Project Summary
Despite over a century of research on the causative agent of syphilis Treponema pallidum, many
questions associated with T. pallidum biology, and by extension syphilis pathology, remain unanswered due to
the previous lack of an in vitro culture system. Syphilis, a quickly growing global public health concern, is a
sexually transmitted, multiphasic disease with varied and devasting symptoms that can be fatal. As T. pallidum
lacks many classical virulence factors (i.e., lipopolysaccharide, toxins, secretion systems) and has a paucity of
outer membrane proteins, making it a stealth pathogen, it is unclear how T. pallidum causes syphilis. A growing
body of work has established released peptidoglycan (PG) monomers (i.e., muropeptides) act as toxins and
immune modulators. Many diderm bacteria have a highly conserved PG recycling pathway to transport
muropeptides released during normal growth into the cytoplasm for reuse. When this pathway is disrupted, highly
immunogenic muropeptides are released into the environment. Through bioinformatic analysis I discovered T.
pallidum lacks this recycling pathway. Modifications to PG composition and architecture affect immune
recognition and response, and our lab has identified unusual PG modifications in related pathogenic spirochetes,
Borrelia burgdorferi (Lyme disease) and Treponema denticola (periodontal disease). Additionally, the release of
unique muropeptides from B. burgdorferi during infection plays a causal role in the development of Lyme arthritis.
Thus, this proposal aims to test the hypothesis that T. pallidum naturally secretes unusual muropeptides that
drive syphilis pathogenesis. To test this hypothesis, I will first determine the biochemical features of T. pallidum
PG as well as the fate and identity of released muropeptides via liquid chromatography tandem mass
spectrometry (LCMS) and hNOD2 reporter assays. Second, I will elucidate the role of released PG in T. pallidum
pathogenesis by creating a novel CRISPR platform to target T. pallidum lytic transglycosylases and testing the
ability of CRISPR mutants to induce inflammation, induce chancre formation, and establish infection in vivo with
a rabbit model. Collectively this proposal will increase our understanding of T. pallidum biology and syphilis
pathology as well as create a new tool for the syphilis research field.