Dental caries is the most common infectious disease in the world caused in large part by the Gram-positive bacterium Streptococcus mutans. Associated annual health care costs tens of billions of dollars, and rates of childhood caries in the U.S. are rising. There is a clear imperative to address this unmet health care need and identify new approaches to stem caries pathogenesis. Organisms that cause cavities form recalcitrant biofilms, generate acids from dietary sugars, and tolerate acid end products. It is recently recognized that micro- organisms can produce functional amyloids that are integral to biofilm development. In this proposal we show that the S. mutans cell surface-localized adhesin called P1 (Antigen I/II, PAc) is an amyloid forming protein. This conclusion is based on defining properties of amyloids including uptake of the amyloidophilic dyes Congo red and Thioflavin T, visualization of amyloid fibers by transmission electron microscopy, and green birefringent properties of Congo red-stained protein aggregates when viewed under cross-polarized light. Amyloid is present in human dental plaque and is produced by both lab strains and clinical isolates. We provide further evidence that amyloid formation is not limited to P1, as bacterial colonies without this adhesin demonstrate residual green birefringence. However, S. mutans lacking sortase, the transpeptidase enzyme that mediates covalent linkage of it substrates to the cell wall peptidoglycan, including P1 and five other proteins, is not birefringent when stained with Congo red and does not form biofilms. We also show that biofilm formation is inhibited when S. mutans is cultured in the presence of known inhibitors of amyloid fibrillization. Amyloid represents an evolutionarily conserved fibrillar, cross 2-sheet quaternary structure in which the 2-sheets laterally self-assemble to form fibers. We recently showed that crystalline P1 demonstrates a unique tertiary structure in which two globular 2-sandwich domains lie on either side of an extended hybrid alpha-polyproline II helix. In this study we will continue biophysical confirmation of P1 amyloidogenesis including its X-ray diffraction pattern, and capitalize on the new tertiary structure information utilizing solid state NMR, electron paramagnetic resonance (EPR), and cryo-electron microscopy methodologies to elucidate P1's ultrastructure particularly as related to its amyloid forming properties (Aim 1).
In Aim 2 we will identify and then characterize additional amyloid forming proteins of S. mutans using a similar approach as that followed for P1. Genes encoding amyloid forming proteins will be deleted singly and in combination.
In Aim 3 we will evaluate amyloid formation with respect to biofilm development using both static and flow models, and by assessing the effects of known amyloid inhibitors on S. mutans biofilm formation by wild-type and mutant strains. We will then confirm our results using the biophysical assays described in Aim 1. Together these Aims will reveal the contribution of amyloid formation to S. mutans biology, and demonstrate the feasibility of amyloid inhibition as a therapeutic approach to preventing biofilm formation by this pathogen.

Public Health Relevance

The bacteria named Streptococcus mutans cause cavities by living in sticky layers (biofilms) adhered to teeth and then destroying tooth enamel by the acid they make from dietary sugars. Amyloids are aggregates of proteins that form fibers. We show that biofilm formation by Streptococcus mutans involves amyloid fibrils built by the microbe;therefore, we will study how this process occurs and how to inhibit it.

Agency
National Institute of Health (NIH)
Institute
National Institute of Dental & Craniofacial Research (NIDCR)
Type
Research Project (R01)
Project #
5R01DE021789-03
Application #
8621984
Study Section
Oral, Dental and Craniofacial Sciences Study Section (ODCS)
Program Officer
Lunsford, Dwayne
Project Start
2012-03-05
Project End
2016-02-28
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
3
Fiscal Year
2014
Total Cost
$694,486
Indirect Cost
$219,486
Name
University of Florida
Department
Dentistry
Type
Schools of Dentistry
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611