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Introduction: The WHO estimates a gap of about 30% between the incident (10.6 million) and notified (7.5 million) cases of tuberculosis (TB). Combined with the growing recognition in prevalence surveys of the high proportion of cases identified who are asymptomatic or paucisymptomatic, these data underscore how current symptom screening approaches and use of diagnostic tests with suboptimal performance on sputum miss large numbers of cases. Thus, the development of sputum-free biomarker-based tests for diagnosis is becoming necessary, which the WHO has already identified as a priority for new TB diagnostics.The objective of this study is to evaluate a combination of exhaled breath condensate (EBC) samples and mycobacterial lipoarabinomannan (LAM) as point-of-care (POC) assays to identify TB patients., Methods and Analysis: This prospective diagnostic accuracy study is conducted at the TB Screening and Treatment Centre of International Center for Diarrhoeal Disease Research, Bangladesh, on a cohort of adults and adolescents >11 years of age. A total of 614 individuals with presumptive pulmonary TB based on TB signs, symptoms and radiography are being recruited from 28 August 2023. Spot sputum is collected for standard reference testing (L-J culture, GeneXpert MTB/Rif, acid-fast Bacilli microscopy) to fine-tune categorisation of TB disease status for each participant, defined as (1) definite TB (at least one positive standard reference test); (2) probable TB (not microbiologically confirmed but under TB treatment); (3) possible TB (no TB treatment but signs, symptoms and radiography suggestive of TB); (4) other respiratory disease (microbiologically not confirmed and no radiography presenting abnormalities compatible with TB); and (5) unknown (no microbiological evidence with normal/no TB abnormalities with radiography). Urine and EBC specimens will be subjected to LAM POC testing and biobanked for further investigation. Statistical analyses will include an assessment of diagnostic accuracy by constructing receiver operating curves and calculating sensitivity and specificity, as well as post-test probabilities., Ethics and Dissemination: The study protocol was approved by the Research Review Committee as well as the Ethical Review Committee of icddr,b and recorded under a protocol reference number, PR-2301. Results will be submitted to open-access peer-reviewed journals, presented at academic meetings, and shared with national and international policymaking bodies., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

DAT 2 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 DAT 2 had been incorrectly assigned, but the correct structure remained elusive. Herein, the correct molecular structure of DAT 2 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 DAT 2 . 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 DAT 2 . Activation of the macrophage inducible Ca 2+ -dependent lectin receptor (Mincle) with all four stereoisomers shows that the natural stereochemistry of mycolipanolic acid / DAT 2 provides the strongest activation, which indicates its high antigenicity and potential application in serodiagnostics and vaccine adjuvants., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

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.

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., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Palčeková et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The bacterial pathogen Mycobacterium tuberculosis binds to the C-type lectin DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin) on dendritic cells to evade the immune system. While DC-SIGN glycoconjugate ligands are ubiquitous among mycobacterial species, the receptor selectively binds pathogenic species from the M. tuberculosis complex ( MTBC ). Here, we unravel the molecular mechanism behind this intriguing selective recognition by means of a multidisciplinary approach combining single-molecule atomic force microscopy with Förster resonance energy transfer and bioassays. Molecular recognition imaging of mycobacteria demonstrates that the distribution of DC-SIGN ligands markedly differs between Mycobacterium bovis Bacille Calmette-Guérin (BCG) (model MTBC species) and Mycobacterium smegmatis (non- MTBC species), the ligands being concentrated into dense nanodomains on M. bovis BCG. Upon bacteria-host cell adhesion, ligand nanodomains induce the recruitment and clustering of DC-SIGN. Our study highlights the key role of clustering of both ligands on MTBC species and DC-SIGN host receptors in pathogen recognition, a mechanism that might be widespread in host-pathogen interactions.

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.

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., (© 2022. The Author(s).)

