Mycobacterium tuberculosis releases an antacid that remodels phagosomes.

Mycobacterium tuberculosis (Mtb) is the world's most deadly pathogen. Unlike less virulent mycobacteria, Mtb produces 1-tuberculosinyladenosine (1-TbAd), an unusual terpene nucleoside of unknown function. In the present study 1-TbAd has been shown to be a naturally evolved phagolysosome disruptor. 1-TbAd is highly prevalent among patient-derived Mtb strains, where it is among the most abundant lipids produced. Synthesis of TbAd analogs and their testing in cells demonstrate that their biological action is dependent on lipid linkage to the 1-position of adenosine, which creates a strong conjugate base. Furthermore, C20 lipid moieties confer passage through membranes. 1-TbAd selectively accumulates in acidic compartments, where it neutralizes the pH and swells lysosomes, obliterating their multilamellar structure. During macrophage infection, a 1-TbAd biosynthesis gene (Rv3378c) confers marked phagosomal swelling and intraphagosomal inclusions, demonstrating an essential role in regulating the Mtb cellular microenvironment. Although macrophages kill intracellular bacteria through phagosome acidification, Mtb coats itself abundantly with antacid.

Mycobacterial Metabolic Syndrome: LprG and Rv1410 Regulate Triacylglyceride Levels, Growth Rate and Virulence in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) mutants lacking rv1411c, which encodes the lipoprotein LprG, and rv1410c, which encodes a putative efflux pump, are dramatically attenuated for growth in mice. Here we show that loss of LprG-Rv1410 in Mtb leads to intracellular triacylglyceride (TAG) accumulation, and overexpression of the locus increases the levels of TAG in the culture medium, demonstrating a role of this locus in TAG transport. LprG binds TAG within a large hydrophobic cleft and is sufficient to transfer TAG from donor to acceptor membranes. Further, LprG-Rv1410 is critical for broadly regulating bacterial growth and metabolism in vitro during carbon restriction and in vivo during infection of mice. The growth defect in mice is due to disrupted bacterial metabolism and occurs independently of key immune regulators. The in vivo essentiality of this locus suggests that this export system and other regulators of metabolism should be considered as targets for novel therapeutics.

Rifampin Resistance Mutations Are Associated with Broad Chemical Remodeling of Mycobacterium tuberculosis.

Global control of tuberculosis has become increasingly complicated with the emergence of multidrug-resistant strains of Mycobacterium tuberculosis First-line treatments are anchored by two antibiotics, rifampin and isoniazid. Most rifampin resistance occurs through the acquisition of missense mutations in the rifampin resistance-determining region, an 81-base pair region encoding the rifampin binding site on the ß subunit of RNA polymerase (rpoB). Although these mutations confer a survival advantage in the presence of rifampin, they may alter the normal process of transcription, thereby imposing significant fitness costs. Because the downstream biochemical consequences of the rpoB mutations are unknown, we used an organism-wide screen to identify the number and types of lipids changed after rpoB mutation. A new mass spectrometry-based profiling platform systematically compared ∼10,000 cell wall lipids in a panel of rifampin-resistant mutants within two genetically distinct strains, CDC1551and W-Beijing. This unbiased lipidomic survey detected quantitative alterations (>2-fold, p < 0.05) in more than 100 lipids in each mutant. By focusing on molecular events that change among most mutants and in both genetic backgrounds, we found that rifampin resistance mutations lead to altered concentrations of mycobactin siderophores and acylated sulfoglycolipids. These findings validate a new organism-wide lipidomic analysis platform for drug-resistant mycobacteria and provide direct evidence for characteristic remodeling of cell wall lipids in rifampin-resistant strains of M. tuberculosis The specific links between rifampin resistance and named lipid factors provide diagnostic and therapeutic targets that may be exploited to address the problem of drug resistance.

Lipidomic analysis links mycobactin synthase K to iron uptake and virulence in M. tuberculosis.

