Mannose-capped Lipoarabinomannan Research Articles

One-Pot Assembly of Mannose-Capped Lipoarabinomannan Motifs up to 101-Mer from the Mycobacterium tuberculosis Cell Wall.

Lipoarabinomannan (LAM) from the Mycobacterium tuberculosis cell envelope represents important targets for the development of new therapeutic agents against tuberculosis, which is a deadly disease that has plagued mankind for a long time. However, the accessibility of long, branched, and complex lipoarabinomannan over 100-mer remains a long-standing challenge. Herein, we report the modular synthesis of mannose-capped lipoarabinomannan 101-mer from the M. tuberculosis cell wall using a one-pot assembly strategy on the basis of glycosyl ortho-(1-phenylvinyl)benzoates (PVB), which not only accelerates the modular synthesis but also precludes the potential problems associated with one-pot glycosylation with thioglycosides. Shorter sequences including 18-mer, 19-mer, and 27-mer are also synthesized for in-depth structure-activity relationship biological studies. Current synthetic routes also highlight the following features: (1) streamlined synthesis of various linear and branched glycans using one-pot orthogonal glycosylation on the combination of glycosyl N-phenyltrifluoroacetimidates, glycosyl ortho-alkynylbenzoates, and glycosyl PVB; (2) highly stereoselective construction of 10 1,2-cis-arabinofuranosyl linkages using 5-O-(2-quinolinecarbonyl)-directing 1,2-cis-arabinofuranosylation via a hydrogen-bond-mediated aglycone delivery strategy; and (3) convergent [(18 + 19) × 2 + 27] one-pot synthesis of the 101-mer LAM polysaccharide. The present work demonstrates that this orthogonal one-pot glycosylation strategy can highly streamline the chemical synthesis of long, branched, and complex polysaccharides.

Microfluidic platform for the enzymatic pretreatment of human serum for the detection of the tuberculosis biomarker mannose-capped lipoarabinomannan.

Tuberculosis (TB) represents a major public health threat, with millions of new cases reported worldwide each year. A major hurdle to curtailing the spread of this disease is the need for low-cost, point-of-care (PoC) diagnostics. Mannose-capped lipoarabinomannan, a significant component of the Mycobacterium tuberculosis bacillus, has been heavily studied as a biomarker for TB, but with little success due to its complexation with endogenous components of body fluids in a manner that sterically interferes with its detection by ELISA and other immunoassays. Recent work by our group and others has shown that complexation can be disrupted with protein-denaturing protocols. By way of followup, we recently described an enzymatic digestion (Proteinase K) sample pretreatment that enables quantitative recovery of ManLAM spiked into healthy human control serum. Herein, we report on the transfer of our benchtop sample pretreatment methodology to an automated microfluidic platform. We show that this platform can be configured to: (1) carry out the pretreatment process with very little user interaction and, (2) yield recoveries for ManLAm spiked into control serum which are statistically indistinguishable from those achieved by the benchtop process. Plans to integrate this device with a portable sample reader as a possible basis for a PoC TB diagnostic system and analyze patient samples are briefly discussed.

Rapid synthesis of α(1,2)-d-mannans on the surface of Mycobacterium tuberculosis and their nitric oxide-inducing activities

The mannose cap motif, which consists of short-chain α(1,2)-d-mannans located on the non-reducing end of Mycobacterium tuberculosis’ mannose-capped lipoarabinomannan (ManLAM), exhibits strong binding affinities toward the lectins on mammalian host cells. The α(1,2)-d-mannans induce the immunosuppressive effects crucial for the survival and virulence of mycobacteria. However, the biological activities of discrete α(1,2)-d-mannans with different chain lengths remain unknown. In this study, the rapid synthesis of α(1,2)-d-mannans was rationally developed to increase the accessibility of α(1,2)-d-mannans. The rapid synthesis enabled simultaneous productions of the protected α(1,2)-d-mannans with chain lengths of 2–5 units, at room temperature (25 °C), within 4 h. After hydrolysis unmasking, different sizes of α(1,2)-d-mannans, and their α(1,6)-d-mannan counterparts were obtained and biologically evaluated. In summary, this work led to the optimally-designed low-steric-hindrance bicyclic orthoester monomer suitable for the gram-scale rapid synthesis of α(1,2)-d-mannans at 25 °C. This provided a basis for further development of more efficient syntheses for glycans with (1,2)-glycosidic linkages. Notably, the improved accessibility to these glycan motifs allowed us to discover that the α(1,2)-d-mannobioside could significantly enhance LPS-induced nitric oxide production in macrophages, suggesting its potential use as an immunomodulator and implying the existence of an unknown pathway or receptor responsible for this effect.

