Supramolecular organization and dynamics of mannosylated phosphatidylinositol lipids in the mycobacterial plasma membrane.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB), a disease that claims ~1.6 million lives annually. The current treatment regime is long and expensive, and missed doses contribute to drug resistance. Therefore, development of new anti-TB drugs remains one of the highest public health priorities. Mtb has evolved a complex cell envelope that represents a formidable barrier to antibiotics. The Mtb cell envelop consists of four distinct layers enriched for Mtb specific lipids and glycans. Although the outer membrane, comprised of mycolic acid esters, has been extensively studied, less is known about the plasma membrane, which also plays a critical role in impacting antibiotic efficacy. The Mtb plasma membrane has a unique lipid composition, with mannosylated phosphatidylinositol lipids (phosphatidyl-myoinositol mannosides, PIMs) comprising more than 50% of the lipids. However, the role of PIMs in the structure and function of the membrane remains elusive. Here, we used multiscale molecular dynamics (MD) simulations to understand the structure-function relationship of the PIM lipid family and decipher how they self-organize to shape the biophysical properties of mycobacterial plasma membranes. We assess both symmetric and asymmetric assemblies of the Mtb plasma membrane and compare this with residue distributions of Mtb integral membrane protein structures. To further validate the model, we tested known anti-TB drugs and demonstrated that our models agree with experimental results. Thus, our work sheds new light on the organization of the mycobacterial plasma membrane. This paves the way for future studies on antibiotic development and understanding Mtb membrane protein function.

Role of succinyl substituents in the mannose-capping of lipoarabinomannan and control of inflammation in Mycobacterium tuberculosis infection.

The covalent modification of bacterial (lipo)polysaccharides with discrete substituents may impact their biosynthesis, export and/or biological activity. Whether mycobacteria use a similar strategy to control the biogenesis of its cell envelope polysaccharides and modulate their interaction with the host during infection is unknown despite the report of a number of tailoring substituents modifying the structure of these glycans. Here, we show that discrete succinyl substituents strategically positioned on Mycobacterium tuberculosis (Mtb) lipoarabinomannan govern the mannose-capping of this lipoglycan and, thus, much of the biological activity of the entire molecule. We further show that the absence of succinyl substituents on the two main cell envelope glycans of Mtb, arabinogalactan and lipoarabinomannan, leads to a significant increase of pro-inflammatory cytokines and chemokines in infected murine and human macrophages. Collectively, our results validate polysaccharide succinylation as a critical mechanism by which Mtb controls inflammation.

Asymmetric Total Synthesis and Structural Revision of DAT2, an Antigenic Glycolipid from Mycobacterium tuberculosis.

DAT2 is a member of the diacyl trehalose family (DAT) of antigenic glycolipids located in the mycomembrane of Mycobacterium tuberculosis (Mtb). Recently it was shown that the molecular structure of DAT2 had been incorrectly assigned, but the correct structure remained elusive. Herein, the correct molecular structure of DAT2 and its methyl-branched acyl substituent mycolipanolic acid is determined. For this, four different stereoisomers of mycolipanolic acid were prepared in a stereoselective and unified manner, and incorporated into DAT2. A rigorous comparison of the four isomers to the DAT isolated from Mtb H37Rv by NMR, HPLC, GC, and mass spectrometry allowed a structural revision of mycolipanolic acid and DAT2. Activation of the macrophage inducible Ca2+-dependent lectin receptor (Mincle) with all four stereoisomers shows that the natural stereochemistry of mycolipanolic acid / DAT2 provides the strongest activation, which indicates its high antigenicity and potential application in serodiagnostics and vaccine adjuvants.

Development of a novel target-based cell assay, reporter of the activity of Mycobacterium tuberculosis protein-O-mannosyltransferase.