The prolonged survival of Mycobacterium tuberculosis (M. tb) in the host fundamentally depends on scavenging essential nutrients from host sources. M. tb scavenges non-heme iron using mycobactin and carboxymycobactin siderophores, synthesized by mycobactin synthases (Mbt). Although a general mechanism for mycobactin biosynthesis has been proposed, the biological functions of individual mbt genes remain largely untested. Through targeted gene deletion and global lipidomic profiling of intact bacteria, we identify the essential biochemical functions of two mycobactin synthases, MbtK and MbtN, in siderophore biosynthesis and their effects on bacterial growth in vitro and in vivo. The deletion mutant, ΔmbtN, produces only saturated mycobactin and carboxymycobactin, demonstrating an essential function of MbtN as the mycobactin dehydrogenase, which affects antigenicity but not iron uptake or M. tb growth. In contrast, deletion of mbtK ablated all known forms of mycobactin and its deoxy precursors, defining MbtK as the essential acyl transferase. The mbtK mutant showed markedly reduced iron scavenging and growth in vitro. Further, ΔmbtK was attenuated for growth in mice, demonstrating a non-redundant role of hydroxamate siderophores in virulence, even when other M. tb iron scavenging mechanisms are operative. The unbiased lipidomic approach also revealed unexpected consequences of perturbing mycobactin biosynthesis, including extreme depletion of mycobacterial phospholipids. Thus, lipidomic profiling highlights connections among iron acquisition, phospholipid homeostasis, and virulence, and identifies MbtK as a lynchpin at the crossroads of these phenotypes.

Molecular profiling of Mycobacterium tuberculosis identifies tuberculosinyl nucleoside products of the virulence-associated enzyme Rv3378c.

To identify lipids with roles in tuberculosis disease, we systematically compared the lipid content of virulent Mycobacterium tuberculosis with the attenuated vaccine strain Mycobacterium bovis bacillus Calmette-Guérin. Comparative lipidomics analysis identified more than 1,000 molecular differences, including a previously unknown, Mycobacterium tuberculosis-specific lipid that is composed of a diterpene unit linked to adenosine. We established the complete structure of the natural product as 1-tuberculosinyladenosine (1-TbAd) using mass spectrometry and NMR spectroscopy. A screen for 1-TbAd mutants, complementation studies, and gene transfer identified Rv3378c as necessary for 1-TbAd biosynthesis. Whereas Rv3378c was previously thought to function as a phosphatase, these studies establish its role as a tuberculosinyl transferase and suggest a revised biosynthetic pathway for the sequential action of Rv3377c-Rv3378c. In agreement with this model, recombinant Rv3378c protein produced 1-TbAd, and its crystal structure revealed a cis-prenyl transferase fold with hydrophobic residues for isoprenoid binding and a second binding pocket suitable for the nucleoside substrate. The dual-substrate pocket distinguishes Rv3378c from classical cis-prenyl transferases, providing a unique model for the prenylation of diverse metabolites. Terpene nucleosides are rare in nature, and 1-TbAd is known only in Mycobacterium tuberculosis. Thus, this intersection of nucleoside and terpene pathways likely arose late in the evolution of the Mycobacterium tuberculosis complex; 1-TbAd serves as an abundant chemical marker of Mycobacterium tuberculosis, and the extracellular export of this amphipathic molecule likely accounts for the known virulence-promoting effects of the Rv3378c enzyme.

Biomarkers for Tuberculosis Based on Secreted, Species-Specific, Bacterial Small Molecules.

Improved biomarkers are needed for tuberculosis. To develop tests based on products secreted by tubercle bacilli that are strictly associated with viability, we evaluated 3 bacterial-derived, species-specific, small molecules as biomarkers: 2 mycobactin siderophores and tuberculosinyladenosine. Using liquid chromatography-tandem mass spectrometry, we demonstrated the presence of 1 or both mycobactins and/or tuberculosinyladenosine in serum and whole lung tissues from infected mice and sputum, cerebrospinal fluid (CSF), or lymph nodes from infected patients but not uninfected controls. Detection of the target molecules distinguished host infection status in 100% of mice with both serum and lung as the target sample. In human subjects, we evaluated detection of the bacterial small molecules (BSMs) in multiple body compartments in 3 patient cohorts corresponding to different forms of tuberculosis. We detected at least 1 of the 3 molecules in 90%, 71%, and 40% of tuberculosis patients' sputum, CSF, and lymph node samples, respectively. In paucibacillary forms of human tuberculosis, which are difficult to diagnose even with culture, detection of 1 or more BSM was rapid and compared favorably to polymerase chain reaction-based detection. Secreted BSMs, detectable in serum, warrant further investigation as a means for diagnosis and therapeutic monitoring in patients with tuberculosis.