Proteinase K Pretreatment for the Quantitative Recovery and Sensitive Detection of the Tuberculosis Biomarker Mannose-Capped Lipoarabinomannan Spiked into Human Serum.

This paper reports on an investigation of an enzymatic pretreatment protocol using proteinase K (ProK) for the analysis of human serum samples spiked with mannose-capped lipoarabinomannan (ManLAM). ManLAM is an antigenic biomarker found in the serum, urine, and other body fluids of individuals infected with tuberculosis (TB). Immunometric measurements of ManLAM are compromised by steric effects due to its complexation with high-molecular-weight components in these matrices that interfere with its capture and/or labeling. Recent work has shown that deproteinization of these types of samples by perchloric acid acidification or ProK digestion releases ManLAM from complexation. Releasing ManLAM greatly improves its detectability and, as a result, its utility as a TB biomarker. The work detailed herein examined how different ProK reaction conditions (e.g., enzyme concentration and digestion time and temperature) affect the recovery and detectability of ManLAM in human serum. As measured by enzyme-linked immunosorbent assay (ELISA), we show that using the optimal set of digestion conditions to free ManLAM, which also yield a small, quantitatively reproducible level of sample concentration, it is possible to achieve a spiked ManLAM recovery of 98 ± 13% and a limit of detection of 10 pg/mL (0.6 pM). Experiments also demonstrated that the ELISA responses measured for a given ManLAM concentration in serum after pretreatment were statistically indistinguishable from those directly determined for the same amounts of ManLAM added to an innocuous buffered solution. Possible adaptations of the digestion protocol for use in point-of-care TB testing are also briefly discussed.

Polysaccharides and glycolipids of Mycobacterium tuberculosis and their induced immune responses.

Tuberculosis (TB) is a chronic infectious disease mainly caused by Mycobacterium tuberculosis (M. tuberculosis). The structures of polysaccharides and glycolipids at M. tuberculosis cell wall vary among different strains, which affect the physiology and pathogenesis of mycobacteria by activating or inhibiting innate and acquired immunity. Among them, some components such as lipomannan (LM) and lipoarabinomannan (LAM) activate innate immunity by recognizing some kinds of pattern recognition receptors (PRRs) like Toll-like receptors, while other components like mannose-capped lipoarabinomannan (ManLAM) could prevent innate immune responses by inhibiting the secretion of pro-inflammatory cytokines and maturation of phagosomes. In addition, many glycolipids can activate natural killer T (NKT) cells and CD1-restricted T cells to produce interferon-γ (IFN-γ). Furthermore, humoral immunity against cell wall components, such as antibodies against LAM, plays a role in immunity against M. tuberculosis infection. Cell wall polysaccharides and glycolipids of M. tuberculosis have potential applications as antigens and adjuvants for novel TB subunit vaccines.

Protective Effect of the T1212C Macrophage Mannose Receptor Gene Polymorphism on Pulmonary Tuberculosis

The interaction between the mannose receptor, which is encoded by the macrophage mannose receptor (MMR) gene, and the most virulent antigen (the mannose-capped lipoarabinomannan) cell wall of virulent strains of Mycobacterium tuberculosis trigger an innate and adaptive immune response. It also produces pro and anti-inflammatory cytokines, which play a role in the pathogenesis of tuberculosis (TB) infection. Therefore, MMR gene polymorphism is a risk factor and is associated with the prognosis for active pulmonary TB. This study aimed to determine the correlation between MMR gene polymorphism and active or latent pulmonary tuberculosis. In this phase, MMR gene polymorphism was analyzed using a case-control design consisting of 74 control group subjects (patients with latent TB) and 74 case groups (patients with active pulmonary TB). The subject’s MMR gene DNA sequencing examination. The study was conducted at the Teaching Hospital, Faculty of Medicine, Universitas Padjadjaran Bandung, from February 2014 to January 2015. The statistical analysis used chi-square and odds ratio. The study’s result has shown the MMR gene polymorphism factor that correlated to the incidence of active pulmonary TB was T1212C (OR=0.253; 95% CI=0.111−0.575; p=0.001). There was an MMR gene in one SNP in the control group (C1323T) only and five single nucleotide polymorphisms (SNPs) in both groups (C1303T, C1221, T1212C, G1186A, and G1195A). Therefore, it can be concluded that MMR gene polymorphism on the T1212C site correlated with the incidence of active pulmonary tuberculosis and was protective.