Glycolipids constitute a major part of the cell envelope of Mycobacterium tuberculosis (Mtb). They are potent immunomodulatory molecules recognized by several immune receptors like pattern recognition receptors such as TLR2, DC-SIGN and Dectin-2 on antigen-presenting cells and by T cell receptors on T lymphocytes. The Mtb glycolipids lipoarabinomannan (LAM) and its biosynthetic relatives, phosphatidylinositol mannosides (PIMs) and lipomannan (LM), as well as other Mtb glycolipids, such as phenolic glycolipids and sulfoglycolipids have the ability to modulate the immune response, stimulating or inhibiting a pro-inflammatory response. We explore here the downmodulating effect of Mtb glycolipids. A great proportion of the studies used in vitro approaches although in vivo infection with Mtb might also lead to a dampening of myeloid cell and T cell responses to Mtb glycolipids. This dampened response has been explored ex vivo with immune cells from peripheral blood from Mtb-infected individuals and in mouse models of infection. In addition to the dampening of the immune response caused by Mtb glycolipids, we discuss the hyporesponse to Mtb glycolipids caused by prolonged Mtb infection and/or exposure to Mtb antigens. Hyporesponse to LAM has been observed in myeloid cells from individuals with active and latent tuberculosis (TB). For some myeloid subsets, this effect is stronger in latent versus active TB. Since the immune response in individuals with latent TB represents a more protective profile compared to the one in patients with active TB, this suggests that downmodulation of myeloid cell functions by Mtb glycolipids may be beneficial for the host and protect against active TB disease. The mechanisms of this downmodulation, including tolerance through epigenetic modifications, are only partly explored., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Correia-Neves, Nigou, Mousavian, Sundling and Källenius.)

Mycobacterium abscessus is an emerging and difficult-to-manage mycobacterial species that exhibits smooth (S) or rough (R) morphotypes. Disruption of glycopeptidolipid (GPL) production results in transition from S to R and severe lung disease. A structure-activity relationship study was undertaken to decipher the role of GPL glycosylation in morphotype transition and pathogenesis. Deletion of gtf3 uncovered the prominent role of the extra rhamnose in enhancing mannose receptor-mediated internalization of M. abscessus by macrophages. In contrast, the absence of the 6-deoxy-talose and the first rhamnose in mutants lacking gtf1 and gtf2, respectively, affected M abscessus phagocytosis but also resulted in the S-to-R transition. Strikingly, gtf1 and gtf2 mutants displayed a strong propensity to form cords and abscesses in zebrafish, leading to robust and lethal infection. Together, these results underscore the importance and differential contribution of GPL monosaccharides in promoting virulence and infection outcomes., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Adhesion is crucial for the infective lifestyles of bacterial pathogens. Adhesion to non-living surfaces, other microbial cells, and components of the biofilm extracellular matrix are crucial for biofilm formation and integrity, plus adherence to host factors constitutes a first step leading to an infection. Adhesion is, therefore, at the core of pathogens' ability to contaminate, transmit, establish residency within a host, and cause an infection. Several mycobacterial species cause diseases in humans and animals with diverse clinical manifestations. Mycobacterium tuberculosis , which enters through the respiratory tract, first adheres to alveolar macrophages and epithelial cells leading up to transmigration across the alveolar epithelium and containment within granulomas. Later, when dissemination occurs, the bacilli need to adhere to extracellular matrix components to infect extrapulmonary sites. Mycobacteria causing zoonotic infections and emerging nontuberculous mycobacterial pathogens follow divergent routes of infection that probably require adapted adhesion mechanisms. New evidence also points to the occurrence of mycobacterial biofilms during infection, emphasizing a need to better understand the adhesive factors required for their formation. Herein, we review the literature on tuberculous and nontuberculous mycobacterial adhesion to living and non-living surfaces, to themselves, to host cells, and to components of the extracellular matrix.

Upon infection with Mycobacterium tuberculosis (Mtb) the host immune response might clear the bacteria, control its growth leading to latent tuberculosis (LTB), or fail to control its growth resulting in active TB (ATB). There is however no clear understanding of the features underlying a more or less effective response. Mtb glycolipids are abundant in the bacterial cell envelope and modulate the immune response to Mtb, but the patterns of response to glycolipids are still underexplored. To identify the CD45 + leukocyte activation landscape induced by Mtb glycolipids in peripheral blood of ATB and LTB, we performed a detailed assessment of the immune response of PBMCs to the Mtb glycolipids lipoarabinomannan (LAM) and its biosynthetic precursor phosphatidyl-inositol mannoside (PIM), and purified-protein derivate (PPD). At 24 h of stimulation, cell profiling and secretome analysis was done using mass cytometry and high-multiplex immunoassay. PIM induced a diverse cytokine response, mainly affecting antigen-presenting cells to produce both pro-inflammatory and anti-inflammatory cytokines, but not IFN-γ, contrasting with PPD that was a strong inducer of IFN-γ. The effect of PIM on the antigen-presenting cells was partly TLR2-dependent. Expansion of monocyte subsets in response to PIM or LAM was reduced primarily in LTB as compared to healthy controls, suggesting a hyporesponsive/tolerance pattern derived from Mtb infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Silva, Sundling, Folkesson, Fröberg, Nobrega, Canto-Gomes, Chambers, Lakshmikanth, Brodin, Bruchfeld, Nigou, Correia-Neves and Källenius.)