Lipidomic discovery of deoxysiderophores reveals a revised mycobactin biosynthesis pathway in Mycobacterium tuberculosis.

To measure molecular changes underlying pathogen adaptation, we generated a searchable dataset of more than 12,000 mass spectrometry events, corresponding to lipids and small molecules that constitute a lipidome for Mycobacterium tuberculosis. Iron is essential for M. tuberculosis survival, and the organism imports this metal using mycobactin and carboxymycobactin siderophores. Detection of an unexpected siderophore variant and deletions of genes for iron scavenging has led to a revised mycobactin biosynthesis model. An organism-wide search of the M. tuberculosis database for hypothetical compounds predicted by this model led to the discovery of two families of previously unknown lipids, designated monodeoxymycobactins and monodeoxycarboxymycobactins. These molecules suggest a revised biosynthetic model that alters the substrates and order of action of enzymes through the mycobactin biosynthetic pathway. We tested this model genetically by solving M. tuberculosis lipidomes after deletion of the iron-dependent regulator (ideR), mycobactin synthase B (mbtB), or mycobactin synthase G (mbtG). These studies show that deoxymycobactins are actively regulated during iron starvation, and also define essential roles of MbtG in converting deoxymycobactins to mycobactin and in promoting M. tuberculosis growth. Thus, lipidomics is an efficient discovery tool that informs genetic relationships, leading to a revised general model for the biosynthesis of these virulence-conferring siderophores.

Discovery of deoxyceramides and diacylglycerols as CD1b scaffold lipids among diverse groove-blocking lipids of the human CD1 system.

Unlike the dominant role of one class II invariant chain peptide (CLIP) in blocking MHC class II, comparative lipidomics analysis shows that human cluster of differentiation (CD) proteins CD1a, CD1b, CD1c, and CD1d bind lipids corresponding to hundreds of diverse accurate mass retention time values. Although most ions were observed in association with several CD1 proteins, ligands binding selectively to one CD1 isoform allowed the study of how differing antigen-binding grooves influence lipid capture. Although the CD1b groove is distinguished by its unusually large volume (2,200 Å(3)) and the T' tunnel, the average mass of compounds eluted from CD1b was similar to that of lipids from CD1 proteins with smaller grooves. Elution of small ligands from the large CD1b groove might be explained if two small lipids bind simultaneously in the groove. Crystal structures indicate that all CD1 proteins can capture one antigen with its hydrophilic head group exposed for T-cell recognition, but CD1b structures show scaffold lipids seated below the antigen. We found that ligands selectively associated with CD1b lacked the hydrophilic head group that is generally needed for antigen recognition but interferes with scaffold function. Furthermore, we identified the scaffolds as deoxyceramides and diacylglycerols and directly demonstrate a function in augmenting presentation of a small glycolipid antigen to T cells. Thus, unlike MHC class II, CD1 proteins capture highly diverse ligands in the secretory pathway. CD1b has a mechanism for presenting either two small or one large lipid, allowing presentation of antigens with an unusually broad range of chain lengths.

Deciphering the role of CD1e protein in mycobacterial phosphatidyl-myo-inositol mannosides (PIM) processing for presentation by CD1b to T lymphocytes.

Lipids are important antigens that induce T cell-mediated specific immune responses. They are presented to T lymphocytes by a specific class of MHC-I like proteins, termed CD1. The majority of the described CD1-presented mycobacterial antigens are presented by the CD1b isoform. We previously demonstrated that the stimulation of CD1b-restricted T cells by the hexamannosylated phosphatidyl-myo-inositol (PIM(6)), a family of mycobacterial antigens, requires a prior partial digestion of the antigen oligomannoside moiety by α-mannosidase and that CD1e is an accessory protein absolutely required for the generation of the lipid immunogenic form. Here, we show that CD1e behaves as a lipid transfer protein influencing lipid immunoediting and membrane transfer of PIM lipids. CD1e selectively assists the α-mannosidase-dependent digestion of PIM(6) species according to their degree of acylation. Moreover, CD1e transfers only diacylated PIM from donor to acceptor liposomes and also from membranes to CD1b. This study provides new insight into the molecular mechanisms by which CD1e contributes to lipid immunoediting and CD1-restricted presentation to T cells.

The polyketide Pks1 contributes to biofilm formation in Mycobacterium tuberculosis.

