SFB796 - Research programme

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Reprogramming of host cells by microbial effectors

The long-term goal of CRC796 is to significantly contribute to the understanding of the molecular and structural basis of pathogen-host interactions. By studying viral and bacterial pathogens infecting plants and humans, respectively, we aim at elucidating common and specific microbial strategies to colonize host cells. Although zooming into the bio-medical and plant-oriented research projects reveals different overall research goals, the underlying basic concepts and technical challenges are surprisingly similar, rendering a comparative approach highly appealing. For example, central cellular responses including protein kinase cascades, ubiquitin-mediated protein degradation or vesicular trafficking are common processes which are targeted by microbial effector proteins.

To reach the long-term research goals, the CRC796 is built on highly motivated scientists with complementary expertise and a central technology platform providing the tool-box and experimental and computational know-how to successfully approach the challenging research aims. This unique and integrative scientific environment enables us to study communication between microorganisms and their host cells from the molecular up to the organism level. The concept of the CRC796 meets the challenges of research areas in molecular medicine and molecular plant pathology, which have benefited tremendously from the technological developments of the post-genomic era. The newly developed tools allow the in-depth molecular and structural analysis of effector-host target interactions, which will lay the ground for the rational design of future drugs. Therefore, the CRC796 will identify valuable targets for medical and agricultural applications and will provide impulses for translational research in medicine. The generated knowledge may eventually lead to the development of novel, presently not anticipated, strategies for disease prevention and treatment.

The research programme is divided into three project areas (A-C), all of which make use of core technologies provided in the central Z1 project. The overall structure of CRC796 is shown in figure 1.

 

Figure 1: Modular structure of CRC796; OICE, Optical Imaging Center Erlangen.

 

The analysis of structure-function relationships of already known effectors and their interactions with specific host targets are within the focus of project area A (structural basis of molecular interactions). During the first funding period project A2 (H. Sticht) was very instrumental in developing and providing computational tools for the prediction of protein-protein interaction domains. In the second funding period project A2 will continue to improve computational predictions of short linear interaction motifs by including motif-specific flanking sequences and will use a combination of molecular modeling, docking, and molecular dynamics simulations to help studying interactions between globular protein interaction domains. In project A3 (Y. Muller) structure-function studies on individual proteins that play central roles during the infection of plant cells by potyviruses, will be continued and complemented with studies on the infection of animal cells by cytomegaloviruses. Thereby, project A3 bridges between project B3 (human virus) and C2 (plant virus). Project A4 (T. Wittenberg) aims at further developing image processing routines with a focus on the automated splitting of touching, overlapping and even overlying cells. At the same time improvements in gaining spatial and temporal information from multi-image datasets are being sought. These developments significantly support progress in several research projects across all project areas (in particular A6, B1, B2, B3, B6, C3 and C7). Inclusion of the new project A6 (N. Tschammer) extends the effector repertoire to viral proteins interfering with host signalling cascades by manipulating G-protein coupled receptor composition. Viruses such as the human cytomegalovirus (HCMV) encode for viral G-protein coupled receptors, which by heterodimerization with host receptors, most likely influence signal perception of infected host cells. The function of these effectors will be tested by synthetic effector molecules which will allow pinning down the role of these receptors during virus infection and propagation.

