Specific Aims
Alpha Amylace (-Amylase) which is the most abundant enzyme in human saliva contributes to the formation of plaque (film of bacteria and mucus deposited on teeth that encourages the formation of dental caries). The alpha-amylase does this by acting as an ion or a molecule for the binding of Amylace-Binding Streptococci (ABS) to teeth.
The aim of this project is to address the association of ABS on dental plaque which can be applied in future studies and clinical applications. The association will use quantitative real-time PCR (qRT-PCR) to determine the amount of ABS relative to the total bacteria in a sample. The assertions made will be corroborated by using tests on model biofilms on calcium hydroxyapatite discs (HA).
Background and significance
Alpha amylase is not only the most abundant in saliva and its function is to catalyze the hydrolysis of starch into substances that can be utilized by the body. It breaks down starch into glucose, maltose, maltotriose and maltodextrins. However, the amylase adheres to several species of oral commensal streptococci such as Streptococcus gordonii, Streptococcus australis, Streptococcus salivarius, Streptococcus mitis, , Streptococcus cristatus and Streptococcus sanguinis. The adhesion is reliant on an in-vitro amylace-ligand binding phenomenon. In its structure, ABS possesses membrane proteins which attach the amylase to the bacterial cell surface called Amylase-Binding Proteins (ABPs) of which the most studied ABP is called Amylace Binding Protein A (AbpA). In particular, the AbpA of the S. gordonii which is an early colonizing bacterium in dental plaque interacts with the salivary amylase and it may lead to the formation of dental plaque.
Previous studies have suggested that the ABS evolutionary adjusted itself to the conditions in the mouth as well as the increased starchy diets common to many people. The ABS does this using the amylase in the salivary pellicle which acts as an adhesion (receptor) in order to enhance the action of bacteria on mucus and starch to form plaque. Amylase forms naturally as a constituent of the complex glycoproteinacious pellicle which forms on cleaned teeth. The pellicle (thin protective membrane) is the surface on which dental plaque forms. The formation of dental plaque leads to the increment in the ABS as well as the secondary colonizing bacteria. The colonizing bacteria in turn release metabolic by-products which when combined with the plaque lead to various oral problems such as inflammation, dental caries, periodontitis and gingivitis. Going by the results if previous studies, there is a heavy association between salivary amylase and the subsequent ABS expression of AbpA. Apparently, the higher the level of salivary amylase, the more the ABS attaches itself to teeth surfaces leading to formation of more plaque.
Preliminary Studies/ Progress Report
There are numerous studies going on in to research the issue of salivary amylase and the formation of dental plaque. One of these happens at Dr. Scannapieco’s laboratory where they are investigating the evolution of ABS. The study shows that a screen of the supernatants of 76 strains representing 13 streptococcal species detected several ABPs. Six of the species contained AbpA-like proteins based on the in-vitro amylase-binding phenomenon discussed earlier. The study showed AbpA-like sequences that varied in size from 20 to 30kDa. The study has sequenced (DNA-seq), assembled and annotated the genomes of 18 strains that represent the streptococcal species. At least five different N-terminal consensus sequences for genes encoding AbpA-like proteins have been identified out of the genomic sequences. It has also been established that besides streptococci, there is no other bacterial species that possesses AbpA. Going forward, the study will make use of the consensus consequences as a basis to design primers to detect ABS to be used in qRT-PCR.
