- Project 1:
Liquid phase electron microscopy of bacterial macromolecular complexes in action
Project Leaders: Dwayne Miller, Eike Schulz and Martin Aepfelbacher
The type three secretion system (T3S) of pathogenic bacteria like Salmonella, Shigella and Yersinia is a multimolecular complex made up by 200 proteins and a total size of approximately 4 MDa. It is assembled in different phases and consists of several parts, i.e. a basal body embedded within the bacterial membranes and an extrabacterial needle like structure, which are distinguishable by conventional cryo electron microscopy. While static structures of T3S’s have been visualized by cryo EM in different states of assembly and in action, there are no recordings of the T3S dynamics beyond the resolution of light microscopy (around 200-400 nm). It’s a longtime dream to conduct electron microscopy (EM) as simple as light microscopy. Liquid phase EM (liquid EM) comes close to this dream.
In liquid EM the specimens are not dried or frozen, but retained in solution, which uniquely offers to observe not only the structure but also the motions of molecules in solution. These dynamic structure changes are key to a complete understanding of macromolecular function and therefore could also provide major new information as to the function of the bacterial type three secretion system.
- Project 2:
Role of prion protein membrane-anchoring in transmission and spreading of prion disease
Project Leaders: Markus Glatzel, Dmitri Svergun
Many fatal neurodegenerative deceases are caused by a misfolded and aggregated form of prion protein, but the cell-to-cell transmission and spreading of these diseases remain elusive. This project will deal with unravelling the molecular and structural mechanisms of how do infectious prion proteins damage the brain. It is known that attachment of prions to membranes and cell-derived vesicles called exosomes plays an important role in the prion fibrillation and in formation of neurotoxic oligomeric species. We shall comprehensively characterize the process of prion aggregation in the presence and absence of exosomes in real time utilizing most novel developments in high brilliance synchrotron X-ray scattering. The scattering studies will be able to detect and identify the transient oligomeric intermediates during prion aggregation, and the structural results will be correlated with the neurotoxicity assays. The results will help understanding potential intracellular protective mechanisms against neurodegenerative diseases.
- Project 3:
Structural enzymology at high spatial and temporal resolution to fight antibiotic resistance
Project Leaders: Holger Rohde, Christian Betzel, Markus Perbandt, Florian Maurer, Matthias Wilmanns,
Arwen Pearson and Martin Aepfelbacher
The rapid worldwide emergence of multi-drug resistant bacteria is a key threat to modern medicine. Resistance emerges through expression of enzymes that modify and inactivate antibiotics, or complex changes in bacterial physiology induced by host environmental factors. Molecular understanding of resistance mechanisms is an essential prerequisite for development of sophisticated (e.g. anti-resistance, anti-virulence) strategies to combat multi-resistant bacteria. In this consortium, combined cutting-edge expertise in molecular biology, protein chemistry, structural biology, and imaging, enzymatic and phenotypic mechanisms of bacterial resistance will be resolved at ultra-high spatial and temporal resolution. Thereby, central knowledge gaps in our general understanding of bacterial multi-resistance will be closed, building basis for the targeted development of tailored strategies to overcome current limitation in treatment options.
- Project 4:
Bio-Nano-Opto-Electronics: Analyzing T cell activation using integrated electro-optical metamaterials
Project Leaders: Andreas Guse and Robert Blick
Infections with pathogenic microorganisms activate the adaptive immune system. T cell activation is the central switch in this process. Upon activation of T cells by pathogenic (antigenic) peptides, intracellular signaling is activated resulting in transcriptional activation, proliferation and differentiation into effector T cells. To analyze these processes in real time with high precision, currently fluorescence confocal microscopy, cell counter, or fluorescence-activated cell sorter (FACS) are in use. These methods are limited regarding resolution and process speed. Previously, we reported use of microtubes with embedded coplanar waveguides for dielectric spectroscopy of T cells (1). Goal of our project is a significant improvement of combined electrical and optical (T) cell analyses, using micro- and nano-structured materials, e.g. micro tubes and/or metamaterials. The basic approach is to develop an integrated Lab-on-Chip (LOC) approach for simultaneous electro-optical testing with significantly drastically enhanced resolution and process velocity.
- Project 5:
Structural approaches to develop new therapeutic strategies against clinically relevant human polyomaviruses
Project Leaders: Arwen Pearson, Eike Schulz and Nicole Fischer Within this projects we apply novel structural approaches to depict two central processes in the pathogenesis of MCPyV and BKV. For MCPyV we will apply in vivo crystallization together with free-electron laser-based serial crystallography to depict the structure of the viral oncoproteins, Large T-Antigen and small T-Antigen, which are the major drivers of MCC tumorigenesis (1PhD student). To target BKV induced pathogenesis (which is dependent on massive viral replication and viral capsid formation) we will address the highly dynamic process of viral capsid formation by applying liquid electron microscopy (1 Postdoc). Applying these novel cutting edge structural approaches we will understand viral pathogenesis in higher resolution which will result in novel therapeutic strategies.
Human polyomaviruses (HPyV) are highly abundant, persist for life time and can induce severe, life threatening complications in elderly and immunosuppressed patients. In particular, Merkel cell polyomavirus (MCPyV) and BK virus (BKV) cause devastating diseases. MCPyV is the etiological agent of a rare but highly aggressive skin cancer, Merkel cell carcinoma (MCC). BK virus (BKV) causes polyomavirus associated nephropathy (PVAN) in up to 10% of kidney transplant patients and hemorrhagic cystitis (PVHC) in 5-15% of allogenic transplant patients. Due to the complete lack of specific anti-viral therapy, treatment options are mainly restricted to reconstitution of the immune system by alleviation of the immunosuppressive regimen, which in the case of BK associated diseases often results in graft rejection or an exacerbation of the underlying condition. For MCPyV and MCC the lack of a specific therapy results in a 5yr overall survival rate of less than 20%. Considering the clinical relevance of BKV and MCPyV the identification of efficient BKV and MCPyV inhibitors is highly desirable.
