Structure and biological activities of a hexosamine-rich cell wall polysaccharide isolated from the probiotic Lactobacillus farciminis.

Lactobacillus farciminis CIP 103136 is a bacterial strain with recognized probiotic properties. However, the mechanisms underlying such properties have only been partially elucidated. In this study, we isolated and purified a cell-wall associated polysaccharide (CWPS), and evaluated its biological role in vitro. The structure of CWPS and responses from stimulation of (i) human macrophage-like THP-1 cells, (ii) human embryonal kidney (HEK293) cells stably transfected with Toll-like receptors (TLR2 or TLR4) and (iii) human colonocyte-like T84 intestinal epithelial cells, upon exposure to CWPS were studied. The structure of the purified CWPS from L. farciminis CIP 103136 was analyzed by nuclear magnetic resonance (NMR), MALDI-TOF-TOF MS, and methylation analyses in its native form and following Smith degradation. It was shown to be a novel branched polysaccharide, composed of linear backbone of trisaccharide repeating units of: [→6αGlcpNAc1 → 4ßManpNAc1 → 4ßGlcpNAc1→] highly substituted with single residues of αGlcp, αGalp and αGlcpNAc. Subsequently, the lack of pro- or anti-inflammatory properties of CWPS was established on macrophage-like THP-1 cells. In addition, CWPS failed to modulate cell signaling pathways dependent of TLR2 and TLR4 in transfected HEK-cells. Finally, in T84 cells, CWPS neither influenced intestinal barrier integrity under basal conditions nor prevented TNF-α/IFN-γ cytokine-mediated epithelium impairment.

Glycosylation Changes Triggered by the Differentiation of Monocytic THP-1 Cell Line into Macrophages.

The human acute monocytic leukemia cell line THP-1 is widely used as an in vitro phagocytic cell model because it exhibits several immune properties similar to native monocyte-derived macrophages. In this study, we investigated the alteration of N- and O-linked glycans as well as glycosphingolipids, during THP-1 differentiation, combining mass spectrometry, flow cytometry, and quantitative real-time PCR. Mass spectrometry revealed that macrophage differentiation led to a marked upregulation of expression of GM3 ganglioside as well as an increase in complex-type structures, particularly triantennary glycans, occurring at the expense of high-mannose N-glycans. Moreover, we observed a slight decrease in the proportion of multifucosylated N-glycans and α2,6-sialylation. The uncovered changes in glycosylation correlated with variations of gene expression of relevant glycosyltransferases and glycosidases including sialyltransferases, ß-N-acetylglucosaminyltransferases, fucosyltransferases, and neuraminidase. Furthermore, using flow cytometry and antibodies directed against glycan structures, we confirmed that the alteration of glycosylation occurs at the cell surface of THP-1 macrophage-like cells. Altogether, we established that macrophagic maturation of THP-1 induces dramatic modifications of the surface glycosylation pattern that may result in differential interaction of monocytic and macrophagic THP-1 with immune or bacterial lectins.

Structural determination and Toll-like receptor 2-dependent proinflammatory activity of dimycolyl-diarabino-glycerol from Mycobacterium marinum.

Although it was identified in the cell wall of several pathogenic mycobacteria, the biological properties of dimycolyl-diarabino-glycerol have not been documented yet. In this study an apolar glycolipid, presumably corresponding to dimycolyl-diarabino-glycerol, was purified from Mycobacterium marinum and subsequently identified as a 5-O-mycolyl-ß-Araf-(1→2)-5-O-mycolyl-α-Araf-(1→1')-glycerol (designated Mma_DMAG) using a combination of nuclear magnetic resonance spectroscopy and mass spectrometry analyses. Lipid composition analysis revealed that mycolic acids were dominated by oxygenated mycolates over α-mycolates and devoid of trans-cyclopropane functions. Highly purified Mma_DMAG was used to demonstrate its immunomodulatory activity. Mma_DMAG was found to induce the secretion of proinflammatory cytokines (TNF-α, IL-8, IL-1ß) in human macrophage THP-1 cells and to trigger the expression of ICAM-1 and CD40 cell surface antigens. This activation mechanism was dependent on TLR2, but not on TLR4, as demonstrated by (i) the use of neutralizing anti-TLR2 and -TLR4 antibodies and by (ii) the detection of secreted alkaline phosphatase in HEK293 cells co-transfected with the human TLR2 and secreted embryonic alkaline phosphatase reporter genes. In addition, transcriptomic analyses indicated that various genes encoding proinflammatory factors were up-regulated after exposure of THP-1 cells to Mma_DMAG. Importantly, a wealth of other regulated genes related to immune and inflammatory responses, including chemokines/cytokines and their respective receptors, adhesion molecules, and metalloproteinases, were found to be modulated by Mma_DMAG. Overall, this study suggests that DMAG may be an active cell wall glycoconjugate driving host-pathogen interactions and participating in the immunopathogenesis of mycobacterial infections.