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., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jøntvedt Jørgensen, Nore, Aass, Layre, Nigou, Mortensen, Tasken, Kvale, Jenum, Tonby and Dyrhol-Riise.)

Francisella tularensis is one of the most virulent pathogenic bacteria causing the acute human respiratory disease tularemia. While the mechanisms underlying F. tularensis pathogenesis are largely unknown, previous studies have shown that a F. novicida transposon mutant with insertions in a gene coding for a putative lysine decarboxylase was attenuated in mouse spleen, suggesting a possible role of its protein product as a virulence factor. Therefore, we set out to structurally and functionally characterize the F. novicida lysine decarboxylase, which we termed LdcF. Here, we investigate the genetic environment of ldcF as well as its evolutionary relationships with other basic AAT-fold amino acid decarboxylase superfamily members, known as key actors in bacterial adaptative stress response and polyamine biosynthesis. We determine the crystal structure of LdcF and compare it with the most thoroughly studied lysine decarboxylase, E. coli LdcI. We analyze the influence of ldcF deletion on bacterial growth under different stress conditions in dedicated growth media, as well as in infected macrophages, and demonstrate its involvement in oxidative stress resistance. Finally, our mass spectrometry-based quantitative proteomic analysis enables identification of 80 proteins with expression levels significantly affected by ldcF deletion, including several DNA repair proteins potentially involved in the diminished capacity of the F. novicida mutant to deal with oxidative stress. Taken together, we uncover an important role of LdcF in F. novicida survival in host cells through participation in oxidative stress response, thereby singling out this previously uncharacterized protein as a potential drug target.

The very high content of structurally diverse and biologically active lipids of exotic structures is the hallmark of Mycobacteria. As such the lipid composition is commonly used to characterize mycobacterial strains at the species and type-species levels. The present chapter describes the methods that allow the purification of the most commonly isolated biologically active lipids and those used for analyzing extractable lipids and their constituents, cell wall-linked mycolic acids (MA), and lipoarabinomannan (LAM). These involve various chromatographic techniques and analytical procedures necessary for structural and metabolic studies of mycobacterial lipids. In addition, as the use of physical methods has brought important overhang on chemical structures of the very-long-chain MA, which typify mycobacteria, NMR and mass spectrometry data of these specific fatty acids are included.

Mycobacterium abscessus is a prevalent pathogenic mycobacterium in cystic fibrosis (CF) patients and one of the most highly drug resistant mycobacterial species to antimicrobial agents. It possesses the property to transition from a smooth (S) to a rough (R) morphotype, thereby influencing the host innate immune response. This transition from the S to the R morphotype takes place in patients with an exacerbation of the disease and a persistence of M. abscessus . We have previously shown that the exacerbation of the Toll-like receptor 2 (TLR2)-mediated inflammatory response, following this S to R transition, is essentially due to overproduction of bacilli cell envelope surface compounds, which we were able to extract by mechanical treatment and isolation by solvent partition in a fraction called interphase. Here, we set up a purification procedure guided by bioactivity to isolate a fraction from the R variant of M. abscessus cells which exhibits a high TLR2 stimulating activity, referred to as TLR2-enriched fraction (TLR2eF). As expected, TLR2eF was found to contain several lipoproteins and proteins known to be stimuli for TLR2. Vaccination with TLR2eF showed no protection toward an M. abscessus aerosol challenge, but provided mild protection in ΔF508 mice and their FVB littermates when intravenously challenged by M. abscessus . Interestingly however, antibodies against TLR2eF compounds were detected during disease in CF patients. In conclusion, we show the potential for compounds in TLR2eF as vaccine and diagnostic candidates, in order to enhance diagnosis, prevent and/or treat M. abscessus -related infections., (Copyright © 2020 Le Moigne, Roux, Jobart-Malfait, Blanc, Chaoui, Burlet-Schiltz, Gaillard, Canaan, Nigou and Herrmann.)

