Aspergillus Fumigatus-induced Phagocytosis Process Of Host Cell Phospholipase D Activation And Its Function

Objective To study the possible phospholipase D (PLD) activation and its role in the invasion of A.fumigutas sporus into host cells; to investigate the formation of phaogocytic vesicle and its relation with the cellular actin cytoskeleton rearrangement and translocation of PLD during the invasionMethods The human lung endothelial cells (A549) were incubated with A.fumigutas at different multiplicity of infection for different time, the invasion efficiency of A.fumigutas into cells and the activation of PLD stimulated by A.fumigutas in the cells were detected; the activation of PLD stimulated by A.fumigutas of different conditions(rest condidia, swallon condidia, hypae) in the cells were detected; with the immuno-fluorescent staining and green-fluorescent-protein-expressing techniques, cellular actin cytoskeleton rearrangement, translocation of PLD and formation of phagocytotic vesicle of A.fumigutas sporus were observed under the laser confocal scanning microscope; A549 cells were pretreated with inhibitor of PLD(1- butanol), the invasion of A.fumigutas into host cells were detected to evaluate the role of PLD in A.fumigutas entry into host cells; By expression of both PLD1 and PLD2 lipase-inactive mutants or silence of endogenous PLDs through RNA interference technique in A549 cells , the PLD activity was detected; so did upregulation by overexpression of wild-type PLDs.Results The invasion of A.fumigutas into A549 cells was significantly enhanced with the increase of MOI and the duration of infection(p<0.05); The internalization of A.fumigutas into A549 cells was associated with strong activation of host PLD(p<0.05); A.fumigutas of different conditions could active the host PLD, swallon condidia was induced the most strongest activation of host PLD(p<0.05); the cellular actin cytoskeleton rearrangement was induced intensively and the phagocytotic vesicle were formed, with more likely translocation of PLD in the invasion site; swallon condidia was induced the most strongest activation of host PLD; the pretreatment of these cells with 1-butanol, resulted in the reductions of invasion of A.fumigutas into the cells; Down regulation of PLD activity by expression of both PLD1 and PLD2 lipase-inactive mutants or silence of endogenous PLDs brought clear decrease on A.fumigutas -internalization(p<0.05), so did interestingly upregulation by overexpression of wild-type PLD(p<0.05).Conclusion The A.fumigutas-induced internalization into A549 cells were associated with cellular actin cytoskeleton rearrangement and activation of host PLD; The inhibition of A.fumigutas-stimulated PLD activity might depress the invasion of A.fumigutas into host cells,Down regulation of PLD activity or upregulation were not resulted in the inductions of invasion of A.fumigutas into the cells. The exact regulatory mechanism of PLD on A.fumigutas entry into host cells need be further clarified.