Members of the murine Guanylate-binding-protein (mGBP) family are induced by Interferon-γ (IFNγ) and have been shown to be important factors in cell-autonomous immunity required for control of the intracellular pathogen Toxoplasma gondii. T. gondii, an intracellularly replicating parasite infects ca. 40-50 % of the world population. Previously, we have identified mGBP2 and mGBP7 to be essential effector proteins for mediating immunity against T. gondii infections. Both proteins assemble at the parasitophorous vacuole membrane (PVM) of T. gondii, mediate its disruption or permeabilization and, subsequently, can directly target the plasma membrane of the parasite.
In the first funding period of CRC 1208 we analyzed the molecular mechanisms by which mGBP2 and mGBP7 impair the vital functions of the membranes of the PV of T. gondii. Thereby, the main focus was laid on mGBP7, since mGBP7 deficient mice show a complete failure of immune defense against T. gondii during the acute phase of infection. In multiplexed live cell imaging studies, we have observed different kinetics of recruitment of mGBP2 and mGBP7 to the PVM of T. gondii, since mGBP7 recruitment to the PV and penetration into the PV lumen and even into the parasite occurred mostly subsequent to mGBP2 recruitment. In lipid-binding assays we have additionally identified specific affinities of mGBP2 and mGBP7 to specific lipids, which could be unambiguously confirmed by fluorescence microscopy using giant unilamellar vesicles (GUV) with respective lipid compositions. Finally, using co-immunoprecipitation and mass-spectrometry we have identified interaction partners of mGBP7 in host cells and determined that mGBP7 is post-translationally modified (PTM).
The work focus of the next funding period will be based on these findings. In particular, we aim at unravelling the function of the identified interaction candidates from the host proteome and from T. gondii. Generation of deletion and knock-out cell lines of the mGBP7 interacting proteins will reveal their distinct roles in T. gondii infection and their influence on mGBP localization, membrane biology, and function.
Moreover, we will analyze the binding and interaction properties of mGBPs to membranes and membrane lipids in detail, thereby uncovering the motifs of mGBP7 responsible for membrane interaction. Using the GUV technology, we will study the influence of mGBP2 and mGBP7 membrane binding on membrane integrity. Furthermore, the colocalization of mGBP7 with specific lipids in cells will be studied using lipid sensors. Finally, we aim to understand the biological significance of PTM of mGBPs and their role in pathogen defense.
Taken together, this project will deliver important insights in the intricate role of mGBPs in cell-autonomous immunity and the biology of membranes at the host-pathogen interface.
Project leaders: Dr. Daniel Degrandi, , and Prof. Dr. Klaus Pfeffer, ,
Researchers: Sophia Kasbrink,