Lipid Droplets In Macrophages Research Articles

Identification of a 1-acyl-glycerol-3-phosphate acyltransferase from Mycobacterium tuberculosis, a key enzyme involved in triacylglycerol biosynthesis.

Latent tuberculosis, caused by dormant Mycobacterium tuberculosis (Mtb), poses a threat to global health through the incubation of undiagnosed infections within the community. Dormant Mtb, which is phenotypically tolerant to antibiotics, accumulates triacylglycerol (TAG) utilizing fatty acids obtained from macrophage lipid droplets. TAG is vital to mycobacteria, serving as a cell envelope component and energy reservoir during latency. TAG synthesis occurs by sequential acylation of glycerol-3-phosphate, wherein the second acylation step is catalyzed by acylglycerol-3-phosphate acyltransferase (AGPAT), resulting in the production of phosphatidic acid (PA), a precursor for the synthesis of TAG and various phospholipids. Here, we have characterized a putative acyltransferase of Mtb encoded by Rv3816c. We found that Rv3816c has all four characteristic motifs of AGPAT, exists as a membrane-bound enzyme, and functions as 1-acylglycerol-3-phosphate acyltransferase. The enzyme could transfer the acyl group to acylglycerol-3-phosphate (LPA) from monounsaturated fatty acyl-coenzyme A of chain length 16 or 18 to produce PA. Complementation of Escherichia coli PlsC mutant invivo by Rv3816c confirmed that it functions as AGPAT. Its active site mutants, H43A and D48A, were incapable of transferring the acyl group to LPA invitro and were not able to rescue the growth defect of E. coli PlsC mutant invivo. Identifying Rv3816c as AGPAT and comparing its properties with other AGPAT homologs is not only a step toward understanding the TAG biosynthesis in mycobacteria but has the potential to explore it as a drug target.

A Persistent Tuberculosis Outbreak in the UK Is Characterized by Hydrophobic fadB4-Deficient Mycobacterium tuberculosis That Replicates Rapidly in Macrophages.

The genetic diversity of Mycobacterium tuberculosis can influence disease severity and transmissibility. To better understand how this diversity influences individuals and communities, we phenotyped M. tuberculosis that was causing a persistent outbreak in the East Midlands, United Kingdom. Compared to nonoutbreak isolates, bacilli had higher lipid contents and more hydrophobic cell surfaces. In macrophage infection models, the bacteria increased more rapidly, provoked the enhanced accumulation of macrophage lipid droplets and enhanced the secretion of IL-1β. Natural deletions in fadB4, nrdB, and plcC distinguished the outbreak isolates from other lineage 3 isolates in the region. fadB4 is annotated with a putative role in cell envelope biosynthesis, so the loss of this gene has the potential to alter the interactions of bacteria with immune cells. Reintroduction of fadB4 to the outbreak strain led to a phenotype that more closely resembled those of nonoutbreak strains. The improved understanding of the microbiological characteristics and the corresponding genetic polymorphisms that associate with outbreaks have the potential to inform tuberculosis control. IMPORTANCE Tuberculosis (TB) killed 1.5 million people in 2020 and affects every country. The extent to which the natural genetic diversity of Mycobacterium tuberculosis influences disease manifestation at both the individual and epidemiological levels remains poorly understood. Insights into how pathogen polymorphisms affect patterns of TB have the potential to translate into clinical and public health practice. Two distinct lineage 3 strains isolated from local TB outbreaks, one of which (CH) was rapidly terminated and the other of which (Lro) persistently transmitted for over a decade, provided us with an opportunity to study these issues. We compared genome sequences, microbiological characteristics, and early immune responses that were evoked upon infection. Our results indicate that the natural lack of fadB4 in the Lro strain contributes to its unique features.

Phenylarsine oxide inhibits the accumulation of lipid droplets in macrophages.

Phenylarsine oxide inhibits the accumulation of lipid droplets in macrophages.