The focus of project area B (reprogramming cellular processes) is the elucidation and detailed understanding of mechanisms used by microbial effectors to reprogram cellular processes, including signal transduction pathways (B1), nuclear processes (B1, B2, B3), innate (B6) and adaptive (B7) immune responses and phagosome maturation (B6) and cell death regulation including apoptosis (B8) and necroptosis (B9). These studies include viral effectors (B1, B2, B3, B9) and bacterial effectors of the type IV and type VI secretion systems (B7, B8). In addition, further bacterial effectors of known (cord factor) and unknown nature are under study (B6). Project B1 (Armin Ensser) has started out to analyse Tip-mediated modifications of the STAT signalling pathway. These studies will be continued and extended to reveal Tip-dependent epigenetic genome modifications in response to differential STAT activation. Since the viral effector Orf3, a tegument protein capable of selective SP100 degradation, is presumably also involved in modifying the epigenetic signature of the virus and host cell genome, the effector will additionally be studied. SP100 is part of the ND10 complex, which is thoroughly investigated in project B3. In project B2 (Alexander Steinkasserer) replication of HSV-1 and hCMV in mature dendritic cells (mDC) is investigated. Research of the second funding period will concentrate on molecular factors restricting viral replication in mDC and on CD83. Preliminary experiments suggest that nuclear egress may be inefficient and thereby limit viral replication. Nuclear egress is also studied in project C3 and molecular processes occurring at the nuclear envelope are analysed in projects C2 and C3. During the first funding period, project B3 (Thomas Stamminger) could demonstrate that IE1 is involved in deSUMOylation of PML and SP100, leading to disruption of the ND10 nuclear complex. In collaboration with the Z project the structure of the primate IE1 protein could be resolved at a resolution of 2.3A. With help of the structural data B3 will continue to analyse IE1 function and in collaboration with project A3 will investigate structure-function relationships of IE1 in ND10 degradation. The glycolipid trehalose-dimycolate (TDM, also known as cord factor) is a well-known trigger for innate immune response. TDM is recognized by the C-type lectin receptor Mincle leading to the initiation of defence. In addition TDM is required to overcome degradation in macrophages. Project B6 (Roland Lang) aims at deciphering the mechanism by which TDM supports the intracellular survival of mycobacteria in host cells, focussing on inhibition of phagosomal maturation and impairment of IFNγ-induced macrophage activation. Mechanisms allowing type VI effector molecules to suppress local immune responses of intestinal host cells will be studied in project B7 (Stefan Wirtz and Markus Neurath). In the first funding period it could be shown that Type VI mutants of Citrobacter rodentium are compromised in their ability to colonize the mouse gut. Therefore, project B7 aims at characterizing the function of selected type VI effectors in newly developed organoid culture systems as well as in macrophage cell lines and primary dendritic cells. Projects B8 (Anja Lührmann) and B9 (Christoph Becker and Michael Stürzl) will study microbial strategies to modify programmed cell death of host cells. While project B8 will study the anti-apoptotic activity of bacterial type IV effectors, project B9 will study the potential regulation of the riptosome by viral FLIP proteins. The riptosome is a major regulatory complex of necroptosis, a form of programmed necrosis. Stable adhesion complexes are crucial for maintenance of the cell-to-cell integrity in healthy epithelia and represent the first barrier for microbial pathogens. Adhesion complexes might be disrupted by a novel secreted effector from Helicobacter and Campylobacter, the protease HtrA. Project B10 (Steffen Backert) is designed to characterise the HtrA secretion route, identify cleavage sites in E-cadherin, pinpoint novel HtrA targets of host cells and study downstream signalling events. Results will provide a potential novel mechanism how pathogens can destroy cellular junctions in order to get access to deeper tissues and trigger disease.

In project area C (replication structures and transport processes) microbial strategies to use and partially reprogram, cellular structures for invasion, replication and propagation are under study. Processes under study include, reprogramming of the nuclear envelope (C2, C3), the manipulation of the cytoskeleton and vesicular trafficking (C6, C7) and the cell-to-cell transport (C2, C6).

In the first funding period project C2 (Uwe Sonnewald) in collaboration with project C3 could demonstrate that the herpes virus pUL50 protein is able to target plant nuclear envelopes and that it interacts with a plant host protein of so far unknown function. The host protein localizes to the plant nucleus and is a potential structural component. Together with project A2, interaction between a viral movement protein and WW-domain containing helicases could be predicted and experimentally verified. Plant mutants, deficient in the targeting of the viral movement protein to cell-to-cell connections, revealed a membrane protein, which is essential for efficient trafficking of the movement protein. Furthermore, elucidating the molecular function of molecular chaperones in viral movement and replication revealed that partial degradation of viral capsid proteins is a necessity for efficient viral replication and that this degradation is mediated by the hsp40/hsp70 complexes and the ubiquitin system. In the seconds funding period nuclear targeting of pUL50 (together with C3), the role of WW-domain proteins in virus infection (together with A2), the molecular interaction between hsp40 and capsid proteins (together with A3) and the host factors required for proper movement protein targeting will be investigated. In project C3 (Manfred Marschall) the transient destabilization of components of the nuclear envelope by herpesviral effector proteins is under study. This process is required for the egress of viral capsids from the nucleus. Together with host proteins, viral effectors form the so called HCMV nuclear egress complex (NEC) which will be further characterized during the second funding period. The transmission of human T-cell lymphotropic virus type 1 (HTLV-1) is in the focus of project C6 (Andrea Thoma-Kreß). HTLV-1 is transmitted via cell-to-cell connections between host cells. These connections are virus induced and require remodelling of the cytoskeleton. The aim of the project is to unravel the mechanisms by which the viral proteins Tax and p8 are able to reprogram the cytoskeleton and the cell-to-cell interactions. The project nicely allows direct collaborations and comparative analysis with results obtained in plant systems including cell-to-cell transport of viruses (C2) and modification of the cytoskeleton (C7) and therefore may bridge between plant and human host responses. The aim of project C7 (Benedikt Kost) is to unravel the importance of Rac/Rop GTPases during the interaction of plant cells with bacterial pathogens and to identify bacterial type III effectors that directly target the activity of plant Rac/Rop GTPases. In animal cells, Rac/Rop GTPases are well known targets for microbial effectors and therefore the analysis will enable to unravel common and specific roles of Rac/Rop GTPases in host pathogen interactions.