The Protein-O-mannosyltransferase is crucial for the virulence of Mycobacterium tuberculosis, the etiological agent of tuberculosis. This enzyme, called MtPMT (Rv1002c), is responsible for the post-translational O-mannosylation of mycobacterial proteins. It catalyzes the transfer of a single mannose residue from a polyprenol phospho-mannosyl lipidic donor to the hydroxyl groups of selected Ser/Thr residues in acceptor proteins during their translocation across the membrane. Previously, we provided evidence that the loss of MtPMT activity causes the absence of mannoproteins in Mycobacterium tuberculosis, severely impacting its intracellular growth, as well as a strong attenuation of its pathogenicity in immunocompromised mice. Therefore, it is of interest to develop specific inhibitors of this enzyme to better understand mycobacterial infectious diseases. Here we report the development of a "target-based" phenotypic assay for this enzyme, assessing its O-mannosyltransferase activity in bacteria, in the non-pathogenic Mycobacterium smegmatis strain. Robustness of the quantitative contribution of this assay was evaluated by intact protein mass spectrometry, using a panel of control strains, overexpressing the MtPMT gene, carrying different key point-mutations. Then, screening of a limited library of 30 compounds rationally chosen allowed us to identify 2 compounds containing pyrrole analogous rings, as significant inhibitors of MtPMT activity, affecting neither the growth of the mycobacterium nor its secretion of mannoproteins. These molecular cores could therefore serve as scaffold for the design of new pharmaceutical agents that could improve treatment of mycobacterial diseases. We report here the implementation of a miniaturized phenotypic activity assay for a glycosyltransferase of the C superfamily.

Rv0180c contributes to Mycobacterium tuberculosis cell shape and to infectivity in mice and macrophages.

Mycobacterium tuberculosis, the main causative agent of human tuberculosis, is transmitted from person to person via small droplets containing very few bacteria. Optimizing the chance to seed in the lungs is therefore a major adaptation to favor survival and dissemination in the human population. Here we used TnSeq to identify genes important for the early events leading to bacterial seeding in the lungs. Beside several genes encoding known virulence factors, we found three new candidates not previously described: rv0180c, rv1779c and rv1592c. We focused on the gene, rv0180c, of unknown function. First, we found that deletion of rv0180c in M. tuberculosis substantially reduced the initiation of infection in the lungs of mice. Next, we established that Rv0180c enhances entry into macrophages through the use of complement-receptor 3 (CR3), a major phagocytic receptor for M. tuberculosis. Silencing CR3 or blocking the CR3 lectin site abolished the difference in entry between the wild-type parental strain and the Δrv0180c::km mutant. However, we detected no difference in the production of both CR3-known carbohydrate ligands (glucan, arabinomannan, mannan), CR3-modulating lipids (phthiocerol dimycocerosate), or proteins in the capsule of the Δrv0180c::km mutant in comparison to the wild-type or complemented strains. By contrast, we established that Rv0180c contributes to the functionality of the bacterial cell envelope regarding resistance to toxic molecule attack and cell shape. This alteration of bacterial shape could impair the engagement of membrane receptors that M. tuberculosis uses to invade host cells, and open a new perspective on the modulation of bacterial infectivity.

Disruption of the SucT acyltransferase in Mycobacterium smegmatis abrogates succinylation of cell envelope polysaccharides.

Similar to other prokaryotes, mycobacteria decorate their major cell envelope glycans with minor covalent substituents whose biological significance remains largely unknown. We report on the discovery of a mycobacterial enzyme, named here SucT, that adds succinyl groups to the arabinan domains of both arabinogalactan (AG) and lipoarabinomannan (LAM). Disruption of the SucT-encoding gene in Mycobacterium smegmatis abolished AG and LAM succinylation and altered the hydrophobicity and rigidity of the cell envelope of the bacilli without significantly altering AG and LAM biosynthesis. The changes in the cell surface properties of the mutant were consistent with earlier reports of transposon mutants of the closely related species Mycobacterium marinum and Mycobacterium avium harboring insertions in the orthologous gene whose ability to microaggregate and form biofilms were altered. Our findings point to an important role of SucT-mediated AG and LAM succinylation in modulating the cell surface properties of mycobacteria.

Efficacy of Bedaquiline, Alone or in Combination with Imipenem, against Mycobacterium abscessus in C3HeB/FeJ Mice.

Mycobacterium abscessus lung infections remain difficult to treat. Recent studies have recognized the power of new combinations of antibiotics, such as bedaquiline and imipenem, although in vitro data have questioned this combination. We report that the efficacy of bedaquiline-imipenem combination treatment relies essentially on the activity of bedaquiline in a C3HeB/FeJ mice model of infection with a rough variant of M. abscessus The addition of imipenem contributed to clearing the infection in the spleen.

Rational design of adjuvants targeting the C-type lectin Mincle.

