Diversity analysis of the supra and subgingival microbiota and proteins profile of saliva and acquired enamel pellicle of individuals with periodontitis.

Author(s)

Pâmela Pontes Penas Amado

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2019-07-10

Examining board members

Marcia Pinto Alves Mayer; Marinella Holzhausen Caldeira; Renato Corrêa Viana Casarin; Maria Regina Lorenzetti Simionato

• Advisor: Marcia Pinto Alves Mayer

Abstract

Localized aggressive periodontitis (LAgP) is still a poorly understood disease. The subgengival microbiota of LAgP is characterized by the presence of periodontopathogens such as Aggregatibacter actinomycetemcomitans (Aa), and by the reduction of beneficial bacteria, however, the microbiome associated with LAgP has not yet been described. Since the initial adhesion of bacteria to the dental surfaces is dependent on the composition of the acquired enamel pellicle (PAE) formed by proteins present in the whole saliva (WS), the present study evaluated the PAE and WS proteome of LAgP, compared with healthy patients (HLAgP). In addition, the salivary cytokines/chemokines profile was evaluated in both groups. We also aimed to determine the composition of the oral and gut microbiota in patients with LAgP, and compare to the microbiota of HLAgP. Seven women with LAgP, aged between 19-26 years, Afro-descendants, and 8 healthy controls with the same profile were selected. Samples of supra (SP) and subgingival biofilme of shallow sites (SH) and affected medium/deep sites (MD), WS, AEP and feces were collected. The oral and gut microbiome were analyzed by sequencing of 16S rRNA, and the presence of the Aa JP2 clone was determined in oral biofilme (OB) samples. WS and AEP proteome was analyzed by mass spectrometry, levels of NO in WS were dosed by Griess colorimetric reaction, levels of cytokines/chemokines in WS were quantified in multiplex assay. The proteolytic activity of WS was evaluated through the degradation of histatins 1 and 5 in different time-points. Spearmans correlation was applyed to evaluate correlations between different variables. qPCR analysis revealed that all LAgP patients harbored Aa, unlike HLAgP, however, the JP2 clone was detected in only 1 patient. Oral and gut microbiome analysis revealed no differences in the alpha diversity indexes. Beta-diversity analysis revealed that samples of OB and MD of LAgP were different from OB and SH of HLAgP, respectively. In LAgP, there was a reduction of the abundance of benefical bacteria, and increase of putative pathogens such as Aa, Porphyromonas, Tannerela and Treponema. The oral dysbiosis was acompained by imbalance in the gut microbiota, with higher abundance of Desulfovibrio in LAgP than in HLAgP. The WS of LAgP patients presented lower levels of CCL2 and CCL25, and higher levels of CCL17 and CCL27 than HLAgP, correlated to levels of Aa, Acidovorax ebreus and Helicobacter pylori in the OB. The AEP of LAgP-affected individuals presented undetectable levels of some proteins involved in immune response, antimicrobial activity and anti-inflammatory molecules, differing from the PAE of the HLAgP. AEP proteins such as ALMS1 and Cystatin-S and in WS such as alpha-enolase, profilin-1, dystonin, A2ML1, alpha-actinin-4 and IGHA1 were present differentially between LAgP and HLAgP. The proteolytic activity of WS was more intense in LAgP than in HLAgP. The data revealed new aspects of LAgP and collaborate with the understanding of mechanisms that may be involved in the development and progression of the disease, and indicate that new treatment strategies aiming at reestablishing the balance of the microbiota could be developed. (AU)

• FAPESP's process: 15/00259-0 - Microbial diversity of supra and subgingival biofilms and protein profile of saliva and acquired pellicle of subjects with aggressive periodontitis
• Grantee: Pâmela Pontes Penas Amado
• Support Opportunities: Scholarships in Brazil - Doctorate