OHSU researchers develop new model to study S. mutans behavior at protein level

Jack Ferracane

Breakthrough innovation doesn’t happen overnight, and it doesn’t happen without money.

Early in their development, new concepts, materials, and methods are not strong candidates for extramural funding. The OHSU Core Pilot Grants provide researchers with funds to develop early-stage innovations. The University Shared Resources pilot program is supported by the Office of the Senior Vice President for Research and the School of Medicine.

In 2016, Core Pilot Grants supported early research by 22 OHSU researchers with more than $200,000.

Pilot funding supported Jack Ferracane, Ph.D., professor and chair of the Department of Restorative Dentistry, in the development of new dental restorative materials, readying the innovation for a competing continuation grant application to the National Institute of Dental and Craniofacial Research.

Ferracane’s team has been developing antimicrobial and remineralizing dental materials containing bioactive glass. The vast majority of adults and 60 to 90 percent of children are affected by tooth decay, making it a significant public health issue. The materials being developed by Ferracane enhance the durability and longevity of dental restorations.

Until now, the rate of bacterial colonization in gaps in dental restorations compared to colonization on the surface of the same type of material has not been demonstrated. Ferracane used a model that has never before been presented or tried to conduct novel experiments on the behavior of Streptococcus mutans bacteria on the surface of a restoration and within a small gap space. S. mutans is the main cause of dental decay and the challenge here was to collect enough protein to study differences in bacterial behavior at the protein level.

Ferracane, and his collaborators Kirsten Lampi, Ph.D., professor of Integrative Biosciences, and Justin Merritt, Ph.D., associate professor of Restorative Dentistry, worked closely with Larry David, Ph.D., and Ashok Reddy, Ph.D., director and associate director, respectively, of the Proteomics Shared Resource facility, to develop and test a methodology to recover and digest the small amounts of proteins present in the recovered bacterial biofilm.

The team used the high-resolution Thermo Scientific™ Orbitrap Fusion™ mass spectrometer in the Proteomics Shared Resource to measure relative changes in protein abundance. Using Tandem Mass Tagging™ technology, the technique allows 10 individual samples to be simultaneously analyzed, increasing the speed and accuracy of the analysis.

Reddy and David worked closely with Ferracane, Merritt, and Lampi to develop and test a methodology to recover and digest the small amounts of protein present in the recovered bacterial biofilm. Their developed methodology used a combination of shearing the bacterial biofilm by intense shaking in the presence of glass beads (bead beating), followed by digestion with trypsin with the aid of ultrafiltration membranes.

Ferracane’s new model draws on a technique developed by Peter Barr-Gillespie in his work on the hair cells that allow humans to perceive sound. The image above is from Barr-Gillespie’s analysis of the proteome of hair-cell stereocilia.

Establishing the protocol took some trial and error, but demonstrated why having core facilities on campus is essential when establishing a new protocol like this.

In this particular case, the use of ultrafiltration membranes to assist protein digestion was a technique borrowed from Peter Barr-Gillespie, Ph.D., professor of otolaryngology and associate vice president for basic research, who uses it when digesting extremely small samples of hair bundle proteins for proteomic analysis in the Proteomics Shared Resource facility. Reddy realized that combining the bead beating technique from Ferracane’s lab and the digestion method from Barr-Gillespie’s lab was the perfect solution to analyze very small samples of bacterial biofilms.

The new model successfully demonstrated that the rate of colonization was lower in the gap than on the surface of the restorative material. Additionally, the model was able to show that certain dental composites with ionreleasing properties show some inhibiting effect on bacterial colonization of gaps.

The University Shared Resources pilot funds, along with scientists and technology at the Proteomics Shared Resource, allowed Ferracane’s team to collect preliminary data sufficient to begin writing the NIH grant application and a manuscript for publication.

The pilot funds, launched in 2016, have supported 45 scientists with more than $360,000. University Shared Resources intends to begin the application process later this year for 2018 funding. If you have worked on a project made possible by the pilot funds, contact Andy Chitty to share your experience.