A human epithelial co-culture system reveals distinct host cell interaction behaviours for 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, the absence of human cell-based models limits clinical relevance and mechanistic understanding of host–pathogen interactions. Here, we established a co-culture system using human epithelial cell lines capable of supporting T. pallidum growth in vitro. Six epithelial or epithelial-like cell lines from diverse tissues were evaluated under microaerophilic conditions using standard cultivation medium. 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 were quantified over extended culture. Using live-cell imaging, we defined two distinct host-cell interaction behaviours: surface-associated crawling and stable single-polar attachment. Both behaviours were observed across all tested cell lines but persisted only in those 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 mechanistic studies of syphilis pathogenesis and vaccine target discovery.