Currently funded:

Acquisition of the Human Oral Microbiome

The Human Microbiome Project has revealed the composition of adult human microbial communities at the level of species, but how they are acquired and how stable they are is not well understood. Community sequencing studies have shown relative levels of taxa to fluctuate over time at multiple body sites, but in the gut and urinary tract, membership of human microbial communities shows considerable stability over the long term. This suggests that the human microbiome is a stable feature with implications for disease and health similar to that of our human genome. This important potential determinant of oral health needs to be investigated starting with the initial acquisition of the oral microbiota. We have recently demonstrated a robust assembly of a common core set of species in human infants that is resistant to disruption by a number of environmental perturbations. This remarkable specificity demonstrates a surprisingly robust process and suggests a biologic blueprint for acquisition of the oral microbiome, and some potential drivers have a human genetic basis. We hypothesize that each human harbors a set of strains/species that is particularly well adapted to his or her specific genetically determined environment, and that these bacteria are a better fit for genetic offspring than those harbored by unrelated individuals. If so, the human microbiome may be another form of heritable genetic material passed from parent to offspring — another sort of “epigenetics”. In aim 1 we will elucidate the natural history of acquisition of the oral microbiome from infancy to adolescence and measure stability of microbial community membership over two years. In aim 2 we will determine the contribution of genetics to bacterial transmission from mothers to babies by comparing biologic and adoptive mother-child dyads. We will determine relative levels of bacterial species using 454 pyrosequencing of the 16S rRNA gene. Comprehensive analysis of strains has just become technically feasible, and this will allow us to elucidate stability, assembly of the oral microbiome and transmission at a new level of resolution. We will determine strain variation by metagenomic sequencing and mapping of bacterial sequences to reference genomes and determining single nucleotide polymorphisms (SNPs). The proposed studies will elucidate the assembly of oral microbial communities from birth through adolescence. They will reveal the complexity of the microbiome at the level of species and strain, the extent to which humans share a core set of species and strains, their stability over time and in response to perturbations, and to what extent the human oral microbiome is a heritable feature, similar to our human genome. These findings will have major implications for disease and health including possible determinants of disease susceptibility, risk assessment and feasibility of strategies for prebiotic/probiotic therapies.

Culturing of the uncultured: reverse genomics and multispecies consortia in oral

The human oral cavity hosts at least 600 microbial species, many of which are uncultivated and whose roles in human health or disease remain unclear. This is especially true in the subgingival crevice where microbial communities interact through complex food webs forming structured metabolic guilds. These communities potentially lead to diseases of the mouth such as periodontitis, which affects approximately one third of the adult population. Recent, open-ended studies utilizing DNA-based methods have shed new light on the complex nature of subgingival microbial community structure. However, without advances in understanding the physiology of potentially key, uncultivated groups, treatment and more importantly, prevention of diseases such as periodontitis will remain difficult. To address this, we propose a hypothesis driven, targeted approach to selectively bring heretofore uncultivated, disease-associated microorganisms (e. g. candidate phyla TM7 and SR1) into study through advanced cell isolation and high-throughput cultivation techniques. We will employ a proven, immunological-based method, which has successfully been shown to capture rare and uncultivated microorganism from natural environments. This method leverages genomic information to synthesize surface antigens expressed by selected organisms thus avoiding a random approach to enrichment/cultivation. Novel isolates will be studied individually but more importantly, in combination with other species potentially serving as metabolite facilitators or syntrophs found in the same niche and health/disease state. Highly controllable microenvironments will be established using microfluidic systems that allow biofilm cultivation in conjunction with real-time, advanced imaging techniques. Model living hosts will also be established in Drosophila to simulate complex interactions in a biological system. These platforms will enable microcultivation of novel oral microbiota needed to establish integrated comparative and functional genomic studies, and will advance our understanding of the intimate interactions between microbes inhabiting the subgingival environment and their role in disease.

The Oral Microbiome in HIV-associated Oral Warts and Candidiasis

Human immunodeficiency virus (HIV) has infected over 30 million individuals worldwide. Left untreated, over 50% of these individuals will incur an oral manifestation associated with HIV. Currently, the most common oral manifestations of HIV disease are oropharyngeal candidiasis (OPC) and oral warts. In addition, HPV-associated head and neck squamous cell carcinomas (HNSCC) are increased in HIV+ individuals and have not seen a reduction in prevalence with the advent of anti-retroviral therapy (ART). The factors which determine an HIV+ individual’s predisposition to OPC, HPV and other oral manifestations are poorly understood as well as how treatments for HIV influence the oral cavity and/or infections/diseases. A realistic description of the composition and diversity of the oral microflora (microbiome) is essential to understanding health and disease in the oral cavity. However, how the oral microbiome is impacted by HIV has not been described, but based on preliminary data, is expected to be significant. Moreover, ART used to treat HIV also likely impacts/alters the oral microbiome. We will use PCR-amplification of 16S rRNA genes and massive parallel sequencing to test the hypothesis that both HIV infection and ART alter the composition and diversity of bacterial species in the oral cavity and such composition can be associated with risk of oral manifestations such as HIV-associated OPC and ART-associated oral warts and HNSCC.

Polymicrobial oral bacterial host interactions in a high throughput model

Chronic periodontitis affects half of adults in the US, and pathogenesis results from polymicrobial-host interactions that are not completely understood. Using next generation sequencing technology many new candidate species have been associated with disease, and others with health. But epidemiologic data does not establish causality, so the role of most of these species in periodontitis remains hypothetical. In attempting to understand pathogenesis, a holistic view that includes contributions from both microbes and host is needed. If we are to unravel host interactions with highly complex bacterial communities, we will need to systematically screen in a high throughput system that models host-microbe interplay. And for clinical relevance it is important to ground studies in human epidemiologic data to target candidates from the large number of possible combinations. Deep-sequencing data generated by our group on species prevalence in health and disease, and co-occurrence in individuals, will be used to guide the selection of candidates for the proposed studies. The genetically tractable animal, Drosophila melanogaster, shares many mammalian innate immune response features, has been well developed as a model host-microbe system, and has been adapted for oral microbiology research by our group. The natural infection (feeding) model provides a high- throughput, powerful approach that allows us to detect and understand in vivo interactions between oral polymicrobial communities and the host. The Drosophila model also allows comprehensive and simultaneous monitoring of bacteria and host gene expression profiles during infection, using RNA-seq. The aims of this project are to identify virulent and beneficial oral species and to identify virulence-enhancing and -attenuating interactions between oral species, in the Drosophila model. Then candidate bacterial and host factors that are important for these interactions will be identified using RNA-seq, and tested using Drosophila gene knockout mutants and/or RNAi knock-down lines. This project will be a considerable step forward in understanding the pathogenesis of periodontitis in a holistic view that includes interactions of microbes with one another and the host. Ultimately this work could lead to therapeutic interventions on the host or bacterial side. If beneficial interactions are discovered, translation to chairside as probiotic therapies could occur very rapidly. Strategies to target virulence mechanisms would have a longer horizon, but are highly likely to be suggested by the project.