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In this study, we analyzed the impact of immunization with the peptidorhamnomannan (PRM) from the cell wall of the fungus Scedosporium (Lomentospora) prolificans in a murine model of invasive scedosporiosis. Immunization with PRM decreased the survival of mice infected with S. prolificans. Immunization of mice with PRM led to decreased secretion of pro-inflammatory cytokines and chemokines but did not affect the secretion of IL-10. Mice immunized with PRM showed an increase in IgG1 secretion, which is an immunoglobulin linked to a nonprotective response. Splenocytes isolated from mice infected with S. prolificans and immunized with PRM showed no differences in the percentages of Th17 cells and no increase in the frequency of the CD4(+)CD62L(Low) T cell population. PRM-immunized mice showed a significant increase in the percentage of Treg cells. In summary, our results indicated that immunization with PRM did not assist or improve the immunological response against S. prolificans infection. PRM exacerbated the infection process by reducing the inflammatory response, thereby facilitating colonization, virulence and dissemination by the fungus.

Peptidorhamnomannans (PRMs), rhamnomannans and α-glucans are especially relevant for the architecture of the Scedosporium/Pseudallescheria boydii cell wall, but many of them are immunologically active, with great potential as regulators of pathogenesis and the immune response of the host. In addition, some of them can be specifically recognised by antibodies from the sera of patients, suggesting that they could also be useful in diagnosis of fungal infections. Their primary structures have been determined, based on a combination of techniques including gas chromatography, electrospray ionization - mass spectrometry (ESI-MS), (1)H-COSY and TOCSY, (13)C and (1)H/(13)C NMR spectroscopy. Using monoclonal antibodies to PRM, we showed that it is involved in germination and viability of P. boydii conidia, in the phagocytosis of P. boydii conidia by macrophages and non-phagocytic cells and in the survival of mice with P. boydii infection. Also, components of the fungal cell wall, such as α-glucans, are involved. Rhamnomannans are immunostimulatory and participate in the recognition and uptake of fungal cells by the immune system. These glycosylated polymers, being present in the fungal cell wall, are mostly absent from mammalian cells, and are excellent targets for the design of new agents capable of inhibiting fungal growth and differentiation of pathogens.

Hot aqueous extraction of conidia of Scedosporium prolificans gave a heterogeneous glycoprotein (RMP-Sp-Coni) with 41% protein and 2MeRha, Rha, Ara, Man, Gal, Glc, and GlcNH2 in a 2:18:3:47:9:15:6 M ratio, the first report of 2-O-methylrhamnose in fungi. Methylation analysis showed nonreducing end- (10%), 2-O- (11%), and 3-O-substituted Rhap (7%), nonreducing end- (8%), 2-O- (12%), 3-O- (16%), and 2,6-di-O-substituted Manp (9%), nonreducing end- (4%), 3-O- (7%), and 4-O-substituted Glcp (7%), and nonreducing end-units of Galp (9%). Mild reductive β-elimination of RMP-Sp-Coni cleaved O-linked structures to give a mixture of oligosaccharides, of which 2MeRha capping groups were present in 2MeRhaRha2Hex2Hex-ol, 2MeRhaRha2Hex-Hex-ol, 2MeRhaRha2HexHex-ol, and 2MeRhaRha2Hex-ol (ESI-MS–MS). The mixture was fractionated by Biogel P-2 column chromatography and the two predominant isolates were β- d -Galp-(1 → 6)-[2Me-α- l -Rhap-(1 → 3)-α- l -Rhap-(1 → 3)-α- d -Manp-(1 → 2)]- d -Man-ol, and another lacking the β-Galp unit. Neither was formed from mycelial glycoprotein, although β- d -Galp-(1 → 6)-[α- l -Rhap-(1 → 3)-α- l -Rhap-(1 → 3)-α- d -Manp-(1 → 2)]- d -Man-ol was a common component.

In this study, we analyze the importance of O-linked oligosaccharides present in peptidorhamnomannan (PRM) from the cell wall of the fungus Scedosporium prolificans for recognition and phagocytosis of conidia by macrophages. Adding PRM led to a dose-dependent inhibition of conidia phagocytosis, whereas de-O-glycosylated PRM did not show any effect. PRM induced the release of macrophage-derived antimicrobial compounds. However, O-linked oligosaccharides do not appear to be required for such induction. The effect of PRM on conidia-induced macrophage killing was examined using latex beads coated with PRM or de-O-glycosylated PRM. A decrease in macrophage viability similar to that caused by conidia was detected. However, macrophage killing was unaffected when beads coated with de-O-glycosylated PRM were used, indicating the toxic effect of O-linked oligosaccharides on macrophages. In addition, PRM triggered TNF-α release by macrophages. Chemical removal of O-linked oligosaccharides from PRM abolished cytokine induction, suggesting that the O-linked oligosaccharidic chains are important moieties involved in inflammatory responses through the induction of TNF-α secretion. In summary, we show that O-glycosylation plays a role in the recognition and uptake of S. prolificans by macrophages, killing of macrophages and production of pro- inflammatory cytokines.

