Lipid Trafficking during Mycobacteria Infection
Tuberculosis (Tb) is caused by Mycobacterium tuberculosis (Mtb) and remains one of the most deadly infectious diseases. The World Health Organization (WHO) estimates that in 2016, Tb killed 1.6 million people emphasizing the importance to develop new drugs, vaccines and diagnostic tools to reduce this burden in the future.
Mycobacteria utilize host lipids as carbon and energy source during infection, but precise knowledge of how Mtb and other pathogenic mycobacteria re-program lipid metabolic and lipid transport pathways to hijack host lipids is scarce.
Using the Dictyostelium/M. marinum infection system, we found that mycobacteria access host lipid droplets (LDs) to build up their own lipid storage organelles and exploit ER-derived phospholipids when LDs are lacking (Barisch et al., 2015; Barisch & Soldati, 2017). Moreover, we observed that mycobacteria that escaped from the Mycobacterium-containing vacuole (MCV) into the cytosol recruit LD-derived enzymes and regulatory proteins on their hydrophobic surface.
Mycobacteria utilize host lipids as carbon and energy source during infection, but precise knowledge of how Mtb and other pathogenic mycobacteria re-program lipid metabolic and lipid transport pathways to hijack host lipids is scarce.
Using the Dictyostelium/M. marinum infection system, we found that mycobacteria access host lipid droplets (LDs) to build up their own lipid storage organelles and exploit ER-derived phospholipids when LDs are lacking (Barisch et al., 2015; Barisch & Soldati, 2017). Moreover, we observed that mycobacteria that escaped from the Mycobacterium-containing vacuole (MCV) into the cytosol recruit LD-derived enzymes and regulatory proteins on their hydrophobic surface.
Dgat2-GFP-labelled LDs interact with cytosolic mycobacteria (from Barisch and Soldati, 2017).
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LD interaction with cytosolic mycobacteria leads to the re-distribution of LD proteins on the mycobacterial surface (from Barisch and Soldati, 2017).
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Previous work indicated that these pathogens not only hijack LDs, but also lipid metabolic enzymes and various components of the lipid trafficking machinery. However, a comprehensive understanding of the underlying molecular principles and their relevance for mycobacterial infection is missing at present.
Therefore, the research focus of the Molecular Infection Biology division is to decipher the molecular mechanisms by which pathogenic mycobacteria manipulate the lipid metabolism and intracellular lipid transport pathways of their host cell. The group combines the use of functional lipids to analyse lipid dynamics at different stages of infection with state-of-the-art microscopy and lipid biochemical tools as well as mass spectrometry lipidomics.
Therefore, the research focus of the Molecular Infection Biology division is to decipher the molecular mechanisms by which pathogenic mycobacteria manipulate the lipid metabolism and intracellular lipid transport pathways of their host cell. The group combines the use of functional lipids to analyse lipid dynamics at different stages of infection with state-of-the-art microscopy and lipid biochemical tools as well as mass spectrometry lipidomics.
Functional impact of lipid logistics during mycobacteria infection (by Stevanus Listian):
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This project aims to identify lipid metabolic pathways that are hijacked by intracellular mycobacteria to exploit lipids from the host. To monitor alterations in lipid levels, Stevanus establishes mass spectrometry lipidomics and thin layer chromatography for the Dictyostelium/M. marinum system. In addition, he plans to disrupt lipid flows from the host to the pathogen using genetics or drugs. To analyse the impact of these disruptions on host-to-pathogen lipid flows, Stevanus uses fluorescent and clickable lipid probes. Finally, he will determine the consequences of blocking specific lipid supply routes on various stages of the mycobacterial infection course. Collectively, these efforts may uncover novel therapeutic targets to fight mycobacteria infection.
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The Mce1 and Mce4 transporters enable mycobacteria to take up fatty acids, but also sterols from their environment.
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Establishment of a drug screening pipeline (by Edwin Ufelmann):
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This project aims to unravel the role of sphingolipids in mycobacteria infections.