Abstract 13967: Macrophages Store Low Density Lipoprotein-Delivered Microbial Small RNAs in Lipid Droplets

Low-density lipoproteins (LDL) are causal factors in atherosclerosis. Inflammation is a driving force for both atherogenesis and advanced lesion progression. LDL stimulates inflammation in the lesion; however, the mechanism by which LDL activates macrophages and promotes atherosclerosis inflammation remains to be fully defined. Recently, we reported that LDL is enriched with microbial small RNAs (msRNA) derived from bacteria in the microbiome and environment. Most importantly, we showed that LDL-delivered msRNA activated toll-like receptor 8 (TLR8) in recipient macrophages and inhibition of this pathway reduced atherosclerosis. Nevertheless, the processes by which macrophages store and catabolize exogenous LDL-delivered msRNA are unknown. We hypothesize that LDL-delivered msRNA is stored in lipid droplets of macrophages . LDL was found to transport approximately 37μg of total RNA per mg of protein, and macrophages were found to take up LDL-msRNAs through an LDL receptor-independent process (pinocytosis). Confocal microscopy and RNA quantification of purified organelles indicate that macrophage lipid droplets (LD) contain RNA and that LDL treatment increases LD-associated RNA. Macrophages transfected with msRNA fluorescence-labeled (msRNA-Cy5) contain signal in both the organic and aqueous phases after Bligh-Dyer lipid extraction, indicating a potential acyl esterification of the msRNA. To evaluate LDL-msRNA delivery to macrophages in vitro , cells were treated with reconstituted LDL (rLDL) loaded with msRNA-Cy5. Utilizing confocal microscopy, rLDL was found to deliver msRNA-Cy5 to macrophages. Ongoing research aims to determine the subcellular processing of LDL-delivered msRNA in macrophages; however, results from microscopy studies support that the endo-lysosome and LD compartments are involved. Moreover, rLDL provides a powerful tool for the study of LDL-msRNA delivery to macrophages by reducing the heterogeneity of lipid cargo that may confound analyses, while enabling the enrichment of specified RNA cargo for cellular delivery. These results support that i) LDL can transfer pro-inflammatory msRNA to macrophages, ii) LDL treatment increases LD-associated RNA, and iii) RNA can be isolated by organic lipid extraction.

Cobalt Chloride Induces Macrophage Foam Cell Formation: A Chemical Hypoxia Model for Anti-Atherosclerotic Drug Screening.

Macrophages would engulf circulating oxidized (ox)- low-density lipoprotein and form lipid droplet-laden foam cells. Macrophage foam cells are considered an important therapeutic target of atherosclerosis. The aim of the study was to investigate a hypoxic foam cell model for anti-atherosclerotic drug screening using the chemical hypoxia-mimicking agent cobalt chloride (CoCl2). The oil red O stating results showed that treatment with CoCl2 could induce lipid accumulation and lead to cell transformation to spindle-shaped and lipid-rich foam cells in RAW 264.7 macrophages. Incubation with 150 μM CoCl2 for 24 h significantly increased the area of intracellular lipid droplets in macrophages, compared with the control group. Our findings indicate that CoCl2-triggered macrophage foam cells should be a potential in vitro hypoxia model for atherosclerosis drug discovery.

Analysis and Quantification of Oxidized Low-Density Lipoprotein-Induced Lipid Droplets in Macrophages Through High-Content Screening: Application for Antiatherogenic Drugs Discovery.

In vascular systems, macrophages can engulf circulating oxidized (ox) low-density lipoprotein (LDL), leading to the accumulation of intracellular lipid droplets, which forms foam cells. Macrophage-derived foam cells are an important therapeutic target for atherosclerosis. However, quantifying intracellular lipid droplets in macrophages is difficult. The purpose of this study was to use high-content screening (HCS) and fluorescence staining to analyze and quantify accumulation of intracellular lipid droplets in macrophages. A murine macrophage cell line RAW 264.7 was seeded in a 96-well black plate and treated with ox-LDL. After fixation, the cells were stained with the lipophilic and nuclear fluorescent dyes briefly. The number and mean fluorescence intensity of the intracellular lipid droplets in the macrophages were detected by an HCS reader. Using HCS to quantify lipid droplets in macrophages could be applied for antiatherogenic drug discovery, and its sensitivity is much higher than that of oil red O staining.

Necrosis Driven Triglyceride Synthesis Primes Macrophages for Inflammation During Mycobacterium tuberculosis Infection.

