Invasion Mechanism Of Aspergillus Fumigatus And PCR-SSCP Analyses Candida. As A Molecular Epidemiological Method

ObjectiveTo 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 cup and its relation with the cellular actin cytoskeleton rearrangement and translocation of PLD during the invasion; to evaluate the feasibility and optimal condition of PCR-single strand conformation polymorphism (PCR-SSCP) as a molecular epidemiological method for clinical Candida spp. strains.MethodsThe human lung endothelial cells (A549) and murine macrophage cells(J774.Al) 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; A549 cells and J774.A1 cells were pretreated with inhibitor of PLD, the invasion of A.fumigutas into host cells were detected to evaluate the role of PLD in A.fumigutas entry into host cells; further, with the immuno-fluorescent staining and green-fluorescent-protein-expressing techniques, cellular actin cytoskeleton rearrangement, translocation of PLD and formation of phagocytic cup of A.fumigutas sporus were observed under the laser confocal scanning microscope. At the end, the electrophoresis conditions of PCR-SSCP analysis, such as amplified region, temperature, concentration of glycerol and so on, were compared and optimized.ResultsThe invasion of A.fumigutas into A549 cells was significantly enhanced with the increase of MOI and the duration of infection, whereas the invasion of A.fumigutas into J774.A1 cells was decreased with the duration of infection. The internalization of A.fumigutas into A549 cells was associated with strong activation of host PLD. Meanwhile, the cellular actin cytoskeleton rearrangement was induced intensively and the phagocytic cups were formed, with more likely translocation of PLD in the invasion site. Moreover, the pretreatment of both of these cells with 1-butanol, resulted in the reductions of invasion of A.fumigutas into the cells. In addition, PCR-SSCP might be a suitable molecular typing method for clinical Candida.spp when it targeted on ITS I regions and was performed in the presence of 5% glycerol at 15℃ConclusionBoth of the A.fumigutas-induced internalization into A549 cells and initiative phagocytosis by J774.A1 cells are 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. Under the appropriate condition, PCR-SSCP analysis is a quick, simple, and reliable method for molecular epidemiological study on clinical Candida spp.