PROJECT SUMMARY For bacterial pathogens, the first studies that begin to define the microorganism’s functional genetic nature on a genome-wide scale, frequently represent a landmark and highly impactful achievement. The major human pathogen Chlamydia trachomatis is a bacterium for which such studies have not been performed, in large part due to Chlamydia’s obligate intracellular nature and historical intractability to modern genetic manipulation. With the development of two major genetic tools for Chlamydia by our group—transposon mutagenesis and lateral gene transfer chimeras—functional genomic investigations in Chlamydia are now possible. Our long term goal is to globally define and functionally characterize the genetic correlates to C. trachomatis infection and pathogenesis. Using the complementary approaches of transposon mutagenesis and interspecies chimeras, we propose to: (i) identify the major genetic determinants of Chlamydia infection using a clinically relevant mouse model and in vitro assays; (ii) determine the Chlamydia genes associated with host adaptation by exploiting interspecies lateral gene transfer within Chlamydia and the signature host tropisms of the parental strains; (iii) characterize the mechanisms of host cell subversion by the secreted inclusion membrane effector proteins IncU and IncS. These efforts are essential for gaining a functional appreciation of the role that unique chlamydial genes play in the organism’s growth and development, and also Chlamydia’s evolutionary adaptation to successfully maintain infection and pathogenesis in complex mammalian hosts. We anticipate that Chlamydia will serve as a model obligate intracellular bacterium, and, therefore, the knowledge obtained from this work will broadly extend to other pathogens that have evolved an obligate intracellular niche in their mammalian hosts. Identification of these loci is critical for our understanding of the mechanisms through which Chlamydia have adapted to mammalian hosts, and represents an important step towards elucidating the major virulence correlates of C. trachomatis in humans.