KLK12 Regulates MMP-1 and MMP-9 via Bradykinin Receptors: Biomarkers for Differentiating Latent and Active Bovine Tuberculosis.

It has been established that kallikrein12 (KLK12) expression is closely related to bovine tuberculosis (bTB) development. Herein, we sought to clarify the regulatory mechanism of KLK12 and its application in tuberculosis diagnosis. KLK12 knockdown macrophages were produced by siRNA transfection. Bradykinin receptors (BR, including B1R and B2R) were blocked with specific inhibitors. Mannose-capped lipoarabinomannan (ManLAM) was extracted from Mycobacterium bovis (M. bovis) and used to study the mechanism of KLK12 activation. In addition, we constructed different mouse models representing the latent and active stages of M. bovis infection. Mouse models and clinical serum samples were used to assess the diagnostic value of biomarkers. Through the above methods, we confirmed that KLK12 regulates MMP-1 and MMP-9 via BR. KLK12 upregulation is mediated by the M. bovis-specific antigen ManLAM. KLK12, MMP-1, and MMP-9 harbor significant value as serological markers for differentiating between latent and active bTB, especially KLK12. In conclusion, we identified a novel signaling pathway, KLK12/BR/ERK/MMPs, in M. bovis-infected macrophages, which is activated by ManLAM. From this signaling pathway, KLK12 can be used as a serological marker to differentiate between latent and active bTB. Importantly, KLK12 also has enormous potential for the clinical diagnosis of human tuberculosis (TB).

The Rv2623-Rv1747 interaction influences phosphatidyl-myo-inositol levels on the cell envelope of Mycobacterium tuberculosis

Abstract Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), has a complex cell envelope that provides a barrier of protection to various environments. Peripheral lipids on the M.tb cell envelope act as virulent factors influencing the host immune response to infection. Of these, some phosphatidyl-myo-inositol mannosides (PIMs) and their associated lipoglycans (lipomannan; mannose-capped lipoarabinomannan) influence M.tb-host interactions by increasing phagocytosis, blocking the maturation of M.tb-containing phagosomes, and increasing the anti-inflammatory response of infected macrophages. However, there is limited knowledge regarding how M.tb regulates PIMs levels, and how this regulation influences infection outcomes. In our previous studies, we hypothesized that Rv2623 and Rv1747 work together to regulate PIMs on the M.tb cell surface. Indeed, deleting the universal stress protein Rv2623 increases PIMs levels, growth, and virulence in vivo; while deleting the ABC transporter Rv1747 decreases PIMs levels. Here we investigate the mechanistic regulation of M.tb PIMs levels and its impact on growth and pathogenesis in vitro by assessing multiple M.tb strains with Rv2623 or Rv1747 mutated at different amino acids. Depending on the mutation, Rv2623 interacts with Rv1747 to modulate M.tb cell envelope PIMs levels at different degrees. Mutants with increased PIMs levels have increased growth in macrophages, and PIMs levels on the M.tb cell envelope may also correlate to resistance to certain anti-TB drugs. Overall, our data indicate that Rv2623 negatively regulates Rv1747 to modulate PIMs levels on the M.tb cell envelope, ultimately influencing growth and M.tb-host interactions in vitro and in vivo. Supported by grants from NIH/NIAID (1R01AI146340-01A1)

Lactosylceramide-enriched microdomains mediate human neutrophil immunological functions via carbohydrate-carbohydrate interaction