Infections caused by biofilms are abundant and highly persistent, displaying phenotypic resistance to high concentrations of antimicrobials and modulating host immune systems. Tuberculosis (TB), caused by Mycobacterium tuberculosis, shares these qualities with biofilm infections. To identify genetic determinants of biofilm formation in M. tuberculosis, we performed a small-scale transposon screen using an in vitro pellicle biofilm assay. We identified five M. tuberculosis mutants that were reproducibly attenuated for biofilm production relative to that of the parent strain H37Rv. One of the most attenuated mutants is interrupted in pks1, a polyketide synthase gene. When fused with pks15, as in some M. tuberculosis isolates, pks1 contributes to synthesis of the immunomodulatory phenolic glycolipids (PGLs). However, in strains such as H37Rv with split pks15 and pks1 loci, PGL is not produced and pks1 has no previously defined role. We showed that pks1 complementation restores biofilm production independently of the known role of pks1 in PGL synthesis. We also assessed the relationship among biofilm formation, the pks15/1 genotype, and M. tuberculosis phylogeography. A global survey of M. tuberculosis clinical isolates revealed surprising sequence variability in the pks15/1 locus and substantial variation in biofilm phenotypes. Our studies identify novel M. tuberculosis genes that contribute to biofilm production, including pks1. In addition, we find that the ability to make pellicle biofilms is common among M. tuberculosis isolates from throughout the world, suggesting that this trait is relevant to TB propagation or persistence.

A Mycobacterium tuberculosis fingerprint in human breath allows tuberculosis detection.

An estimated one-third of tuberculosis (TB) cases go undiagnosed or unreported. Sputum samples, widely used for TB diagnosis, are inefficient at detecting infection in children and paucibacillary patients. Indeed, developing point-of-care biomarker-based diagnostics that are not sputum-based is a major priority for the WHO. Here, in a proof-of-concept study, we tested whether pulmonary TB can be detected by analyzing patient exhaled breath condensate (EBC) samples. We find that the presence of Mycobacterium tuberculosis (Mtb)-specific lipids, lipoarabinomannan lipoglycan, and proteins in EBCs can efficiently differentiate baseline TB patients from controls. We used EBCs to track the longitudinal effects of antibiotic treatment in pediatric TB patients. In addition, Mtb lipoarabinomannan and lipids were structurally distinct in EBCs compared to ex vivo cultured bacteria, revealing specific metabolic and biochemical states of Mtb in the human lung. This provides essential information for the rational development or improvement of diagnostic antibodies, vaccines and therapeutic drugs. Our data collectively indicate that EBC analysis can potentially facilitate clinical diagnosis of TB across patient populations and monitor treatment efficacy. This affordable, rapid and non-invasive approach seems superior to sputum assays and has the potential to be implemented at point-of-care.

Deciphering sulfoglycolipids of Mycobacterium tuberculosis.

For 4 decades, in vivo and in vitro studies have suggested that sulfoglycolipids (SGLs) play a role in the virulence or pathogenesis of the tubercle bacilli. However, the SGL structure and biosynthesis pathway remain only partially elucidated. Using the modern tools of structural analysis, including MALDI-time-of-flight MS, MS/MS, and two-dimensional NMR, we reevaluated the structure of the different SGL acyl (di-, tri-, and tetra-acylated) forms of the reference strain Mycobacterium tuberculosis H37Rv, as well as those produced by the mmpL8 knockout strains previously described to intracellularly accumulate di-acylated SGL. We report here the identification of new acyl forms: di-acylated SGL esterified by simple fatty acids only, as well as mono-acylated SGL bearing a hydroxyphthioceranoic acid, which were characterized in the wild-type strain. In a clinical strain, a complete family of mono-acylated SGLs was characterized in high abundance for the first time. For the mmpL8 mutant, SGLs were found to be esterified i) by an oxophthioceranoic acid, never observed so far, and ii) at nonconventional positions in the case of the unexpected tri-acylated forms. Our results further confirm the requirement of MmpL8 for the complete assembly of the tetra-acylated forms of SGL and also provide, by the discovery of new intermediates, insights in terms of the possible SGL biosynthetic pathways.

Targeted Lipidomics of Mycobacterial Lipids and Glycolipids.

