Research

Chlamydia trachomatis is a gram-negative bacterial pathogen of tremendous public health concern. Ocular serovars lead to trachoma and genital serovars are the leading cause of bacterial sexually transmitted disease in developed countries. Vaccines are not available and despite the implementation of C. trachomatis screening programs, and the effectiveness of antibiotics to treat trachoma and uncomplicated sexually transmitted chlamydial infection, case rates are not declining and reinfection rates are increasing.

Chlamydia are characterized by a biphasic developmental cycle that occurs exclusively in the host cell (Fig.1).  Once internalized, Chlamydia reside in a membrane bound compartment, named the inclusion and alternate between an infectious form (Elementary Body, EB) and a replicative form (Reticulate Body, RB). The cycle lasts two to three days depending upon the species.

Chlamydia are characterized by a biphasic developmental cycle that occurs exclusively in the host cell

Figure 1 Chlamydia developmental cycle

If our knowledge of the cellular processes that are targeted by Chlamydia has greatly increased, we have only begun to identify the bacterial and host factors required for bacterial development.

In the past, genetic intractability of Chlamydia has made it challenging to fully dissect the role of virulence factors involved in pathogenesis, but tremendous advances have recently occurred and genetic tools (transformation, mutagenesis) are emerging. We have contributed to the field through the development of cloning vectors, the generation of Chlamydia strains expressing various fluorescent proteins, a conditional gene expression system and most recently a fluorescent reporter to monitor RB-to-EB transition (Fig.2).

Figure 2: Confocal micrograph of HeLa cells infected for 28 (A), 40 (B), 52 (C) hours with C. trachomatis expressing mCherry from a constitutive promoter and GFP from a late promoter. Scale Bar: 20μm.

Figure 2: Confocal micrograph of HeLa cells infected for 28 (A), 40 (B), 52 (C) hours with C. trachomatis expressing mCherry from a constitutive promoter and GFP from a late promoter. Scale Bar: 20μm.

 

 

 

 

 

 

 

 

We have also contributed to the field through the identification of host factors involved in Chlamydia infection using the RNAi methodology. One of our candidates led us to uncover that, in addition to vesicular trafficking, C. trachomatis hijacks the non-vesicular lipid transfer machinery at zone of close apposition (10-50 nm) between the ER and the inclusion membrane. These specialized platforms are referred to as ER-Inclusion membrane contact sites (MCSs) (Fig.3).

Figure 2 ER - Chlamydia Inclusion Membrane Contact Sites

Figure 3 ER – Chlamydia Inclusion Membrane Contact Sites. Part of the Chlamydia inclusion membrane makes close contact with the endoplasmic reticulum. These platforms are termed ER-Inclusion membrane contact sites. A schematic representation with the ER in green (left) and an electron micrograph (right) are shown.

Our overarching goal is to better understand the molecular mechanisms involved in the formation and function of ER-Inclusion MCS and elucidate their role in infection and pathogenesis. One of our approaches has been to identify and characterize the molecular components of ER-Inclusion MCS.

We have identified several molecular components of ER-Inclusion MCS. More specifically, we have uncovered a role for Inc proteins (a subset of Chlamydia specific Type III translocated effectors that are imbedded into the inclusion membrane) in the subversion of the endoplasmic reticulum. The recurrent theme is that, at ER-Inclusion MCS, distinct Inc proteins mediate the recruitment of and associate with specific host factors (Fig.4).

Our current view of ER-Inclusion MCS

Figure 4: Our current view of ER-Inclusion MCS

The inclusion membrane protein IncD interacts with the host ceramide transfer protein CERT, which interacts with ER-resident VAP proteins. Combined with data from the Engel Lab at UCSF, it is proposed that this complex is involved in lipid transfer to the inclusion. Recently, we have shown that through molecular mimicry of 2 eukaryotic FFAT motifs, IncV interacts with VAPs and is involved in tethering the ER with the inclusion membrane. Our lab has also shown that the ER calcium sensor protein STIM1, which is usually found at ER-PM MCS, is also enriched at the ER-inclusion MCS. We have unpublished data indicating that another Inc, that we named IncS, mediates STIM1 recruitment to ER-Inclusion MCS. The role of the IncS-STIM1 complex is under investigation.

Our research will further our understanding of the molecular mechanisms involved in the infection process and may reveal drug targets to facilitate the translational research development of tools to prevent, treat and control Chlamydia infection.