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

Isolation of utricle hair bundles from Peter Barr-Gillespie's analysis of the proteome of hair-cell stereocilia. Ferracane’s new model draws on Barr-Gillespie's technique.

Isolation of utricle hair bundles from Peter Barr-Gillespie’s analysis of the proteome of hair-cell stereocilia. Ferracane’s new model draws on Barr-Gillespie’s technique.

Breakthrough innovation doesn’t happen overnight, and it doesn’t happen without money. OHSU Core Pilot Grants provide OHSU researchers with funds to develop new concepts or methods and to strengthen extramural grant proposals. The program is made possible by University Shared Resources, the School of Medicine, and the Office of the Senior Vice President for Research.

In 2016, the University Shared Resources pilot funds provided more than $200,000 for early research by 22 OHSU researchers. With this funding, a project by Jack Ferracane, Ph.D., professor and chair of the Department of Restorative Dentistry, was made ready for a competing continuation grant application to the National Institute of Dental and Craniofacial Research.

Lead author of the study, Kirsten J Lampi, Ph.D., a professor in the School of Dentistry, will present a poster of the research at the 2017 International Association for Dental Research meeting.

Ferracane’s work focuses on the structure, function, and clinical performance of dental restorative materials and his team has been developing antimicrobial and remineralizing dental materials containing bioactive glass to enhance the durability and longevity of dental restorations. The vast majority of adults and 60–90 percent of children are affected by tooth decay, making it a significant public health issue.

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 shown. 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.

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 PSR 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 PSR, 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.

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Casey Williamson writes about research at OHSU.

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