A complement inhibitor (CI) produced by Aspergillus fumigatus selectively inhibited the alternative pathway. This was the first observation of a CI produced by a fungus which causes human disease. Aspergillus CI might enhance pathogenicity of Aspergillus in the human host by impairing opsonization and diminishing generation of the chemoattractant for polymorphonuclear neutrophils and monocytes, C5a. CI will be chromatographically purified, and homogeneity of the final material will be judged using SDS-PAGE and isoelectric focusing. Following purification, aminoacid and monosaccharide content will be determined. The mechanism of alternative pathway inhibition will be clarified by adding graded concentrations of purified C3, Factor B, and Factor D to CI in a cryptococcal alternative pathway system. Binding between the relevant complement protein and CI will be assessed using a combination of gel filtration and immunoelectrophoresis. In vivo production of CI will be examined using labeled anti-CI IgG F(ab')2 in the cortisone-treated mouse model of invasive aspergillosis, and in available human tissues. ELISA will be employed to check for CI in the sera of infected mice and of patients with various forms of aspergillosis. A CI- mutant will be selected following ultraviolet irradiation of CI+ wild-type A.fumigatus. This mutant will allow an assessment of the in vivo significance of CI by comparison of LD50s for inhaled inocula of both isolates in the mouse model. The role of CI in inhibition of C3-mediated clot lysis will also be examined using an in vitro test. The potential usefulness of CI for treatment of human diseases in which complement becomes pathologically activated will be explored in mouse models of neutrophil-mediated lung injury and systemic lupus erythematosus, as well as the Shwartzman reaction. If these in vivo treatment and toxicity studies require larger quantities of CI than can be obtained chromatographically, Aspergillus CI DNA will eventually be cloned. This will be approached using the plasmid vector pBR322 first in E. coli K12; if necessary, the yeast Saccharomyces cerevisiae could be employed as an alternative to E. coli for production of this eukaryotic gene product.
| Washburn, R G; Bryant-Varela, B J; Julian, N C et al. (1991) Differences in Cryptococcus neoformans capsular polysaccharide structure influence assembly of alternative complement pathway C3 convertase on fungal surfaces. Mol Immunol 28:465-70 |
| Washburn, R G; DeHart, D J; Agwu, D E et al. (1990) Aspergillus fumigatus complement inhibitor: production, characterization, and purification by hydrophobic interaction and thin-layer chromatography. Infect Immun 58:3508-15 |
