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Bioprocess development generates extensive datasets from different unit operations and sources (e.g. time series, quality measurements). The development of such processes can be accelerated by evaluating all data generated during the experimental design. This can only be achieved by having a clearly defined data logging and analysis strategy. The latter is described in this manuscript. It consists in a combination of a feature based approach along with principal component analysis and partial least square regression. Application of this combined strategy is illustrated by applying it in an upstream processing (USP) case study. Data from the development and optimization of an animal component free USP of Sabin inactivated poliovirus vaccine (sIPV) was evaluated. During process development, 26 bioreactor runs at scales ranging from 2.3 to 16 L were performed. Several operational parameters were varied, and data was routinely analyzed following a design of experiments (DoE) methodology. With the strategy described here, it became possible to scrutinize all data from the 26 runs in a single data study. This included the DoE response parameters, all data generated by the bioreactor control systems, all offline data, and its derived calculations. This resulted in a more detailed, reliable and exact view on the most important parameters affecting bioreactor performance. In this case study, the strategy was applied for the analysis of previously produced data. Further development will use this data analysis methodology for continuous enhancing and accelerating process development, intensified DoE and integrated process modelling.

Helminth parasites control host-immune responses by secreting immunomodulatory glycoproteins. Clinical trials and mouse model studies have demonstrated the potential of helminth-derived glycoproteins for the treatment of immune-related diseases, like allergies and autoimmune diseases. Studies are however hampered by the limited availability of native parasite-derived proteins. Moreover, recombinant protein production systems have thus far been unable to reconstitute helminth-like glycosylation essential for the functionality of some helminth glycoproteins. Here we exploited the flexibility of the N-glycosylation machinery of plants to reconstruct the helminth glycoproteins omega-1 and kappa-5, two major constituents of immunomodulatory Schistosoma mansoni soluble egg antigens. Fine-tuning transient co-expression of specific glycosyltransferases in Nicotiana benthamiana enabled the synthesis of Lewis X (LeX) and LDN/LDN-F glycan motifs as found on natural omega-1 and kappa-5, respectively. In vitro and in vivo evaluation of the introduction of native LeX motifs on plant-produced omega-1 confirmed that LeX on omega-1 contributes to the glycoprotein’s Th2-inducing properties. These data indicate that mimicking the complex carbohydrate structures of helminths in plants is a promising strategy to allow targeted evaluation of therapeutic glycoproteins for the treatment of inflammatory disorders. In addition, our results offer perspectives for the development of effective anti-helminthic vaccines by reconstructing native parasite glycoprotein antigens.

The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, remains an important worldwide health threat. Although TB is one of the oldest infectious diseases of man, a detailed understanding of the mycobacterial mechanisms underlying pathogenesis remains elusive. Here, we studied the role of the α(1→2) mannosyltransferase MptC in mycobacterial virulence, using the Mycobacterium marinum zebrafish infection model. Like its M. tuberculosis orthologue, disruption of M. marinum mptC (mmar_3225) results in defective elongation of mannose caps of lipoarabinomannan (LAM) and absence of α(1→2)mannose branches on the lipomannan (LM) and LAM mannan core, as determined by biochemical analysis (NMR and GC-MS) and immunoblotting. We found that the M. marinum mptC mutant is strongly attenuated in embryonic zebrafish, which rely solely on innate immunity, whereas minor virulence defects were observed in adult zebrafish. Strikingly, complementation with the Mycobacterium smegmatis mptC orthologue, which restored mannan core branching but not cap elongation, was sufficient to fully complement the virulence defect of the mptC mutant in embryos. Altogether our data demonstrate that not LAM capping, but mannan core branching of LM/LAM plays an important role in mycobacterial pathogenesis in the context of innate immunity.

Fascioliasis, caused by the liver fluke Fasciola hepatica, is a neglected tropical disease infecting over 1 million individuals annually with 17 million people at risk of infection. Like other helminths, F. hepatica employs mechanisms of immune suppression in order to evade its host immune system. In this study the N-glycosylation of F. hepatica’s 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 F. hepatica 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., Author Summary Fascioliasis, caused by the liver fluke Fasciola hepatica, is a neglected tropical disease infecting over 1 million individuals annually with 17 million people at risk of infection. These worms infect the liver and can survive for many years in its animal or human host because they supress the host’s immune system that is important in clearing worm infection. Worms are similar to humans in that they are made of proteins, fats and sugars, and while there are many studies on worm proteins, few studies have examined the sugars. We are interested in the sugars because we believe that they help the parasite survive for many years within its host. To examine this, we have used a technique called mass spectrometric analysis to characterise the sugars present in F. hepatica. We also have developed systems in the laboratory to test if these sugars can suppress the host’s immune system. We conclude that F. hepatica sugars are crucial in suppressing its host’s immune system; however, the exact way the sugars can do this requires further studies. These studies are important for the development of worm vaccines or therapies.

