Elucidating the Contribution of Genetic Recombination in Treponema pallidum to Syphilis Pathogenesis - ABSTRACT The spirochete Treponema pallidum subspecies pallidum (TP) causes syphilis, a multi-stage sexually transmitted infection. Cases of TP are rapidly increasing in high income countries and remain persistently high worldwide, with a total of 8 million incident cases globally each year. TP can disseminate to nearly any tissue, leading to significant morbidity in advanced stages of disease, and can be vertically transmitted, causing congenital syphilis. Despite its protean manifestations, TP has a minimalist genome with limited genetic diversity and accumulates single nucleotide changes slowly. In contrast, TP readily undergoes inter-strain or intra-chromosomal homologous recombination (HR) to generate novel variants. For example, intra- chromosomal HR between 53 defined donor sites and seven extracellular loops of the Treponema pallidum repeat protein K (TprK) generates novel epitopes and underlies immune escape. Additionally, we recently examined samples from a patient with uncontrolled HIV and co-infected with two strains of TP, with HR observed between the strains in a penicillin-binding protein and a fatty acid transporter, with unknown fitness effects. In spite of considerable evidence that TP uses genetic recombination as a survival strategy, little is known about mechanisms of HR in TP. Building on our published and unpublished findings, here we will identify the TP genes involved in HR and the response to DNA damage and examine TP genomic changes following the induction of recombination. This will be achieved via two complementary Specific Aims: In Aim 1, we will induce DNA damage and HR directly by treatment with genotoxic agents or indirectly with sublethal penicillin treatment. Then, we will examine longitudinal transcriptional responses to identify gene networks, such as homologs of the SOS Response pathway, involved in HR. We will also identify putative transcription factor binding sites among differentially expressed TP genes. In Aim 2, we will use whole genome sequencing and targeted amplicon long-read sequencing to observe the genomic alterations caused by HR between Nichols and SS14 strains during longitudinal co-infection in vitro and in vivo (Aim 2.1) and between TP and HR donor plasmids following DNA damage or sublethal penicillin treatment (Aim 2.2). We will determine the TP genes that undergo HR most frequently when supplied with donor template, focusing particularly on penicillin binding proteins and extracellular loops of outer membrane proteins being considered for inclusion in an eventual syphilis vaccine. This proposal makes use of simple and elegant experiments using our established protocols for in vitro culture, rabbit infection, TP RNAseq, and whole genome and long-read amplicon sequencing. Insights gained from these experiments will have implications for antibiotic stewardship and help inform selection of antigens for syphilis vaccine development. Together, these studies provide crucial insight into a previously unexplored mechanism of TP pathogenesis.