The innate immune system of mammalian cells is the first line of defense against pathogenic microorganisms. Phagocytes, which play the central role in this system, engulf microorganisms by a mechanism that involves pattern recognition receptors on their own surface and pathogen-associated molecular patterns (PAMPs) expressed by the microorganism. Components of PAMPs include glycans (polysaccharides) and glycoconjugates (carbohydrates covalently linked to other biological molecules). Pathogenic microorganisms display specific binding affinity to various types of glycosphingolipids (sphingosine-containing glycolipids; GSLs), and GSLs are involved in host-pathogen interactions. We observed that lactosylceramide (LacCer), a neutral GSL, binds directly to certain pathogen-specific molecules (e.g., Candida albicans-derived β-glucans, mycobacterial lipoarabinomannan) via carbohydrate-carbohydrate interaction. LacCer is expressed highly on human neutrophils, and forms membrane microdomains. Such LacCer-enriched microdomains mediate several important neutrophil functions, including chemotaxis, phagocytosis, and superoxide generation. Human neutrophils phagocytose pathogenic mycobacteria (including Mycobacterium tuberculosis) through carbohydrate-carbohydrate interaction between LacCer on their own surface and mannose-capped lipoarabinomannan on the bacterium. During recognition of pathogen-specific glycans, direct association of LacCer-containing C24 fatty acid chain with Lyn (a Src family kinase) is necessary for signal transduction from the neutrophil exterior to interior. Pathogenic mycobacteria utilize a similar interaction to avoid killing by neutrophils. We describe here the mechanisms whereby LacCer mediates neutrophil immune systems via carbohydrate-carbohydrate interaction.

The Regulation of ManLAM-Related Gene Expression in Mycobacterium tuberculosis with Different Drug Resistance Profiles Following Isoniazid Treatment.

IntroductionTuberculosis (TB) caused by Mycobacterium tuberculosis (MTB) remains a global health concern because of the development of drug resistance. The adaptability of MTB in response to a variety of environmental stresses is a crucial strategy that supports their survival and evades host defense mechanisms. Stress regulates gene expression, particularly virulence genes, leading to the development of drug tolerance. Mannose-capped lipoarabinomannan (ManLAM) is a critical component of the cell wall, functions as a virulence factor and influences host defense mechanisms.PurposeThis study focuses on the effect of isoniazid (INH) stress on the regulation of ManLAM-related genes, to improve our understanding of virulence and drug resistance development in MTB.Materials and MethodsMTB with distinct drug resistance profiles were used for gene expression analysis. Multiplex-real time PCR assay was performed to monitor stress-related genes (hspX, tgs1, and sigE). The expression levels of ManLAM-related genes (pimB, mptA, mptC, dprE1, dprE2, and embC) were quantified by qRT-PCR. Sequence analysis of drug resistance-associated genes (inhA, katG, and rpoB) and ManLAM-related genes were performed to establish a correlation between genetic variation and gene expression.ResultsINH treatment activates the stress response mechanism in MTB, resulting in a distinct gene expression pattern between drug resistance and drug-sensitive TB. In response to INH, hspX was up-regulated in RIF-R and MDR. tgs1 was strongly up-regulated in MDR, whereas sigE was dramatically up-regulated in the drug-sensitive TB. Interestingly, ManLAM-related genes were most up-regulated in drug resistance, notably MDR (pimB, mptA, dprE1, and embC), implying a role for drug resistance and adaptability of MTB via ManLAM modulation.ConclusionThis study establishes a relationship between the antibiotic stress response mechanism and the expression of ManLAM-related genes in MTB samples with diverse drug resistance profiles. The novel gene expression pattern in this work is valuable knowledge that can be applied for TB monitoring and treatment in the future.

Bioinspired design of mannose-decorated globular lysine dendrimers promotes diabetic wound healing by orchestrating appropriate macrophage polarization

