Participation of two-component regulatory systems in the formation of S. mutans biofilm on the surface of the teeth of children and adults

The aim of the planned research is to detect the presence of 13 two-component regulatory systems (TCS) identified in the genome of S. mutans UA159 (ATCC 700610) and to investigate the role of four of them, by constructing deletion mutants of S. mutans clinical isolates, in the formation of dental plaque in children and adults, taking into account two types of teeth (permanent and milk). The obtained strains with inactivated genes encoding sensor domains will be analyzed in terms of the produced biomass and the structure of the created biofilm. As part of this project, it is also planned to assess the drug susceptibility of clinical strains of S. mutans and the obtained mutants to selected antibiotics and chemotherapeutic agents. Swabs from the tooth surfaces of children and adults will be taken and inoculated onto Mitis Salivarius Agar supplemented with sucrose, bacitracin and potassium tellurite, and then incubated for 48 h at 37 ° C in 5% CO2 concentration. The identification will involve two steps: identification based on biochemical features and genetic identification using the nested-PCR method. The project will then rely on basic molecular biology techniques (ie isolation of genetic material, PCR, Multiplex PCR, restriction analysis, etc.). Multiplex PCR and / or classical PCR will be used to identify 13 TCS. In turn, the method of ligating mutagenesis will be used to inactivate genes encoding two-component systems. The ability and intensity of in vitro biofilm formation on polystyrene titration plates, both by clinical isolates and mutant strains, will be assessed by crystal violet staining and by MTT staining using a spectrophotometer. The biofilm culture will be established on BHI medium and incubated at 37 ° C in 5% CO concentration for 24 hours to 48 hours. In turn, the structure of the biofilm will be examined using scanning electron microscopy. The assessment of drug susceptibility of clinical strains of S. mutans and mutants will be carried out with the use of the Vitek-2 Compact system. S. mutans is one of the most important species of microorganisms among the microflora inhabiting the oral cavity of adults and children, directly related to the formation of caries in permanent and deciduous teeth. Caries is a very serious and common infectious disease, and despite the advances in dentistry and hygiene, it remains a significant problem in our society. One of the important factors in the virulence of carcinogenic bacteria, including S. mutans, is the ability to create plaque, the so-called natural biofilm. In a diverse environment, which is the oral cavity, bacteria living in the structure of the biofilm are exposed to various adverse environmental factors, such as changes in temperature, nutritional conditions and oxygen intensity. Their ability to adapt, survive and cause such a serious disease as caries indicates the need for an in-depth analysis of the molecular mechanisms of the response of bacteria living in the biofilm to the stress conditions encountered. As demonstrated in the case of S. mutans, the response to environmental changes is mediated by two-component regulatory systems. Therefore, an in-depth understanding of the structure and functioning of TCS seems to be necessary for the complete elimination or modification of the characteristics of this microorganism, the more so as recent years' research has shown that two-component regulatory systems may constitute new potential sites of drug action.