Identification of Antigenic Glycans from Schistosoma mansoni by Using a Shotgun Egg Glycan Microarray.

Infection of mammals by the parasitic helminth Schistosoma mansoni induces antibodies to glycan antigens in worms and eggs, but the differential nature of the immune response among infected mammals is poorly understood. To better define these responses, we used a shotgun glycomics approach in which N-glycans from schistosome egg glycoproteins were prepared, derivatized, separated, and used to generate an egg shotgun glycan microarray. This array was interrogated with sera from infected mice, rhesus monkeys, and humans and with glycan-binding proteins and antibodies to gather information about the structures of antigenic glycans, which also were analyzed by mass spectrometry. A major glycan antigen targeted by IgG from different infected species is the FLDNF epitope [Fucα3GalNAcß4(Fucα3)GlcNAc-R], which is also recognized by the IgG monoclonal antibody F2D2. The FLDNF antigen is expressed by all life stages of the parasite in mammalian hosts, and F2D2 can kill schistosomula in vitro in a complement-dependent manner. Different antisera also recognized other glycan determinants, including core ß-xylose and highly fucosylated glycans. Thus, the natural shotgun glycan microarray of schistosome eggs is useful in identifying antigenic glycans and in developing new anti-glycan reagents that may have diagnostic applications and contribute to developing new vaccines against schistosomiasis.

Differential expression of anti-glycan antibodies in schistosome-infected humans, rhesus monkeys and mice.

Schistosomiasis is a debilitating parasitic disease of humans, endemic in tropical areas, for which no vaccine is available. Evidence points to glycan antigens as being important in immune responses to infection. Here we describe our studies on the comparative humoral immune responses to defined schistosome-type glycan epitopes in Schistosoma mansoni-infected humans, rhesus monkeys and mice. Rhesus anti-glycan responses over the course of infection were screened on a defined glycan microarray comprising semi-synthetic glycopeptides terminating with schistosome-associated or control mammalian-type glycan epitopes, as well as a defined glycan microarray of mammalian-type glycans representing over 400 glycan structures. Infected rhesus monkeys generated a high immunoglobulin G (IgG) antibody response to the core xylose/core α3 fucose epitope of N-glycans, which peaked at 8-11 weeks post infection, coinciding with maximal ability to kill schistosomula in vitro. By contrast, infected humans generated low antibody levels to this epitope. At 18 months following praziquantel therapy to eliminate the parasite, antibody levels were negligible. Mice chronically infected with S. mansoni generated high levels of anti-fucosylated LacdiNAc (GalNAcß1, 4(Fucα1, 3)GlcNAc) IgM antibodies, but lacked a robust response to the core xylose/core α3 fucose N-glycan antigens compared with other species studied, and their sera demonstrated an intermediate level of schistosomula killing in vitro. These differential responses to parasite glycan antigens may be related to the ability of rhesus monkeys to self-cure in contrast to the chronic infection seen in humans and mice. Our results validate defined glycan microarrays as a useful technology to evaluate diagnostic and vaccine antigens for schistosomiasis and perhaps other infections.

Increased pathology in lungs of mice after infection with an alpha-crystallin mutant of Mycobacterium tuberculosis: changes in cathepsin proteases and certain cytokines.

Latency and reactivation are a significant problem that contributes to the incidence, transmission and pathogenesis of tuberculosis. The mechanisms involved in these processes, at the level of both the bacillus and the host, are poorly understood. In Mycobacterium tuberculosis the alpha-crystallin (acr) gene has been linked to latency, because it is highly expressed during hypoxic growth conditions. Deletion of the acr gene in M. tuberculosis H37Rv (Deltaacr strain) was previously shown to reduce the intracellular growth of bacilli in macrophages; however, its impact on pathogenesis in vivo was unknown. This study demonstrated that infection of C57BL6 mice with Deltaacr results in lung bacillary loads 1-2 log units higher in comparison to parental H37Rv. Haematoxylin/eosin staining of lungs revealed exacerbated pathology characterized by extensive obliteration of alveolar air spaces by granulomatous inflammation. RT-PCR analysis and immunostaining of lungs showed that infection with either H37Rv or Deltaacr results in the differential expression of lysosomal cathepsin proteases. A slight increase in the expression of the matrix-degrading acidic-type cathepsins B, D and H was noted in Deltaacr-infected mice and was associated with clusters of macrophages within lung granulomas. Deltaacr-infected mice also showed high serum levels of TNF-alpha, IFN-gamma and G-CSF, suggesting that Acr may play a role in modulating the host response to infection.

The down-regulation of cathepsin G in THP-1 monocytes after infection with Mycobacterium tuberculosis is associated with increased intracellular survival of bacilli.

Cathepsin G (CatG) is a serine protease found in the azurophilic granules of monocytes that is known to have antimicrobial properties, but its role in Mycobacterium tuberculosis infection is unknown. We found that M. tuberculosis infection of human THP-1 monocytic cells induced the down-regulation of CatG mRNA expression, as demonstrated by gene array analysis and reverse transcription-PCR. This was associated with a concomitant decrease in CatG protein and enzymatic activity. In contrast, the expression of lysosomal cathepsins B and D was up-regulated in infected cells. This effect was also observed when THP-1 cells were induced to differentiate into adherent macrophages by exposure to bacterial lipopolysaccharide (LPS). In agreement with this, CatG expression was null in adherent macrophages isolated from bronchoalveolar lavages and normal blood. We wanted to determine if the down-regulation of CatG would be relevant to M. tuberculosis infection. First, we found that addition of CatG to THP-1 cells prior to infection resulted in decreased bacillary viability, presumably due to extracellular killing of bacilli. However, pretreatment of cells with LPS, which decreases intracellular CatG expression, resulted in increased bacillary viability. Second, we found that CatG cationic peptides killed M. tuberculosis bacilli and were five- to sevenfold more bactericidal than full-length CatG. These observations suggest that M. tuberculosis infection of human monocytic cells results in a "cathepsin switch" with down-regulation of CatG rendering M. tuberculosis bacilli more viable. Therefore, the down-regulation of CatG in macrophages is advantageous to M. tuberculosis bacilli and possibly is an important mechanism by which M. tuberculosis is able to evade the host immune defenses.

