A terpene nucleoside from M. tuberculosis induces lysosomal lipid storage in foamy macrophages
Melissa Bedard, Sanne van der Niet, Elliott M. Bernard, Gregory Babunovic, Tan-Yun Cheng, Beren Aylan, Anita E. Grootemaat, Sahadevan Raman, Laure Botella, Eri Ishikawa, Mary P. O’Sullivan, Seónadh O’Leary, Jacob A. Mayfield, Jeffrey Buter, Adriaan J. Minnaard, Sarah M. Fortune, Leon O. Murphy, Daniel S. Ory, Joseph Keane, Sho Yamasaki, Maximiliano G. Gutierrez, Nicole van der Wel, D. Branch Moody
Melissa Bedard, Sanne van der Niet, Elliott M. Bernard, Gregory Babunovic, Tan-Yun Cheng, Beren Aylan, Anita E. Grootemaat, Sahadevan Raman, Laure Botella, Eri Ishikawa, Mary P. O’Sullivan, Seónadh O’Leary, Jacob A. Mayfield, Jeffrey Buter, Adriaan J. Minnaard, Sarah M. Fortune, Leon O. Murphy, Daniel S. Ory, Joseph Keane, Sho Yamasaki, Maximiliano G. Gutierrez, Nicole van der Wel, D. Branch Moody
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Research Article Infectious disease Microbiology

A terpene nucleoside from M. tuberculosis induces lysosomal lipid storage in foamy macrophages

Abstract

Induction of lipid-laden foamy macrophages is a cellular hallmark of tuberculosis (TB) disease, which involves the transformation of infected phagolysosomes from a site of killing into a nutrient-rich replicative niche. Here, we show that a terpenyl nucleoside shed from Mycobacterium tuberculosis, 1-tuberculosinyladenosine (1-TbAd), caused lysosomal maturation arrest and autophagy blockade, leading to lipid storage in M1 macrophages. Pure 1-TbAd, or infection with terpenyl nucleoside–producing M. tuberculosis, caused intralysosomal and peribacillary lipid storage patterns that matched both the molecules and subcellular locations known in foamy macrophages. Lipidomics showed that 1-TbAd induced storage of triacylglycerides and cholesterylesters and that 1-TbAd increased M. tuberculosis growth under conditions of restricted lipid access in macrophages. Furthermore, lipidomics identified 1-TbAd–induced lipid substrates that define Gaucher’s disease, Wolman’s disease, and other inborn lysosomal storage diseases. These data identify genetic and molecular causes of M. tuberculosis–induced lysosomal failure, leading to successful testing of an agonist of TRPML1 calcium channels that reverses lipid storage in cells. These data establish the host-directed cellular functions of an orphan effector molecule that promotes survival in macrophages, providing both an upstream cause and detailed picture of lysosome failure in foamy macrophages.

Authors

Melissa Bedard, Sanne van der Niet, Elliott M. Bernard, Gregory Babunovic, Tan-Yun Cheng, Beren Aylan, Anita E. Grootemaat, Sahadevan Raman, Laure Botella, Eri Ishikawa, Mary P. O’Sullivan, Seónadh O’Leary, Jacob A. Mayfield, Jeffrey Buter, Adriaan J. Minnaard, Sarah M. Fortune, Leon O. Murphy, Daniel S. Ory, Joseph Keane, Sho Yamasaki, Maximiliano G. Gutierrez, Nicole van der Wel, D. Branch Moody

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Figure 6

M. tuberculosis–produced 1-TbAd induces lipid accumulation in human macrophages.

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M. tuberculosis–produced 1-TbAd induces lipid accumulation in human mac...
(A) Human M1 macrophages were infected with M. tuberculosis or MtbΔRv3378c for 4 days, as reported previously (32), and were then subjected to anti-CD63 staining and annotated. Scale bars: 200 nm. (B) In a separate infection with WT M. tuberculosis, representative TEM images taken over 4 days showed lysosomal swelling. (C–E) Immunofluorescence images of human M1 macrophages infected for 4 days were stained with Hoechst (blue), anti–M. tuberculosis protein (green), and lipids with Nile red (red). The Nile red images were captured in excitation/emission detection windows that allowed broad detection of lipids (wide-field, 532–538 nm/570 nm), as well as detection of neutral lipids (515 nm/585 nm) and phospholipids (554 nm/638 nm). Wide-field Nile red puncta were quantified in 2 experiments with 35–56 cells for each infection condition. P values in panel D were determined by a least-squares means post test with adjustment by Tukey’s method after fitting a generalized linear mixed model and factorial ANOVA (overall P < 0.001 for strain). Data from 2 experiments were pooled after determining that the model fit was unchanged. In panel E, CLEM analysis of human macrophages infected for 4 days identified infected compartments and the limiting membranes of infected phagosomes with visible bacilli, along with staining for lipids (Nile red) and anti–M. tuberculosis antisera. Scale bars: 5 μm (B, C, and E). FM, fluorescence microscopy. (F and G) Human macrophages were infected with M. tuberculosis for 4 days, followed by staining with anti-PLIN2 immunogold. High-magnification images (insets 1 and 2) show a membrane bilayer and monolayer, respectively. In 2 independent experiments, 3,661 electron-lucent compartments stained with (PLIN2+) and without (PLIN2–) immunogold were counted in 9–17 cells per condition.