During infection with the blood fluke Schistosoma mansoni, glycan motifs present on glycoproteins of the parasite's eggs mediate immunomodulatory effects on the host. The recognition of these glycan motifs is primarily mediated by C-type lectin receptors on dendritic cells and other cells of the immune system. However, it is not yet known which individual glycoproteins interact with the different C-type lectin receptors, and which structural components are involved. Here we investigated the structural basis of the binding of two abundant egg antigens, kappa-5 and IPSE/α1, by the C-type lectin receptor dendritic cell-specific ICAM3-grabbing non-integrin, macrophage galactose-type lectin and mannose receptor. In the natural soluble form, the secretory egg glycoprotein IPSE/α1 interacts with dendritic cells mainly via mannose receptors. Surprisingly, in plate-based assays mannose receptors preferentially bound to mannose conjugates, while in cell-based assays, IPSE/α1 is bound via the fucosylated Galβ1-4(Fucα1-3)GlcNAc (LeX) motif on diantennary N-glycans. Kappa-5, in contrast, is bound by dendritic cells via all three C-type lectin receptors studied and for a minor part also via other, non-C-type lectin receptors. Kappa-5 interacts with macrophage galactose-type lectins via the GalNAcβ1-4GlcNAc antenna present on its triantennary N-glycans, as well as the GalNAcβ1-4(Fucα1-3)GlcNAc antennae present on a minor N-glycan subset. Dendritic cell-specific ICAM3-grabbing non-integrin binding of kappa-5 was mediated via the GalNAcβ1-4(Fucα1-3)GlcNAc antennae, whereas binding of mannose receptors may involve either GalNAcβ1-4(Fucα1-3)GlcNAc antennae or the fucosylated and xylosylated chitobiose core. This study provides a molecular and structural basis for future studies of the interaction between C-type lectin receptors and other soluble egg antigen glycoproteins and their effects on the host immune response. © 2012 Australian Society for Parasitology Inc..

Schistosome ribonuclease Omega-1 primes DCs to generate Th2 responses by binding and internalization by the mannose receptor and by subsequently impairing protein synthesis., Omega-1, a glycosylated T2 ribonuclease (RNase) secreted by Schistosoma mansoni eggs and abundantly present in soluble egg antigen, has recently been shown to condition dendritic cells (DCs) to prime Th2 responses. However, the molecular mechanisms underlying this effect remain unknown. We show in this study by site-directed mutagenesis of omega-1 that both the glycosylation and the RNase activity are essential to condition DCs for Th2 polarization. Mechanistically, we demonstrate that omega-1 is bound and internalized via its glycans by the mannose receptor (MR) and subsequently impairs protein synthesis by degrading both ribosomal and messenger RNA. These experiments reveal an unrecognized pathway involving MR and interference with protein synthesis that conditions DCs for Th2 priming.

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.

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 threeα(1→2)-linked mannosyl residues, the mannose cap, producing ManLAM. ManLAM has been associated with a range of different immunomodulatory properties ofMycobacterium tuberculosisduring 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 theMycobacterium marinumorthologue ofRv1565c.MMAR_2380encodes a predicted transmembrane acyltransferase. InM. marinumΔMMAR_2380, the LAM arabinan domain is still intact, but the mutant LAM lacks the mannose cap. Additional effects of mutation ofMMAR_2380on 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 ofM. marinumΔMMAR_2380, the deficiency in the mannose cap on LAM and changes at the cell surface, could be reversed by complementing the mutant strain withMMAR_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.