A large number of cytokines or growth factors have been used in the treatment of inflammation. However, they are highly dependent on an optimal delivery system with sufficient loading efficiency and protection of growth factors from proteolytic degradation. To develop the immunotherapy capacity of peptide dendrimers themselves, inspired by the structure and immunoregulatory functions of mannose-capped lipoarabinomannan (ManLAM), we thus propose a hypothesis that mannose-decorated globular lysine dendrimers (MGLDs) with precise molecular design can elicit anti-inflammatory activity through targeting and reprogramming macrophages to M2 phenotype. To achieve this, a series of mannose-decorated globular lysine dendrimers (MGLDs) was developed. Size-controlled MGLDs obtained were spherical with positive surface charges. The mean size ranged from 50-200 nm in varying generations and modification degrees. The initial screening study revealed that MGLDs have superior biocompatibility. When cocultured with MGLDs, mouse bone marrow-derived macrophages (BMDMs) acquired an anti-inflammatory M2 phenotype characterized by significant mannose receptor (MR) clustering on the cell surface and the elongated shape, an increased production of transforming growth factor (TGF)-β1, interleukin (IL)-4 and IL-10, a downregulated secretory of IL-1β, IL-6, and tumor necrosis factor (TNF)-α, and increased ability to induce fibroblast proliferation. Then in vivo studies further demonstrated that topical administration of optimized MGLDs accelerates wound repair of full-thickness cutaneous defects in type 2 diabetic mice via M2 macrophage polarization. Mechanistically, MGLDs treatment showed an enhanced closure rate, collagen deposition, and angiogenesis, along with mitigated inflammation modulated by a suppressed secretory of pro-inflammation cytokines, and increased production of TGF-β1. These findings provide the first evidence that the bioinspired design of MGLDs can direct M2 macrophage polarization, which may be beneficial in the therapy of injuries and inflammation.

Aptamer Detection of Mycobaterium tuberculosis Mannose-Capped Lipoarabinomannan in Lesion Tissues for Tuberculosis Diagnosis

Tuberculosis (TB) is the leading infectious cause of mortality worldwide. However, the diagnosis of TB, especially extrapulmonary TB (EPTB) diagnosis from lesion tissues, remains a challenge. Nucleic acid aptamers are analogous to antibodies and have advantages of easier modification, high specificity, and affinity. Mannose-capped lipoarabinomannan (ManLAM) is a unique surface lipoglycan component or constantly released from mycobacterium tuberculosis (M.tb) cell wall, which makes it a perfect candidate biomarker for TB diagnosis. Our present study aims to establish M.tb ManLAM aptamer-based immunohistochemistry (IHC) method for TB diagnosis. We performed TB diagnosis using 263 formalin-fixed paraffin-embedded tissue samples including 213 TB samples (pulmonary TB (PTB) and EPTB), and 8 samples from latent TB infection (LTBI) high risk subjects, and 42 samples from other non-TB patients with ManLAM aptamer-based IHC and routine laboratory TB diagnostic methods parallelly. The sensitivity and specificity of the ManLAM aptamer-based IHC were 86.38% and 92.86%, with much higher sensitivity than those of mycobacterial culture (9.66%) and acid-fast staining (AFS) (43.01%) and comparability to Interferon-gamma Release Assay (IGRA) (84.38%) and GeneXpert (79.31%). High agreement between ManLAM based-IHC and IGRA or GeneXpert for TB diagnosis were observed. Furthermore, ManLAM aptamer-based IHC combination with other routine TB laboratory diagnostic methods significantly increased the sensitivity up to 88.64%–97.92%. As our knowledge, this is the first report about aptamer-based IHC for disease diagnosis. Thus, ManLAM aptamer-based IHC has potentials for TB diagnosis, including PTB, and EPTB, and assists the diagnosis of LTBI with high effectiveness, feasibility, and easy production.

The cyanobacterial lectin, microvirin-N, enhances the specificity and sensitivity of lipoarabinomannan-based TB diagnostic tests.

Tuberculosis (TB) is one of the top ten causes of death globally, despite being treatable. The eradication of TB disease requires, amongst others, diagnostic tests with high specificity and sensitivity that will work at the point of care (POC) in low-resource settings. The TB surface glycolipid antigen, mannose-capped lipoarabinomannan (ManLAM) currently serves as the only POC molecular diagnostic biomarker suitable for use in low cost immunoassays. Here, we demonstrate the high affinity and exceptional specificity of microvirin-N (MVN), a 14.3 kDa cyanobacterial lectin, toward H37Rv TB ManLAM and utilize it to develop a novel on-bead ELISA. MVN binds to ManLAM with sub-picomolar binding affinity, but does not bind to other variants of LAM expressed by non-pathogenic mycobacteria – a level of binding specificity and affinity that current commercially available anti-LAM antibodies cannot achieve. An on-bead ELISA was subsequently developed using MVN-functionalized magnetic beads which allows for the specific capture of ManLAM from human urine with a limit of detection (LOD) of 1.14 ng mL−1 and no cross-reactivity when tested with PILAM, a variant of LAM found on non-pathogenic mycobacteria.