Edwin uses high-throughput imaging and a plate reader to develop a drug screening pipeline for the Dictyostelium/M. marinum infection system. Image: Drug targets in sphingolipid biosynthesis. Modified from Kroll et al., 2018. |
Induction of membrane contact sites during mycobacteria infection (by Aby Anand):
Membrane contact sites connect organelles that are not part of the vesicular transport system.
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An emerging concept is that bulk lipid transport inside eukaryotic cells largely occurs at membrane contact sites (MCSs), i.e. specialized microcompartments where two organelles are closely apposed to facilitate a functional integration of compartmentalized cellular processes. MCSs are typically enriched in lipid biosynthetic enzymes and transport machinery, notably cytosolic lipid transfer proteins (LTPs) that enable lipids to reach their destination independent of vesicular trafficking.
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Intriguingly, pathogens like tombusviruses and Chlamydia have been reported to co-opt LTPs from their hosts to build MCSs between the inclusion membrane and host organelles for the acquisition of lipids needed for their replication.
Strikingly, we have evidence that mycobacteria induce the formation of MCS between biogenic host organelles and the MCV to get access to host sterols. To monitor MCS, Aby currently establishes advanced imaging methods such as correlative light and electron microscopy (CLEM). - This project is part of the SFB944.
Strikingly, we have evidence that mycobacteria induce the formation of MCS between biogenic host organelles and the MCV to get access to host sterols. To monitor MCS, Aby currently establishes advanced imaging methods such as correlative light and electron microscopy (CLEM). - This project is part of the SFB944.
Analysing the expression level of LTPs (by Anna-Carina Mazur)
The subcellular localization of proteins in Dictyostelium is typically monitored in GFP-overexpressors. To analyse the localization of endogenous LTPs during infection, Anna Mazur currently generates GFP knock-in strains. This will also help to monitor the expression level of LTPs under various conditions and during infection.
Cloning strategy to generate knock-ins by homologous recombination. From Paschke et al. 2018 (https://doi.org/10.1371/journal.pone.0196809)
ID of interaction partners & lipid ligands of lipid transfer proteins
(by Iris Hube and Deise Schäfer):
(by Iris Hube and Deise Schäfer):
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To recruit LTPs to the membranes of their vacuoles, intracellular pathogens such as Chlamydia and Salmonella secret so called effector proteins. If such a mechanism exists during mycobacteria infections is so far poorly understood. To identify potential effector proteins of M. marinum, Iris Hube uses GFP-traps coupled with mass spectrometry. In her second project she analyses the expression level of host LTPs and previously identified mycobacterial effector proteins by qRT-PCR.
LTPs typically extract a lipid from a donor organelle, shield it in a hydrophobic pocket from the aqueous cytosol and deposit the lipid at a target organelle. To identify the mode of action of selected LTPs and their lipid ligands, Deise Schäfer uses recombinantly expressed proteins combined with liposome assays and bifunctional lipid probes. |
Structure of Dd OSBP8 generated with Swissmodel.org
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Balancing Act: How a pertubation in fatty acid homeostasis impacts on the phagosomal escape of mycobacteria (by Sylvana Hüttel):
The complex cell wall lipids of Mycobacterium tuberculosis are crucial for the survival and persistence of the bacteria in professional phagocytes. To synthesize membrane lipids during infection, Mtb utilizes mainly host-derived fatty acids (FAs). However, FAs need to be modified by fatty acyl-CoA ligases (FACLs) and fatty acyl-AMP ligases (FAALs) to provide activated FAs for protein acylation, energy generation, phospholipid and membrane lipid synthesis, respectively. FA metabolism of Mtb plays a key role during infection, however, so far it is still unknown how Mtb exploits and activates FAs.
Photoactivatable lipid probes to monitor FA flows from the host to intracellular mycobacteria (à la Haberkant, ACS Chem. Biol. (2016)).
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Sylvana's project aims to characterize the function of FA-activating enzymes in lipid synthesis and phagosomal escape of mycobacteria using the Dictyostelium/M. marinum system. To characterize FA flow and metabolism in host and bacteria mutants depleted in FA-activating enzymes, Sylvana establishes a protocol that combines the use of bifunctional FA probes with expansion microscopy and lipidomics. - This project is part of the SPP2225.
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