description:ObjectivesThe present study aimed to evaluate the potential of the salivary pellicle (SP) formed on titanium (Ti) surfaces to modulate the formation of a biofilm composed of Streptococcus gordonii, Actinomyces naeslundii, Fusobacterium nucleatum, and Porphyromonas gingivalis.Materials and methodsTi substrates were incubated for 2 h with a pool of saliva samples obtained from 10 systemically and periodontally healthy subjects. Enamel substrates were included as a biological reference. Scanning electron microscopy (SEM) and Raman spectroscopy analysis were used to analyze the formation of the salivary pellicle. After the SP formation, the surfaces were incubated for 12 h with a mix of Streptococcus gordonii, Actinomyces naeslundii, Fusobacterium nucleatum, and Porphyromonas gingivalis. The number of bacterial cells attached to each surface was determined by the XTT assay while bacterial viability was analyzed by fluorescence microscopy using the LIVE/DEAD® BacLightTM kit.ResultsThe SEM and Raman spectroscopy analysis confirmed the presence of a salivary pellicle formed on the tested surfaces. Regarding the biofilm formation, the presence of the SP decreases the number of the bacterial cells detected in the test surfaces, compared with the uncover substrates. Even more, the SP-covered substrates showed similar bacterial counts in both Ti and enamel surfaces, meaning that the physicochemical differences of the substrates were less determinant than the presence of the SP. While on the SP-uncover substrates, differences in the bacterial adhesion patterns were directly related to the physicochemical nature of the substrates.ConclusionsThe salivary pellicle was the main modulator in the development of the biofilm consisting of representative oral bacteria on the Ti substrates.Clinical relevanceThe results of this study provide valuable information on the modulatory effect of the salivary pellicle on biofilm formation; such information allows us to understand better the events involved in the formation of oral biofilms on Ti dental implants.

Project description:Shewanella oneidensis is an important model organism for bioremediation studies because of its diverse respiratory capabilities. In recent years, biofilm development of S. oneidensis has been extensively studied because it is essential to reduce solid metals. As a special form of biofilm, however, pellicles are largely overlooked. The goal of this work was to understand requirements of S. oneidensis pellicle formation and the molecular basis of pellicle formation. We demonstrated that successful pellicle formation and survival was likely to require the threshold level of cell density and higher concentration of oxygen. Proteinase K and EDTA were potent pellicle disrupter. DNA microarray experiments were used to study the gene expression profile of young air–liquid interface pellicle relative to planktonic cells, which indicated that the air–liquid interface pellicle was more metabolically active than the planktonic cells. Most notably, consistently up-regulation of iron or heme uptake and transportation proteins was observed in the S. oneidensis MR-1 pellicle. However, neither the hmuT nor hugA heme transport mutant was defective in pellicle formation. An examination of the influence of several metal cations on the anti-pellicle activity of EDTA showed that Ca (II), Mn(II), Cu(II), and Zn(II) fully protected S. oneidensis MR-1 pellicle against EDTA treatment while additional of iron enabled the initiation of pellicle formation but maturation was significantly impaired. Collectively, iron was less important than other metals with respect to pellicle formation in S. oneidensis. A fresh colony grown overnight on a LB plate was used to inoculate 50 ml LB and incubated in a shaker (200 rpm) to an OD600 of 0.8 at the room temperature. This culture was then diluted 500-fold with fresh LB, resulting in the starting cultures. Aliquots of 30ml starting cultures were transferred to 50-ml Pyrex beakers and allowed to develop pellicles at the room temperature. When a complete but thin (young pellicle) at the interface were formed (about 30h hours), planktonic culture and pellicle were separated and applied to centrifugation at 8000 rpm for 3 min at room temperature. 3 parallel starting cultures were used and 3 samlpes of pellicle cells or planktonic cells were collected at 30h. RNA from the pellicle cells was fluorescently labeled with Cy3, and that from the planktonic was labeled with Cy5.

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Formation of salivary-mucosal pellicle: the role of transglutaminase.

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