Pathogenic mycobacteria have the ability to persist in phagocytic cells and to suppress the immune system. The glycolipid lipoarabinomannan (LAM), in particular its mannose cap, has been shown to inhibit phagolysosome fusion and to induce immunosuppressive IL-10 production via interaction with the mannose receptor or DC-SIGN. Hence, the current paradigm is that the mannose cap of LAM is a crucial factor in mycobacterial virulence. However, the above studies were performed with purified LAM, never with live bacteria. Here we evaluate the biological properties of capless mutants of Mycobacterium marinum and M.bovis BCG, made by inactivating homologues of Rv1635c. We show that its gene product is an undecaprenyl phosphomannose-dependent mannosyltransferase. Compared with parent strain, capless M.marinum induced slightly less uptake by and slightly more phagolysosome fusion in infected macrophages but this did not lead to decreased survival of the bacteria in vitro, nor in vivo in zebra fish. Loss of caps in M.bovis BCG resulted in a sometimes decreased binding to human dendritic cells or DC-SIGN-transfected Raji cells, but no differences in IL-10 induction were observed. In mice, capless M.bovis BCG did not survive less well in lung, spleen or liver and induced a similar cytokine profile. Our data contradict the current paradigm and demonstrate that mannose-capped LAM does not dominate the Mycobacterium-host interaction. © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd.

Infection with the helminth Schistosoma mansoni drives the development of IL-10-producing regulatory B cells (Breg cells) which suppress experimental allergic airway inflammation. Here we investigated whether schistosomal egg antigens (SEA) directly interact with B cells and induce development of IL-10-producing Breg cells. Splenic B cell cytokine production and regulatory capacity was analysed after repeated intraperitoneal injection of SEA into C57BL/6 mice. To identify cell types that bind SEA in vivo, mice were intravenously injected with fluorescently labeled SEA and spleens analysed by immunohistochemistry and flow cytometry. For in vitro assays, naive CD19 sorted splenic B cells were cultured with SEA to study binding as well as B cell cytokines and regulatory capacity. SEA treatment upregulated IL-10, CD40 and CD86 expression by splenic marginal zone (MZ) B cells in vivo, and MZ B cells as well as MZ macrophages bound SEA. Importantly, SEA could also induce Breg cell development in vitro which was further increased by CD40 ligation, whereas TLR2- and TLR4-signaling was not involved. SEA-induced Breg cells triggered regulatory T cell development and suppressed T cell IFN-γ production in co-culture experiments. Macrophages were dispensable for Breg induction as shown by macrophage depletion in SEA-treated mice. In conclusion, schistosomal egg antigens can drive Breg cell development by targeting B cells directly. Current work focuses on the identification of relevant molecules in SEA and the role of pattern-recognition receptors to provide new insights in the generation of Breg cells. This may form a basis for novel therapies against allergic disorders by enhancing Breg cell activity.

The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, remains an important worldwide health threat. Although TB is one of the oldest infectious diseases of man, a detailed understanding of the mycobacterial mechanisms underlying pathogenesis remains elusive. Here, we studied the role of the α(1→2) mannosyltransferase MptC in mycobacterial virulence, using the Mycobacterium marinum zebrafish infection model. Like its M. tuberculosis orthologue, disruption of M. marinum mptC (mmar_3225) results in defective elongation of mannose caps of lipoarabinomannan (LAM) and absence of α(1→2)mannose branches on the lipomannan (LM) and LAM mannan core, as determined by biochemical analysis (NMR and GC-MS) and immunoblotting. We found that the M. marinum mptC mutant is strongly attenuated in embryonic zebrafish, which rely solely on innate immunity, whereas minor virulence defects were observed in adult zebrafish. Strikingly, complementation with the Mycobacterium smegmatis mptC orthologue, which restored mannan core branching but not cap elongation, was sufficient to fully complement the virulence defect of the mptC mutant in embryos. Altogether our data demonstrate that not LAM capping, but mannan core branching of LM/LAM plays an important role in mycobacterial pathogenesis in the context of innate immunity.

Cyanovirin-N (CV-N) is a mannose-binding lectin that inhibits HIV-1 infection by blocking mannose-dependent target cell entry via C-type lectins. Like HIV-1, Mycobacterium tuberculosis expresses mannosylated surface structures and exploits C-type lectins to gain cell access. In this study, we investigated whether CV-N, like HIV-1, can inhibit M. tuberculosis infection. We found that CV-N specifically interacted with mycobacteria by binding to the mannose-capped lipoglycan lipoarabinomannan. Furthermore, CV-N competed with the C-type lectins DC-SIGN and mannose receptor for ligand binding and inhibited the binding of M. tuberculosis to dendritic cells but, unexpectedly, not to macrophages. Subsequent in vivo infection experiments in a mouse model demonstrated that, despite its activity, CV-N did not inhibit or delay M. tuberculosis infection. This outcome argues against a critical role for mannose-dependent C-type lectin interactions during the initial stages of murine M. tuberculosis infection and suggests that, depending on the circumstances, M. tuberculosis can productively infect cells using different modes of entry.

