The natural release of Treponema pallidum peptidoglycan as a pathogenic determinant of syphilis

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.