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Proteomic characterization of the mucosal pellicle formed in vitro on a cellular model of oral epithelium

ABSTRACT: The oral mucosal pellicle is a thin lubricating layer generated by the binding of saliva proteins on epithelial oral cells. The protein composition of this biological structure has been to date studied by targeted analyses of specific salivary proteins. In order to perform a more exhaustive proteome characterization of pellicles, we used TR146 cells expressing or not the transmembrane mucin MUC1 and generated pellicles by incubation with human saliva and washing to remove unbound proteins. A suitable method was established for the in vitro isolation of the mucosal pellicle by “shaving” it from the cells using trypsin. Comparison of pellicle and saliva compositions evidenced the adsorption of proteins not previously reported as pellicle constituents such as proteins of the PLUNC family. Pellicles formed on TR146 and TR146/MUC1 were also analyzed and compared by protein label-free quantification.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell, Saliva

SUBMITTER: Gianluigi Cabiddu

LAB HEAD: Gianluigi Cabiddu

PROVIDER: PXD017268 | Pride | 2020-05-06

REPOSITORIES: Pride

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Proteomic characterization of the mucosal pellicle formed in vitro on a cellular model of oral epithelium.

Cabiddu Gianluigi G Maes Pauline P Hyvrier Franck F Olianas Alessandra A Manconi Barbara B Brignot Hélène H Canon Francis F Cabras Tiziana T Morzel Martine M

Journal of proteomics 20200428

The oral mucosal pellicle is a thin lubricating layer generated by the binding of saliva proteins on epithelial oral cells. The protein composition of this biological structure has been to date studied by targeted analyses of specific salivary proteins. In order to perform a more exhaustive proteome characterization of pellicles, we used TR146 cells expressing or not the transmembrane mucin MUC1 and generated pellicles by incubation with human saliva and washing to remove unbound proteins. A sui ...[more]

PMID: 32360370

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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.

Project description:Human saliva has been commonly used as protein source in in vitro microbiological and biological assays to mimic the protein pellicle formation, termed acquired salivary pellicle, that precedes microbial and cell adhesion on surfaces exposed to the oral environment. However, saliva requires previous processing to remove food debris, microorganisms, and other molecules prior its use in microbiological and biological in vitro assays. For this purpose, 0.22 μm filtration, 0.45 μm filtration, and pasteurization methods have been commonly used, but the effect of these processing methods on the proteomic profile of saliva has not been tested experimentally. Stimulated human saliva was collected from 8 healthy volunteers and submitted to the following processes: non-processing (control), 0.22 μm filtration, 0.45 μm filtration, and pasteurization. The proteomic profile of non-processed saliva was compared with 0.22 μm filtered-, 0.45 μm filtered-, and pasteurized-saliva by liquid chromatography-mass spectrometry. The effect of processed saliva in microbial adhesion was tested using bacterial and fungus species, and in biological cell behavior using HaCaT immortalized human keratinocytes. Two hundred seventy-eight proteins were identified in non-processed saliva, 54 proteins (≈19%) were exclusive. Saliva processing reduced identified proteins to 222 (≈80%) for the 0.22 μm filtered saliva, 219 (≈79%) for the 0.45 μm filtered saliva, and 201 (≈72%) for the pasteurized saliva, compared to non-processed saliva. Although there were slight differences in the protein composition, the proteomic profile showed similar molecular functions and biological processes. The different saliva processing methods did not alter microbial adhesion (ANOVA, p>0.05). Interestingly, pasteurized saliva reduced keratinocytes cell viability. Saliva processing methods tested reduced the proteomic profile diversity of saliva, but maintained similar molecular functions and biological processes mediated by remaining proteins, not interfering on microbial adhesion and cell viability, except for pasteurization, which reduced cell viability.

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	A2NUT2.mgf	Mgf
	A2NUT2.prot.xml	Xml
	A2NUT2.zip	Other
	A8K2U0.mgf	Mgf