Dataset Information

Ultra-deep and quantitative saliva proteome reveals dynamics of the oral microbiome

ABSTRACT: The oral cavity is home to one of the most diverse microbial community of the human body and a major entry portal for pathogens. Its homeostasis is maintained by saliva, which fulfills key functions including lubrication of food, predigesting and bacterial defense. Consequently, disruptions in saliva secretion contributes to conditions such as tooth decay and respiratory tract infections. Here we used recent improvements in mass spectrometry (MS)-based proteomics to develop a rapid workflow for mapping to map the saliva proteome quantitatively and at great depth. Microgram protein amounts retrieved from cotton swabs were processed in a single-run format, resulting in more than 3,700 quantified human proteins in 100 min measurements gradients. After separation into eight fractions, this increased to 5,500 human proteins. Remarkably, our measurements also quantified more than 2,000 microbial proteins and we find peptide evidence for more than 70 bacterial genera without any microbial culture. Co-analysis of the proteomics results with next generation sequencing data as well as MALDI Biotyper revealed strong agreement. The oral microbiome differs between individuals and changes drastically upon eating and tooth brushing. Rapid and robust shotgun technology can now simultaneously characterize the human and microbiome contributions to the proteome of a body fluid.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Saliva

SUBMITTER: Niklas Grassl

LAB HEAD: Matthias Mann

PROVIDER: PXD003028 | Pride | 2016-03-18

REPOSITORIES: Pride

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			Action	DRS
	20141116_QEp1_LC7_PhGe_4_53_M1_2.raw	Raw
	20141116_QEp1_LC7_PhGe_4_53_M1_3.raw	Raw
	20141116_QEp1_LC7_PhGe_4_53_M1_4.raw	Raw
	20141116_QEp1_LC7_PhGe_4_53_M2_1.raw	Raw
	20141116_QEp1_LC7_PhGe_4_53_M2_2.raw	Raw

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Ultra-deep and quantitative saliva proteome reveals dynamics of the oral microbiome.

Grassl Niklas N Kulak Nils Alexander NA Pichler Garwin G Geyer Philipp Emanuel PE Jung Jette J Schubert Sören S Sinitcyn Pavel P Cox Juergen J Mann Matthias M

Genome medicine 20160421 1

<h4>Background</h4>The oral cavity is home to one of the most diverse microbial communities of the human body and a major entry portal for pathogens. Its homeostasis is maintained by saliva, which fulfills key functions including lubrication of food, pre-digestion, and bacterial defense. Consequently, disruptions in saliva secretion and changes in the oral microbiome contribute to conditions such as tooth decay and respiratory tract infections. Here we set out to quantitatively map the saliva pr ...[more]

PMID: 27102203

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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.

Project description:Background. Hematopoietic cell transplantation (HCT) is a potentially curative therapy for a wide range of pediatric malignant and nonmalignant diseases. However, complications, including blood stream infection (BSI) remain a major cause of morbidity and mortality. While certain bacteria that are abundant in the oral microbiome, such as S. mitis, can cause BSI, the role of the oral microbial community in the etiology of BSI is not well understood. The finding that the use of xylitol wipes, which specifically targets the cariogenic bacteria S. mutans is associated with reduced BSI in pediatric patients, lead us to investigate dental caries as a risk factor for BSI. Methods. A total of 41 pediatric patients admitted for allogenic or autologous HCT, age 8 months to 25 years, were enrolled. Subjects with high dental caries risk were identified as those who had dental restorations completed within 2 months of admission for transplant, or who had untreated decay. Fisher’s exact test was used to determine if there was a significant association between caries risk and BSI. Dental plaque and saliva were collected on a cotton swab from a subset of 4 high caries risk (HCR) and 4 low caries risk (LCR) children following pretransplant conditioning. 16SrRNA sequencing was used to compare the microbiome of HCR and LCR subjects and to identify microbes that were significantly different between the 2 groups. Results. There was a statistically significant association between caries risk and BSI (p<0.035) (Fisher’s exact test). Multivariate logistic regression analysis showed children in the high dental caries risk group were 21.39 times more likely to have BSI, with no significant effect of age or mucositis severity. HCR subjects showed significantly reduced microbial alpha diversity as compared to LCR subjects. LEfse metagenomic analyses, showed the oral microbiome in HCR children enriched in order Lactobacillales. This order includes Streptococcus and Lactobacillus, both which contain bacteria primarily associated with dental caries. Discussion. These findings support the possibility that the cariogenic microbiome can enhance the risk of BSI in pediatric populations. Future metagenomic analyses to measure microbial differences at, before, and after conditioning related to caries risk, may further unravel the complex relationship between the oral microbiome, and whether it affects health outcomes such as BSI.