ABSTRACT Treponema pallidum subsp. pallidum is the causative agents of syphilis, a sexually transmitted infection with an estimated worldwide incidence of 16-32 million new cases per year. The incidence of syphilis in the United States has increased 6-fold over the past two decades. Although this important human pathogen was identified over 100 years ago, the inability to grow T. pallidum in vitro had greatly hampered research into its biology and mechanisms of pathogenesis. In 2018, our group reported continuous long-term multiplication of T. pallidum in co-culture with rabbit epithelial cells and a complex medium. We now have T. pallidum cultures that have been maintained over five years. The in vitro cultivation system provides an excellent animal-free model system as the in vitro cultured organisms fully retain “wild-type” motility, morphology, virulence and generation times. Transcriptional profiles of T. pallidum grown in vitro are quite similar to organisms harvested from infected rabbits. We have successfully used this in vitro culture system to isolate clonal populations of T. pallidum, to conduct systematic nutritional analysis of nutrients required for growth in vitro, and to analyze patterns of peptidoglycan synthesis and division sites. In recent studies, conditions have been identified that supported limited multiplication of T. pallidum under axenic conditions (i.e. without the presence of mammalian cells). In the continuation of this project, we propose to extend and expand the study of factors and genes affecting T. pallidum multiplication using both metabolomic and mutational approaches. The specific aims are to 1) define T. pallidum nutritional requirements and develop an empirically validated metabolic map with the goal of achieving axenic growth; and 2) perform a global mutational analysis of T. pallidum genes involved in in vitro growth and pathogenesis through transposon mutagenesis and other genetic approaches. The further development of the T. pallidum in vitro culture system and the creation of a publicly available library of T. pallidum mutants will likely facilitate many aspects of T. pallidum research, including studies of physiology, structure, antimicrobial susceptibility, pathogenesis, and host immunity.