Pulmonary tuberculosis (TB) exhibits granulomatous inflammation, a site of controlling bacterial dissemination at the cost of host tissue damage. Intrigued by the granuloma type-dependent expression of inflammatory markers in TB, we sought to investigate underlying metabolic changes that drive amplification of inflammation in TB. Here, we show an association of higher inflammation in necrotic granulomas with the presence of triglyceride (TG)-rich foamy macrophages. The conspicuous absence of these macrophages in solid granulomas identified a link between the ensuing pathology and the metabolic programming of foamy macrophages. Consistent with in vivo findings, in vitro infection of macrophages with Mycobacterium tuberculosis (Mtb) led to increase in TG synthesis only under conditions of ~60% necrosis. Genetic and pharmacologic intervention that reduced necrosis prevented this bystander response. We further demonstrate that necrosis independent of Mtb also elicits the same bystander response in human macrophages. We identified a role for the human enzyme involved in TG synthesis, diacylglycerol O-acyltransferase (DGAT1), in this phenomenon. The increased TG levels in necrosis-associated foamy macrophages promoted the pro-inflammatory state of macrophages to infection while silencing expression of diacylglycerol O-acyltransferase (DGAT1) suppressed expression of pro-inflammatory genes. Our data thus invoke a role for storage lipids in the heightened host inflammatory response during infection-associated necrosis. Our data provide a functional role to macrophage lipid droplets in host defense and open new avenues for developing host-directed therapies against TB.

Lipids Affect the Cryptococcus neoformans-Macrophage Interaction and Promote Nonlytic Exocytosis.

Many microbes exploit host cellular lipid droplets during the host-microbe interaction, but this phenomenon has not been extensively studied for fungal pathogens. In this study, we analyzed the role of lipid droplets during the interaction of Cryptococcus neoformans with macrophages in the presence and the absence of exogenous lipids, in particular, oleate. The addition of oleic acid increased the frequency of lipid droplets in both C. neoformans and macrophages. C. neoformans responded to oleic acid supplementation by faster growth inside and outside macrophages. Fungal cells were able to harvest lipids from macrophage lipid droplets. Supplementation of C. neoformans and macrophages with oleic acid significantly increased the rate of nonlytic exocytosis while having no effect on lytic exocytosis. The process for lipid modulation of nonlytic exocytosis was associated with actin changes in macrophages. In summary, C. neoformans harvests lipids from macrophages, and the C. neoformans-macrophage interaction is modulated by exogenous lipids, providing a new tool for studying nonlytic exocytosis.

The mycobacterial Rv1551 glycerol-3-phosphate acyltransferase enhances phospholipid biosynthesis in cell lysates of Escherichia coli

Latent tuberculosis is caused by dormant Mycobacterium tuberculosis (Mtb) that is phenotypically tolerant to antibiotics. Dormant Mtb accumulates triacylglycerol (TAG) utilizing fatty acids obtained from macrophage lipid droplets. The Rv1551 (PlsB1) gene is annotated as a putative glycerol-3-phosphate acyltransferase (GPAT) in the Mtb genome. GPAT catalyzes the first step of the glycerophospholipid biosynthetic pathway that synthesizes the lipid precursors for triacylglycerol biosynthesis. Although triacylglycerol biosynthesis is associated with Mtb dormancy, the functionality of the Rv1551 acyltransferase has not been investigated. We cloned the open reading frame of the Rv1551 acyltransferase and expressed it in Escherichia coli to study its function. We observed that E. coli cell lysates expressing Rv1551 displayed increased synthesis of phosphatidylglycerol, phosphatidylethanolamine and cardiolipin from radiolabeled glycerol-3-phosphate and fatty acyl-coenzyme A precursors. When cultured in medium supplemented with long-chain fatty acids, E. coli expressing Rv1551 exhibited significantly higher viable cell counts during the exponential and stationary phases. These results suggest that Rv1551 displays function as a GPAT by enhancing the synthesis of phospholipids from exogenously provided fatty acids in E. coli cell lysates. This is the first report showing that Rv1551 is a functional GPAT that catalyzes the initial step of glycerophospholipid biosynthesis in the mycobacterial cell.

Phosphoinositide phosphatase Sac3 regulates the cell surface expression of scavenger receptor A and formation of lipid droplets in macrophages

The findings of this study suggest that the phosphoinositide phosphatase Sac3 maintains the protein level of scavenger receptor A (SR-A) and regulates foam cell formation. RAW264.7 macrophages were transfected with short hairpin RNAs that target Sac3. The knockdown decreased the level of the cell surface SR-A and suppressed the acetylated low density lipoprotein-induced foam cell formation. The associated regulator of PIKfyve (ArPIKfyve) is a scaffold protein that protects Sac3 from proteasome-dependent degradation. The knockdown of ArPIKfyve decreased Sac3, cell surface SR-A, and foam cell formation. The knockdown of PIKfyve had no effect on SR-A protein levels. These results suggest that the ArPIKfyve-Sac3 complex regulates SR-A protein levels independently of its effect on PIKfyve activity.