The advances in subunit vaccines development have intensified the search for potent adjuvants, particularly adjuvants inducing cell-mediated immune responses. Identification of the C-type lectin Mincle as one of the receptors underlying the remarkable immunogenicity of the mycobacterial cell wall, via recognition of trehalose-6,6'-dimycolate (TDM), has opened avenues for the rational design of such molecules. Using a combination of chemical synthesis, biological evaluation, molecular dynamics simulations, and protein mutagenesis, we gained insight into the molecular bases of glycolipid recognition by Mincle. Unexpectedly, the fine structure of the fatty acids was found to play a key role in the binding of a glycolipid to the carbohydrate recognition domain of the lectin. Glucose and mannose esterified at O-6 by a synthetic α-ramified 32-carbon fatty acid showed agonist activity similar to that of TDM, despite their much simpler structure. Moreover, they were seen to stimulate proinflammatory cytokine production in primary human and murine cells in a Mincle-dependent fashion. Finally, they were found to induce strong Th1 and Th17 immune responses in vivo in immunization experiments in mice and conferred protection in a murine model of Mycobacterium tuberculosis infection. Here we describe the rational development of new molecules with powerful adjuvant properties.

Mycobacterium tuberculosis inhibits human innate immune responses via the production of TLR2 antagonist glycolipids.

Mycobacterium tuberculosis is a major human pathogen that is able to survive inside host cells and resist immune clearance. Most particularly, it inhibits several arms of the innate immune response, including phagosome maturation or cytokine production. To better understand the molecular mechanisms by which M. tuberculosis circumvents host immune defenses, we used a transposon mutant library generated in a virulent clinical isolate of M. tuberculosis of the W/Beijing family to infect human macrophages, utilizing a cell line derivative of THP-1 cells expressing a reporter system for activation of the transcription factor NF-κB, a key regulator of innate immunity. We identified several M. tuberculosis mutants inducing a NF-κB activation stronger than that of the wild-type strain. One of these mutants was found to be deficient for the synthesis of cell envelope glycolipids, namely sulfoglycolipids, suggesting that the latter can interfere with innate immune responses. Using natural and synthetic molecular variants, we determined that sulfoglycolipids inhibit NF-κB activation and subsequent cytokine production or costimulatory molecule expression by acting as competitive antagonists of Toll-like receptor 2, thereby inhibiting the recognition of M. tuberculosis by this receptor. Our study reveals that producing glycolipid antagonists of pattern recognition receptors is a strategy used by M. tuberculosis to undermine innate immune defense. Sulfoglycolipids are major and specific lipids of M. tuberculosis, considered for decades as virulence factors of the bacilli. Our study uncovers a mechanism by which they may contribute to M. tuberculosis virulence.

C-type lectin receptor DCIR modulates immunity to tuberculosis by sustaining type I interferon signaling in dendritic cells.

Immune response against pathogens is a tightly regulated process that must ensure microbial control while preserving integrity of the infected organs. Tuberculosis (TB) is a paramount example of a chronic infection in which antimicrobial immunity is protective in the vast majority of infected individuals but can become detrimental if not finely tuned. Here, we report that C-type lectin dendritic cell (DC) immunoreceptor (DCIR), a key component in DC homeostasis, is required to modulate lung inflammation and bacterial burden in TB. DCIR is abundantly expressed in pulmonary lesions in Mycobacterium tuberculosis-infected nonhuman primates during both latent and active disease. In mice, we found that DCIR deficiency impairs STAT1-mediated type I IFN signaling in DCs, leading to increased production of IL-12 and increased differentiation of T lymphocytes toward Th1 during infection. As a consequence, DCIR-deficient mice control M. tuberculosis better than WT animals but also develop more inflammation characterized by an increased production of TNF and inducible NOS (iNOS) in the lungs. Altogether, our results reveal a pathway by which a C-type lectin modulates the equilibrium between infection-driven inflammation and pathogen's control through sustaining type I IFN signaling in DCs.

Potential Plasticity of the Mannoprotein Repertoire Associated to Mycobacterium tuberculosis Virulence Unveiled by Mass Spectrometry-Based Glycoproteomics.

To date, Mycobacterium tuberculosis (Mtb) remains the world's greatest infectious killer. The rise of multidrug-resistant strains stresses the need to identify new therapeutic targets to fight the epidemic. We previously demonstrated that bacterial protein-O-mannosylation is crucial for Mtb infectiousness, renewing the interest of the bacterial-secreted mannoproteins as potential drug-targetable virulence factors. The difficulty of inventorying the mannoprotein repertoire expressed by Mtb led us to design a stringent multi-step workflow for the reliable identification of glycosylated peptides by large-scale mass spectrometry-based proteomics. Applied to the differential analyses of glycoproteins secreted by the wild-type Mtb strain-and by its derived mutant invalidated for the protein-O-mannosylating enzyme PMTub-this approach led to the identification of not only most already known mannoproteins, but also of yet-unknown mannosylated proteins. In addition, analysis of the glycoproteome expressed by the isogenic recombinant Mtb strain overexpressing the PMTub gene revealed an unexpected mannosylation of proteins, with predicted or demonstrated functions in Mtb growth and interaction with the host cell. Since in parallel, a transient increased expression of the PMTub gene has been observed in the wild-type bacilli when infecting macrophages, our results strongly suggest that the Mtb mannoproteome may undergo adaptive regulation during infection of the host cells. Overall, our results provide deeper insights into the complexity of the repertoire of mannosylated proteins expressed by Mtb, and open the way to novel opportunities to search for still-unexploited potential therapeutic targets.

