Intracellular processing of Borrelia burgdorferi in human macrophages
Final Report Abstract
The project aimed to identify the molecular mechanisms that regulate intracellular processing of Borrelia burgdorferi, by primary human macrophages. We followed three lines of investigation: i) analysis of Borrelia-macrophage interaction by high resolution microscopy, ii) a candidate approach of Borrelia phagosome regulation, where we investigated the role of the kinesin KIF16B, and iii) an unbiased approach based on purification of phagosomes and mass spectrometry, which led to the identification of the tyrosine kinase AXL. i) Using FIB/SEM-based microscopy and 3D reconstruction, we could show that the interaction of borreliae with macrophages is more complex than previously believed. Borreliae are captured at the macrophage surface by multiple protrusions, including coiling pseudopods and membrane folds. Our analyses also showed that contact with borreliae can lead to the formation of partially empty nascent phagosomes that we termed “membrane tunnels”. Tunnels are likely formed by partial extrication of the highly motile borreliae, showing that the interaction with macrophages is not only driven by the host cell but probably more like a tug-of-war. We also showed for the first time that membrane tubulation at fully formed phagosomes, which leads to gradual reduction of the phagosome surface and compaction of borreliae, is a frequent phenomenon. Finally, both tunnels and phagosomes were shown to develop distinct contact sites with the endoplasmic reticulum that are positive for the ER marker STIM1. A respective manuscript was published in Journal of Cell Science . ii) In the candidate approach, we could identify the kinesin KIF16B as a novel regulator of borreliae phagosome positioning and maturation in macrophages. We could show that KIF16B is recruited early upon internalization of borreliae to respective phagosomes, which is based on its direct binding to the phospholipid PI(3)P in the phagosomal coat. Using pharmacological intervention and siRNA-based depletion, we could show that KIF16B retains borreliae phagosomes in the cell periphery until their full compaction. Upon depletion of PI(3)P, KIF16B dissociates from phagosomes and enables centripetal movement of phagosomes by dynein, which is accompanied by acidification and maturation of phagosomes. We could thus identify KIF16B as a regulator of borreliae phagosome maturation, by establishing a checkpoint for phagosome compaction. KIF16B retains incompletely compacted, i.e. PI(3)P positive, phagosomes in the cell periphery, ensuring that only fully compacted phagosomes enter the pathway to further maturation. A respective publication is currently being finalized. iii) In an unbiased approach, using purification of magnetically labelled phagosomes and subsequent mass spectrometry, we could identify the receptor tyrosine kinase AXL as a potential regulator of Borrelia phagocytosis. We could show that both endogenous and overexpressed forms of AXL prominently colocalize with borreliae phagosomes. SiRNA-based depletion further showed that AXL is a positive regulator of both internalization and compaction of borreliae. We are currently performing rescue experiments to confirm this, using siRNA-insensitive constructs of wild type, kinase-dead/deleted and AP2-binding deficient mutants to elucidate AXL´s mode of action. Using BioID labelling, we could further identify several potential interaction partners of AXL, in particular the H+/Cl- channel CLC7, which colocalizes with AXL at phagosomes and apparently regulates phagosome maturation. Further experiments addressing AXL´s role in phagosome maturation and intracellular processing of borreliae are currently conducted and are expected to lead to a respective publication within the next 12 months.
Publications
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FIB/SEM-based analysis of Borrelia intracellular processing by human macrophages. Journal of Cell Science.
Klose, Matthias; Scheungrab, Maximilian; Luckner, Manja; Wanner, Gerhard & Linder, Stefan
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Molecular Mechanisms of Borrelia burgdorferi Phagocytosis and Intracellular Processing by Human Macrophages. Biology, 10(7), 567.
Woitzik, Philipp & Linder, Stefan
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Get a grip: Podosomes as potential players in phagocytosis. European Journal of Cell Biology, 102(4), 151356.
Linder, Stefan & Barcelona, Bryan