O-linked oligosaccharide groups ranging from di- to hexasaccharide were beta-eliminated by mild alkaline treatment under reducting conditions from the peptidogalactomannan of Aspergillus fumigatus mycelial cell wall. The resulting reduced oligosaccharides, which were the minor components of the peptidogalactomannan fraction, were fractionated to homogeneity by successive gel filtration and high-performance liquid chromatography. Their primary structures were determined based on a combination of techniques including gas chromatography, ESI-QTOF-MS, H-1 COSY and TOCSY, and H-1-C-13 HMQC NMR spectroscopy and methylation analysis, to be: alpha-Glcp-(1 --> 6)-Man-ol, beta-Galf-(1 --> 6)-alpha-Manp-(1 --> 6)-Man-ol, beta-Galf-(1 --> 5)-beta-Galf-(1 --> 6)-alpha-Manp-(1 --> 6)-Man-ol and beta-Galf-(1 --> 5)-[beta-Galf-(1 --> 5](3)-beta-Galf-(1 --> 6)-Man-ol. The beta-Galf containing oligosaccharides have not been previously described as fungal O-linked oligosaccharides. The peptidogalactomannan is antigenic and was recognized by human sera of patients with aspergillosis when probed by ELISA, but de-O-glycosylation rendered a 50% decrease in its reactivity. Furthermore, when tested in a hapten inhibition test, the isolated oligosaccharide alditols were able to block, on a dose-response basis, recognition between human sera and the intact peptidogalactomannan. The immunodominant epitopes were present in the tetra- and hexasaccharide, which contain a beta-Galf-(1 --> 5)-beta-Galf terminal group. These results suggest that the O-glycosidically linked oligosaccharide chains, despite being the less abundant carbohydrate component of the A. fumigatus peptidogalactomannan, may account for a significant part of its antigenicity, other than the known activity associated with the galactomannan component.

Pseudallescheria boydii (Scedosporium apiospermum) is a saprophytic fungus widespread in the environment, and has recently emerged as an agent of localized as well as disseminated infections, particularly mycetoma, in immunocompromised and immunocompetent hosts. We have previously shown that highly purified α-glucan from P. boydii activates macrophages through Toll-like receptor TLR2, however, the mechanism of P. boydii recognition by macrophage is largely unknown. In this work, we investigated the role of innate immune receptors in the recognition of P. boydii. Macrophages responded to P. boydii conidia and hyphae with secretion of proinflammatory cytokines. The activation of macrophages by P. boydii conidia required functional MyD88, TLR4, and CD14, whereas stimulation by hyphae was independent of TLR4 and TLR2 signaling. Removal of peptidorhamnomannans from P. boydii conidia abolished induction of cytokines by macrophages. A fraction highly enriched in rhamnomannans was obtained and characterized by NMR, high performance TLC, and GC-MS. Preparation of rhamnomannans derived from P. boydii triggered cytokine release by macrophages, as well as MAPKs phosphorylation and IκBα degradation. Cytokine release induced by P. boydii-derived rhamnomannans was dependent on TLR4 recognition and required the presence of non-reducing end units of rhamnose of the rhamnomannan, but not O-linked oligosaccharides from the peptidorhamnomannan. These results imply that TLR4 recognizes P. boydii conidia and this recognition is at least in part due to rhamnomannans expressed on the surface of P. boydii.

