This proposal from the University of California, San Francisco seeks supports for a K08 Mentored Clinical Scientist Research Career Development Award to provide the necessary training, practical experience, and knowledge for the candidate to characterize the microbial etiology of refractory growth following treatment of carious lesions with silver diamine fluoride (SDF).

The research plan proposes to test the hypothesis that refractory growth of carious lesions after SDF treatment occurs because genetic regulation of heavy metal detoxification proteins enable cariogenic bacteria to penetrate through an SDF-induced structural barrier. The Aims of this proposal are to: (Aim 1) Identify differentially abundant bacterial species in SDF-refractory carious lesion growth; (Aim 2) Test the relationship of bacterial SDF tolerance to penetration through SDF-treated carious lesions; and (Aim 3) Elucidate the role of metal resistance genes in SDF tolerance.

Dental caries occurs when dental plaque bacteria ferment dietary sugars into acids that dissolve the tooth. Caries is the most prevalent disease, is a leading cause of emergency room visits, and is one of the most expensive diseases in the U.S. Caries therapies are used with the goals of eradicating the bacteria and strengthening the weakened tooth structures. Topical application of SDF twice per year arrests 68-89% of cavitated dentin lesions (lesions stop growing and harden). Yet the 11-32% that continue to grow will amount to hundreds of millions of cavities that will not respond to this treatment. The occurrence of caries arrest and refractory growth in the same patients suggests that tooth-level differences such as bacterial constituents of the carious lesion contribute to treatment response.

The research strategy addresses substantial gaps in existing knowledge of SDF. The multi-disciplinary project builds from clinical response to SDF, to metagenomic determination of differentially abundant bacteria in clinical samples with caries arrest or refractory growth, to validation of differential SDF-tolerance in the abundant species isolated from the same samples, to assessment of the isolates in an ex vivo carious lesion model, to genetic and transcriptional assessment of laboratory phenotypes. The mid-term objective of this research is to develop prognostics to predict which lesions will respond to SDF, and the long-term objective is to use the mechanisms discovered to develop better therapies beyond SDF. The goal of the career development award is to transition the candidate into an independent investigator with work combining clinical and laboratory studies in support of improved oral health and prevention of disease.

The research will lead to improved treatment and generalizable knowledge impacting treatment of tooth decay and provide data in support of independent research grants. The career development aspect permits the candidate to gain the skills to contribute to the next generation of advances in dental caries management.

This K99/R00 resubmission requests support for Jeremy A. Horst for mentored research and career development with the goal of becoming an independent investigator. The multi-disciplinary research will employ a combination of simple and state-of-the-art physical imaging and bacteriological techniques to identify failure modes of SDF in the treatment of cavities. During the K99 phase, under the combined mentorship of world class experts in infectious disease microbiology, clinical studies of dental caries interventions, and enamel structure (mentors), and caries bacteriology, dentin structure, physical imaging, and SDF clinical use (collaborators), the candidate, will accomplish three key goals:

First, participate in didactic education and mentored skill acquisition in the disciplines of the mentors and collaborators, visit the laboratories of 3 of the collaborators each for a week each year during the K-phase, take courses, perform literature reviews on caries lesion microstructure and bacteriology, and participate in scholarly activities including journal clubs and research meetings. Second, conduct microbiological (Aim 1) and physical (Aim 2) assessment of clinical samples of teeth and dental plaque, to test for associations of quantifiable physical structures, microbial behaviors, and genetic elements to SDF treatment failure. Third, perform inductive laboratory studies on microbial resistance to SDF (Aim 1), validate a laboratory model of SDF treatment failure, and use this model to evaluate alterations that mitigate undesirable qualities of SDF (Aim 3).

Silver diamine fluoride (SDF) stops 81% of dentinal caries lesions (cavities), is inexpensive, and is simple to apply. Yet the 19% that continue to grow amount to hundreds of millions of cavities that will not respond to this treatment. The occurrence of treatment failure and success for different cavities in the same patients suggests that tooth-level differences contribute to treatment response. Biological and structural characterization of treatment failure and establishment of laboratory models thereof will lead to improved treatment and avoidance of suffering by anticipating failure.

The research strategy addresses substantial gaps in existing knowledge of SDF, which will lead to improved treatment and generalizable knowledge to impact the treatment of tooth decay and provide data in support of an independent research support. The career development aspect will permit the candidate to gain the skills necessary to contribute to the next generation of advances in dental caries management. Short term goals following this project are to test the ability of available dental products to address the structural and/or bacterial correlates of SDF treatment failure identified in the proposed research. The long-term goal of this project is to transition the candidate into a career combining clinical and laboratory studies to drive the development of therapeutics for dental caries.

The primary goals of this proposal are to transition the applicant into a productive faculty role advancing oral health through research, education, and service, and to identify predictive factors for the success of dental cavity treatment using a brush-on medication called silver diamine fluoride.