A plasmonic fiberoptic absorbance biosensor for mannose-capped lipoarabinomannan based tuberculosis diagnosis

Tuberculosis (TB) is a resurgent infectious disease affecting a large number of people in the developing countries. An on-site, affordable diagnostic screening at an early-stage for an immediate anti-TB treatment is known to tremendously minimize the high mortality rates. Lipoarabinomannan (LAM), a surface glycolipid, has been identified as a potential TB biomarker present in urine at ultra-low concentrations of a few fg/mL. Here, we report a plasmonic fiber optic absorbance biosensor (P-FAB) strategy for mannosylated LAM (Man-LAM or Mtb LAM) detection down to attomolar concentrations. It involves a plasmonic sandwich immunoassay on a U-bent fiber optic probe with gold plasmonic (AuNP) labels functionalized with anti-Mtb LAM immunoglobulin M (IgM) and anti-Mtb LAM IgG respectively. The Mtb LAM is quantified in terms of absorption of light passing through the fiber probe using a green LED and a photodetector. The choice of fiber optic probes (fused silica versus polymer), the optimum size (20, 40, 60 and 80 nm) and concentration (2 × , 10 × , and 20 × ) of AuNP labels were investigated to obtain high sensitivity and lower limits of analyte detection (LoD). P-FAB with a simple LED-photodetector pair, 200 μm fused silica U-bent fiber probe and 60 nm (20 × ) AuNP labels gave LoDs down to 1 fg/mL and 10 fg/mL in the buffer and synthetic urine respectively. Moreover, the anti-Mtb LAM IgM bound sensor probes and the AuNP reagent stored at 4 °C were stable up to 45 days. P-FAB based Mtb LAM sensor demonstrates its potential for an on-site TB diagnosis.

Detection of Mycobacterial Mannose‐capped Lipoarabinomannan via Electrochemical Biosensor

Mycobacterium tuberculosis (M.tb) is one of the world’s most prevalent bacterial pathogens. However, Nontuberculous Mycobacterium (NTM) are less recognized than M.tb while remaining a pressing public health risk. Often, NTM lung disease is confused with that of a tubercle infection leading to unreliable diagnosis. Additionally, the time spent to fully diagnose a mycobacterial disease is extensive and dependent on the species growth rate due to the necessity of culturing for identification. Thus, designing a sensitive, electrochemical DNA‐based (E‐DNA) biosensor as a diagnostic tool for NTM would dramatically decrease the timeline of diagnosis and therefore could improve patient outcomes. In this work, we pursued detection of the cell envelope of NTM, which includes many complex glycolipids and glycopeptidolipids—many of which are believed to have immunopathogenic mechanisms. As mannose‐capped lipoarabinomannan (ManLAM) is one of the most prevalent of the glycolipids, we adapted it as a biomarker for the sensitive detection of NTM. An aptamer specific to ManLAM was incorporated into our E‐DNA biosensor, and exhibits significant current change under square wave voltammetric interrogation.

Merged experimental guided computational strategy toward tuberculosis treatment mediated by alveolar macrophages mannose receptor

Macrophage mannose receptor (MMR) is a C-type lectin that regulates the phagocytosis and phagocytosis-lysosome (P-L) fusion in tuberculosis. Mannose-capped lipoarabinomannan, a lipoglycan present at the surface of Mycobacterium tuberculosis, is an important factor in phagocyte attachment and internalization that is specific for MMR. Based on this idea, herein we have designed our experiment to understand the better site-specific delivery against tuberculosis. An experimental outcome was used as a basis to revisit the reverse experimental strategy for tuberculosis management. Stearic mannose was prepared from stearic acid incubation with the D-mannose. Interestingly, stearic mannose explained its internalization via stimulating actin-mediated phagocytic pathway of MMR experimentally. Following, an in silico strategy towards hypothetical designing of various mannose-stearyl conjugates (SBKK1-7) against tuberculosis, as binding promoter of MMR (PDB: 1EGI), was carried out using molecular docking and dynamics approaches. Overall, SPKK-5 viz. ortho stearic mannose showed a higher binding affinity with notable H-bonding and hydrophobic interactions. Pharmacokinetic and toxicity examinations illustrated an ideal range of descriptors values for apex screened compounds. Molecular dynamics simulations have confirmed its significant intactness with the MMR. Ultimately, the whole effort led to the identification of promising hit (SBKK-5), which positively correlates with the experimental work and furthermore need to explore its novel drug delivery systems with improved anti-tubercular therapy.Communicated by Ramaswamy H. Sarma

Mannose-capped lipoarabinomannan-induced B10 cells decrease severity of dextran sodium sulphate-induced inflammatory bowel disease in mice.