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., (Copyright © 2020 American Society for Microbiology.)

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.

Complete Freund's adjuvant (CFA) has historically been one of the most useful tools of immunologists. Essentially comprised of dead mycobacteria and mineral oil, we asked ourselves what is special about the mycobacterial part of this adjuvant, and could it be recapitulated synthetically? Here, we demonstrate the essentiality of N-glycolylated peptidoglycan plus trehalose dimycolate (both unique in mycobacteria) for the complete adjuvant effect using knockouts and chemical complementation. A combination of synthetic N-glycolyl muramyl dipeptide and minimal trehalose dimycolate motif GlcC14C18 was able to upregulate dendritic cell effectors, plus induce experimental autoimmunity qualitatively similar but quantitatively milder compared to CFA. This research outlines how to substitute CFA with a consistent, molecularly-defined adjuvant which may inform the design of immunotherapeutic agents and vaccines benefitting from cell-mediated immunity. We also anticipate using synthetic microbe-associated molecular patterns (MAMPs) to study mycobacterial immunity and immunopathogenesis.

Autophagy is an important innate immune defense mechanism that controls Mycobacterium tuberculosis ( Mtb ) growth inside macrophages. Autophagy machinery targets Mtb -containing phagosomes via xenophagy after damage to the phagosomal membrane due to the Type VII secretion system Esx-1 or via LC3-associated phagocytosis without phagosomal damage. Conversely, Mtb restricts autophagy-related pathways via the production of various bacterial protein factors. Although bacterial lipids are known to play strategic functions in Mtb pathogenesis, their role in autophagy manipulation remains largely unexplored. Here, we report that the lipid virulence factors sulfoglycolipids (SLs) and phthiocerol dimycocerosates (DIMs) control autophagy-related pathways through distinct mechanisms in human macrophages. Using knock-out and knock-in mutants of Mtb and Mycobacterium bovis BCG (Bacille Calmette Guerin) and purified lipids, we found that (i) Mtb mutants with DIM and SL deficiencies promoted functional autophagy via an MyD88-dependent and phagosomal damage-independent pathway in human macrophages; (ii) SLs limited this pathway by acting as TLR2 antagonists; (iii) DIMs prevented phagosomal damage-independent autophagy while promoting Esx-1-dependent xenophagy; (iv) and DIMs, but not SLs, limited the acidification of LC3-positive Mtb compartments. In total, our study reveals an unexpected and intricate role for Mtb lipid virulence factors in controlling autophagy-related pathways in human macrophages, thus providing further insight into the autophagy manipulation tactics deployed by intracellular bacterial pathogens., Competing Interests: The authors declare no conflicts of interest.

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., (© 2019 Palčeková et al.)

Dectin-2 is a C-type lectin involved in the recognition of several pathogens such as Aspergillus fumigatus, Candida albicans, Schistosoma mansonii, and Mycobacterium tuberculosis that triggers Th17 immune responses. Identifying pathogen ligands and understanding the molecular basis of their recognition is one of the current challenges. Purified M. tuberculosis mannose-capped lipoarabinomannan (ManLAM) was shown to induce signaling via Dectin-2, an activity that requires the (α1 → 2)-linked mannosides forming the caps. Here, using isogenic M. tuberculosis mutant strains, we demonstrate that ManLAM is a bona fide and actually the sole ligand mediating bacilli recognition by Dectin-2, although M. tuberculosis produces a variety of cell envelope mannoconjugates, such as phosphatidyl-myo-inositol hexamannosides, lipomannan or manno(lipo)proteins, that bear (α1 → 2)-linked mannosides. In addition, we found that Dectin-2 can recognize lipoglycans from other bacterial species, such as Saccharotrix aerocolonigenes or the human opportunistic pathogen Tsukamurella paurometabola, suggesting that lipoglycans are prototypical Dectin-2 ligands. Finally, from a structure/function relationship perspective, we show, using lipoglycan variants and synthetic mannodendrimers, that dimannoside caps and multivalent interaction are required for ligand binding to and signaling via Dectin-2. Better understanding of the molecular basis of ligand recognition by Dectin-2 will pave the way for the rational design of potent adjuvants targeting this receptor.

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., Competing Interests: The authors declare no conflict of interest.