LppM impact on the colonization of macrophages by Mycobacterium tuberculosis.

Mycobacterium tuberculosis produces several bacterial effectors impacting the colonization of phagocytes. Here, we report that the putative lipoprotein LppM hinders phagocytosis by macrophages in a toll-like receptor 2-dependent manner. Moreover, recombinant LppM is able to functionally complement the phenotype of the mutant, when exogenously added during macrophage infection. LppM is also implicated in the phagosomal maturation, as a lppM deletion mutant is more easily addressed towards the acidified compartments of the macrophage than its isogenic parental strain. In addition, this mutant was affected in its ability to induce the secretion of pro-inflammatory chemokines, interferon-gamma-inducible protein-10, monocyte chemoattractant protein-1 and macrophage inflammatory protein-1α. Thus, our results describe a new mycobacterial protein involved in the early trafficking of the tubercle bacillus and its manipulation of the host immune response.

Mannodendrimers prevent acute lung inflammation by inhibiting neutrophil recruitment.

Mycobacterium tuberculosis mannose-capped lipoarabinomannan inhibits the release of proinflammatory cytokines by LPS-stimulated human dendritic cells (DCs) via targeting the C-type lectin receptor DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN). With the aim of mimicking the bioactive supramolecular structure of mannose-capped lipoarabinomannan, we designed and synthesized a set of poly(phosphorhydrazone) dendrimers grafted with mannose units, called mannodendrimers, that differed by size and the number and length of their (α1→2)-oligommanoside caps. A third-generation dendrimer bearing 48 trimannoside caps (3T) and a fourth-generation dendrimer bearing 96 dimannosides (4D) displayed the highest binding avidity for DC-SIGN. Moreover, these dendrimers inhibited proinflammatory cytokines, including TNF-α, production by LPS-stimulated DCs in a DC-SIGN-dependent fashion. Finally, in a model of acute lung inflammation in which mice were exposed to aerosolized LPS, per os administration of 3T mannodendrimer was found to significantly reduce neutrophil influx via targeting the DC-SIGN murine homolog SIGN-related 1. The 3T mannodendrimer therefore represents an innovative fully synthetic compound for the treatment of lung inflammatory diseases.

Bacterial protein-O-mannosylating enzyme is crucial for virulence of Mycobacterium tuberculosis.

A posttranslational protein O-mannosylation process resembling that found in fungi and animals has been reported in the major human pathogen Mycobacterium tuberculosis (Mtb) and related actinobacteria. However, the role and incidence of this process, which is essential in eukaryotes, have never been explored in Mtb. We thus analyzed the impact of interrupting O-mannosylation in the nonpathogenic saprophyte Mycobacterium smegmatis and in the human pathogen Mtb by inactivating the respective putative protein mannosyl transferase genes Msmeg_5447 and Rv1002c. Loss of protein O-mannosylation in both mutant strains was unambiguously demonstrated by efficient mass spectrometry-based glycoproteomics analysis. Unexpectedly, although the M. smegmatis phenotype was unaffected by the lack of manno-proteins, the Mtb mutant had severely impacted growth in vitro and in cellulo associated with a strong attenuation of its pathogenicity in immunocompromised mice. These data are unique in providing evidence of the biological significance of protein O-mannosylation in mycobacteria and demonstrate the crucial contribution of this protein posttranslational modification to Mtb virulence in the host.

A glycomic approach reveals a new mycobacterial polysaccharide.