Within this projects we apply novel structural approaches to depict two central processes in the pathogenesis of MCPyV and BKV. For MCPyV we will apply in vivo crystallization together with free-electron laser-based serial crystallography to depict the structure of the viral oncoproteins, Large T-Antigen and small T-Antigen, which are the major drivers of MCC tumorigenesis (1PhD student). To target BKV induced pathogenesis (which is dependent on massive viral replication and viral capsid formation) we will address the highly dynamic process of viral capsid formation by applying liquid electron microscopy (1 Postdoc). Applying these novel cutting edge structural approaches we will understand viral pathogenesis in higher resolution which will result in novel therapeutic strategies.
- Project 6:
The mechanism of protein secretion in pathogenic slow and fast growing mycobacteria
Project Leaders: Matthias Wilmanns, Annabel Parret and Florian Maurer
Abstract: Mycobacteria are among the most threatening bacterial pathogens affecting humans both in the developing and developed parts of the world. Due to their extraordinary capacity to survive under hostile conditionals in the human host, mycobacterial infections especially of multi-drug resistant strains are difficult and occasionally even impossible to treat. The mycobacterial Type VII secretion system (T7SS) is a highly specialized molecular machine allowing to transport virulence proteins into host cells and thus is of central interest to understand pathogenesis. Our group has recently provided first structural insight into the underlying structure of this complex machine crossing the inner mycobacterial membrane (Beckham et al. Nature Microbiology, 2017). Apart from slow growing Mycobacterium tuberculosis (Mtb) causing tuberculosis, other more specialized, fast pathogenic mycobacteria are increasingly recognized to cause severe infections. M. abscessus (Mab) is a major cause for pulmonary infections in patients with chronic conditions such as cystic fibrosis. In this project, we aim at a comparative functional and structural characterisation of the T7SS of both Mab and Mtb to obtain an in-depth understanding of the role of these molecular complexes in the pathogenesis of mycobacterial infections. Due to the highly divergent expertise of the participating partners (integrative structural biology, biochemistry, genetics, microbiology, access to and diagnostic screening of patient samples) this project is ideally suited to unravel a broad understanding of the role of T7SS in mycobacterial pathogenicity and will thus provide a profound basis for future translational research
- Project 7:
In vivo crystallization of proteins mediating virulence and/or antibiotic resistance in infectious diseases
Scientists of the Infectophysics research group 7 will utilize the potential of living cells, particularly the baculovirus Sf9 insect cell system, to produce from proteins relevant in infection biology bulk amounts of micro- and nanosized protein-crystals (Figure 1).
Figure 1. In vivo grown crystals of PAK4:iBox in Sf9 insect cells. (A) Light microscope image of in vivo crystals growing inside living Sf9 cells after 5-6 days post infection. (B) Suspension of purified in vivo grown crystals, ready for diffraction data collection.
Suitable crystals will be applied for the novel method of serial diffraction data collection established recently at PETRA III (DESY) and the European XFEL. Diffraction data obtained will be used to analyse the three-dimensional structure of proteins at high resolution and will deliver crucial information to support drug discovery investigations to target for example bacterial antibiotic resistance (Figure 2).
Figure 2. Scheme for serial diffraction data collection and structure based drug discovery investigations.
Till now conventional crystallography requires in principle only one µm sized crystal to collect diffraction data. However, the crystal needs to be shock frozen at 100 K to reduce radiation damage as possible. SFX instead requires approx. 10-9, micro-crystals, which is challenging to produce in vitro, but opens opportunities to avoid radiation damage and to perform time resolved experiments at room temperature to follow for example in 3D enzymes in action.
The Infecto research collaboration 7 will aim to produce X-ray suitable crystals in vivo in high amounts for time resolved studies and will team up with research group 3 to combine efforts to analyse structure and dynamics of infecto relevant proteins at high resolution.
In this context, one major goal of this project will be to obtain more and detailed insights in and about the in vivo crystallization process in cells to establish a platform allowing first to identify promising proteins timely and second allowing to upscale routinely the production of in vivo grown crystals.
Project specific references:
Baskaran, Y. et al. An in cellulo-derived structure of PAK4 in complex with its inhibitor Inka1.
Nature communications 6, 8681, doi:10.1038/ncomms9681 (2015).
Gati, C. et al. Serial crystallography on in vivo grown microcrystals using synchrotron
radiation. IUCrJ 1, 87-94, doi:10.1107/s2052252513033939 (2014).
Jakobi, A. J. et al. In cellulo serial crystallography of alcohol oxidase crystals inside yeast
cells. IUCrJ 3, 88-95, doi:10.1107/s2052252515022927 (2016).
Redecke, L. et al. Natively inhibited Trypanosoma brucei cathepsin B structure determined
by using an X-ray laser. Science (New York, N.Y.) 339, 227-230, doi:10.1126/science.1229663 (2013).
Schönherr, R. et al. Real-time investigation of dynamic protein crystallization in living cells.
Structural dynamics (Melville, N.Y.) 2, 041712, doi:10.1063/1.4921591 (2015).
Tsutsui, H. et al. A diffraction-quality protein crystal processed as an autophagic cargo.
Molecular cell 58, 186-193, doi:10.1016/j.molcel.2015.02.007 (2015).