Scedosporium apiospermum is part of the Pseudallescheria-Scedosporium complex. Peptidorhamnomannans (PRMs) are cell wall glycopeptides present in some fungi, and their structures have been characterized in S. apiospermum, S. prolificans and Sporothrix schenckii. Prior work shows that PRMs can interact with host cells and that the glycopeptides are antigenic. In the present study, three monoclonal antibodies (mAbs, IgG1) to S. apiospermum derived PRM were generated and their effects on S. apiospermum were examined in vitro and in vivo. The mAbs recognized a carbohydrate epitope on PRM. In culture, addition of the PRM mAbs increased S. apiospermum conidia germination and reduced conidial phagocytosis by J774.16 macrophages. In a murine infection model, mice treated with antibodies to PRM died prior to control animals. Thus, PRM is involved in morphogenesis and the binding of this glycopeptide by mAbs enhanced the virulence of the fungus. Further insights into the effects of these glycopeptides on the pathobiology of S. apiospermum may lead to new avenues for preventing and treating scedosporiosis., Author Summary The incidence of fungal infections has increased dramatically over the last 50 years, largely because of the increasing size of the population at risk, which especially includes immunocompromised hosts. Scedosporium apiospermum is a filamentous fungus that causes a variety of infections, ranging from localized disease to life-threatening disseminated infections. Glycoproteins are molecules present in the fungal surface and are comprised of carbohydrate and protein components. They are involved in different important functions in the fungal cell. Monoclonal antibodies can be used as therapeutic agents for infectious disease, but some factors involved in their efficacy are often not well understood. We found that monoclonal antibodies to glycoproteins present in fungal surface can be nonprotective and can even enhance the disease. The administration of these antibodies can affect functions of the fungal cell and the immune cells, resulting in a survival advantage for the fungus during interactions with the host.

A peptidogalactomannan was isolated from mycelia of Cladosporium (Hormoconis) resinae and characterized using methylation–fragmentation analysis, partial acid hydrolysis and 1 H and 13 C-NMR spectroscopy. The galactomannan component was a branched structure and consisted of a main chain containing (1→6)-linked α- d -Man p residues substituted at O-2 by side chains containing (1→2)-linked α- d -Man p residues. β- d -Gal f residues were present as side chains of 3–4 units that are (1→5)-interlinked. This structure is very similar to a pGM isolated from Aspergillus fumigatus and differs from that of Cladosporium werneckii (currently named Hortaea werneckii ), with this pGM and other fungal galactomannans having single terminal (1→6)-linked β-Gal f residues. The importance of the carbohydrate moiety of Cladosporium resinae pGM in immunoassays was also demonstrated. On FACS examination, a decrease (60%) in rabbit serum anti- C. resinae binding to C. resinae conidia occurred when this serum had been previously incubated with pGMs from C. resinae and A. fumigatus or with mannoprotein from Candida parapsilosis , suggesting the presence of cross-reactive determinants in these fungi.

The host response to fungi is in part dependent on activation of evolutionarily conserved receptors, including toll-like receptors and phagocytic receptors. However, the molecular nature of fungal ligands responsible for this activation is largely unknown. Herein, we describe the isolation and structural characterization of an alpha-glucan from Pseudallescheria boydii cell wall and evaluate its role in the induction of innate immune response. These analyses indicate that alpha-glucan of P. boydii is a glycogen-like polysaccharide consisting of linear 4-linked alpha-D-Glcp residues substituted at position 6 with alpha-D-Glcp branches. Soluble alpha-glucan, but not beta-glucan, led to a dose-dependent inhibition of conidia phagocytosis. Furthermore, a significant decrease in the phagocytic index occurred when alpha-glucan from conidial surface was removed by enzymatic treatment with alpha-amyloglucosidase, thus indicating an essential role of alpha-glucan in P. boydii internalization by macrophages. alpha-Glucan stimulates the secretion of inflammatory cytokines by macrophages and dendritic cells; again this effect is abolished by treatment with alpha-amyloglucosidase. Finally, alpha-glucan induces cytokine secretion by cells of the innate immune system in a mechanism involving toll-like receptor 2, CD14, and MyD88. These results might have relevance in the context of infections with P. boydii and other fungi, and alpha-glucan could be a target for intervention during fungal infections.

Our objective was to evaluate the effect of stress-related dose of dopamine (DA) on the in vitro proliferation and cytokine production in polyclonally-activated T cells from healthy individuals or individuals with generalized anxiety disorder (GAD). Our results demonstrated that cell cultures from GAD group proliferated less following T cell activation, as compared with control group. The addition of DA reduced the proliferative response in cell cultures from healthy but not from GAD individuals. The cytokine profile in GAD individuals revealed Th1 and Th2 deficiencies associated with a dominant Th17 phenotype, which was enhanced by DA. A similar DA-induced immunomodulation was also observed in PPD-activated cell cultures from GAD individuals. Unlike the control, DA-enhanced Th17 cytokine production in GAD individuals was not affected by glucocorticoid. In conclusion, our results show that the T cell functional dysregulation in GAD individuals is significantly amplified by DA. These immune abnormalities can have impact in increasing the susceptibility of individuals with anxiety disorders to infectious diseases and inflammatory/autoimmune disorders.