Influence of Streptococcus mutans cell wall and cell membrane turnover on the bridging of extracellular matrix components and biofilm organization

• Grant number: 23/09612-0
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: August 01, 2023
• End date: January 31, 2027
• Field of knowledge: Health Sciences - Dentistry
• Principal Investigator: Marlise Inêz Klein Furlan
• Grantee: Emilio Alberto Ponce Fuentes
• Host Institution: Faculdade de Odontologia de Piracicaba (FOP). Universidade Estadual de Campinas (UNICAMP). Piracicaba , SP, Brazil
• Associated research grant: 21/06801-1 - Extracellular matrix: from biology to strategies for controlling cariogenic biofilms, AP.JP2

Abstract

Dental caries results from dysbiosis in biofilm derived from microbial metabolism of dietary carbohydrates. These carbohydrates are utilized to produce extracellular matrix components and organic acids that are trapped by the matrix inside the biofilm, and over time, these acids demineralize the surfaces and lead to teeth structure loss. The main matrix components are exopolysaccharides (soluble and insoluble), extracellular DNA, and lipoteichoic acids. The association of these components restricts the diffusion of substances in and out of biofilms. The main goal is to determine how Streptococcus mutans (the main known matrix producer species) cell wall and membrane turnover affects the bridging of extracellular matrix components and consequent configuration (creation of microniches). Additional aims include: to investigate whether there is an association between functional GtfB (insoluble glucan synthesis) and DltB (membrane channel for LTA secretion) in constructing biofilms; and, to evaluate the cell surface charge (hydrophobicity) change caused by the deletion of genes. Therefore, the strain UA159 and its deletion strains lytS, lytT, dltA, dltD, and ”gtfB will be grown as planktonic cultures; the cells will be harvested at mid-log and stationary growth phases for transmission electron microscopy (TEM), Scanning Electron Microscope (SEM), Fatty Acid Analysis (FAME), chemical analysis of LTA and charge (hydrophobicity). Next, biofilms of UA159, dltD, and ”gtfB will be formed on saliva-coated hydroxyapatite discs. The biofilms will be removed at distinct growth phases for confocal microscopy (structure and function via microniches evaluation), FAME, chemical analysis of LTA, and transcriptomics (RNA-seq). Also, the cell membrane microvesicles will be evaluated to determine their contribution to EPS production, in addition to eDNA. Also, to investigate whether there is an association between functional GtfB (insoluble exopolysaccharide synthesis) and DltB (membrane channel for LTA secretion) in the creation of cariogenic biofilms, a conditional mutant for the expression of both genes will be evaluated. Furthermore, the mechanism by which LTA is synthesized has been described for some species but not for S. mutans. Previous homology analysis demonstrated that SMU_775c was a candidate for LTA synthase or ltaS. Deletion mutation was lethal; thus, a conditional mutant will be constructed and subjected to distinct growth conditions and downstream assays to verify its phenotype. Quantitative data will be analyzed via descriptive and inferential statistics (±=0.05). (AU)