Chlamydiae are obligate intracellular bacterial pathogens that cause disease in human and animal populations worldwide. C. trachomatis is the most prominent cause of both bacterial sexually transmitted disease and infectious blindness in the world. Peptidoglycan (PG) plays a critical role in the physiology of all bacteria. It determines cell morphology, provides protection against osmotic stress, and plays a critical role in cell division. Fragments of PG are also recognized by mammalian receptors and stimulate the host inflammatory response during bacterial infection. Although recent metabolic labeling techniques finally succeeded in demonstrating the presence of PG in Chlamydia, large gaps in our knowledge still remain. What role does PG play in cell division? How are PG glycan chains assembled in the chlamydial periplasm? How does Chlamydia limit the production of degradation fragments that can stimulate the innate immune response? Chlamydia infection in both the ocular as well as genital niches induces a severe inflammatory response that leads to tissue damage including blindness and pelvic inflammatory disease. It is unclear to what extent components of Chlamydia PG are responsible for this response. We need more insight into how Chlamydia synthesizes and degrades its peptidoglycan to better understand the pathologic processes of Chlamydia disease. The long-term goal is to determine the functions of peptidoglycan in Chlamydia physiology, specifically in cell division and development. The central hypothesis to be tested is that the PG synthesized by Chlamydia plays critical roles in cell division and the host immune response. A major focus will be on PG assembly, degradation, and recycling, which we hypothesize are central to these processes. The interrelated Specific Aims of this proposal are: 1. Elucidate the role played by PG in chlamydial cell division 2. Identify and characterize the genes involved in assembly/polymerization and degradation of chlamydial PG 3. Determine the fate and immunostimulatory potential of chlamydial PG muropeptides subsequent to their degradation Breakthrough technologies in experimental manipulation of Chlamydia make our proposal feasible: genetic transformation of Chlamydia, complementation vectors, inducible promoter constructs for controlled gene expression in Chlamydia, and allelic exchange/knockout mutagenesis. We will also employ metabolic labeling of PG, immune-reporter assays, mass spectroscopy, and superresolution microscopy. This research will fill in critical gaps in our understanding of Chlamydia growth and its recognition by host cells. The knowledge gained will provide information that may translate into new drugs to inhibit chlamydial PG synthesis or disrupt the PG fragments that can trigger the severe inflammation that accompanies Chlamydia infection.
Chlamydia cause severe genital, pulmonary, and ocular diseases with serious consequences, in particular for women's reproductive health. The aims of this project will lead to a better understanding of cell wall peptidoglycan synthesis and degradation and elucidate the role of peptidoglycan in Chlamydia cell division. Since enzymes in these pathways have historically provided the best targets for antimicrobial discovery, these studies can identify new targets for effective prevention and treatment of Chlamydia infections.