A novel pan-proteome array for high-throughput profiling of the humoral response to Treponema pallidum.

Given the resurgence of syphilis, research endeavors to improve current assays for serological diagnosis and management of this disease are a priority. A proteome-scale platform for high-throughput profiling of the humoral response to Treponema pallidum (T. pallidum) proteins during infection could identify antigens suitable to ameliorate the performance and capabilities of treponemal tests for syphilis. Additionally, because infection-induced immunity is partially protective, profiling the response to T. pallidum outer membrane proteins (OMPs) could help select vaccine candidates. Therefore, we developed a pan-proteome array (PPA) based on the Nichols and SS14 strain complete proteomes and used it to define the immunoglobulin M (IgM) and IgG humoral response to T. pallidum proteins in sera collected longitudinally from long-term infected rabbits and from rabbits that were infected, treated, and re-infected. We identified antigens that could facilitate early diagnosis and immunity to a core set of OMP that could explain protection upon reinfection.

Evaluation of a minimal array of Treponema pallidum antigens as biomarkers for syphilis diagnosis, infection staging, and response to treatment.

IMPORTANCE: This manuscript explores the host humoral response to selected antigens of the syphilis agent during infection to evaluate their potential use as diagnostic tests and markers for treatment.

In Vitro Transformation and Selection of Treponema pallidum subsp. pallidum.

Although the isolation of Treponema pallidum subsp. pallidum (T. pallidum) from a syphilis patient dates to 1912, for the duration of the 20th century, this pathogen has remained an exceedingly difficult organism to study due to the lack of a system to support its viability in vitro. This limitation, in turn, has precluded the application of genetic engineering techniques via transformation and subsequent selection of T. pallidum transformants. A recently described method for in vitro cultivation of T. pallidum, however, has made it possible for us to experiment with transformation and selection methods. Here we describe the approach that we adopted to successfully transform T. pallidum with foreign DNA and select the resulting recombinant strain using kanamycin. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Transformation of T. pallidum Support Protocol 1: Quantification of T. pallidum in suspensions using dark-field microscopy Support Protocol 2: Counting cells using a hemacytometer Basic Protocol 2: Selection, initial passaging, and expansion of transformed cultures Basic Protocol 3: Isolation of a clonal strain through limiting dilution.