Nutrient acquisition and cyclic nucleotide signaling in Treponema pallidum as underpinnings of stealth pathogenicity - Abstract Syphilis rates in the United States and globally have skyrocketed since their nadir in the early 2000s, creating a pressing need for novel containment strategies rooted in improved understanding of the basic biology of Treponema pallidum (TPA). For three principal reasons, our understanding of syphilis pathogenesis has lagged behind most other bacterial infections. First and foremost was the inability to genetically manipulate TPA in vitro. Second was the limited knowledge regarding the genetic programs that enable syphilis spirochetes to persist in humans. Last was the poor understanding of TPA's outer membrane protein (OMP) repertoire, which constitutes the host-pathogen interface during human infection. This proposal leverages recent breakthroughs in basic syphilis research to investigate fundamental aspects of disease pathogenesis – nutrient acquisition and environmental sensing. Using the in vitro cultivation system developed by Edmondson and Norris, we generated data supporting physiologic/virulence-related functions for three of the five `atypical' FadL-like orthologs encoded by TPA. These findings led us to postulate that these OMPs form a new functional class of transporters that import poorly soluble nutrients required for growth of TPA, an extreme auxotroph. In Aim 1, we will phenotypically characterize TPA mutants containing insertions in one or more FadL loci to determine their respective contributions to growth in vitro, adherence to mammalian cells, and infectivity in the rabbit model. Our mining of the TPA genome also revealed regulatory pathways linking host-derived small molecules with the production of the cyclic nucleotide second messangers cAMP and c-di-GMP. In our working model, ligand binding by the periplasmic CACHE domain for TPA's adenylate cyclase TP0485 activates synthesis of cAMP by the protein's cytosolic cyclase domain. cAMP, in turn, enables DNA binding by TPA's canonical CRP transcription factor (TP0262), whose regulon includes members of the Tpr OMP family. We further postulate that cAMP serves as an activating ligand for the spirochete's cytoplasmic diguanylate cyclase (TP0981) GAF domain, leading to the production of c-di-GMP; c-di-GMP exerts its signaling function via TPA's sole PilZ protein (TP0086), an allosteric effector potentially mediating changes in transcription and/or motility potentially involved in persistence. In Aim 2, we will generate TPA strains defect in cyclic nucleotide signaling to assess their contributions to growth of TPA in vitro and infectivity in the rabbit model. Using complementary approaches, we will also examine crosstalk between the cAMP and c-d-GMP signaling pathways. The research described in this proposal will yield the first systems biology level understanding of physiologic, regulatory, and pathogenic processes that enable the stealth pathogen to establish persistence in individuals and flourish in at-risk populations.
