Lysosomal acid lipase regulates cholesterol metabolism during phagosomal maturation
Description
Phagocytosis, a central process in innate immunity, depends on dynamic lipid remodelling, yet how cholesterol accumulates on late phagosomes remains unresolved. Here, we identify lysosomal acid lipase (LAL) as a key cholesterol ester (CE) hydrolase driving cholesterol mobilization during phagosomal maturation. Using integrated lipidomics, chemoproteomics, and biochemical assays, we show that LAL exhibits acidic CE hydrolase activity enriched on late phagosomes, generating free cholesterol essential for lipid raft formation. Pharmacologically inhibiting and genetically depleting LAL disrupts cholesterol-rich lipid raft assembly, impairs phagosomal trafficking, and alters pathogen fate – enhancing Staphylococcus aureus persistence, while restricting Mycobacterium tuberculosis survival. These findings reveal that LAL couples cholesterol metabolism to phagocytosis, defining a mechanistic link between lipid catabolism and antimicrobial defence. By positioning CE hydrolysis as a critical determinant of phagosomal dynamics, our work uncovers a metabolic checkpoint in innate immunity and identifies LAL as a potential therapeutic node in infection and inflammation. Major findings of the study: Lalistat2/Lysosomal acid lipase knockdown has a pronounced effect on the levels of cholesterol and cholesteryl esters on the phagosomes.
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Steps to reproduce
Briefly, lipid extracts of Late Phagosomes (LPs) and Lalistat treated LPs were extracted, dried under nitrogen and reconstituted in 200 μL of chloroform:methanol (2:1, v/v). From this solution, 10 μL was injected into an Agilent 6545 quadrupole time-of-flight (Q-TOF) liquid chromatography–mass spectrometry (LC-MS) system operated in high-resolution auto MS/MS mode for semiquantitative lipid profiling. Separation of lipid species was achieved using a Gemini 5U C-18 column (Phenomenex) coupled with a Gemini guard cartridge (4 × 3 mm, Phenomenex security cartridge).The mobile phases consisted of Buffer A (95:5,water:methanol) and Buffer B (60:35:5, isopropanol:methanol:water).To facilitate ionization, 0.1% ammonium hydroxide (v/v) was added to both buffers for negative ion mode analysis, whereas 0.1% formic acid (v/v) supplemented with 10 mM ammonium formate was included in both buffers for positive ion mode analysis. A 60-minute gradient program was used for untargeted lipid measurements: 100% buffer A at a flow rate of 0.3 ml/min for 5 minutes, followed by a linear gradient to 100% buffer B over 40 minutes at 0.5 ml/min, an isocratic hold at 100% buffer B for 10 minutes, and re-equilibration with 100% buffer A for 5 minutes at 0.5 ml/min. All LC-MS analyses were performed using an electrospray ionization (ESI) source under the following optimized conditions: drying and sheath gas temperature, 320 °C; drying and sheath gas flow rate, 10 L/min; fragmentor voltage, 150 V; capillary voltage, 4 kV; nebulizer gas pressure, 45 Ψ; and nozzle voltage, 1 kV. For lipid identification, a curated Personal Compound Database and Library (PCDL) was employed. Peaks were validated based on accurate mass, relative retention time, and MS/MS fragmentation patterns. Quantification of individual lipid species was carried out by calculating the peak areas, normalizing them against internal standards spiked into the samples, and further adjusting values relative to either total protein content or tissue weight, depending on the sample type. All "pos.d" files are the samples run in positive mode All "neg.d" files are the samples run in negative mode
Institutions
- Indian Institute of Science Education Research PuneMaharashtra, Pune