Development of an sRNA-mediated conditional knockdown system for .

We describe a new protein depletion method that uses an engineered small RNA (sRNA) to inhibit translation of a target gene. In proof-of-principle experiments, we induced functional knockdown of IncA, a fusion-mediating inclusion membrane protein, as shown with Western blots, loss of IncA staining at the inclusion membrane, and production of multiple chlamydial inclusions within an infected cell. These effects were titratable and reversible.

Chlamydia trachomatis induces disassembly of the primary cilium to promote the intracellular infection.

Chlamydia trachomatis is a clinically important bacterium that infects epithelial cells of the genitourinary and respiratory tracts and the eye. These differentiated cells are in a quiescent growth state and have a surface organelle called a primary cilium, but the standard Chlamydia cell culture infection model uses cycling cells that lack primary cilia. To investigate if these differences are relevant, we performed infections with host cells that have a primary cilium.

A Reverse Genetic Approach for Studying sRNAs in Chlamydia trachomatis.

sRNAs are noncoding transcripts that play critical roles in posttranscriptional regulation in prokaryotes. In the intracellular bacterium Chlamydia, sRNAs have been identified, but functional studies have been limited to an E. coli heterologous system.

Differential Effects of Small Molecule Inhibitors on the Intracellular Chlamydia Infection.

Chlamydia are obligate intracellular bacteria that reside within a membrane-bound compartment called the chlamydial inclusion inside a eukaryotic host cell. These pathogens have a complex biphasic developmental cycle, which involves conversion between a replicating, but noninfectious, reticulate body (RB) and an infectious elementary body (EB). Small molecule inhibitors have been reported to have deleterious effects on the intracellular Chlamydia infection, but these studies have typically been limited in terms of assays and time points of analysis.

Chlamydia and HPV induce centrosome amplification in the host cell through additive mechanisms.

Based on epidemiology studies, Chlamydia trachomatis has been proposed as a co-factor for human papillomavirus (HPV) in the development of cervical cancer. These two intracellular pathogens have been independently reported to induce the production of extra centrosomes, or centrosome amplification, which is a hallmark of cancer cells. We developed a cell culture model to systematically measure the individual and combined effects of Chlamydia and HPV on the centrosome in the same host cell.