The role of the bi-functional Chlamydia trachomatis protein, Ct663, in regulating the essential, developmental cycle between infectious elementary bodies (EBs) and actively dividing, reticulate bodies (RBs) is not well-understood at the molecular level. The model proposed for Ct663's functions is concurrent dual activity, which suggests a regulatory mechanism involving stoichiometric control of Ct663 as a type III secretion system chaperone and an RNA polymerase s66-dependent transcriptional inhibitor. Because Ct663 has two essential functions and is unique to C. trachomatis, it is a novel target for developing anti- virulence drugs. In order to develop drugs targeting the developmental cycle of C. trachomatis, the struc- tural and thermodynamic mechanisms controlling Ct663's chaperone function and its interaction with its protein partners must first be determined. The long-term goal of the proposed research is to screen and design drugs targeting Ct663's functions, which would prevent the bacteria from spreading;likely curing C. trachomatis directly and certainly mitigating the development of antibiotic-resistant strains, when com- bined with current therapies. The overall objective of this application is to develop a model of Ct663 that includes the 3-dimensional high-resolution structure of Ct663 and characterization of Ct663's interactions with the chaperone, Scc1, and the essential virulence factor, CopN. The central hypothesis is that Ct663's stoichiometry and complex formation regulates its dual activity. The rationale for the proposed research is that disrupting Ct663's interactions and functions is an attractive therapy because it is highly likely to prevent the bacteria from differentiating into infectious EB's. The central hypothesis and objec- tive of this application will be tested and attained by pursuing two specific aims: (1) develop a model of (Ct663:Scc1):CopN assembly by determining the stoichiometry, dissociation constants, and quaternary structure of the complexes and (2) determine the 3D structure of Ct663 and identify its Scc1-binding in- terface. It is anticipated that these aims will yield the expected outcome of a model of Ct663 and its complexes for rational drug design. This outcome is expected to have an important positive impact be- cause a structural and functional model of Ct663 will provide a new target for therapeutic interventions in addition to fundamentally advancing the fields of structural biology and infectious disease biology. This contribution is significant because it is the first step in a continuum of research that is expected to lead to understanding of Ct663's dual functions and contribute to the treatment of the most common, sexually transmitted bacterial disease. The proposed research is innovative, in my opinion, because it represents a new and substantive departure from the status quo, namely the structural and biochemical characteri- zation of a unique bi-functional protein from C. trachomatis using NMR to target bacterial virulence as a therapeutic strategy.
The proposed research is relevant to public health because Chlamydia trachomatis is the most common, sexually transmitted bacterial disease in the United States and can cause reproductive health complications and pneumonia in infants born to infected women. A novel enzyme of Chlamydia trachomatis, called Ct663, is an attractive drug target because it has two functions that contribute to the pathogenesis of this disease. Developing a structural model of Ct663 and its protein complexes is an essential step toward the rational design of drugs and to fundamentally advance the field of microbiology.