Decades of study have highlighted the richness and uniqueness of the repertoire of lipid and glycolipid families produced by mycobacteria. Many of these families potently regulate host immune responses, in stimulatory or suppressive ways. Thus, the global study of this repertoire in different genetic backgrounds or under model conditions of infection is gaining interest. Despite the difficulties associated with the specificities of this repertoire, the field of mass spectrometry-based lipidomics of mycobacteria has recently made considerable progress, particularly at the analytical level. There is still considerable scope for further progress, especially with regard to the development of an efficient bioinfomatics pipeline for the analysis of the large datasets generated. This chapter describes an HPLC-MS methodology allowing the simultaneous screening of more than 20 of the lipid families produced by mycobacteria and provides recommendations to analyze the generated data given the state-of-the-art.

Plasma LOX-Products and Monocyte Signaling Is Reduced by Adjunctive Cyclooxygenase-2 Inhibitor in a Phase I Clinical Trial of Tuberculosis Patients.

Introduction: Eicosanoids and intracellular signaling pathways are potential targets for host-directed therapy (HDT) in tuberculosis (TB). We have explored the effect of cyclooxygenase 2 inhibitor (COX-2i) treatment on eicosanoid levels and signaling pathways in monocytes. Methods: Peripheral blood mononuclear cells isolated from TB patients included in a randomized phase I clinical trial of standard TB treatment with (n=21) or without (n=18) adjunctive COX-2i (etoricoxib) were analyzed at baseline, day 14 and day 56. Plasma eicosanoids were analyzed by ELISA and liquid chromatography-mass spectrometry (LC-MS), plasma cytokines by multiplex, and monocyte signaling by phospho-flow with a defined set of phospho-specific antibodies. Results: Lipoxygenase (LOX)-derived products (LXA4 and 12-HETE) and pro-inflammatory cytokines were associated with TB disease severity and were reduced during TB therapy, possibly accelerated by adjunctive COX-2i. Phosphorylation of p38 MAPK, NFkB, Erk1/2, and Akt in monocytes as well as plasma levels of MIG/CXCL9 and procalcitonin were reduced in the COX-2i group compared to controls. Conclusion: COX-2i may reduce excess inflammation in TB via the LOX-pathway in addition to modulation of phosphorylation patterns in monocytes. Immunomodulatory effects of adjunctive COX-2i in TB should be further investigated before recommended for use as a HDT strategy.

The assembly of CD1e is controlled by an N-terminal propeptide which is processed in endosomal compartments.

CD1e displays unique features in comparison with other CD1 proteins. CD1e accumulates in Golgi compartments of immature dendritic cells and is transported directly to lysosomes, where it is cleaved into a soluble form. In these latter compartments, CD1e participates in the processing of glycolipid antigens. In the present study, we show that the N-terminal end of the membrane-associated molecule begins at amino acid 20, whereas the soluble molecule consists of amino acids 32-333. Thus immature CD1e includes an N-terminal propeptide which is cleaved in acidic compartments and so is absent from its mature endosomal form. Mutagenesis experiments demonstrated that the propeptide controls the assembly of the CD1e alpha-chain with beta(2)-microglobulin, whereas propeptide-deleted CD1e molecules are immunologically active. Comparison of CD1e cDNAs from different mammalian species indicates that the CD1e propeptide is conserved during evolution, suggesting that it may also optimize the generation of CD1e molecules in other species.

Trafficking of Mycobacterium tuberculosis Envelope Components and Release Within Extracellular Vesicles: Host-Pathogen Interactions Beyond the Wall.

Components of Mycobacterium tuberculosis (Mtb) envelope such as lipoproteins, lypoglycans, lipids, and glycolipids act as Pathogen Associated Molecular Patterns and/or antigens, hence contributing in different ways to the bacillus recognition, phagocytosis, and to immune responses modulation. However, Mtb envelope components are not only encountered at the bacillus-host direct contact but can act remotely from the bacillus envelope. Indeed, they are also released from the bacillus envelope and are detected in different compartments such as the infected cells endosomal compartments or in extracellular vesicles produced by the bacillus itself or by infected cells. Characterizing their trafficking is of main importance for our understanding of their role in host-pathogen interactions and consequently for their potential use as vaccine components. This review aims at providing an overview of the current knowledge of the nature of Mtb envelope components shuttled within extracellular vesicles, the interaction of these vesicles with host immune cells and the remaining black holes.

Host-Derived Lipids from Tuberculous Pleurisy Impair Macrophage Microbicidal-Associated Metabolic Activity.

