UC Irvine

Chlamydia are obligate intracellular bacterial pathogens with a significant impact on human health. Chlamydia infect eukaryotic cells, and a wide range of effects on the host cell have been attributed to a chlamydial protease, CPAF, through cleavage or degradation of numerous host proteins. We discovered that this reported proteolysis was an in vitro phenomenon that occurred during preparation of infected cells for protein analysis. We showed that CPAF activity was induced by detachment of Chlamydia-infected cells from a cell culture monolayer and that this protease remained active in cell lysates resulting in artifactual proteolysis. Chlamydial phenotypes attributed to CPAF were still observed when we took precautions to prevent in vitro proteolysis. Our findings challenged the prevailing model about the function of CPAF during the chlamydial infection and its intracellular targets. In a second project, we studied the unusual chlamydial developmental cycle with a novel three-dimensional electron microscopy (3D-EM) method. During the intracellular infection, the bacterium converts within the chlamydial inclusion from an infectious, but non-dividing, elementary body (EB) into a reticulate body (RB) that divides repeatedly by binary fission before converting back into EBs to infect new cells. We analyzed each Chlamydia-infected cell with several hundred two dimensional EM slices and used computational methods to reconstruct the three-dimensional images of the chlamydial inclusions. The results provided the first comprehensive analysis of the developmental cycle, including the number, size, and location of each EB, RB, and intermediates of RB replication and RB-to-EB conversion. We described for the first time that RB volume progressively decreases as RB number increases, indicating that RBs divide before doubling in size. However, we did not observe RBs below a minimum size, leading us to postulate that RBs below a size threshold convert into an EB. We propose a new model of chlamydial development in which RB-to-EB conversion is regulated by RB size because several rounds of RB replication are required for the RB to become small enough to convert into an EB. In this model, RB size acts as a timer and provides a mechanism to delay RB-to-EB conversion until the RB pool has expanded.