Mycobacterium tuberculosis lipoarabinomannan (LAM) and biosynthetically related lipoglycans and glycans play an important role in host-pathogen interactions. Therefore, the elucidation of the complete biosynthetic pathways of these important molecules is expected to afford novel therapeutic targets. The characterization of biosynthetic enzymes and transporters involved in the formation and localization of these complex macromolecules in the bacterial cell envelope largely relies on genetic manipulation of mycobacteria and subsequent analyses of lipoglycan structural alterations. However, lipoglycans are present in relatively low amounts. Their purification to homogeneity remains tedious and time-consuming. To overcome these issues and to reduce the biomass and time required for lipoglycan purification, we report here the development of a methodology to efficiently purify lipoglycans by sodium deoxycholate-polyacrylamide gel electrophoresis. This faster purification method can be applied on a small amount of mycobacterial cells biomass (10-50 mg), resulting in tens of micrograms of purified lipoglycans. This amount of purified products was found to be sufficient to undertake structural analyses of lipoglycans and glycans carbohydrate domains by a combination of highly sensitive analytical procedures, involving cryoprobe NMR analysis of intact macromolecules and chemical degradations monitored by gas chromatography and capillary electrophoresis. This glycomic approach was successfully applied to the purification and structural characterization of a newly identified polysaccharide, structurally related to LAM, in the model fast-growing species Mycobacterium smegmatis.

Lipoarabinomannan mannose caps do not affect mycobacterial virulence or the induction of protective immunity in experimental animal models of infection and have minimal impact on in vitro inflammatory responses.

Mannose-capped lipoarabinomannan (ManLAM) is considered an important virulence factor of Mycobacterium tuberculosis. However, while mannose caps have been reported to be responsible for various immunosuppressive activities of ManLAM observed in vitro, there is conflicting evidence about their contribution to mycobacterial virulence in vivo. Therefore, we used Mycobacterium bovis BCG and M. tuberculosis mutants that lack the mannose cap of LAM to assess the role of ManLAM in the interaction of mycobacteria with the host cells, to evaluate vaccine-induced protection and to determine its importance in M. tuberculosis virulence. Deletion of the mannose cap did not affect BCG survival and replication in macrophages, although the capless mutant induced a somewhat higher production of TNF. In dendritic cells, the capless mutant was able to induce the upregulation of co-stimulatory molecules and the only difference we detected was the secretion of slightly higher amounts of IL-10 as compared to the wild type strain. In mice, capless BCG survived equally well and induced an immune response similar to the parental strain. Furthermore, the efficacy of vaccination against a M. tuberculosis challenge in low-dose aerosol infection models in mice and guinea pigs was not affected by the absence of the mannose caps in the BCG. Finally, the lack of the mannose cap in M. tuberculosis did not affect its virulence in mice nor its interaction with macrophages in vitro. Thus, these results do not support a major role for the mannose caps of LAM in determining mycobacterial virulence and immunogenicity in vivo in experimental animal models of infection, possibly because of redundancy of function.

Impact of Methylthioxylose Substituents on the Biological Activities of Lipomannan and Lipoarabinomannan in Mycobacterium tuberculosis.

Two lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM), play various, albeit incompletely defined, roles in the interactions of mycobacteria with the host. Growing evidence points to the modification of LM and LAM with discrete covalent substituents as a strategy used by these bacteria to modulate their biological activities. One such substituent, originally identified in Mycobacterium tuberculosis (Mtb), is a 5-methylthio-d-xylose (MTX) sugar, which accounts for the antioxidative properties of LAM. The widespread distribution of this motif across Mtb isolates from several epidemiologically important lineages have stimulated interest in MTX-modified LAM as a biomarker of tuberculosis infection. Yet, several lines of evidence indicate that MTX may not be restricted to Mtb and that this motif may substitute more acceptors than originally thought. Using a highly specific monoclonal antibody to the MTX capping motif of Mtb LAM, we here show that MTX motifs not only substitute the mannoside caps of LAM but also the mannan core of LM in Mtb. MTX substituents were also found on the LM and LAM of pathogenic, slow-growing nontuberculous mycobacteria. The presence of MTX substituents on the LM and LAM from Mtb enhances the pro-apoptotic properties of both lipoglycans on LPS-stimulated THP-1 macrophages. A comparison of the cytokines and chemokines produced by resting and LPS-activated THP-1 cells upon exposure to MTX-proficient versus MTX-deficient LM further indicates that MTX substituents confer anti-inflammatory properties upon LM. These findings add to our understanding of the glycan-based strategies employed by slow-growing pathogenic mycobacteria to alter the host immune response to infection.

Differential arabinan capping of lipoarabinomannan modulates innate immune responses and impacts T helper cell differentiation.