Exposure of mycobacteria to cell wall-inhibitory drugs decreases production of arabinoglycerolipid related to Mycolyl-arabinogalactan-peptidoglycan metabolism.

The "cell wall core" consisting of a mycolyl-arabinogalactan-peptidoglycan (mAGP) complex represents the hallmark of the mycobacterial cell envelope. It has been the focus of intense research at both structural and biosynthetic levels during the past few decades. Because it is essential, mAGP is also regarded as a target for several antitubercular drugs. Herein, we demonstrate that exposure of Mycobacterium bovis Bacille Calmette-Guérin or Mycobacterium marinum to thiacetazone, a second line antitubercular drug, is associated with a severe decrease in the level of a major apolar glycolipid. This inhibition requires MmaA4, a methyltransferase reported to participate in the activation process of thiacetazone. Following purification, this glycolipid was subjected to detailed structural analyses, combining gas-liquid chromatography, mass spectrometry, and nuclear magnetic resonance. This allowed to identify it as a 5-O-mycolyl-ß-Araf-(1→2)-5-O-mycolyl-α-Araf-(1→1)-Gro, designated dimycolyl diarabinoglycerol (DMAG). The presence of DMAG was subsequently confirmed in other slow growing pathogenic species, including Mycobacterium tuberculosis. DMAG production was stimulated in the presence of exogenous glycerol. Interestingly, DMAG appears structurally identical to the terminal portion of the mycolylated arabinosyl motif of mAGP, and the metabolic relationship between these two components was provided using antitubercular drugs such as ethambutol or isoniazid known to inhibit the biosynthesis of arabinogalactan or mycolic acid, respectively. Finally, DMAG was identified in the cell wall of M. tuberculosis. This opens the possibility of a potent biological function for DMAG that may be important to mycobacterial pathogenesis.

Mycobacterium bovis BCG infection alters the macrophage N-glycome.

Macrophage glycosylation is essential to initiate the host-immune defense but may also be targeted by pathogens to promote infection. Indeed, the alteration of the cell-surface glycosylation status may affect the binding of lectins involved in cell activation and adhesion. Herein, we demonstrate that infection by M. bovis BCG induces the remodeling of the N-glycomes of both human primary blood monocyte-derived macrophages (MDM) and macrophage-cell line THP1. MALDI-MS based N-glycomic analysis established that mycobacterial infection induced increased synthesis of biantennary and multifucosylated complex type N-glycans. In contrast, infection of macrophages by M. bovis BCG did not modify the glycosphingolipids composition of macrophages. Further nano-LC-MSn glycotope-centric analysis of total N-glycans demonstrated that the increased fucosylation was due to an increased expression of the Lex (Galß1-4[Fucα1-3]GlcNAc) epitope, also known as stage-specific embryonic antigen-1. Modification of the surface expression of Lex was further confirmed in both MDM and THP-1 cells by FACS analysis using an α1,3-linked fucose specific lectin. Activation with the mycobacterial lipopeptide Pam3Lp19, an agonist of toll-like receptor 2, did not modify the overall fucosylation pattern, which suggests that the infection process is required to modify surface glycosylation. These results pave the way toward the understanding of infection-triggered cell-surface remodeling of macrophages.

Identification of the Mycobacterium marinum Apa antigen O-mannosylation sites reveals important glycosylation variability with the M. tuberculosis Apa homologue.

The 45/47 kDa Apa, an immuno-dominant antigen secreted by Mycobacterium tuberculosis is O-mannosylated at multiple sites. Glycosylation of Apa plays a key role in colonization and invasion of the host cells by M. tuberculosis through interactions of Apa with the host immune system C-type lectins. Mycobacterium marinum (M.ma) a fish pathogen, phylogenetically close to M. tuberculosis, induces a granulomatous response with features similar to those described for M. tuberculosis in human. Although M.ma possesses an Apa homologue, its glycosylation status is unknown, and whether this represents a crucial element in the pathophysiology induced by M.ma remains to be addressed. To this aim, we have identified two concanavalin A-reactive 45/47 kDa proteins from M.ma, which have been further purified by a two-step anion exchange chromatography process. Advanced liquid chromatography-nanoESI mass spectrometry-based proteomic analyses of peptides, derived from either tryptic digestion alone or in combination with the Asp-N endoproteinase, established that M.ma Apa possesses up to seven distinct O-mannosylated sites with mainly single mannose substitutions, which can be further extended at the Ser/Thr/Pro rich region near the N-terminus. This opens the way to further studies focussing on the involvement and biological functions of Apa O-mannosylation using the M.ma/zebrafish model.