Development and utilization of
Treponema pallidum
expressing 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: November 2024

Syphilis is a sexually transmitted infection caused by the highly invasive and immunoevasive spirochetal pathogen
Treponema pallidum
subsp.
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 in
in vitro
cultivation and genetic manipulation of syphilis spirochetes to engineer a
TPA
strain that constitutively expresses green fluorescent protein (GFP). GFP
+
TPA
grew identically to the Nichols parent strain
in vitro
and exhibited wild-type infectivity in the rabbit model. We then used the GFP
+
strain to visualize
TPA
interactions with host cells during co-cultivation
in vitro
, within infected rabbit testes, and following opsonophagocytosis by murine bone marrow-derived macrophages. The 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 disruption
in vitro
. Notably, we observed greater OM disruption of GFP
+
TPA
with sera from immune rabbits infected with the
TPA
Nichols 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 of
TPA
during syphilitic infection. The availability of fluorescent
TPA
strains 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.

IMPORTANCE

Syphilis, a sexually transmitted infection caused by
Treponema pallidum
(
TPA
), remains a pressing threat to global public health.
TPA
has a remarkable and still poorly understood ability to disseminate rapidly from the site of inoculation and establish persistent infection throughout the body. Recent advances in
in vitro
cultivation and genetic manipulation of syphilis spirochetes enabled the development of fluorescent
TPA
. In the study, we generated and characterized an infectious
TPA
strain that constitutively expresses green fluorescent protein and used this strain to visualize the interaction of
TPA
with host cells and functionally characterize antibodies directed against treponemal outer membrane proteins. Most notably, we assessed the ability of surface-bound antibodies to inhibit the growth of
TPA in vitro
and/or disrupt the spirochete’s fragile outer membrane. Fluorescent
TPA
strains 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.