Pulmonary granulomatous inflammation: From sarcoidosis to tuberculosis.

Granulomatous inflammation of the lung is characterized by the recruitment and organization of activated macrophages and lymphocytes in discrete lesions laced in a network of matrix proteins. These lesions, termed granulomas, represent an important defense mechanism against infectious organisms such as fungi and mycobacteria, but also can be elicited by noninfectious agents. Occasionally, this inflammatory reaction can develop for unknown reasons, causing a systemic illness termed sarcoidosis. The mechanisms involved in granuloma formation in the lung have not been elucidated entirely. However, studies performed in animal models of granuloma formation and in humans suggest important roles for specific soluble mediators (eg, cytokines, chemokines) produced by monocytic cells. If uncontrolled, granulomatous inflammation leads to excessive tissue remodeling, causing fibrosis and/or cavitation as seen in tuberculosis. This review summarizes our current understanding of the factors involved in granuloma formation in the lung with particular attention to their role in sarcoidosis and tuberculosis.

Molecular cloning and expression of a novel glycolipid sulfotransferase in Mycobacterium tuberculosis.

Sulfated trehalose glycolipids are among the most characteristic cell wall molecules of virulent strains of Mycobacterium tuberculosis. They comprise a family of trehalose-2-sulfate esters with an array of acyl fatty acids at various positions of the trehalose moiety. Although their structure has been well characterized, most of the enzymes involved in their biosynthesis, such as sulfotransferases, are unknown. It is demonstrated here by metabolic labelling with 35S abundant incorporation into sulfolipids of M. tuberculosis strains, in comparison to Mycobacterium avium, Mycobacterium bovis BCG and Mycobacterium smegmatis. The most abundant sulfolipid, sulfolipid I, is present in virulent strains H37Rv and Erdman, but absent in attenuated H37Ra. Sulfotransferase assays with the donor substrate 3'-phosphoadenosine-5'-[35S]phosphosulfonate and whole cell lysates of H37Ra resulted in the synthesis of four major sulfolipids (I, II, IV and VI). A search for sulfotransferase gene sequences in M. tuberculosis yielded gene Rv1373, a 981 bp gene slightly homologous (24% identity) to eukaryotic aryl-sulfotransferases. Rv1373 was cloned by PCR and expressed as a 39 kDa recombinant his-tagged protein. The recombinant M. tuberculosis aryl-sulfotransferase exhibited activity towards the cerebroside glycolipids glucosyl- and galactosylceramide. No activity was detected with sulfatide (3'-sulfated galactosylceramide), suggesting that sulfation of galactosylceramide may occur at C-3 of the galactose. Treatment of sulfated products with ceramide glycanase resulted in the release of 35S-labelled material showing that sulfation was at the saccharide moiety (galactose or glucose) of the ceramide. Assays with the M. tuberculosis aryl-sulfotransferase and total H37Ra glycolipids showed one major product corresponding to sulfolipid IV. These results demonstrate that Rv1373 encodes a novel glycolipid sulfotransferase with activity towards typical ceramide glycolipids and mycobacterial trehalose glycolipids.

M. tuberculosis induction of matrix metalloproteinase-9: the role of mannose and receptor-mediated mechanisms.

Mycobacterium tuberculosis (Mtb) infection induces the expression of matrix metalloproteinase-9 (MMP-9) in mouse lungs. In cultured human monocytic cells, Mtb bacilli and the cell wall glycolipid lipoarabinomannan (LAM) stimulate high levels of MMP-9 activity. Here, we explore the cellular mechanisms involved in the induction of MMP-9 by Mtb. We show that infection of THP-1 cells with Mtb caused a fivefold increase in MMP-9 mRNA that was associated with increased MMP-9 activity. MMP-9 induction was dependent on microtubule polymerization and protein kinase activation and was associated with increased DNA binding by the transcription factor activator protein-1 (AP-1), which appeared to be important for MMP-9 expression. We then explored the surface molecules potentially involved in Mtb induction of MMP-9, focusing on ligands of the mannose and beta-glucan receptors. MMP-9 activity was induced by the mannose receptor ligands mannan, zymosan, and LAM, whereas the beta-glucan receptor ligand laminarin was not effective. The most active inducers of MMP-9 activity were the particulate ligand zymosan and LAM. Pretreatment of cells with an anti-mannose receptor monoclonal antibody, but not anti-complement receptor 3, decreased the induction of MMP-9 activity by Mtb bacilli. Together, these results suggest that MMP-9 induction by Mtb occurs by receptor-mediated signaling mechanisms involving the binding of mannosylated ligands to mannose receptors, the modulation by cytoskeletal elements such as microtubules, the activation of protein kinases, and transcriptional activation by AP-1.