Development and utilization ofTreponema pallidumexpressing green fluorescent protein to study spirochete-host interactions and antibody-mediated clearance: expanding the toolbox for syphilis research
Authors: Delgado Kristina N. , Vicente Crystal F. , Hennelly Christopher M. , Aghakhanian Farhang , Parr Jonathan B. , Claffey Kevin P. , Radolf Justin D. , Hawley Kelly L. , Caimano Melissa J. 
Published: October 2024

Syphilis is a sexually transmitted infection caused by the highly invasive and immunoevasive spirochetal pathogenTreponema pallidumsubsp.pallidum(TPA). Untreated syphilis can lead to infection of multiple organ systems, including the central nervous system. The alarming increase in syphilis cases globally underscores the importance of developing novel strategies to understand the complexities of syphilis pathogenesis. In this study, we took advantage of recent advances inin vitrocultivation and genetic manipulation of syphilis spirochetes to engineer aTPAstrain that constitutively expresses green fluorescent protein (GFP). GFP+TPAgrew identically to the Nichols parent strainin vitroand exhibited wild-type infectivity in the rabbit model. We then used the GFP+strain to visualizeTPAinteractions with host cells during co-cultivationin vitro, within infected rabbit testes, and following opsonophagocytosis by murine bone marrow-derived macrophages. Development of fluorescent strain also enabled us to develop a flow cytometric-based assay to assess antibody-mediated damage to the spirochete’s fragile outer membrane (OM), demonstrating dose-dependent growth inhibition and OM disruptionin vitro. Notably, we observed greater OM disruption of GFP+TPAwith sera from immune rabbits infected with theTPANichols strain compared to sera generated against the genetically distinct SS14 strain. These latter findings highlight the importance of OM protein-specific antibody responses for clearance ofTPAduring syphilitic infection. The availability of fluorescentTPAstrains paves the way for future studies investigating spirochete-host interactions as well as functional characterization of antibodies directed treponemal OM proteins, the presumptive targets for protective immunity.ImportanceSyphilis, a sexually transmitted infection caused byTreponema pallidum(TPA), remains a pressing threat to global public health.TPAhas a remarkable and still poorly understood ability to disseminate rapidly from the site of inoculation and establish persistent infection throughout the body. Recent advances inin vitrocultivation and genetic manipulation of syphilis spirochetes enabled the development of fluorescentTPA. In the study, we generated and characterized an infectiousTPAstrain that constitutively expresses green fluorescent protein and used this strain to visualize interaction ofTPAwith host cells and functionally characterize antibodies directed against treponemal outer membrane proteins. Most notably, we assessed the ability of surface-bound antibodies to inhibit growth ofTPA in vitroand/or disrupt the spirochete’s fragile outer membrane. FluorescentTPAstrains provide a powerful new tool for elucidating host-pathogen interactions that enable the syphilis spirochete to establish infection and persistent long-term within its obligate human host.