We study how obligate intracellular bacterial pathogens like the Chlamydiae exploit the cell biology of their host to replicate, disseminate and, ultimately, to cause disease. Some of the main projects currently under being pursued in our laboratory include:
1. Genomic Tools for the Analysis of Gene function in Chlamydia trachomatis.
Since Chlamydia is not amenable to traditional “loss-of-function” genetics, we tackled this biological problem by a “gain-of-function” approach. In short, we systematically express individual Chlamydia ORFs as epitope-tagged proteins in yeast and mammalian cells to identify proteins that disrupt conserved eukaryotic cellular processes. These ordered collections of expression strains represent the foundation of our research program to understand the biology of Chlamydia and how it interacts with its host.
i. Expression Systems for the Functional Analysis of Chlamydia Proteins.
We use the yeast Saccharomyces cerevisiae as a platform for chlamydial protein expression and phenotype-based screens. Because of the conserved nature of many basic mechanisms of lipid and membrane transport, signaling and cytoskeletal function this approach is broadly applicable to mammalian cell systems. Furthermore, we reasoned that the genetic, biochemical and genomic tools available to study yeast cell biology would greatly facilitate the characterization of chlamydial proteins and their eukaryotic targets. These strains are being screened for chlamydial proteins that impair various yeast cellular functions or that display tropism towards eukaryotic organelles. We have also constructed comprehensive protein expression libraries directly in HeLa cells. The advantages of screening chlamydial proteins directly in mammalian cells included the potential of identifying targeted pathways that are not conserved in yeast cells (e.g. immune related functions) or that are divergent enough that the Chlamydia effector protein does not recognize the yeast counterpart. Overall, these studies led to the identification of several putative chlamydial effector proteins, the function of which is under current investigation.
ii. The Chlamydia Interactome
In addition to GFP tags, we have constructed libraries of RFP-tagged ORFs for co-localization studies, GST-, 6xHis- and Protein A-tagged ORFs for biochemical assays and affinity purification, and Gal4-AD (activator domain) and DBD (DNA binding domain)-tagged ORFs to identify protein-protein interactions by Yeast Two-Hybrid (YTH) analysis. In 2006, we initiated a project to map all protein-protein interactions in C. trachomatis by YTH. The impetus for this approach was the failure of previous genetic and biochemical approaches to define functional interactions between chlamydial proteins. We have initially focused on identifying proteins that interact with the conserved core of the Type III secretion translocon. In this manner we sought to identify components of the secretion machinery, regulatory factors, secretion chaperones and novel targets of secretion. We identified a network of 36 proteins that interact with various components of the secretion apparatus (Spaeth et al, in preparation).
This “omics” approach is the most straightforward means, given the experimental limitations of Chlamydia, to delineate how the TTS apparatus intersects with metabolism, physiology and cell architecture. Already this approach has yielded unexpected results: our preliminary screens detected interaction between components of the protein translation machinery and the TTS translocon, raising the possibility that virulence protein secretion is coupled with translational controls.
(Left: Network of interaction proteins identified by Y2H. Core TTS components (red) interact with effectors proteins (yellow) and secretion chaperones (blue). ORFs of unknonw fucntion are denoted in green.
2. The Cell Biology of Chlamydia Infections
Our phenotypic screens in yeast suggested that chlamydial proteins target cytoskeletal, nuclear, mitochondrial and neutral lipid storage functions. Because our interest in the cell biology of Chlamydia infections stem from the possibility of identifying new examples of the complexity of interactions between a pathogen and its host, our research efforts are focused on two unique features of Chlamydia infection: the hijacking of host lipid droplets and the engagement of the host cytoskeleton to modulate the dynamics of the inclusion membrane.
i. The molecular basis of Chlamydia co-option of mammalian Lipid Droplets.
We identified several chlamydial proteins that bind avidly to Lipid Droplets (LDs), a ubiquitous but poorly characterized eukaryotic organelle. Many of these LD-associated (Lda) proteins are secreted into the cytoplasm of infected cells and localize to the vicinity of the inclusion membrane. LDs are complex but poorly understood organelles that participate in lipid storage, membrane traffic and signaling. We characterized the interaction between Chlamydia and LDs and determined that C. trachomatis i) alters the lipid and protein composition of host cell LDs, ii) inclusions associate with LDs and recruit LD markers and iii) depends on LD biogenesis for replication.
