Preliminary Research On Maturation Of A. Fumigatus-containing Phagosomes In Macrophages

Objective Macrophages play a key role in host defense against virtually all pathogenic microorganisms. Professional phagocytes have as a primary function the engulfment of microbial invaders within a membrane-bound compartment called a phagosome. Phagosomes go through a series of maturation steps, culminating in the destruction of the internalized cargo. In this research, we investigated the mechasims of the phagocytosis of A. fumigatus by macrophage; the basic maturation time point of conidia-containing phagosomes; and applied a method of isolating Aspergillus fumigatus conidia containing phagosomes by ultracentrifugation on a sucrose density gradient.Methods By challenging macrophage with GFP-expressing conidia, we constructed a model for phagocytosis research. Internalization of conidia by macrophages was revealed by scanningelectron microscopy. Conidia killing rate was determined by antibiotic protecting assay. Cell activity was measured by microplate-MTT colorimetric assay; maturation of A. fumigatus-containing phagosomes was revealed by Immunofluorescence microscopy assay. Phagosomes containing A. fumigatus conidia was isolated by ultracentrifugation on a sucrose density gradient. Conidia pigment mutants were obtained by exposure to UV and isolated by the color of colony.Results The rate of phagocytosis of conidia by macrophages are slower than phagocytosis of latex beads. Macrophagus began to kill A. fumigatus conidia within one hour. Either A. fumigatus conidia or swllen conidia inhibits activity of macrophages but not hyphae. The conidia of white mutat strain did not inhibit activity of macrophage. We used antibodies specifically recognizing various membrane markers of the endocytic pathway such as CatD, EEA1, LAMP1 to determine the extent to which the A. fumigatus-containing phagosomes interacted with compartments of the endocytic pathway. At early time points, a typical phagosome marker of CatD was observed on 30'/0', with occasionally a larger ring like structure. Betweena 30'/0'and 30'/30', the early phagosome marker of EEA1 was observed. At 30'/60', the late phagosome marker of LAMP1 was observed. The last, in our in-depth investigation of the method of phagosome isolation, we adapted the method to purify conidia-containing phagosomes from infected macrophages.Conclusion The surfaces of A. fumigatus conidia have something which can inhibit phagocytosis. Macrophage began to kill conidia within 1 h and 50% of conidia were killed in 2h, 80% of conidia were killed after 6h. Either A. fumigatus conidia or swllen conidia can inhibit oxidation activity of macrophages. White conidia and hyphae don't inhibit activity of macrophages. Melanin has been established as an important virulence factor, inhibition oxidation activity of macrophages. Phagosome acquire microbicidal and degradative capabilities on 30'/60'.