ATP synthase is a validated drug target for the treatment of tuberculosis, and ATP synthase inhibitors are promising candidate drugs for the treatment of infections caused by other slow-growing mycobacteria, such as Mycobacterium leprae and Mycobacterium ulcerans. ATP synthase is an essential enzyme in the energy metabolism of Mycobacterium tuberculosis; however, no biochemical data are available to characterize the role of ATP synthase in slow-growing mycobacterial strains. Here, we show that inverted membrane vesicles from the slow-growing model strain Mycobacterium bovis BCG are active in ATP synthesis, but ATP synthase displays no detectable ATP hydrolysis activity and does not set up a proton-motive force (PMF) using ATP as a substrate. Treatment with methanol as well as PMF activation unmasked the ATP hydrolysis activity, indicating that the intrinsic subunit ɛ and inhibitory ADP are responsible for the suppression of hydrolytic activity. These results suggest that the enzyme is needed for the synthesis of ATP, not for the maintenance of the PMF. For the development of new antimycobacterial drugs acting on ATP synthase, screening for ATP synthesis inhibitors, but not for ATP hydrolysis blockers, can be regarded as a promising strategy.

Publisher Summary 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.

Mycobacterium tuberculosis possesses a variety of immunomodulatory factors that influence the host immune response. When the bacillus encounters its target cell, the outermost components of its cell envelope are the first to interact. Mycobacteria, including M. tuberculosis, are surrounded by a loosely attached capsule that is mainly composed of proteins and polysaccharides. Although the chemical composition of the capsule is relatively well studied, its biological function is only poorly understood. The aim of this study was to further assess the functional role of the mycobacterial capsule by identifying host receptors that recognize its constituents. We focused on α-glucan, which is the dominant capsular polysaccharide. Here we demonstrate that M. tuberculosis α-glucan is a novel ligand for the C-type lectin DC-SIGN (dendritic cell-specific ICAM-3-grabbing nonintegrin). By using related glycogen structures, we show that recognition of α-glucans by DC-SIGN is a general feature and that the interaction is mediated by internal glucosyl residues. As for mannose-capped lipoarabinomannan, an abundant mycobacterial cell wall-associated glycolipid, binding of α-glucan to DC-SIGN stimulated the production of immunosuppressive IL-10 by LPS-activated monocyte-derived dendritic cells. By using specific inhibitors, we show that this IL-10 induction was DC-SIGN-dependent and also required acetylation of NF-κB. Finally, we demonstrate that purified M. tuberculosis α-glucan, in contrast to what has been reported for fungal α-glucan, was unable to activate TLR2.

The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is the major receptor on DCs for mycobacteria of theMycobacterium tuberculosiscomplex. Recently, we have shown that although the mannose caps of the mycobacterial surface glycolipid lipoarabinomannan (ManLAM) are essential for the binding to DC-SIGN, genetic removal of these caps did not diminish the interaction of whole mycobacteria with DC-SIGN and DCs. Here we investigated the role of the structurally related glycolipids phosphatidylinositol mannosides (PIMs) as possible ligands for DC-SIGN. In a binding assay with both synthetic and natural PIMs, DC-SIGN exhibited a high affinity for hexamannosylated PIM6, which contains terminal α(1→2)-linked mannosyl residues identical to the mannose cap on ManLAM, but not for di- and tetramannosylated PIM2and PIM4, respectively. To determine the role of PIM6in the binding of whole mycobacteria to DC-SIGN, a mutant strain ofM. bovisbacillus Calmette-Guérin deficient in the production of PIM6(ΔpimE) was created, as well as a double knockout deficient in the production of both PIM6and the mannose caps on LAM (ΔpimEΔcapA). Compared to the wild-type strain, both mutant strains bound similarly well to DC-SIGN and DCs. Furthermore, the wild-type and mutant strains induced comparable levels of interleukin-10 and interleukin-12p40 when used to stimulate DCs. Hence, we conclude that, like ManLAM, PIM6represents a bona fide DC-SIGN ligand but that other, as-yet-unknown, ligands dominate in the interaction between mycobacteria and DCs.