YC-1 induces lipid droplet formation in RAW 264.7 macrophages.

Background3-(5'-Hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) is a potential anticancer drug that may activate soluble guanylyl cyclase (sGC) and increase the level of cyclic guanosine monophosphate (cGMP). The aim of this study was to explore the effects of YC-1 on lipid droplet accumulation and foam cell formation in macrophages.ResultsHuman-oxidized low density lipoprotein (ox-LDL) was used to induce accumulation of lipid droplets in a murine macrophage cell line, RAW 264.7. Oil red O staining showed that treatment with 20 μM YC-1 for 24 h increased the area of intracellular lipid droplets in macrophages. The results of high content screening (HCS) with the AdipoRed™ assay further revealed that YC-1 enhanced ox-LDL-induced foam cell formation. This was evidenced by an increase in the total area of lipid droplets and the mean fluorescence intensity per cell. Inhibition of cGMP-dependent protein kinase (PKG) using KT5823 significantly reduced YC-1-enhanced lipid droplet formation in ox-LDL-induced macrophage foam cells.ConclusionYC-1 induces lipid droplet formation in macrophages, possibly through the sGC/cGMP/PKG signaling pathway. This chemical should be tested with caution in future clinical trials.

Automobile diesel exhaust particles induce lipid droplet formation in macrophages in vitro

Exposure to diesel exhaust particles (DEP) has been associated with adverse cardiopulmonary health effects, which may be related to dysregulation of lipid metabolism and formation of macrophage foam cells. In this study, THP-1 derived macrophages were exposed to an automobile generated DEP (A-DEP) for 24h to study lipid droplet formation and possible mechanisms. The results show that A-DEP did not induce cytotoxicity. The production of reactive oxygen species was only significantly increased after exposure for 3h, but not 24h. Intracellular level of reduced glutathione was increased after 24h exposure. These results combined indicate an adaptive response to oxidative stress. Exposure to A-DEP was associated with significantly increased formation of lipid droplets, as well as changes in lysosomal function, assessed as reduced LysoTracker staining. In conclusion, these results indicated that exposure to A-DEP may induce formation of lipid droplets in macrophages in vitro possibly via lysosomal dysfunction.

Purple Perilla frutescens extracts enhanced cholesterol efflux and inhibited ER stress in macrophages exposed to oxidized LDL (831.8)

There is growing evidence that excess lipids can stimulate endoplasmic reticulum (ER) stress in atherosclerosis. However, the pathophysiological importance and the underlying mechanisms of this phenomenon remain unknown. The current study tested that oxidized LDL promoted ER stress in macrophages, which was blocked by purple Perilla frutescens extracts (PPE). The ER stressor tunicamycin was employed as a positive control. Treating oxidized LDL for 24 h accumulated lipid droplets in macrophages and formed foam cells, evidenced by Oil red O staining. PPE suppressed foam cell formation, but enhanced ATP‐binding cassette (ABC) transporter A1 and G1, cholesterol efflux markers. Oxidized LDL as well as tunicamycin induced the ER stress markers of activating transcription factor 6 and this effect was blocked by 蠅5 μg/ml PPE, indicating that intracellular toxic lipids in macrophages initiated ER stress. PPE also down‐regulated the induction of the ER resident chaperone BiP/Grp78. The LXR ligand TO‐0131917 increased ABCA1 expression that was blunted by the ER stressor. Accordingly, an upstream mechanism that links toxic lipids to ER stress may entail the action of lipid chaperones through inhibition of LXR. Purple Perilla frutescens extract (PPE) is an annual herb of mint family, which has possessed against inflammation and cancer. Therefore, PPE enhanced cholesterol efflux and inhibited ER stress in macrophages exposed to lipotoxic signals associated with atherosclerosis.Grant Funding Source: Supported by Ministry for Food, Agriculture, Forestry and Fisheries and Brain Korea 21 plus

ChemInform Abstract: Phenochalasins, Inhibitors of Lipid Droplet Formation in Mouse Macrophages, Produced by Phomopsis sp. FT-0211.

