Salivary amylase provides an excellent example of a salivary protein that has the potential to modulate the bacterial colonization in the oral cavity. Although its binding to bacteria in solution may result in bacterial clearance (protective) its presence in the enamel pellicle may facilitate Dental plaque formation (harmful). In the last granting period, the structure-function relationships of amylase were examined in the context of its physiological role in starch hydrolysis, hydroxyapatite binding and bacterial binding on plaque formation. We will continue our studies on the three functions of amylase through molecular approaches. Several mutants of amylase will be designed to abolish starch binding and hydrolysis, amylase binding to hydroxyapatite and S. gordonii protein AbpA. These mutants will be generated using an established baculovirus expression system. Individually, these mutants will be utilized to illustrate the interrelationships among the three functions and to test hypotheses related to the localization of starch binding, HAp binding and bacterial binding sites in amylase. In addition, using these mutants, we plan to study the role of amylase in the basic mechanisms responsible for the colonization of a pioneer bacterium S. gordonii and a cariogenic bacterium such as S. mutans. Given its stature as a major protein in human saliva and its ability to interact with proteins from S. gordonii (AbpA) and S. mutans (Gtf), amylase may play a central role in the enamel pellicle. Through amylase, S. mutans may bind to an established S. gordonii biofilm either directly or through an interaction with A. naeslundii: Our goal is to define the role of amylase in these complex intergeneric interactions. A well established flow cell model incorporating co-cultured S. gordonii, A. naeslundii and S. mutans bathed in amylase or saliva will be used to examine these relationships. The second generation amylase mutants proposed here will eventually lead to the design of strategies to manipulate human salivary amylase-bacterial interactions that favor the host and thus reduce the potential for Dental diseases mediated by biofilms. Finally, we are excited that our approach in dissecting the contribution of amylase to foster the colonization of S. mutans may serve as a model to study the role of other salivary proteins in the oral cavity.
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