Our research projects

The major specific aim of our lab is to understand the function of individual effectors of Chlamydia and Legionella. This will contribute to further our knowledge of the virulence mechanisms used by these bacteria, and could provide novel insights into the cell biology of eukaryotic cells or reveal novel drug targets.

We are well aware that effectors delivered by a single bacterium into a host cell act together and often have redundant functions. Therefore, analyses of the function of single effectors can be misleading and extremely difficult. However, we believe that continuous efforts to understand the molecular and cellular function of single effectors will eventually provide a comprehensive picture of virulence mechanisms of bacteria injecting host cells with effector proteins.

Project 1. Identification and characterization of novel C. trachomatis T3SS effectors (Inês S. Pereira, Inês Leal, Irina S. Franco)

We previoulsy identified several potential novel C. trachomatis T3SS effectors using Yersinia enterocolitica as a heterologous host. As a follow-up of this study, we are now using the recently described methods for transformation, ectopic expession, and gene knock-out in C. trachomatis to test if the proteins we singled-out are indeed translocated effectors and what is their function during the Chlamydia life-cycle. We showed that the type III secretion substrates CT142, CT143, and CT144 are secreted into the inclusion lumen, and work is in progress to further characterize these and other candidate effectors.

Project 2. Functional analyses of Inc proteins of C. trachomatis (Maria P. Luís)

Inc proteins are a large group of chlamydiae-specific proteins (representing about 6% of the coding capacity of chlamydial genomes) and play an important role in Chlamydia-host cell interactions. For example, the genome of C. trachomatis might encode for ≈60 Inc proteins. We recently found that some Inc proteins could contribute to the unique tropism and invasiveness of C. trachomatis lymphogranuloma venereum (LGV) strains.

We now are focused at identifying the molecular mode of action of Inc proteins. The overall approach being followed is: (i) identification of host cells targets of Inc proteins; (ii) validation of host cell targets found; (iii) characterization of the biological function of Inc host cell targets during C. trachomatis infection; (iv) structure biology of the Inc-host cell factor interactions; (v) gain-of-function screens to search for functions for Inc proteins. Using these approaches we described that C. trachomatis Inc CT288 binds a host centrosomal protein (CCDC146) that is recruited to the inclusion membrane. Ongoing studies are aimed at furthering the understanding of how CT288 and other Inc proteins function.

Project 3. The molecular and cellular function of Legionella effectors (Irina S. Franco)

VipA (339 amino acid residues) is a Legionella effector which functions as an actin nucleator that alters organelle trafficking when expressed in Saccharomyces cerevisiae. This suggested that VipA might function by controlling trafficking of the LCV through interaction with actin. We recently found that: (i) the C-terminal region of VipA (amino acid residues 206-339) is necessary and sufficient for actin-binding; (ii) the N-terminal region of VipA (amino acid residues 1-133) could be involved in targeting VipA to early endosomes; (iii) both the C-terminal and N-terminal regions of VipA are required for interference with organelle trafficking in yeast.

As a follow-up of these studies we are currently: (i) investigating the role of VipA during Legionella infection of macrophage cells; (ii) testing the possible role of the N-terminal region of VipA in mediating interactions with early endosomes; (iii) identifying novel VipA binding partners.

We are also studying additional effector proteins identified based on the genome of the L. pneumophila strain that caused a major outbreak of Legionnaire's disease in Vila Franca de Xira, Portugal, in 2014.