The proposal describes use of multidisciplinary techniques to approach the research question. Experiential learning with these techniques will prepare the applicant to lead investigations throughout his career.

Silver diamine fluoride (SDF) has the potential to become the first drug for dental caries. It works well, is inexpensive, and application is simple. The contemporary paradigm of dentistry does not address the disease entity, does not prevent future disease, is expensive, and for young children requires expensive and dangerous interventions to enable treatment. SDF is a compelling approach to the inherent problem. Yet SDF does not always work. Some cavities stop, while others continue to grow, even in the same patient. No mechanistic work has been done to figure out why this happens, or to avoid suffering by anticipating failure.

In Aim 1, I will characterize changes in the dental plaque microbiota of patients treated with SDF, using massively parallel genetic sequencing. Samples come from participants in a short-term randomized clinical trial and a 18 month cohort, with multiple sites at each timepoint, all designed for this proposal. Sampling in the cohort will occur prior to SDF treatment and 1, 3, 7, 14 days, and 1, 3, 6, 12, and 18 months after. I will report analysis of the trial samples, and of the first month for the cohort, during the K99 phase, and publish a final report on the cohort during the R00 phase. Reports will focus on predictors of treatment success or failure.

In Aim 2, I will characterize the changes to the cavity’s chemical structure in response to SDF, using three-dimensional (3D) nano-radiography, and two-dimensional (2D) chemical nano-imaging. Samples come from teeth that were clinically treated with SDF and then lost by extraction for orthodontic purposes, due to abscess, or naturally exfoliated. As for the dental plaque cohort, I will issue a semi-quantitative and descriptive characterization during the K99 phase, and a comprehensive quantitative report during the R00 phase.

In Aim 3, during the K99 phase I will study the responses to SDF of dental cavity plaque cultured in a cutting-edge laboratory model. In the R00 phase I will validate the laboratory model against the clinical response by assessing dental plaque responses to SDF in the model and in the patient who gave the plaque sample. I will then monitor these patients to validate the short-term laboratory study to disease treatment outcomes.

Finally, I will bring these studies back to clinical relevance during the R00 phase by collating the factors that correlate best with treatment success or treatment failure in these studies into a predictive model, which will be the subject of a collaboratively biomarker trial planning grant application using the predictive model as a diagnostic for SDF treatment outcome.

Despite over 100 years of active research, there are no drugs to treat dental caries (tooth decay). Caries occurs when bacteria ferment simple sugars into acids that dissolve the tooth. Bacteria in dental plaque and carious lesions are wildly heterogeneous, creating the situation of an abundance of poorly characterized organisms and confusion about what to target.

The emergence of efficient whole genome sequencing and expanding interest in the microbiome create the opportunity to assess genetic correlates of caries at the highest biologically encoded resolution.

The genus Lactobacillus offers a compelling study model for caries microbiology because of strong correlations to caries, broad genetic heterogeneity, and the functions of lactic acid production and low pH tolerance being common but varying broadly in magnitude across species and strains.

I hypothesize that caries is caused by controllers of common metabolic acid production pathways that are encoded by genetic elements that vary across strains. To address this hypothesis I will correlate genetic elements to differences in acid production and low pH tolerance in clinical and probiotic isolates of Lactobacilli.

I also hypothesize that metabolite-like small molecule compounds control acid production in cariogenic bacteria. To address this hypothesis I will build a proteome-wide pharmacopeia-wide predictive interaction platform to assess the relationship of all modelable proteins to all human-ingestible metabolite-like compounds, and select compounds to target acid control proteins.

I will then integrate these complimentary data into a testable mechanistic model of the pharmacologically controllable aspects of acid production in cariogenic bacteria. The goal of this work is to identify genes and proteins that can be used as diagnostics and drug targets, and compounds that can be used as therapeutics.

My preliminary results demonstrate a wide range of cariogenic behaviors and genomic composition among clinical and probiotic isolates of Lactobacilli. I used the first proteome-wide pharmacopeia-wide interaction matrix to select extremely safe metabolite-like compounds, predicted to target proteins predicted to control acid production, that actually do decrease acid production by S. mutans. It is likely that I will have similar success in Lactobacillus, and that this project will result in identification of lead inhibitors of caries.

These analyses lay the foundation for the genomic and proteomic study of cariogenic behaviors in dental plaque bacteria. To propel advancement in the field, I will make all relevant data accessible through public data repositories including NCBI, and relate models of pH control through gene sequence to other putative cariogenic genera such as Streptococcus, Actinomyces, Bifidobacterium, and Scardovia. Deliverables include the identification of genes, proteins, and metabolite-like compounds to control acid production and low pH tolerance in cariogenic bacteria – from which diagnostics and therapeutic will be developed in future work.