All in all, major focus areas are intra- and inter cellular trafficking, signaling, nuclear processes, protein degradation and programmed cell death, summarized in figure 2. Inclusion of new projects and adjustment of established projects allowed a coherent and focused research program which will carry the interdisciplinary work to the next level.

 

Figure 2: Major focus areas of CRC796. A, simplified scheme of pathogens. Colour code indicates projects focusing on plants (green), mammalian (red) or both (grey) systems. B, summary of cellular targets and target processes in host cells.

 

The unifying goal of all participating projects is to unravel the modes of action that microbial effectors deploy to reprogram cellular processes in the host cell. For this purpose, it is necessary to understand in detail the basic molecular steps, as well as to identify the structural prerequisites for the interaction between microbial effectors and their host targets. Such in-depth approaches extend well beyond the possibilities and resources of each individual research group. The integration of many different resources offers unique opportunities. Given the fact that many important effector proteins do not belong to any known protein family, we anticipate that clues regarding their function can be derived from structural analyses, which can then be tested in functional assays. By implementing such interdisciplinary approaches, we anticipate to create opportunities for (i) in-depth mechanistic explanations of the modes of action of effector molecules, (ii) the discovery of general virulence mechanisms and, as a long term goal, (iii) the development of new active reagents to disrupt effector function. This is only possible because of the close collaboration between the diverse, yet complementary research groups, the spatial proximity of the different laboratories within a single city, as well as their commitment to focus on a common research goal. Since the CRC796 covers a wide variety of bacterial and viral pathogens acting on different mammalian and plant cells, we anticipate that general principles for the action of microbial effectors can be discovered and that these will hold true for many pathogens, exceeding those studied by the CRC796. Our collaborative research effort certainly has the methodological breadth to analyze individual findings and to develop from these generally applicable insight.

Crucial methods for generating novel insight are provided by the central project (Z1). The central project will continue to reach into all research areas by offering an integrated and state-of-the-art technology platform supporting all groups of the CRC796. The Z1 project is divided into five inter-connected modules.

Module I (Bioinformatics)   will provide a central computational platform for the analysis of microbial effectors and their cellular targets. The work packages are embedded into subproject A2 in which computational methods have been optimized that allow the prediction of linear sequence motifs in microbial effectors that could mediate protein-protein interactions and thus are involved in effector function.

Module II (Protein production and and crystallisation)   will provide a platform for the heterologous production of proteins of interest for in vitro interaction studies, to validate the native-folded state of these proteins and to determine individual conditions for the successful crystallization and subsequent crystal structure determination.

Module III (Bioimaging)   the established CLSM platform will be transferred into the newly established Optical Imaging Centre Erlangen (OICE) which bundles the available expertise in bio-imaging creating an added value for the CRC as researchers will gain access to novel imaging methods and technology platforms previously not available to the consortium. Access to the platform will be organized using the online microscope booking system established during the first funding period. Members of the CRC will also receive scientific support and user training through the OICE.

Module IV (Yeast-two-hybrid screening)   will provide access to existing yeast-two-hybrid libraries and conduct screening and first validations of positive clones.

Module V (Proteomics)   will provide a comprehensive and efficient service in MS-based proteomics analyses. The module will offer mass-spectrometric analyses of proteins with a particular focus on protein identification in purified protein complexes and the characterization of post-translational modifications.