In the last few years there has been renewed interest in the cellular functions of LDs; new findings indicate that these organelles are highly dynamic and may mediate lipid and protein transport functions independently of the classical Golgi-dependent vesicular transport pathways. To our knowledge, our finding represents the first example of a bacterial pathogen co-opting the function of these organelles. We characterized the interaction of LDs with the inclusion in greater detail and made the surprising discovery that LDs were present in the lumen of the inclusion. LDs dock at the surface of the inclusion, penetrate the inclusion membrane and intimately associate with Reticulate Bodies, the replicative form of Chlamydia.
(Left: Lipid Droplets in the inclusion lumen interact with chlamydial RBs. Transmission electron micrographs of mamlchite green fixed cells reveal LD in close apposition to RBs.
Overall, our finding that intact LDs are translocated into the lumen of a parasitophorous vacuole leads us to reassess the extent to which the inclusion membrane can act as barrier to bacterial interactions with cytoplasmic contents. These findings also raise a new series of research questions that we are currently pursuing: What is the molecular basis for LD docking on inclusion membranes? What is the mechanism underlying the involution of LDs into the inclusion lumen and what role do Ldas and host protein play in this process? How is the lipolysis of LDs initiated and how do bacteria utilize these lipids?
Live cell imaging of Lipid Droplets in the process of translocation into the inclusion lumen. Lipid Droplets and inclusion membranes were labeleled by expression of an Lda3-EGFP transgene in Chlamydia-infected cells (28h) and images acquired by laser scanning confocal microscopy over a span of 30min.
ii. The function and regulation of cytoskeletal re-arrangements at the host-pathogen interface.
LIve video micropscopy revealed that the inclusion retains its shape throughout the bacterial life cycle (See Movie). As such, we predicted that the cytoskeleton would play a role in regulating the shape of the inclusion. The major components of the mammalian cytoskeleton, microtubules, actin microfilaments and intermediate filaments (IF), differ widely in their structural and biochemical properties. Both actin filaments and microtubules perform functions ranging from providing structural stability to regulating intracellular vesicle trafficking, cell migration and division. In contrast, IFs form static scaffolds that provide mechanical support to metazoan cells
We determined that the inclusion is encased in a dynamic network of IFs, and that these structures act cooperatively with actin microfilaments to impart an unusual degree of structural stability upon the inclusion.
Disruption of F-actin or IFs with pharmacological inhibitors or by genetic ablation leads to a loss of inclusion integrity, increased formation of inclusion membrane-derived fibers and spillage of bacteria and LPS into the cytoplasm. The consequence of this is the robust increase in IL-8 expression, presumably as a result of the activation of cytoplasmic microbe pattern recognition receptors.
(Left: Cages of F-actin (red) and intermediate filaments(green) encase the inclusion.
Although a model wherein IFs and actin stabilize the inclusion was appealing, the concept of a static IF cage surrounding this dynamic organelle is inconsistent with the need for vacuolar expansion to accommodate logarithmic bacterial replication. Chlamydia has solved this problem by altering the structural properties of the IF network. We determined that the secreted bacterial protease CPAF (Chlamydia Proteosomal-like Activity Factor) cleaves the Head domain of vimentin and other IFs. As infection progresses, the IF network at the surface of the inclusion is increasingly sensitive to detergent extraction and fails to sediment after centrifugation. These results suggest that the scaffolding properties of the inclusion-associated IF cage change throughout infection. In this manner Chlamydia strike a balance between growth in an inherently fragile organelle while minimizing the leakage of bacterial products that may alert innate immune responses.
This works has opened a new set of research question that we are currently pursuing. For example, what are the host and microbial contributors to the recruitment and assembly of F-actin adn IF? Since this pathway does not follow the canonical stress fiber assembly path, we predict that chlamydial proteins help orchestrate these events.
For Additional ongoing research projects see Lab Members section.
