Breaking fat! How mycobacteria and other intracellular pathogens manipulate host lipid droplets

Tuberculosis (Tb) is a lung infection caused by Mycobacterium tuberculosis (Mtb). With one third of the world population latently infected, it represents the most prevalent bacterial infectious diseases worldwide. Typically, persistence is linked to so-called "dormant" slow-growing bacteria, which have a low metabolic rate and a reduced response to antibiotic treatments. However, dormant bacteria regain growth and virulence when the immune system is weakened, leading again to the active form of the disease. Fatty acids (FAs) released from host triacylglycerols (TAGs) and sterols are proposed to serve as sole carbon sources during infection. The metabolism of FAs requires beta-oxidation as well as gluconeogenesis and the glyoxylate shunt. Interestingly, the Mtb genome encodes more than hundred proteins involved in the five reactions of beta-oxidation, clearly demonstrating the importance of lipids as energy source. FAs have also been proposed to play a role during resuscitation, the resumption of replicative activities from dormancy. Lipid droplets (LDs) are energy and carbon reservoirs and have been described in all domains. TAGs and sterol esters (SEs) are stored in their hydrophobic core, surrounded by a phospholipid monolayer. Importantly, host LDs have been described as crucial for several intracellular bacterial pathogens and viruses and specifically translocate to the pathogen-containing vacuole (PVC) during mycobacteria infection. FAs released from host LDs are used by the pathogen as energy source and as building blocks for membrane synthesis. Despite their essential role, the mechanisms by which pathogenic mycobacteria induce the cellular redistribution of LDs and gain access to the stored lipids are still poorly understood. This review describes recent evidence about the dual interaction of mycobacteria with host LDs and membrane phospholipids and integrates them in a broader view of the underlying cellular processes manipulated by various intracellular pathogens to gain access to host lipids

Bacteria accumulate ILIs in the <i>dgat</i> KO mutants.

<p>Cells of (A) wild type, (B) <i>dgat1&2</i> DKO, (C) <i>dgat1</i> KO and (D) <i>dgat2</i> KO and were infected with mCherry-expressing <i>M</i>. <i>marinum</i>. At 3 hpi bacteria are lean (asterisks) whereas at 21 hpi bacteria harbour many ILIs in all cell types (arrows). Cells were fed with FAs prior to infection. At the indicated time points samples were fixed with PFA/picric acid, and MCVs visualized by staining for p80. Bacterial ILIs were stained with Bodipy 493/503. Scale bar, 5 μm.</p

Excess FAs leads to ER-membrane proliferation in <i>dgat1&2</i> DKO cells.

<p>A. LDs are formed in wild type and <i>dgat2</i>, but not in <i>dgat1</i> KO cells. Instead of LDs, massive Bodipy-positive structures were observed in the <i>dgat1&</i>2 DKO (arrowheads). FAs were added to the culture medium and a time-lapse movie was recorded with 10 minute frame intervals. Shown are maximum z-projections of 6 sections 1.5 μm apart taken after 180 min. Scale bars, 10 μm. B. The neutral lipid structures in the <i>dgat1&2</i> DKO (arrowheads) are not of endosomal nature. Wild type or <i>dgat1&2</i> DKO cells expressing AmtA-mCherry were incubated with FAs and a time-lapse movie with 5 min frame intervals was recorded. Shown is a representative image taken after 70 min. Scale bar, 10 μm; Zoom 5 μm. C. The neutral lipid structures in the <i>dgat1&2</i> DKO are formed by ER-membranes. <i>Dictyostelium</i> was fed 3 hours with FAs before fixation with glutaraldehyde. Asterisks label mitochondria that have been seen close to the ER-membrane-proliferations. Arrowheads point to long ER-strands. D. GFP-HDEL accumulates in the ER-membrane proliferations in the <i>dgat1&2</i> DKO. Images of <i>Dictyostelium</i> expressing GFP-HDEL were taken under normal conditions (-FAs) and after 3 hrs incubation with FAs (+FAs). Shown are maximum z-projections. Arrowheads point to ER-membrane proliferations. Scale bar, 5 μm.</p

Dynamics of Dgat2-GFP-LDs during infection.

