Objectives:
Our present study aimed to analyze the major glycoconjugate present on the F. oxysporum cell surface by a combination of chemical and spectroscopic methods, including one and two-dimensional nuclear magnetic resonance (1D and 2D NMR) spectroscopic analysis and how the glycosylation of F. oxysporum glycoconjugate influences the recognition and uptake of F. oxysporum by macrophages as well as its role in the production of proinflammatory cytokines. The reactivity of the glycoconjugate was evaluated by ELISA using sera from patients with invasive fusariosis and invasive aspergillosis.

Methods:
F.oxysporum glycocojungate was extracted with 0.05 M phosphate buffer, pH 7.2 at 100◦C for 2 h. Monosacharide composition in PGM was determined based on the chemical analysis and GC-MS analyses. A combination of chemical and spectroscopic methods, including 1D and 2D NMR were made to obtain a structural characterization of a PGM from F. oxysporum. The reactivate of human sera from the Mycology Laboratory of the University Hospital (Federal University of Rio de Janeiro) against PGM were tested by ELISA. The macrophage effector functions were evaluated by phagocytic and cytokine assay.

Results:
The galactomannan component consists of a main chain containing (1→6)-linked β-D-galactofuranose residues with side chains containing (1→2)-linked α-D-Glcp, (1→2)-linked -β-D-Manp (1→2) and β-D-Manp terminal nonreducing end units and differs from that of Aspergillus fumigatus and Cladosporium resinae A strong decrease in reactivity was also observed with de-O-glycosylated PGM. In addition, de-O-glycosylated PGM was not able to inhibit F. oxysporum phagocytosis, suggesting that macrophages recognize and internalize F. oxysporum via PGM. F. oxysporum PGM triggered TNF-α release by macrophages. Chemical removal of O-linked oligosaccharides from PGM led to a significant increase of TNF-α cytokine levels, suggesting that their removal could exposure another PGM motifs able to induce a higher secretion of TNF-α levels. Interestingly, F. oxysporum conidia, intact and de-O-linked PGM were not able to induce IL-10 cytokine release. The difference in patient serum reativity using a PGM from F. oxysporum characterized in the present study as compared with a PGM from C. resinae, that presents the same epitopes recognized by serum from patients with aspergillosis.

Conclusion:
The outcome of invasive aspergillosis has improved in the last decade, and one of the reasons is the early diagnosis of the disease, achieved after the introduction of serum galactomannan testing. With this regard, a test that is sensitive and specific, and appears in the serum before the clinical manifestations of invasive fusariosis may have an impact in decreasing the mortality of this devastating disease. The PGM characterized in the present study may be a candidate, and should be tested with more sera.

ß–1,6-linked Galactofuranose-rich Peptidogalactomannan of Fusarium oxysporum is Important in the Activation of Macrophage Mechanisms and as a Potential Diagnostic Antigen