My laboratory's main interest is to understand how obligate intracellular bacterial pathogens like Chlamydia trachomatis, exploit the cell biology of their hosts to replicate, disseminate and cause disease. C. trachomatis replicates within a membrane bound compartment (“inclusion”) by manipulating the actin cytoskeleton, co-opting microtubule-based motors, inhibiting lysosomal recognition of the inclusion, activating signaling pathways, re-routing lipid transport, and prevents the onset of programmed cell death. Remarkably, all of these functions are achieved with a genome that encodes < 900 proteins, suggesting that the Chlamydiae have streamlined their genomes to enhance intracellular survival and dissemination.

Unfortunately, because the Chlamydiae do not replicate outside eukaryotic cells, are not amenable to genetic manipulation , and a large fraction of their genomes encode for proteins with no known functional homologues, a molecular understanding of how these pathogens co-opt eukaryotic cellular functions is very limited.   We approached this biological problem by establishing a yeast-based expression system to screen for chlamydial proteins that interfere with conserved eukaryotic cellular functions.   In this manner, we have identified thirty-four chlamydial proteins, including previously characterized virulence factors.  

By using a combination of cell biological techniques, including advanced cell imaging techniques, microinjection, biochemistry, proteomics and molecular biology my lab is focused on determining the function of these virulence factors, their targets in mammalian cells and understanding any novel cell biology that arises from the analysis of this host-pathogen interaction. Example of current projects include:

1 . Characterization of chlamydial virulence factors.   We generated antibodies to several of the chlamydial proteins identified in our phenotypic screens in yeast and determined that these proteins are translocated into the host cytoplasm. The next year will be mostly devoted to characterizing these proteins and focus on those that point to interesting new aspects of chlamydial biology (e.g. regulators of host nuclear, mitochondrial and endosomal function).

2. Chlamydia functional genomics. We have extended the use of chlamydial protein expression arrays to perform additional screens including an immunological-based survey for proteins secreted into the bacteria-containing vacuoles.   This approach identified twenty-three chlamydial proteins, including structural components of the vacuolar membrane.   In addition, we have recloned chlamydial-specific genes into yeast 2H vectors to create a protein-protein interaction map of chlamydial proteins at the host-pathogen interface.   This interaction map will be used to design “dominant-negative” fragments that can interfere with protein complex formation in vivo so that we can assess their role in chlamydial replication and survival in infected cells.

3. The biology of Lipid Droplets.   Our screens in yeast revealed a family of chlamydial proteins that target Lipid Droplets (LDs), a ubiquitous but poorly characterized eukaryotic organelle.   We have determined that these LD- associated (Lda) proteins are secreted into the cytoplasm of infected cells and that LDs are essential for chlamydial replication.   Furthermore, Chlamydia causes a proliferation of LDs, alters their protein composition, and recruits them to the bacteria-containing vacuole.   Because this is the first report of a bacterial pathogen co-opting the function of these organelles we are in the unique position of exploring a completely new filed of study in cellular micorbiology. Our current work in this area centers determining the function of Lda proteins, identifying their targets in mammalian cells and characterizing the cell biology of LD biogenesis in infected cells.