Lipoarabinomannan and related glycoconjugates: structure, biogenesis and role in Mycobacterium tuberculosis physiology and host–pathogen interaction

Approximately one third of the world's population is infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. This bacterium has an unusual lipid-rich cell wall containing a vast repertoire of antigens, providing a hydrophobic impermeable barrier against chemical drugs, thus representing an attractive target for vaccine and drug development. Apart from the mycolyl–arabinogalactan–peptidoglycan complex, mycobacteria possess several immunomodulatory constituents, notably lipomannan and lipoarabinomannan. The availability of whole-genome sequences of M. tuberculosis and related bacilli over the past decade has led to the identification and functional characterization of various enzymes and the potential drug targets involved in the biosynthesis of these glycoconjugates. Both lipomannan and lipoarabinomannan possess highly variable chemical structures, which interact with different receptors of the immune system during host–pathogen interactions, such as Toll-like receptors-2 and C-type lectins. Recently, the availability of mutants defective in the synthesis of these glycoconjugates in mycobacteria and the closely related bacterium, Corynebacterium glutamicum, has paved the way for host–pathogen interaction studies, as well as, providing attenuated strains of mycobacteria for the development of new vaccine candidates. This review provides a comprehensive account of the structure, biosynthesis and immunomodulatory properties of these important glycoconjugates

Mycobacterium marinum MMAR_2380, a predicted transmembrane acyltransferase, is essential for the presence of the mannose cap on lipoarabinomannan

Lipoarabinomannan (LAM) is a major glycolipid in the mycobacterial cell envelope. LAM consists of a mannosylphosphatidylinositol (MPI) anchor, a mannan core and a branched arabinan domain. The termini of the arabinan branches can become substituted with one to three α(1→2)-linked mannosyl residues, the mannose cap, producing ManLAM. ManLAM has been associated with a range of different immunomodulatory properties of Mycobacterium tuberculosis during infection of the host. In some of these effects, the presence of the mannose cap on ManLAM appears to be crucial for its activity. So far, in the biosynthesis of the mannose cap on ManLAM, two enzymes have been reported to be involved: a mannosyltransferase that adds the first mannosyl residue of the mannose caps to the arabinan domain of LAM, and another mannosyltransferase that elongates the mannose cap up to three mannosyl residues. Here, we report that a third gene is involved, MMAR_2380, which is the Mycobacterium marinum orthologue of Rv1565c. MMAR_2380 encodes a predicted transmembrane acyltransferase. In M. marinum ΔMMAR_2380, the LAM arabinan domain is still intact, but the mutant LAM lacks the mannose cap. Additional effects of mutation of MMAR_2380 on LAM were observed: a higher degree of branching of both the arabinan domain and the mannan core, and a decreased incorporation of [1,2-14C]acetate into the acyl chains in mutant LAM as compared with the wild-type form. This latter effect was also observed for related lipoglycans, i.e. lipomannan (LM) and phosphatidylinositol mannosides (PIMs). Furthermore, the mutant strain showed increased aggregation in liquid cultures as compared with the wild-type strain. All phenotypic traits of M. marinum ΔMMAR_2380, the deficiency in the mannose cap on LAM and changes at the cell surface, could be reversed by complementing the mutant strain with MMAR_2380. Strikingly, membrane preparations of the mutant strain still showed enzymic activity for the arabinan mannose-capping mannosyltransferase similar to that of the wild-type strain. Although the exact function of MMAR_2380 remains unknown, we show that the protein is essential for the presence of a mannose cap on LAM

Direct Visualization by Cryo-EM of the Mycobacterial Capsular Layer: A Labile Structure Containing ESX-1-Secreted Proteins

The cell envelope of mycobacteria, a group of Gram positive bacteria, is composed of a plasma membrane and a Gram-negative-like outer membrane containing mycolic acids. In addition, the surface of the mycobacteria is coated with an ill-characterized layer of extractable, non-covalently linked glycans, lipids and proteins, collectively known as the capsule, whose occurrence is a matter of debate. By using plunge freezing cryo-electron microscopy technique, we were able to show that pathogenic mycobacteria produce a thick capsule, only present when the cells were grown under unperturbed conditions and easily removed by mild detergents. This detergent-labile capsule layer contains arabinomannan, α-glucan and oligomannosyl-capped glycolipids. Further immunogenic and proteomic analyses revealed that Mycobacterium marinum capsule contains high amounts of proteins that are secreted via the ESX-1 pathway. Finally, cell infection experiments demonstrated the importance of the capsule for binding to cells and dampening of pro-inflammatory cytokine response. Together, these results show a direct visualization of the mycobacterial capsular layer as a labile structure that contains ESX-1-secreted proteins

