Extraction protocols and chemical characterization of cell wall polysaccharide and intracellular polysaccharide from Streptococcus mutans biofilm

Author(s)

Ana Carolina dos Santos Ré

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2022-05-13

Examining board members

Carolina Patricia Aires Garbellini; Elaine Rosechrer Carbonero; Silmara Aparecida Milori Corona; Marlise Inêz Klein Furlan

• Advisor: Carolina Patricia Aires Garbellini; Antonio Pedro Ricomini Filho

Abstract

Streptococcus mutans is one of the microorganisms related to the development of dental caries, producing a biofilm, a complex three-dimensional structure composed mainly of polysaccharides, which can be classified as: i) extracellular polysaccharides, dispersed throughout the biofilm matrix, protecting bacteria especially from antimicrobial compounds; ii) rhamnose-containing cell wall polysaccharides (RHAP) anchored to peptidoglycan, essential for bacterial virulence and iii) intracellular polysaccharides (IPS), polymers stored inside the cell, acting as an energy reserve. In contrast to extracellular polysaccharides, the chemical structure and the protocol for the extraction of RHAP and IPS is not established in the literature. Thus, considering the biological importance of RHAP and IPS, we aimed to evaluate a protocol for extraction of these polymers and study their structural characteristics in S. mutans biofilm. For this, S. mutans UA159 biofilms were formed on glass slides for 5 days, and were collected and processed to remove the extracellular microbial matrix. The resulting bacterial cells were submitted to chemical (hot alkaline extraction with 0.5 M or 1 M NaOH for 5, 10 or 15 min at 100 °C) or physical (sonication with 1 pulse of 15 s with potency of 5W or 20W in water or saline solution) parameters to obtain the RHAP and IPS. After acid hydrolysis (trifluoracetic acid for 8 h at 100 °C), the monosaccharide composition of all experimental groups was determined by high performance liquid chromatography associated with a refractive index detector, and the extraction protocol with 1M NaOH, 15 min at 100 °C was selected for the chemical characterization of the structures using nuclear magnetic resonance (NMR). The results suggest that while IPS is a glucose polysaccharide with α-(1→4) main chain linked with α-(1→6) branched glucose side chains, RHAP is a rhamnoglucan with α-(1→2) and α-(1→3) rhamnose main chain and α-(1→2) glucose side chains. Both structures are being studied for the first time in a single fraction. Thus, a single alkaline homogenate obtained from cariogenic biofilm helped to clarify the chemical structure by NMR of two molecules: i) the cell wall polysaccharide, which may be key to the design of future therapeutic targets for antimicrobials and ii) the intracellular polysaccharide, which may contribute to a better understanding of the carbohydrate metabolic pathway in S. mutans. (AU)

• FAPESP's process: 16/24042-2 - Efect of antimicrobial on chemical strucuture and metabolism of storage polysaccharides from cariogenic biofilm
• Grantee: Ana Carolina dos Santos Ré
• Support Opportunities: Scholarships in Brazil - Doctorate