PROJECT SUMMARY/ABSTRACT Periodontitis is a highly prevalent disease associated with several debilitating systemic conditions, including vascular and lung diseases, diabetes mellitus, and pre-term birth. The most recent epidemiological evidence suggests that tobacco smoking accounts for the majority of destructive periodontal disease cases in developed nations. Smoking enhances infection rates and enriches numbers of a key periodontal pathogen, Porphyromonas gingivalis. However, the mechanisms underlying this phenomenon are in need of elucidation. In the first iteration of award, facilitated by a two year NIGMS-funding mechanism, we generated an 80,000 colony transposon sequencing library for P. gingivalis strains ATCC 33277 and exploited this resource to: (i) Successfully identify 256 genes that are putatively essential for surviving tobacco-induced stress; (ii) Delineate a “core stress genome” commonly required for tobacco-survival, epithelial colonization and subcutaneous abscess formation; and (iii) Validated several genes as conditionally essential for P. gingivalis to survival of cigarette smoke- induced stress in vitro by generating single gene deletion mutants that exhibit either reduced absolute fitness in competition assays with the parental P. gingivalis strain in smoked medium; (iv) We have also generated a novel murine model of tobacco-exacerbated periodontitis. We now plan to build on these successes via three specific aims, as follow: Aim 1: Further elucidate P. gingival CSE-related survival strategies by generating mutants in single genes that are both CSE-essential (TnSeq data) ;and tobacco-regulated (RNAseq and qPCR); yet not compromised in their ability to colonize oral epithelial cells (TIGK model) or immune resistance (murine abscess model). Aim 2: Determine in vivo relevance of putatively CSE-essential genes using a murine smoke-exposure chamber model in a chronic Baker model of P. gingivalis-induced periodontitis. Aim 3: Establish the broad applicability of data gathered in Aims 1 and 2 by confirming biological function and distribution of CSE essential genes in multiple P. gingivalis strains. Long term advances are likely to include (a) A better understanding of how P. gingivalis thrives in a tobacco-toxin rich environment; (b) Future therapeutic targeting of essential genes to control P. gingivalis infection in smokers (CSE-essentiality data) and in general (multiple disease-relevant conditions, core stress genome data); (c) Alternate treatment regimens for smokers based on mechanistic insight into smoke-induced and/or exacerbated periodontal diseases;and (d) The establishment of an essential gene database for tobacco- enhanced pathogens that will facilitate the identification of common bacterial strategies for surviving tobacco smoke exposure, thus, broadening the significance of the research beyond the oral cavity.