Phomopsis sp. FT-0211, a soil isolate, was found to produce inhibitors of lipid droplet formation in mouse peritoneal macrophages. Structurally related new compounds designated phenochalasins A and B were isolated from the fermentation broth of the producing strain by solvent extraction, ODS column chromatography and preparative HPLC. Phenochalasin A caused a dose-dependent reduction in the number and size of lipid droplets in macrophages without any cytotoxic effect at least up to 20 microm. On the other hand, phenochalasin B showed inhibition of lipid droplet formation with a severe cytotoxic effect on macrophages.

The role of hypoxia in atherosclerosis

It is important to address the factors involved in the progression of atherosclerosis because advanced atherosclerotic lesions are prone to rupture, leading to disability or death. Hypoxic areas are known to be present in human atherosclerotic lesions, and lesion progression is associated with the formation of lipid-loaded macrophages and increased local inflammation. Here we summarize the role of hypoxia in the development of advanced atherosclerotic lesions by promoting lipid accumulation, inflammation, ATP depletion, and angiogenesis. A recent study clearly demonstrated the presence of hypoxia in macrophage-rich regions of advanced human carotid atherosclerotic lesions. We showed that hypoxia increases the formation of lipid droplets in macrophages and promotes increased secretion of inflammatory mediators, and recent evidence indicates that lipid droplets may play a role in mediating the inflammatory response. Hypoxia also promotes lesion progression by exacerbating ATP depletion and lactate accumulation, and the presence of hypoxia in human carotid atherosclerotic lesions correlates with angiogenesis. Recent studies indicate that hypoxia may play a key role in the progression to advanced lesions by promoting lipid accumulation, increased inflammation, ATP depletion, and angiogenesis. Further understanding of the effects of hypoxia in atherosclerotic lesions could indicate potential therapeutic targets.

Inhibition of Lipid Droplet Accumulation in Mouse Macrophages by Stemphone Derivatives

From a study on the biological activity of fungal stemphones and their derivatives, five derivatives having an O-alkyl moiety at C-11 of stemphone C were found to inhibit lipid droplet accumulation in macrophages without any cytotoxic effect. Among the derivatives, those having O-isopropyl and O-isobutyl were the most potent inhibitors by blocking the synthesis of both cholesteryl ester (CE) and triacylglycerol (TG), the main constituents of lipid droplets in macrophages.

Ocimum basilicum ethanolic extract decreases cholesterol synthesis and lipid accumulation in human macrophages

Macrophage lipid accumulation induced by low density lipoproteins (LDL) plays a pivotal role in atherosclerotic plaque development. Previous work showed that Ocimum basilicum extract, used as hypocholesterolemic agent by traditional medicine in Morocco, has hypolipidemic activity in rat acute hyperlipimidemia. This study investigated the effects of ethanolic extract of O. basilicum on lipid accumulation in human macrophages. As modification of LDL increase atherogenicity of the particles we evaluated the effects of the extract on LDL oxidation. The extract caused a dose-related increase of LDL-resistance to Cu(2+)-induced oxidation. Furthermore, at the dose of 60 microg/ml, significantly decreases the accumulation of macrophage lipid droplets induced by modified LDL evaluated as by red-oil staining. Cholesterol esterification and triacylglycerol synthesis in the cells were not affected. Macrophage treatment with 60 microg/ml, but not 20 microg/ml, of the extract reduced newly synthesized unesterified cholesterol by about 60% and decreased scavenger receptors activity by about 20-30%, evaluated by the internalization of cholesterol carried by [(3)H]CE-aggregated-LDL. The results suggest that O. basilicum ethanolic extract has the capability to reduce foam cell formation through the reduction of cholesterol synthesis and the modulation of the activity of surface scavenger receptors.

Discovery and Combinatorial Synthesis of Fungal Metabolites Beauveriolides, Novel Antiatherosclerotic Agents

