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"STRUCTURAL-FUNCTIONAL ANALYSIS OF Streptococcus gordoniiís AMYLASE BINDING PROTEIN A"

by
Prerna Gopal
Oral Biology Program
B.D.S. 2007, SRM University, India
Thesis Advisor: Narayanan Ramasubbu, Ph.D. Professor
Department of Oral Biology

Thursday, June 25, 2015
11:00 A.M., Room: B853, Dental School


Abstract

Streptococcus gordonii is one of the most common initial colonizers of the oral cavity. Its ability to bind to salivary Š amylase with the help of its Amylase binding protein A enhances the bacteriumís survival and colonization in the complex oral ecology. AbpA is known to play critical role in S.gordoniiís attachment to tooth surface and biofilm formation. Interestingly, this protein is present in other initial colonizers as well and possibly plays the same role of aiding bacterial attachment to the tooth surface. Being one of the first inhabitant of the oral cavity, these primary colonizers get to decide the progression of the oral biofilm either by helping the secondary colonizers or preventing their attachment by creating an environment fatal for their successors. Hence this study is focused on understanding AbpA and HSAmy interaction and how this protein interaction can affect the overall survival of the primary colonizers.
Our first aim was to determine the structure function analysis of AbpA. With the help of homology modelling we designed several muteins of the AbpA protein and studied their interaction with HSAmy. We used these muteins in real time binding assays ĖSPR and ITC to find out the binding site on Amylase and were successful in identifying that the bacterial binding site and enzymatic site of amylase are two distinct identities.
The second aim of the study was focused on exploring how amylase and AbpA induce changes in the S.gordoniiís gene expression profile for bacteriaís successful colonization in the oral cavity. We performed RNA Sequencing on wild type and AbpA mutant S.gordonii in the presence of amylase and found that the amylase upregulates genes involved in bacterial metabolic pathways especially galactose metabolism. On further analysis on the role of AbpA, we found that in the presence of amylase and AbpA, the bacteria expresses genes that enhances its attachment, biofilm forming capacity, generates more ammonia that regulates the oral pH and also creates suitable environment for other selective bacteria to attach to S. gordoniiís surface.
This study broadened our understanding of the crucial role that initial colonizers can play in governing the cariogenicity of the dental plaque from a molecular prospective.


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