Research
Unlocking the Secrets of Host-Pathogen Interactions
An increasing number of pathogens are becoming resistant to antibiotics, posing a threat to human health. Therefore, understanding the host-pathogen interaction is necessary and urgent to unravel disease pathogenesis at the molecular level. We focus on the physical barrier of the epithelial surface, autophagy and the immune response, which are mediated by macromolecular complexes or organelles.
Using state-of-the-art cryo-electron tomography (cryo-ET), we aim to dissect the dynamic responses of these macromolecular complexes or organelles in unprecedented detail. By integrating microbiology, immunology, computational biology and engineering approaches, we aim to address the multifaceted nature of host-pathogen interactions from every angle.
A Salmonella bacterium is engulfed by a phagophore and an autophagosome as shown in the tomogram and 3D segmentation of correlative cellular cryo-ET. Read more here.
Cryo-ET method development
Cryo-electron tomography (cryo-ET) is an advanced microscopy technique used in the field of structural biology to visualize the three-dimensional (3D) structures of biological macromolecules and cellular organelles in their native, hydrated state at nanometer-scale resolution. Cryo-ET has become an invaluable tool for structural biologists to study the shape and interactions of macromolecules and organelles in situ. It has applications in various fields, including the exploration of fundamental biological processes,the study of diseases, and the development of therapeutics.
The development of in situ cryo-ET methods is at the forefront of structural biology and microscopy, and has evolved in recent years with advances in instrumentation, sample preparation, data processing and interdisciplinary collaboration. We are using pathogens and their hosts as a model system to develop new methods, studying dynamic and heterogeneous biological events across organisms.
The following is an introduction to in situ cryo-ET methods and their applications:
Correlative cellular cryo-ET workflow
An application example
Correlative cellular cryo-ET workflow used for investigating Salmonella-induced autophagy in HeLa cells. We started by culturing the host cells on grids and then freezing the Salmonella-infected cells. We then used a cryo-fluorescence microscope (cryo-FM) to take images of the grids to identify the location of the cells and the presence of Salmonella within them. We then used cryo-focused ion beam (cryo-FIB) milling to create ~200 nm thick lamellae of cells at these identified locations. The grids were then transferred to a Titan microscope to collect cryo-ET tilt series. After thorough data processing, we visualized phagophore formation during Salmonella clearance in situ, insights into cellular organisation and details of interaction networks. Read more here. Top panel workflow images are modified from Alexander Rigort et al., 2018.
Volume cryo-ET workflow with serial lift-out FIB milling
Isolated tobacco trichomes were first subjected to high pressure freezing to obtain extracellular vitreous samples due to their large size and high water content. Serial lift-out FIB milling is a perfect method of choice, allowing serial preparation of lamellae from a volume sample (developed by the Juergon Plitzko group@MPIB). In this case, we obtained 6 lamellae from a cell wall junction. Subsequent data processing allowed us to observe the architecture of lipid droplets with unprecedented resolution. This work was supervised by Prof. Wolfgang Baumeister at the Max Planck Institute of Biochemistry.