Chlamydia trachomatis is a gram-negative obligate intracellular bacterium that has a significant public health toll. During infection, the bacteria alternate between a metabolically inert, infectious form (elementary body, EB), and a metabolically active, non-infectious form (reticulate body, RB). The process through which the bacteria alternate between the two forms, called the developmental cycle, occurs within the confines of a parasitophorous vacuole termed the inclusion. The inclusion represents an important aspect of chlamydial pathogenesis as it is the mediator between the host cell and the bacteria.

In order to establish and maintain infection, all pathogens must be able to acquire nutrients from the host. Vacuolar intracellular pathogens have developed three ways in which to acquire nutrients: (1) intercept vacuoles from the endosomal/lysosomal pathway or through the autophagosomal pathway, (2) have open channels that allow for free exchange between the host cytoplasm, and (3) possess a transport system. The inclusion of C. trachomatis does not acquire nutrients via fusion of host vesicles within the endosomal/lysosomal or autophagosomal pathways. It also does not possess an open channel as studies have shown the inclusion to exclude molecules larger than 520Da. These studies demonstrate that the two previously mentioned methods of nutrient acquisition are not utilized by C. trachomatis and suggest there exists a transport system within the chlamydial inclusion. No transport system within a bacterial vacuole has been reported, although it has been documented in parasitic vacuoles. It is intriguing to note that RBs align themselves along the inner inclusion membrane. Specifically there is a unique structural morphology indicative of a large macromolecular complex located at the junction between RB and integral to the inclusion membrane. This leads to the hypothesis that the chlamydial inclusion possesses a nutrient transport complex that links the RBs to the inclusion that is essential for nutrient acquisition.

Methionine and glucose analog localization was monitored in C. trachomatis infected HeLa cells. It was found that each analog exhibited a unique localization pattern that was dependent upon host and bacterial factors. Methionine was sequestered in the bacteria, while glucose was found in the lumen of the inclusion. This work was carried out in conjunction with the development of a novel protocol to isolate inclusions from the host cell. This would provide a means for direct manipulation of the inclusion, allowing for more detailed analysis of nutrient acquisition.

Chlamydia trachomatis is an obligate intracellular bacterium that is a public health burden worldwide. The precise strategies that intracellular pathogens use to exit host cells have a direct impact on their ability to disseminate within a host, transmit to new hosts, and manipulate immune responses. Chlamydia exits the host cell by two distinct strategies, lysis and extrusion. Lysis is a sequential rupture of vacuole, nuclear and plasma membranes, culminating in the release of free infectious bacteria. Extrusion is a packaged release of Chlamydia that begins with invagination of the chlamydial inclusion, followed by the pinching of the cell plasma membrane, resulting in a double membrane compartment containing Chlamydia, chlamydial inclusion, host cell cytoplasm, and host plasma membrane.

Host cytokinesis proteins are required for the pinching of the chlamydial inclusion just prior to extrusion, but deeper mechanistic understanding of the host requirements that govern the final severing of the extrusion from the host cell have yet to be elucidated. Abscission, the final stage of cytokinesis that severs two daughter cells, is governed largely by the ESCRT-III family of proteins, which also participate in virus budding. Here, we define a role for host abscission proteins in the release of extrusion from the host cell.

The defining characteristics of extrusions, and advantages gained by Chlamydia within this unique double-membrane structure are not well understood. Results presented here define extrusions as mostly devoid of host organelles, and containing phosphatidylserine on the outer surface of the extrusion membrane. Results further demonstrate that extrusion is highly conserved across Chlamydiae. Extrusions also served as transient, intracellular-like niches for enhanced Chlamydia survival outside the host cell. In addition to enhanced extracellular survival, extrusions are phagocytosed by primary bone marrow-derived macrophages, after which they provide a protective microenvironment for Chlamydia. Extrusion-derived Chlamydia staved off macrophage-based killing, and culminated in the release of infectious EB. Based on these findings, we propose a model in which extrusions serve as ‘trojan horses’ for Chlamydia, by exploiting macrophages as vehicles for dissemination, immune evasion, and potentially transmission.