<p>A. Dgat2-GFP-positive LDs attach to the bacteria when they are exposed to the cytosol (3 and 21 hpi). At a later infection stage, Dgat2-GFP completely surrounds a cytosolic bacterium (45 hpi). White arrows point to bacteria that are inside the p80-positive MCV. Asterisks label bacteria that are partially exposed to the cytosol. The yellow arrow points to a cytosolic bacterium that is completely surrounded by Dgat2-GFP. Scale bars, 10 μm; Zoom, 2 μm. B. Dgat2-GFP-labelled LDs stick to an intracellular mCherry-expressing mycobacterium. A time-lapse movie was recorded at 24 hpi with 5 sec frame intervals. Arrows point to LDs aggregated at the surface of the bacterium. Scale bar, 3 μm. C. Coalescence of a Dgat2-GFP-positive LD with an mCherry-expressing mycobacterium surrounded by Dgat2-GFP (arrows). A time-lapse movie was recorded at 42 hpi with 2 sec frame intervals. Arrows point to an LD that coalescences onto the bacterium. Asterisks label the same LD in the Zoom. Scale bar, 5 μm; Zoom, 2μm. D. Dgat2-GFP surrounds a cytosolic wild type <i>M</i>. <i>marinum</i> negative for AmtA-mCherry. No co-localization was observed with <i>M</i>. <i>marinum</i> ΔRD1. Arrows label intracellular bacteria. Samples were taken at 45 hpi and bacteria stained with Vybrant Ruby. Scale bars, 5 μm. E. Quantification of D. While clusters of Dgat2-GFP-labelled LDs were frequently observed close to wild type <i>M</i>. <i>marinum</i>, only a few LDs associated with the RD1 mutant. Dgat2-GFP-positive bacteria were counted in maximum z-projections. Statistical significance was calculated with an unpaired t-test (* p<0.05, ** p<0.01). Bars represent the mean and SD of two independent experiments. For all the experiments presented in Fig 2 <i>Dictyostelium</i> was fed with FA prior to infection.</p

Localization of Dgat1- and Dgat2-GFP during infection of <i>Dictyostelium</i> with <i>M</i>. <i>marinum</i>.

<p>A. LDs with their typical morphology are formed in cells overexpressing Dgat2-GFP even without FA supplementation. Cells that were treated with and without FAs were fixed and processed for EM. Arrowheads label LDs. Scale bar, 1 μm. B.Dynamics of RFP-Plin and Dgat2-GFP in <i>Dictyostelium</i> treated with exogenous FAs. In axenic medium without FA supplementation, RFP-Plin is cytosolic whereas Dgat2-GFP is located on LDs. Upon treatment with exogenous FAs, RFP-Plin translocates to the surface of LDs where it co-localizes with Dgat2-GFP. <i>Dictyostelium</i> expressing both RFP-Plin and Dgat2-GFP was cultured in medium supplemented with FAs and a time-lapse movie was recorded with 5 min frame intervals. Shown is the maximum z-projection of 6 sections spaced 1.5 μm apart. Scale bars, 10 μm. C. Dgat2-GFP-positive LDs cluster at bacterial poles. <i>Dictyostelium</i> expressing Dgat2-GFP was infected with mCherry-expressing mycobacteria. Samples for live imaging were taken at 3, 24 and 45 hpi. <i>Dictyostelium</i> was fed with FA prior to infection. Arrows point to LD clusters. Scale bars, 10 μm. D. Quantification of C. The number of <i>Dictyostelium</i> cells harbouring bacteria that co-localize with LDs aggregates was stable over the time course of infection. Bacteria surrounded by Dgat2-GFP were only observed at late stages, as judged by quantification using z-projections. The statistical significance was calculated with an unpaired t-test (* p<0.05, ** p<0.01). Bars represent the mean and SD of two independent experiments. E. Dgat1-GFP is enriched at the ER and at the perinuclear ER during infection of <i>Dictyostelium</i> with mCherry-expressing <i>M</i>. <i>marinum</i>. Samples for live imaging were taken at 3, 24 and 45 hpi. <i>Dictyostelium</i> was fed with FA prior to infection. Scale bars, 10 μm.</p

Lipids derived from host phospholipids are transferred to the MCV.

