Abstract
Streptococcus mutans is the leading microorganism associated with dental caries, the most common infectious disease afflicting humans, and it is also considered an important agent in the establishment of infective endocarditis (IE), a serious infection of the heart valves with high mortality rates. Invasive strains of S. mutans mainly of serotypes f and k were found to express a surface collagen-binding protein named Cnm, which avidly binds to collagen and laminin (extracellular matrices) and is associated with an increased ability of S. mutans to colonize human tissues. These findings strongly suggest that adhesion, invasion and persistence of S. mutans in the host's cytoplasm is an important pathogenic trait, hitherto unknown, which characterizes the presence of Cnm as a new virulence factor related to this microorganism. Hence, it was accomplished the molecular characterization of the cnm locus (transcriptional organization of the cnm operon, identification of environmental factors controlling the expression of Cnm), as well as the functional analysis of Cnm in relation to the binding to host's extracellular matrix. Ultimately, its glycosylated nature and biological function were also assessed (unpublished data). Complementary, and because of the need to elucidate the mechanisms by which S. mutans interacts with the host, we proposed in the Ph.D. linked project (FAPESP #2013/25080-7) to determine the prevalence of S. mutans strains expressing Cnm from caries-free and caries-active children and adults and establish a correlation between the presence of Cnm-expressing strains and the caries status of the individuals (A). Moreover, these Cnm-positive strains will be used to further investigate the role of such glycoprotein in the development and severity of root caries lesions in an animal model (B) and in the S. mutans - host interactions (in vitro and ex vivo) (C). In the research experience abroad (University of Rochester, BEPE scholarship requested), we plan to complete the aims A - partially (prevalence study) and C (pathogen-host interactions). In Brazil, the aim A will be continued as a multicenter prevalence study, and the aim C will be completed, thus fulfilling the goals of the Ph.D. linked project. Initially, samples of saliva, dental plaque and carious tissue will be collected from different caries-free and caries-active groups (children and smoker/non-smoker adults) and subjected to tests to identify which isolates express Cnm. Then, the Cnm-positive clinical isolates will be used to investigate the influence of such glycoprotein in the development and severity of root caries lesions in rats. Subsequently, the Cnm-positive isolates and also strains of S. mutans UA159 (non-invasive and Cnm-negative) and OMZ175 (invasive and Cnm-positive), cnm mutants and recombinant strains of Lactobacillus lactis expressing Cnm will be used in the platelet activation assays, including the isolation of platelets from healthy donors and evaluation of bacterial-platelet complexes and platelet activation by flow cytometry. Subsequently, these microorganisms will be assessed for adhesion to heart valves using an ex vivo porcine model and evaluated by Field Emission Scanning Electron Microscopy. The results of this research project are expected to provide new knowledge on the mechanisms linking Cnm to oral diseases, such as tooth decay, and to extra-oral diseases, such as IE. At the end of this project, we will have determined the prevalence of strains expressing Cnm in different age groups and the influence of such glycoprotein in the development and severity of caries. Ultimately, we will have established the role of Cnm in processes related to the pathogenesis of IE such as platelet activation and adherence to cardiac tissues. (AU)
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