Abstract
We found that after prolonged infection in macrophages and dendritic cells, M. tuberculosis translocates from phago-lysosomes to the cytosol and kills the host cell a few days later, while the BCG vaccine strain fails to translocate (Cell; 129:1287). This process is dependent on a novel type VII secretion system (T7SS) (Abdallah AM et al., Nat. Rev. Microbiol. 5:883, Abdallah AM et al., J. Immunol. 2187:4744 and Houben et al., Cell Microbiol. 14:1287).
Since my move to Maastricht University, where I was invited to establish a new Institute (www.maastrichtuniversity.nl/m4i), we use cryo-EM single-particle analysis (SPA) and cryo-EM tomography to investigate recombinant purified proteins of individual gene products from the T7SS. In addition, we are purifying the entire intact T7SS structure using biochemical methods for 3D reconstruction. We are also developing the next generation vitrification machine for proteins and cells which we call the VitroJet. I will provide a status update of our developments, and present structures solved from samples prepared with this device.
We also generate thin lamellae of infected cells using cryo-FIB/SEM technology and electron tomography. The workflow starts with a high precision of localization light microscope, with live cell imaging of cell cultures on an EM carrier, which can be used throughout the complete workflow in order to prevent loss of orientation and sample contamination or destruction. Once a specific event or location of a GFP-tagged protein of the T7SS has been identified by light microscopy and preserved by cryo-fixation using jet freezing, the vitrified sample is re-examined for GFP expression. After transferring the sample to the cryo-FIB/SEM, the identified region is thinned down to approximately 150 nm without artifacts, using the focused ion beam (FIB). Samples are then transferred to the cryo-TEM (Arctica or Krios) for high-resolution cryo-tomography. The cryo-SPA data of the T7SS will then be docked onto the images from vitreous sections or lamellae to construct a macromolecular map of the tubercle bacillus within the host cell.
The broader objective is to gain insight into the structure and function of the mechanism for T7SS-mediated translocation. This knowledge should lay the groundwork for the development of novel antibiotics and better vaccines against tuberculosis, still the most deadly infectious disease.
Please also check my recent TEDx talk: How can cryoEM contribute to have a healthy sexual life.
