Project Summary Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer worldwide with an average 5- year survival rate of only 68% and accounts for over 180,000 deaths annually. Risk factors for OSCC include tobacco and alcohol usage, and human papillomavirus infections. Poor oral hygiene has also been correlated with various cancers and other chronic diseases. The oral microbiome has therefore been hypothesized to play a role in the development of these diseases. In recent years, multiple studies have clearly shown a significant difference in the oral microbiome composition of OSCC patients versus non-OSCC individuals. However, only two oral pathogens, Porphyromonas gingivalis, and Fusobacterium nucleatum, have been shown to have direct effect on OSCC development, while little is known about whether other members of the oral microbiome also contribute to cancer progression. Recently, we discovered that Prevotella intermedia, another well-known periodontal pathogen previously found to have higher abundance in oral cavity of OSCC patients, also contribute to cancer progression. We showed that certain P. intermedia isolates enhanced the proliferation and migration of multiple human OSCC cell lines in part by the secretion of the cysteine proteinase Interpain A (InpA). We further revealed that InpA might enhance OSCC proliferation and migration by interacting with the human protease-activators receptor 2 (PAR2), a known oncoprotein. To test the hypothesis that P. intermedia is a novel oncobacterium, we aim to identify the mechanism by which P. intermedia stimulates OSCC progression. In addition to confirm the role of InpA and PAR2 in P. intermedia-stimulated OSCC progression, we will perform transcriptomic studies to reveal the full extent of P. intermedia’s effect on OSCC gene expression. Identification of P. intermedia genes associated with OSCC progression will be achieved by the establishment of a high-throughput assay to screen for P. intermedia transposon mutants deficient in their ability to enhance cell proliferation and migration. In the second aim, we will determine if polymicrobial interaction plays a role in P. intermedia-stimulated carcinogenesis by in vitro assays and by establishing a murine model of oral tumorigenesis. The studies presented here will reveal for the first time that P. intermedia contributes to OSCC progression. Equally important are the novel high-throughput screening system developed in this study which will provide invaluable tools for other investigators to examine the role of P. intermedia in other diseases. Finally, our study will yield clues to the mechanisms behind oral microbiota facilitated OSCC development.