Inflammatory bowel disease (IBD) is a chronic, non‐specific, inflammatory gastrointestinal disease that mainly consists of Crohn's disease and ulcerative colitis. However, the aetiology and pathogenesis of IBD are still unclear. B10 (IL‐10 producing regulatory B) cells, a subset of regulatory B cells, are known to contribute to intestinal homeostasis and the aberrant frequency of B10 cells is associated with IBD. We have recently reported that B10 cells can be induced by ManLAM (mannose‐capped lipoarabinomannan), a major cell‐wall lipoglycan of M tb (Mycobacterium tuberculosis). In the current study, the ManLAM‐induced B10 cells were adoptively transferred into IL(interleukin)‐10−/− mice and the roles of ManLAM‐induced B10 cells were investigated in DSS (dextran sodium sulphate)‐induced IBD model. ManLAM‐induced B10 cells decrease colitis severity in the mice. The B10 cells downregulate Th1 polarization in spleen and MLNs (mesenteric lymph nodes) of DSS‐treated mice. These results suggest that IL‐10 production by ManLAM‐treated B cells contributes to keeping the balance between CD4+ T cell subsets and protect mice from DSS‐induced IBD.

Mycobacterium tuberculosis Mannose-Capped Lipoarabinomannan Induces IL-10-Producing B Cells and Hinders CD4+Th1 Immunity

SummaryThe importance of Th1/interferon (IFN)-γ-mediated responses in mycobacterial infection has been well established. However, little is known about B cell-mediated immunity during Mycobacterium tuberculosis (Mtb) infection. Interleukin (IL)-10-producing B cells (B10 cells), a subset of B regulatory cells (Bregs), are implicated in modulating the immune response. Herein, we found that B10 cells were significantly increased in patients with tuberculosis. Furthermore, mannose-capped lipoarabinomannan (ManLAM), a major surface lipoglycan component from Mtb, induced a significant increase in B10 cells, which enriched in CD5+ B1a B cells. ManLAM induced IL-10 production mainly by activating MyD88/PI3K/AKT/Ap-1 and K63-linked ubiquitination of NF-κB essential modulator/nuclear factor kappa-light-chain-enhancer of activated B cells signaling pathways in B cells via Toll-like receptor 2. IL-10 production by ManLAM-treated B cells further inhibited CD4+ Th1 polarization, leading to increased susceptibility to mycobacterial infection compared with ManLAM-treated IL-10−/− B group. Thus, we report a new immunoregulation mechanism in which Mtb ManLAM-induced B10 cells negatively regulate host anti-TB cellular immunity.

Deciphering the molecular basis of mycobacteria and lipoglycan recognition by the C-type lectin Dectin-2

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.

Surface‐enhanced resonance Raman scattering for the sensitive detection of a tuberculosis biomarker in human serum

AbstractImprovements in the detection of biomarkers indicative of disease continue to be vital to human health. Toward this end, this paper presents (a) the development and characterization of a biomarker detection strategy based on the inherently stronger signals generated by surface‐enhanced resonance Raman scattering (SERRS), as opposed to surface‐enhanced Raman scattering (SERS); (b) the application of this approach to the detection of mannose‐capped lipoarabinomannan (ManLAM), an antigenic marker indicative of active tuberculosis infection; and (c) a comparison of readout for this SERRS platform to that of the more frequently used approach based on SERS. More specifically, the work detailed herein describes the design and testing of a SERRS immunoassay that incorporates a resonance Raman‐enhanced adlayer of cyanine 5 on a smooth gold capture surface and its application to biomarker detection when “turned on” by the tagging of captured ManLAM with gold nanoparticle labels. The results of these experiments demonstrated an improvement in the detection of ManLAM spiked into human serum in terms of limit of detection by 10× and analytical sensitivity of almost 40× when compared with SERS. Findings also indicate that these improvements arise primarily from the intrinsic increase in signal strength due to the resonance Raman effect and a small but measurable increase in nanoparticle label density. Potential routes to further improve the performance of this approach to immunoassay signal generation are briefly discussed.