Mannose-fucose recognition by DC-SIGN

Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN). DC-SIGN is a C-type lectin receptor that recognizes N-linked high-mannose oligosaccharides and branched fucosylated structures. It is now clear that the biological role of DC-SIGN is two-fold. It is primarily expressed by dendritic cells and mediates important functions necessary for the induction of successful immune responses that are essential for the clearance of microbial infections, such as the capture, destruction, and presentation of microbial pathogens to induce successful immune responses. Yet, on the other hand, pathogens may also exploit DC-SIGN to modulate DC functioning thereby skewing the immune response and promoting their own survival. This chapter presents an overview of the structure of DC-SIGN and its expression pattern among immune cells. The current state of knowledge of DC-SIGN-carbohydrate interactions is discussed and how these interactions influence dendritic cell functioning is examined. The molecular aspects that underlie the selectivity of DC-SIGN for mannose-and fucose-containing carbohydrates are detailed. Furthermore, the chapter discusses the role of DC-SIGN in dendritic cell biology and how certain bacterial pathogens exploit DC-SIGN to escape immune surveillance

<i>Fasciola hepatica</i> Surface Coat Glycoproteins Contain Mannosylated and Phosphorylated N-glycans and Exhibit Immune Modulatory Properties Independent of the Mannose Receptor

<div><p>Fascioliasis, caused by the liver fluke <i>Fasciola hepatica</i>, is a neglected tropical disease infecting over 1 million individuals annually with 17 million people at risk of infection. Like other helminths, <i>F</i>. <i>hepatica</i> employs mechanisms of immune suppression in order to evade its host immune system. In this study the N-glycosylation of <i>F</i>. <i>hepatica’s</i> tegumental coat (FhTeg) and its carbohydrate-dependent interactions with bone marrow derived dendritic cells (BMDCs) were investigated. Mass spectrometric analysis demonstrated that FhTeg N-glycans comprised mainly of oligomannose and to a lesser extent truncated and complex type glycans, including a phosphorylated subset. The interaction of FhTeg with the mannose receptor (MR) was investigated. Binding of FhTeg to MR-transfected CHO cells and BMDCs was blocked when pre-incubated with mannan. We further elucidated the role played by MR in the immunomodulatory mechanism of FhTeg and demonstrated that while FhTeg’s binding was significantly reduced in BMDCs generated from MR knockout mice, the absence of MR did not alter FhTeg’s ability to induce SOCS3 or suppress cytokine secretion from LPS activated BMDCs. A panel of negatively charged monosaccharides (i.e. GlcNAc-4P, Man-6P and GalNAc-4S) were used in an attempt to inhibit the immunoregulatory properties of phosphorylated oligosaccharides. Notably, GalNAc-4S, a known inhibitor of the Cys-domain of MR, efficiently suppressed FhTeg binding to BMDCs and inhibited the expression of suppressor of cytokine signalling (SOCS) 3, a negative regulator the TLR and STAT3 pathway. We conclude that <i>F</i>. <i>hepatica</i> contains high levels of mannose residues and phosphorylated glycoproteins that are crucial in modulating its host’s immune system, however the role played by MR appears to be limited to the initial binding event suggesting that other C-type lectin receptors are involved in the immunomodulatory mechanism of FhTeg.</p></div

Association of monocyte HLA-DR expression over time with secondary infection in critically ill children: a prospective observational study

An impaired immune response could play a role in the acquisition of secondary infections in critically ill children. Human leukocyte antigen-DR expression on monocytes (mHLA-DR) has been proposed as marker to detect immunosuppression, but its potential to predict secondary infections in critically ill children is unclear. We aimed to assess the association between mHLA-DR expression at several timepoints and the change of mHLA-DR expression over time with the acquisition of secondary infections in critically ill children. In this prospective observational study, children < 18 years with fever and/or suspected infection (community-acquired or hospital-acquired) were included at a paediatric intensive care unit in the Netherlands. mHLA-DR expression was determined by flow cytometry on day 1, day 2–3 and day 4–7. The association between delta-mHLA-DR expression (difference between last and first measurement) and secondary infection was assessed by multivariable regression analysis, adjusted for age and Paediatric Logistic Organ Dysfunction-2 score. We included 104 patients at the PICU (median age 1.2 years [IQR 0.3–4.2]), of whom 28 patients (27%) developed a secondary infection. Compared to 93 healthy controls, mHLA-DR expression of critically ill children was significantly lower at all timepoints. mHLA-DR expression did not differ at any of the time points between patients with and without secondary infection. In addition, delta-mHLA-DR expression was not associated with secondary infection (aOR 1.00 [95% CI 0.96–1.04]). Conclusions: Our results confirm that infectious critically ill children have significantly lower mHLA-DR expression than controls. mHLA-DR expression was not associated with the acquisition of secondary infections.What is Known:• An impaired immune response, estimated by mHLA-DR expression, could play an essential role in the acquisition of secondary infections in critically ill children.• In critically ill children, large studies on the association of mHLA-DR expression with secondary infections are scarce.What is New:• Our study confirms that critically ill children have lower mHLA-DR expression than healthy controls.• mHLA-DR expression and change in mHLA-DR was not associated with the acquisition of secondary infection

FhTeg preparation is rich in oligomannose and truncated complex type <i>N-</i>glycans carrying fucose or sulfate/phosphate moieties.

