Chlamydiae species are obligate intracellular bacteria that are the most frequent cause of sexually transmitted disease as well as the leading cause of preventable blindness worldwide. Chlamydiae replicate in a non-acidified vacuole, termed an inclusion, which is actively modified by chlamydiae to prevent lysosomal fusion and promote intracellular survival. The molecular determinants that mediate chlamydial pathogenesis are largely undefined primarily due to the inability to manipulate the chlamydial genome. The overall goal of this research is to identify pathogenic mechanisms utilized by chlamydiae to promote and maintain their intracellular survival. Because chlamydiae remain sequestered within a vacuole, all interactions between chlamydiae and their host must be mediated through the inclusion membrane. We have identified Chlamydia trachomatis-specific proteins (IncD/E/F/G) that are localized to the inclusion membrane. Their intracellular localization makes them potential mediators of host-pathogen interactions via direct interactions with host proteins. To achieve our overall goals, we propose to identify biological functions of IncD/E/F/G through identification and characterization of cellular targets of IncD/E/F/G We have identified mammalian 14-3-3 proteins, as the first and only cellular targets of an inclusion membrane protein, IncG. 14-3-3 proteins are dimeric phosphoserine binding proteins that regulate diverse signal transduction pathways through directed subcellular localization of signaling complexes.
Specific Aim 1 : Experiments are designed to define biological functions of 14-3-3 IncG interactions and determine whether chlamydiae target 14-3-3 proteins to exploit host signal and vesicular-mediated pathways. First, we will disrupt 14-3-3 IncG interactions through expression of 14-3-3 dominant negative mutants and microinjection of anti-IncG antibodies to examine whether 14-3-3's recruitment to the inclusion functions in exploitation of cellular signal transduction and vesicular-mediated pathways. Second, we will use a combination of fluorescence microscopy, yeast tri-hybrid assays and co-immunoprecipitation experiments to determine whether 14-3-3 proteins recruit additional signaling proteins to the inclusion. And third, we will employ co-immunoprecipitation experiments to determine whether chlamydiae alter 14-3-3-dependent signaling pathways by altering host 14-3-3/ligand interactions.
Specific Aim 2 utilizes yeast two-hybrid assays to identify cellular targets of IncD/E/F. Identification of cellular targets of Incs and how these interactions contribute to chlamydial pathogenesis will lead to a better understanding of the complex host-pathogen interactions that facilitate chlamydial intracellular survival.
| Brumell, John H; Scidmore, Marci A (2007) Manipulation of rab GTPase function by intracellular bacterial pathogens. Microbiol Mol Biol Rev 71:636-52 |
