Development and utilization ofexpressing green fluorescent protein to study spirochete-host interactions and antibody-mediated clearance: expanding the toolbox for syphilis research.

Abstract

UNLABELLED: Syphilis is a sexually transmitted infection caused by the highly invasive and immunoevasive spirochetal pathogensubsp.(). 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 incultivation and genetic manipulation of syphilis spirochetes to engineer astrain that constitutively expresses green fluorescent protein (GFP). GFPgrew identically to the Nichols parent strainand exhibited wild-type infectivity in the rabbit model. We then used the GFPstrain to visualizeinteractions with host cells during co-cultivation, 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. Notably, we observed greater OM disruption of GFPwith sera from immune rabbits infected with theNichols 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 ofduring syphilitic infection. The availability of fluorescentstrains 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(), remains a pressing threat to global public health.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 incultivation and genetic manipulation of syphilis spirochetes enabled the development of fluorescent. In the study, we generated and characterized an infectiousstrain that constitutively expresses green fluorescent protein and used this strain to visualize the interaction ofwith 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 ofand/or disrupt the spirochete's fragile outer membrane. Fluorescentstrains 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.