For discovery of a new type of antiatherosclerotic agents, a cell-based assay of lipid droplet accumulation using primary mouse peritoneal macrophages was conducted as a model of macrophage-derived foam cell accumulation, which occurs in the early stage of atherosclerogenesis. During the screening of microbial metabolites for inhibitors of lipid droplet accumulation, 13-membered cyclodepsipeptides, known beauveriolide I and new beauveriolide III, were isolated from the culture broth of fungal Beauveria sp. FO-6979, a soil isolate, by solvent extraction, ODS column chromatography, silica gel column chromatography, and preparative HPLC. The structure including the absolute stereochemistry of beauveriolide III was elucidated as cyclo-[(3 S,4 S)-3-hydroxy-4-methyloctanoyl- l-phenylalanyl- l-alanyl- d-alloisoleucyl] by spectral analyses, amino acid analyses, and synthetic methods. Furthermore, the absolute stereochemistry was confirmed by the total synthesis of beauveriolides. Study on the mechanism of action revealed that beauveriolides inhibited macrophage acyl-CoA:cholesterol acyltransferase (ACAT) activity to block the synthesis of cholesteryl ester (CE), leading to a reduction of lipid droplets in macrophages. There are two ACAT isozymes in mammals, ACAT1 and ACAT2. ACAT1 is ubiquitously expressed in most tissues and cells including macrophages, while ACAT2 is expressed predominantly in the liver (hepatocytes) and the intestine (enterocytes). Interestingly, beauveriolides inhibited both ACAT1 and ACAT2 to a similar extent in an enzyme assay that utilized microsomes but inhibited ACAT1 selectively in intact cell-based assays. Beauveriolides proved orally active in both low-density lipoprotein receptor and apolipoprotein E knockout mice, reducing the atheroma lesion of heart and aorta without any side effects such as diarrhea or cytotoxicity to adrenal tissues as observed for many synthetic ACAT inhibitors. To obtain more potent inhibitors, a focused library of beauveriolide analogues was prepared by combinatorial chemistry in which solid-phase assembly of linear depsipeptides was carried out using a 2-chlorotrityl linker, followed by solution-phase cyclization, yielding 104 beauveriolide analogues. Among them, diphenyl derivatives were found to show 10 times more potent inhibition of CE synthesis in macrophages than beauveriolide III. Furthermore, most analogues showed selective ACAT1 inhibition or inhibition of both ACAT1 and ACAT2, but interestingly certain analogues gave selective ACAT2 inhibition. These data indicated that subtle structural differences of the inhibitors could discriminate the active sites of the ACAT1 and ACAT2 isozymes. Efforts of further analogue synthesis would make it possible to obtain highly selective ACAT1/ACAT2 inhibitors.

Molecular Target of Decursins in the Inhibition of Lipid Droplet Accumulation in Macrophages

During screening for inhibitors of lipid droplet accumulation in mouse peritoneal macrophages, two coumarins identified as decursin and decursinol angelate were isolated from the roots of Angelicae gigantis. The cellular molecular target of these inhibitors in macrophages was studied. Decursin and decursinol angelate inhibited cholesteryl ester (CE) synthesis with IC50 values of 9.7 and 10.1 microM, respectively, whereas they enhanced triacylglycerol (TG) synthesis. Lysosomal metabolism of cholesterol to CE was inhibited by the compounds, indicating that the site of inhibition is one of the steps between the exiting of cholesterol from the lysosomes and CE synthesis in the endoplasmic reticulum. Therefore, acyl-CoA:cholesterol acyltransferase (ACAT) activity in the microsomal fractions prepared from mouse macrophages was studied, and the results showed inhibition of this activity by decursin and decursinol angelate with IC50 values of 43 and 22 microM, respectively. Thus, it was concluded that the compounds inhibit macrophage ACAT activity to decrease CE synthesis, leading to a reduction of lipid droplets in macrophages.

Mycobactin-mediated iron acquisition within macrophages

Restricting the availability of iron is an important strategy for defense against bacterial infection. Mycobacterium tuberculosis survives within the phagosomes of macrophages; consequently, iron acquisition is particularly difficult for M. tuberculosis, because the phagosomal membrane is an additional barrier for its iron access. However, little is known about the iron transport and acquisition pathways adapted by this microbe in vivo. Extracellular iron sources are usually mobilized by hydrophilic siderophores. Here, we describe direct evidence that mycobactins, the lipophilic siderophores of mycobacteria, efficiently extract intracellular macrophage iron. The metal-free siderophore is diffusely associated with the macrophage membrane, ready for iron chelation. Notably, the mycobactin-metal complex accumulates with high selectivity in macrophage lipid droplets, intracellular domains for lipid storage and sorting. In our experiments, these mycobactin-targeted lipid droplets were found in direct contact with phagosomes, poised for iron delivery. The existence of this previously undescribed iron acquisition pathway indicates that mycobacteria have taken advantage of endogenous macrophage mechanisms for iron mobilization and lipid sorting for iron acquisition during infection. The pathway could represent a new target for the control of mycobacterial infection.