Slow-growing human cell lines are a suitable alternative to rabbit Sf1Ep cells for in vitro cultivation of Treponema pallidum

Treponema pallidum subsp. pallidum, the causative agent of syphilis, remains difficult to study owing to long-standing limitations in in vitro cultivation. Although a rabbit epithelial cell co-culture system (Sf1Ep) enabled major advances in recent years, the lack of human cell-based models restricts clinical relevance and mechanistic insight into host-pathogen interactions. Here, we sought to establish a co-culture system that uses human epithelial cell lines capable of supporting T. pallidum growth in vitro. Six human epithelial or epithelial-like cell lines from diverse tissue origins were evaluated under microaerophilic conditions using the standard T. pallidum cultivation medium. Among these, CAL-39 (vulva) and HepG2 (liver) supported T. pallidum survival, replication, characteristic growth behaviours, and long-term passage at levels comparable to Sf1Ep cells. Growth kinetics, attachment dynamics, and motility of T. pallidum were quantified over extended culture periods. Using live-cell imaging in this co-culture system, for the first time we were able to define two distinct T. pallidum host-cell interaction behaviours; surface-associated crawling and stable single-polar attachment. Both behaviours were observed across all tested host cell lines and persisted over time only in cell lines permissive for sustained growth. Together, our findings establish clinically-relevant human epithelial co-culture models for T. pallidum, provide new insights into host-cell-dependent growth and motility, and create a platform for future mechanistic studies of syphilis pathogenesis and vaccine target discovery.