Intracellular processing of Borrelia burgdorferi in human macrophages

• Applicant: Professor Dr. Stefan Linder
• Subject Area: Cell Biology
• Term: from 2020 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 452810684

This project investigates molecular mechanisms that regulate intracellular processing of borreliae by primary human macrophages, with a focus on phagosomal compaction and phagolysosomal maturation. Borrelia burgdorferi is the causative agent of Lyme disease, a multisystemic disorder affecting primarily skin, joints and nervous system. Successful recognition, uptake and elimination of borreliae by human immune cells such as macrophages is thus decisive for the outcome of a respective infection. Our previous work identified the sorting nexin SNX3 as a central hub between phagolysosmal maturation and membrane recycling in macrophages. SNX3 enables docking of Rab5a positive vesicles to the phagosomal coat, through binding of PI(3)P, and recruits a second subset of galectin-9- and flotillin-2 positive vesicles. This pathway leads to the generation and fission of membrane tubules at phagosomes, and thus to phagosome compaction, a prerequisite for phagolysosomal maturation. We will now investigate i) proteins shown by our previous work to be important for borreliae processing, but also ii) pursue an unbiased approach for the identification of novel regulators. Accordingly, the first work package is a candidate-based approach that focuses on the molecular mechanisms of the previously identified regulators galectin-9, flotillin-2, and KIF16B, especially regarding their spatiotemporal interplay. The second work package aims to identify new regulators of phagosome compaction and maturation, based on magnetic labeling of spirochetes and isolation of borreliae-containing phagosomes, coupled with stable isotope labeling of cells (SILAC) and mass spectrometry analysis. The feasibility of these approaches has been demonstrated, and all necessary techniques are established in our lab or in the lab of our collaborator. Investigation of borreliae intracellular processing will be performed by a combination of techniques, including i) molecular biological techniques such as expression of fusion and mutant proteins, siRNA-mediated knockdown, immunoprecipitation GST/MBP pull down, and phosphoinositide binding assays, ii) microscopic techniques such as confocal live cell imaging and STED superresolution microscopy, iii) advanced cell-based assays such as phagosomal compaction, phagolysosomal acidification and proteolysis, borreliae intracellular survival assays, SILAC labelling of primary cells, and proximity ligation assays, and iv), in collaboration with the lab of Dr. Ludger Johannes (Institut Curie, France), preparation of giant unilamellar vesicles (GUVs), and membrane tubulation assays. Collectively, these complementary lines of investigation will not only lead to novel data on borreliae phagosome compaction and phagolysosomal maturation, but also uncover new molecular mechanisms that link intracellular phagosome transport with the endosomal recycling machinery, thus revealing general regulatory principles of pathogen processing in human immune cells.

• DFG Programme: Research Grants