Toll-like receptors (TLRs) recognize pathogens by interacting with pathogen-associated molecular patterns, such as the phosphatidylinositol-based lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM). Such structures are present in several pathogens, including Mycobacterium tuberculosis, being important for the initiation of immune responses. It is well established that the interaction of LM and LAM with TLR2 is a process dependent on the structure of the ligands. However, the implications of structural variations on TLR2 ligands for the development of T helper (Th) cell responses or in the context of in vivo responses are less studied. Herein, we used Corynebacterium glutamicum as a source of lipoglycan intermediates for host interaction studies. In this study, we have deleted a putative glycosyltransferase, NCgl2096, from C. glutamicum and found that it encodes for a novel α(1→2)arabinofuranosyltransferase, AftE. Biochemical analysis of the lipoglycans obtained in the presence (wild type) or absence of NCgl2096 showed that AftE is involved in the biosynthesis of singular arabinans of LAM. In its absence, the resulting molecule is a hypermannosylated (hLM) form of LAM. Both LAM and hLM were recognized by dendritic cells, mainly via TLR2, and triggered the production of several cytokines. hLM was a stronger stimulus for in vitro cytokine production and, as a result, a more potent inducer of Th17 responses. In vivo data confirmed hLM as a stronger inducer of cytokine responses and suggested the involvement of pattern recognition receptors other than TLR2 as sensors for lipoglycans.

A small multidrug resistance-like transporter involved in the arabinosylation of arabinogalactan and lipoarabinomannan in mycobacteria.

The biosynthesis of the major cell envelope glycoconjugates of Mycobacterium tuberculosis is topologically split across the plasma membrane, yet nothing is known of the transporters required for the translocation of lipid-linked sugar donors and oligosaccharide intermediates from the cytoplasmic to the periplasmic side of the membrane in mycobacteria. One of the mechanisms used by prokaryotes to translocate lipid-linked phosphate sugars across the plasma membrane relies on translocases that share resemblance with small multidrug resistance transporters. The presence of an small multidrug resistance-like gene, Rv3789, located immediately upstream from dprE1/dprE2 responsible for the formation of decaprenyl-monophosphoryl-ß-D-arabinose (DPA) in the genome of M. tuberculosis led us to investigate its potential involvement in the formation of the major arabinosylated glycopolymers, lipoarabinomannan (LAM) and arabinogalactan (AG). Disruption of the ortholog of Rv3789 in Mycobacterium smegmatis resulted in a reduction of the arabinose content of both AG and LAM that accompanied the accumulation of DPA in the mutant cells. Interestingly, AG and LAM synthesis was restored in the mutant not only upon expression of Rv3789 but also upon that of the undecaprenyl phosphate aminoarabinose flippase arnE/F genes from Escherichia coli. A bacterial two-hybrid system further indicated that Rv3789 interacts in vivo with the galactosyltransferase that initiates the elongation of the galactan domain of AG. Biochemical and genetic evidence is thus consistent with Rv3789 belonging to an AG biosynthetic complex, where its role is to reorient DPA to the periplasm, allowing this arabinose donor to then be used in the buildup of the arabinan domains of AG and LAM.

A truncated lipoglycan from mycobacteria with altered immunological properties.

Maintenance of cell-wall integrity in Mycobacterium tuberculosis is essential and is the target of several antitubercular drugs. For example, ethambutol targets arabinogalactan and lipoarabinomannan (LAM) biosynthesis through the inhibition of several arabinofuranosyltransferases. Apart from their role in cell-wall integrity, mycobacterial LAMs also exhibit important immunomodulatory activities. Here we report the isolation and detailed structural characterization of a unique LAM molecule derived from Mycobacterium smegmatis deficient in the arabinofuranosyltransferase AftC (AftC-LAM). This mutant LAM expresses a severely truncated arabinan domain completely devoid of 3,5-Araf-branching residues, revealing an intrinsic involvement of AftC in the biosynthesis of LAM. Furthermore, we found that ethambutol efficiently inhibits biosynthesis of the AftC-LAM arabinan core, unambiguously demonstrating the involvement of the arabinofuranosyltransferase EmbC in early stages of LAM-arabinan biosynthesis. Finally, we demonstrate that AftC-LAM exhibits an enhanced proinflammatory activity, which is due to its ability to activate Toll-like receptor 2 (TLR2). Overall, our efforts further describe the mechanism of action of an important antitubercular drug, ethambutol, and demonstrate a role for specific arabinofuranosyltransferases in LAM biosynthesis. In addition, the availability of sufficient amounts of chemically defined wild-type and isogenic truncated LAMs paves the way for further investigations of the structure-function relationship of TLR2 activation by mycobacterial lipoglycans.