Mycobacterium tuberculosis (Mtb) regulates the macrophage metabolic state to thrive in the host, yet the responsible mechanisms remain elusive. Macrophage activation toward the microbicidal (M1) program depends on the HIF-1α-mediated metabolic shift from oxidative phosphorylation (OXPHOS) toward glycolysis. Here, we ask whether a tuberculosis (TB) microenvironment changes the M1 macrophage metabolic state. We expose M1 macrophages to the acellular fraction of tuberculous pleural effusions (TB-PEs) and find lower glycolytic activity, accompanied by elevated levels of OXPHOS and bacillary load, compared to controls. The eicosanoid fraction of TB-PE drives these metabolic alterations. HIF-1α stabilization reverts the effect of TB-PE by restoring M1 metabolism. Furthermore, Mtb-infected mice with stabilized HIF-1α display lower bacillary loads and a pronounced M1-like metabolic profile in alveolar macrophages (AMs). Collectively, we demonstrate that lipids from a TB-associated microenvironment alter the M1 macrophage metabolic reprogramming by hampering HIF-1α functions, thereby impairing control of Mtb infection.

Metabolic anticipation in Mycobacterium tuberculosis.

Humans serve as both host and reservoir for Mycobacterium tuberculosis, making tuberculosis a theoretically eradicable disease. How M. tuberculosis alternates between host-imposed quiescence and sporadic bouts of replication to complete its life cycle, however, remains unknown. Here, we identify a metabolic adaptation that is triggered upon entry into hypoxia-induced quiescence but facilitates subsequent cell cycle re-entry. Catabolic remodelling of the cell surface trehalose mycolates of M. tuberculosis specifically generates metabolic intermediates reserved for re-initiation of peptidoglycan biosynthesis. These adaptations reveal a metabolic network with the regulatory capacity to mount an anticipatory response.

Protective efficacy of a lipid antigen vaccine in a guinea pig model of tuberculosis.

The bacillus Calmette Guérin (BCG) vaccine, the only licensed vaccine against TB, displays partial and variable efficacy, thus making the exploitation of novel vaccination strategies a major priority. Most of the current vaccines in pre-clinical or clinical development are based on the induction of T cells recognizing protein antigens. However, a large number of T cells specific for mycobacterial lipids are induced during infection, suggesting that lipid-based vaccines might represent an important component of novel sub-unit vaccines. Here, we investigated whether immunization with defined mycobacterial lipid antigens induces protection in guinea pigs challenged with M. tuberculosis. Two purified mycobacterial lipid antigens, the diacylated sulfoglycolipids (Ac2SGL) and the phosphatidyl-myo-inositol dimannosides (PIM2) were formulated in biophysically characterized liposomes made of dimethyl-dioctadecyl-ammonium (DDA) and synthetic trehalose 6,6'-dibehenate (TDB). In three protection trials, a reduction of bacterial load in the spleen of inoculated animals was consistently observed compared to the unvaccinated group. Moreover, a reduction in the number of lesions and severity of pathology was detected in the lungs and spleen of the lipid vaccine group compared to unvaccinated controls. As the degree of protection achieved is similar to that observed using protein antigens in the same guinea pig model, these promising results pave the way to future investigations of lipid antigens as subunit vaccines.

Lysosomal Lipases PLRP2 and LPLA2 Process Mycobacterial Multi-acylated Lipids and Generate T Cell Stimulatory Antigens.

Complex antigens require processing within antigen-presenting cells (APCs) to form T cell stimulatory complexes with CD1 antigen-presenting molecules. It remains unknown whether lipids with multi-acylated moieties also necessitate digestion by lipases to become capable of binding CD1 molecules and stimulate T cells. Here, we show that the mycobacterial tetra-acylated glycolipid antigens phosphatidyl-myo-inositol mannosides (PIM) are digested to di-acylated forms by pancreatic lipase-related protein 2 (PLRP2) and lysosomal phospholipase A2 (LPLA2) within APCs. Recombinant PLRP2 and LPLA2 removed the sn1- and sn2-bound fatty acids from the PIM glycerol moiety, as revealed by mass spectrometry and nuclear magnetic resonance studies. PLRP2 or LPLA2 gene silencing in APCs abolished PIM presentation to T cells, thus revealing an essential role of both lipases in vivo. These findings show that endosomal lipases participate in lipid antigen presentation by processing lipid antigens and have a role in T cell immunity against mycobacteria.