<p>A. and B. Topfluor-LysoPC-tagged host lipids first label the membrane of the MCV, accumulate inside the compartment and are finally found inside the bacteria. Phospholipids of wild type (A) and <i>dgat1&2</i> DKO (B) were labelled with Topfluor-LysoPC as described in materials and methods. Cells were infected with mCherry-expressing mycobacteria. Images were taken at the indicated time points. Scale bar, 5 μm; Zoom, 2μm.</p

<i>Mycobacterium marinum</i> Degrades Both Triacylglycerols and Phospholipids from Its <i>Dictyostelium</i> Host to Synthesise Its Own Triacylglycerols and Generate Lipid Inclusions

<div><p>During a tuberculosis infection and inside lipid-laden foamy macrophages, fatty acids (FAs) and sterols are the major energy and carbon source for <i>Mycobacterium tuberculosis</i>. Mycobacteria can be found both inside a vacuole and the cytosol, but how this impacts their access to lipids is not well appreciated. Lipid droplets (LDs) store FAs in form of triacylglycerols (TAGs) and are energy reservoirs of prokaryotes and eukaryotes. Using the <i>Dictyostelium discoideum</i>/<i>Mycobacterium marinum</i> infection model we showed that <i>M</i>. <i>marinum</i> accesses host LDs to build up its own intracytosolic lipid inclusions (ILIs). Here, we show that host LDs aggregate at regions of the bacteria that become exposed to the cytosol, and appear to coalesce on their hydrophobic surface leading to a transfer of diacylglycerol O-acyltransferase 2 (Dgat2)-GFP onto the bacteria. <i>Dictyostelium</i> knockout mutants for both Dgat enzymes are unable to generate LDs. Instead, the excess of exogenous FAs is esterified predominantly into phospholipids, inducing uncontrolled proliferation of the endoplasmic reticulum (ER). Strikingly, in absence of host LDs, <i>M</i>. <i>marinum</i> alternatively exploits these phospholipids, resulting in rapid reversal of ER-proliferation. In addition, the bacteria are unable to restrict their acquisition of lipids from the <i>dgat1&2</i> double knockout leading to vast accumulation of ILIs. Recent data indicate that the presence of ILIs is one of the characteristics of dormant mycobacteria. During <i>Dictyostelium</i> infection, ILI formation in <i>M</i>. <i>marinum</i> is not accompanied by a significant change in intracellular growth and a reduction in metabolic activity, thus providing evidence that storage of neutral lipids does not necessarily induce dormancy.</p></div

LD and ILI dynamics during infection.

<p>A.—D. Bacteria accumulate ILIs in the wild type and the <i>dgat1&2</i> DKO. E. and F. Cytosolic bacteria harbour more ILIs than bacteria inside an MCV. G. Extracellular bacteria are lean. H. and I. LDs translocate to cytosolic bacteria J.—L. LDs are recruited to the vicinity of the MCV early in infection. <i>Dictyostelium</i> wild type (A and C) and <i>dgat1&2</i> DKO cells (B and D) or cells expressing Dgat2-GFP (E-L) were infected with unlabelled <i>M</i>. <i>marinum</i> wild type. At the indicated time points, samples were fixed and further processed for EM. Arrowheads label LDs. a: autophagosomes, c: cytosolic bacteria. Scale bars, 1 μm. M. Quantification of the ILI surface per bacterium as a fraction of the total bacterium surface. For each condition ILIs of 10 to 13 bacteria were quantified using FIJI. The statistical significance was calculated with an unpaired t-test (*** p<0.001, **** p<0.0001). For all the experiments presented in Fig 4 <i>Dictyostelium</i> was fed with FA prior to infection.</p

<i>M</i>. <i>marinum</i> uses host phospholipids to build up TAGs.

<p>A. Topfluor- and Bodipy-labelled lipid standards show a different migration behaviour than unlabelled standards. The migration of BodipyC12, Topfluor-FFAs (C11), Topfluor-TAGs (TAGs*; 18:1, 18:1, C11) and Topfluor-LysoPC was compared to unlabelled standards for TAGs (Triolein) and FAs (oleic acid (OA)). Additionally, the migration of PDIM, phenolic glycolipids (PGL), phosphatidic acid (PA), cholesterol esters (CE), PE, PS and PC was monitored by using the respective standards. B. Scheme showing how lipids of host and pathogen were separated prior to extraction with chloroform/methanol. C. and D. <i>M</i>. <i>marinum</i> incorporates host-derived lipids into TAGs. Wild type and <i>dgat1&2</i> DKO cells were labelled with Topfluor-LysoPC (C) and BodipyC12 (D) as described in materials and methods. Cells were infected with unlabelled <i>M</i>. <i>marinum</i>. At the indicated time points samples were taken for lipid extraction. To separate host (C1 and D1) and bacterial lipids (C2 and D2), cells were lysed with 0.05% TritonX-100 and the lipids of the pellet (bacterial lipids) were extracted with chloroform:methanol (1:2) for 24 hrs. The lipids of the supernatant (host lipids) were directly extracted with chloroform:methanol (1:2). Bands were identified by comparison with lipid standards. PPLs: phospholipids.</p