Structural modification of lipid A, the lipid anchor of LPS, is one of the strategies used by Gram-negative bacteria to evade host innate immunity. Francisella tularensis is a human pathogen that infects and replicates within phagocytic cells. It produces an atypical lipid A, whose structure precludes an efficient recognition by both innate immune players, TLR4 and cationic antimicrobial peptides. Interestingly, a recent report indicates that the lipid A of Francisella (LVS vaccinal strain) undergoes polar modifications when bacteria are grown in human macrophages as compared to in broth. To characterize the structural modifications of lipid A that may be induced intracellularly, Francisella novicida, a surrogate strain for the highly virulent F. tularensis, was submitted to different stress conditions mimicking the harsh environment encountered in the macrophages. To analyze lipid A directly from intact bacteria without any chemical treatment or purification steps, we used a rapid and sensitive MALDI-TOF mass spectrometry approach. Among the many conditions tested, only bacteria exposure to acidic pHs (from 6 to 5) induced a change in lipid A structure. These changes were characterized by an increase in the relative abundance of molecular species bearing an additional hexose unit on the diglucosamine backbone, similar to species present when bacteria are grown under reduced environmental temperature. This lipid A glyco-form, which is observed in trace amounts in normal in vitro growth conditions at 37 °C, may contribute to the intracellular parasitism of macrophages by Francisella., (Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Lipoarabinomannan (LAM) is a lipoglycan found in abundant quantities in the cell envelope of all mycobacteria. The nonreducing arabinan termini of LAM display species-specific structural microheterogeneity that impacts the biological activity of the entire molecule. Mycobacterium tuberculosis, for instance, produces mannoside caps made of one to three α-(1 → 2)-Manp-linked residues that may be further substituted with an α-(1 → 4)-linked methylthio-d-xylose (MTX) residue. While the biological functions and catalytic steps leading to the formation of the mannoside caps of M. tuberculosis LAM have been well established, the biosynthetic origin and biological relevance of the MTX motif remain elusive. We here report on the discovery of a five-gene cluster dedicated to the biosynthesis of the MTX capping motif of M. tuberculosis LAM, and on the functional characterization of two glycosyltransferases, MtxS and MtxT, responsible, respectively, for the production of decaprenyl-phospho-MTX (DP-MTX) and the transfer of MTX from DP-MTX to the mannoside caps of LAM. Collectively, our NMR spectroscopic and mass spectrometric analyses of mtxS and mtxT overexpressors and knockout mutants support a biosynthetic model wherein the conversion of 5'-methylthioadenosine, which is a ubiquitous byproduct of spermidine biosynthesis, into 5'-methylthioribose-1-phosphate precedes the formation of a 5'-methylthioribose nucleotide sugar, followed by the epimerization at C-3 of the ribose residue, and the transfer of MTX from the nucleotide sugar to decaprenyl-phosphate yielding the substrate for transfer onto LAM. The conservation of the MTX biosynthetic genes in a number of Actinomycetes suggests that this discrete glycosyl substituent may be more widespread in prokaryotes than originally thought.

Post-translational modifications (PTMs) are essential processes conditioning the biophysical properties and biological activities of the vast majority of mature proteins. However, occurrence of several distinct PTMs on a same protein dramatically increases its molecular diversity. The comprehensive understanding of the functionalities resulting from any particular PTM association requires a highly challenging full structural description of the PTM combinations. Here, we report the in-depth exploration of the natural structural diversity of the M. tuberculosis (Mtb) virulence associated 19 kDa lipoglycoprotein antigen (LpqH) using intact protein high-resolution mass spectrometry (HR-MS) coupled to liquid chromatography. Combined top-down and bottom-up HR-MS analyses of the purified Mtb LpqH protein allow, for the first time, to uncover a complex repertoire of about 130 molecular species resulting from the intrinsically heterogeneous combination of lipidation and glycosylation together with some truncations. Direct view on the co-occurring PTMs stoichiometry reveals the presence of functionally distinct LpqH lipidation states and indicates that glycosylation is independent from lipidation. This work allowed the identification of a novel unsuspected phosphorylated form of the unprocessed preprolipoglycoprotein totally absent from the current lipoglycoprotein biogenesis pathway and providing new insights into the biogenesis and functional determinants of the mycobacterial lipoglycoprotein interacting with the host immune PRRs.

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.