Chlamydia trachomatis is an obligate intracellular bacterial pathogen responsible for human genital and ocular infections. Species of Chlamydia utilize a type-III secretion system to deliver bacterial effector proteins into the host cell in order to promote invasion and establish residence within a parasitophorous vacuole called an inclusion. The effector protein Tarp has been previously implicated as an important effector for promoting invasion during Chlamydia trachomatis infection by directing the formation of new actin filaments and bundles. Intriguingly, the significance of Tarp mediated cytoskeletal changes has not been fully explored in vivo. Host-pathogen interaction studies that replicate the human infection can be performed with mouse adapted Chlamydia, Chlamydia muridarum. However, the genetic tools to create gene deletions in C. muridarum have been lacking. Recently, our collaborators in the Fields and Wolf Laboratories developed a novel genetic tool for creating Tarp deletion mutants and complement clones in Chlamydia muridarum. Through the use of this tool, we were able to study the significance of Tarp in a murine infection model. In addition to Tarp, two other early effectors TmeA and TmeB are hypothesized to play a role in invasion, but a full account of their involvement remained unknown. In our studies, we were able to determine the roles of TmeA and TmeB in remodeling the host cytoskeleton. Using biochemical crosslinking assays, and actin polymerization studies, we discovered that TmeA has the ability to activate host protein N-Wasp in order to increase Arp2/3-dependent actin polymerization, while TmeB can in turn inhibit Arp2/3-directed actin polymerization via direct interactions with Arp2/3. Collectively, these are important findings as our studies have revealed how a collection of early chlamydial effectors work to modulate the host cytoskeleton to facilitate Chlamydia infections.

This study was motivated by a theoretical formulation on mechanobiology of soft and hard skeletal tissue differentiation. To prove this formulation experimentally, I hypothesized that cartilaginous phenotype can be induced in vitro in a seemingly non-cartilaginous cell source from fibrous tissue. In testing this hypothesis, I have focused on cartilage as a target and fibrous tissue as an origin or the source of cell. Four different trials were pursued with one supposition in common, i.e. hydrostatic pressure is one of the main driving forces for chondroinduction in vitro. The first and second trials pertained to the influence of a relatively short and long duration cyclic hydrostatic compression on rat Achilles tendon fibroblasts. The third trial was to examine the effect of two different drugs on cytoskeletal elements of mesenchymal stem cells or mouse embryonic fibroblast lines in pellet cultures combined with the similar duration and/or frequency of cyclic hydrostatic pressure adopted in the aforesaid trials with no pharmacological agents added. Last, attempts were made to implement an advanced technique in molecular biology called 'PCR array' to further quantify expression levels of eighty four pathway-specific genes in mouse TGFbeta/BMP signaling traffic under the same physiological regimen of hydrostatic compression. Results demonstrated that transdifferentation in phenotype from tendon to fibrocartilage may have occurred in vitro in tendon fibroblasts in pellet cultures exposed to hydrostatic pressure. Experiments on the role of the cytoskeleton in mechanotransduction of the applied level of hydrostatic pressure demonstrated that disruption of microfilaments in the presence of cytochalasin-D did not significantly interfere with the anabolic effect of cyclic pressure. However, disruption of microtubule assembly by nocodazole abolished the pressure-induced stimulation in cartilage marker genes. These findings suggest that microtubules, but not microfilaments, are involved in mechanotransduction of hydrostatic pressure by mesenchymal stem cells.

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