<p>Fh tegumental antigens were digested with trypsin followed by PNGase F treatment. Released N-glycans were subsequently labelled with 2-AA and analysed by MALDI-TOF-MS in the negative ion-reflector mode. Signals are labelled with monoisotopic masses. Most abundant <i>N</i>-glycan structures are annotated in the spectrum while minor peaks are reported in the supplementing material (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004601#pntd.0004601.s004" target="_blank">S1 Table</a>). The signal at <i>m/z</i> 1582.8 [M-H]^- is annotated according to MALDI-TOF/TOF-MS analysis.</p

MALDI-FT-ICR-MS analysis of FhTeg N-glycans indicates the presence of phosphorylated glycan species.

<p><b>A:</b> FT-ICR-MS spectrum indicating the phosphorylated glycans identified by accurate mass determination with external calibration and additional comparison with the internal confirmed oligomannose glycans. <b>B:</b> Zoom region showing the high resolution separation between the Man₆GlcNAc₂ glycan and a phosphorylated glycan of almost identical mass. <b>C:</b> The scatter plot indicates the deviation of the measured <i>m/z</i> from the calculated <i>m/z</i> of phosphorylated glycans in comparison with hypothetical sulphated glycans and the oligomannosyl glycans also present in the spectrum.</p

FhTeg binding to dendritic cells is mediated by MR and is carbohydrate and calcium dependent.

<p><b>A-B:</b> MR-transfected CHO cells (A) and BMDCs (B) were stimulated with and without inhibitors, i.e. EGTA (10mM), anti-MR (1 μg ml^-1), mannan (A: 100 μg ml^-1;B: 1 mg/mL), GalNAc-4S (A: 1mM; B: 25 mM), for 45 min prior to stimulation with fluorescently labelled FhTeg (A: 1–10 μg ml^-1; B: 5 μg/mL) for 45 min. Fluorescently labelled BSA was also used as control. FhTeg binding to cells was assessed by flow cytometry and reported in bar chart format. Data shown is the mean ± SD of one representative experiment; the experiment was repeated 2–3 times, **, <i>p</i> ≤ 0.01; ***, <i>p</i> ≤ 0.001 compared to FhTeg. <b>C-D:</b> BMDCs were stimulated with fluorescently labelled FhTeg (10μg ml^-1, green) or BSA (<u>10g</u> ml^-1, green)) for 45 min prior to paraformaldehyde fixation and mounting with DAPI (blue); Scale bar: 25μm.</p

The immune properties of FhTeg are independent of the MR receptor.

<p><b>A</b>: BMDCs were stimulated with mannan for 30 min prior to incubation with FhTeg for 2.5 h. Total RNA was extracted, and after reverse transcription cDNA was analyzed with qPCR for SOCS3. RNA expression was normalized to GAPDH and actin control genes. <b>B:</b> BMDCs were pre-incubated with mannan prior stimulation with PBS or FhTeg (10μg) before addition of LPS (100ng ml^-1) for 18 h. IL12p70 levels were measured with commercial ELISA kits. Data are presented as the mean ± SEM of two independent experiments. ***<i>p</i> ≤ 0.001; ****<i>p</i> ≤ 0.0001 compared to LPS group. <b>C,D:</b> BMDCs isolated from MR-knockout mice were stimulated with fluorescently labelled FhTeg or BSA (10μg ml^-1, green) for 45 min prior to paraformaldehyde fixation. FhTeg binding to cells was assessed by flow cytometry and reported in bar chart format. <b>E:</b> BMDCs isolated from MR-knockout mice were stimulated with FhTeg (10μg) for 2.5 h. Total RNA was extracted, and after reverse transcription cDNA was analyzed with qPCR for SOCS3. RNA expression was normalized to GAPDH and actin control genes. <b>F.</b> BMDCs derived from MR knockout mice were stimulated with <b>PBS</b> or FhTeg (10μg) before addition of LPS (100ng ml^-1) for 18 h. IL12p70 levels were measured with commercial ELISA kits. Data are presented as the mean ± SEM of two independent experiments. *<i>p</i> ≤ 0.05, **, <i>p</i> ≤ 0.01; ***, <i>p</i> ≤ 0.001.</p