A terpene nucleoside from M. tuberculosis induces lysosomal lipid storage in foamy macrophages
Melissa Bedard, … , Nicole van der Wel, D. Branch Moody
Melissa Bedard, … , Nicole van der Wel, D. Branch Moody
Published February 9, 2023
Citation Information: J Clin Invest. 2023;133(6):e161944. https://doi.org/10.1172/JCI161944.
View: Text | PDF
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

×

Figure 2

1-TbAd causes the accumulation of autophagosomes due to blockage of autophagic flux.

Options: View larger image (or click on image) Download as PowerPoint
1-TbAd causes the accumulation of autophagosomes due to blockage of auto...
(A and B) M1 macrophages treated with chloroquine or 1-TbAd (20 μM) for 2 hours were immunogold labeled for LC3, LAMP1, or both markers. The area (μm2) of electron-lucent compartments was measured and the number of gold particles were counted per compartment. Double-immunogold labeling was scored as no label (<3 particles) or labeled (>3 particles), with subgroups of LAMP1 single positive, LC3B single positive, and LAMP1 AND LC3B double positive. Single LC3 analysis used a linear model with a negative binomial fit, with P values determined by factorial ANOVA and Tukey’s post test. Linear mixed models treated the double label as a random effect variable (χ2 P << 0.0001). For single and double labels, P values were determined by least squares mean post-test after factorial ANOVA and adjustment by Tukey’s method. (C) RAW264.7 macrophages stimulated for 4 hours were analyzed by immunofluorescence for LC3B recruitment to LAMP1+ compartments. Scale bars: 5 μm. One representative experiment of 3 experiments is shown. P values were determined by Browne-Forsythe ANOVA followed by Games-Howell’s multiple comparisons. (D) RAW264.7 macrophages transiently expressing GFP-mCherry-LC3B were treated with vehicle (DMSO), BafA1, 1-TbAd, or N6-TbAd for 4 hours and then fixed. Black bars indicate the mean values and the data are representative of 3 experiments. *P < 0.05, ***P < 0.001, and ****P < 0.0001, by Browne-Forsythe ANOVA followed by Games-Howell’s multiple-comparison test. Scale bars: 10 μm. (E) In 3 experiments, RAW264.7 macrophages were stimulated for 2 hours or 4 hours and then subjected to Western blotting.