The Study Of Aspergillus Fumigatus Phospholipase D Gene Deletion And Pld-Deleted Strain

Objective:Aspergillus fumigatus (A. fumigatus) is one of the most prevalent airborne fungal pathogen inducing serious infections in immunocompromised patients. Phospholipase D (PLD) is a key enzyme in pathogenic microorganism. There is poor study of phospholipase D in A. fumigatus. We explore the pathogenic mechanism of phoshpolipase D in A. fumigatus. Our studies will give a new way in prevention and treatment of A. fumigatus infection.Methods:We have constructed the pld-deleted strain (Apld) using Agrobacterium tumefaciens-mediated transformation (ATMT) by replacing A. fumigatus the phoshpholipase D gene with hygromycin resistance gene (hph). To detect the adhension and invasion ability of the Apld strain in A549 cells in vitro and the virulence in immunosupression mice which were immunosuppressed by hydrocortisone acetate or cyclophosphamide, respectively.Results:We have got Apld and pldc strains proved by Southern blot. PLD activity was confirmed to significantly decrease both intracellular and extracellular. The disruption of pld gene did not alter conidial morphological characteristics, growth, germination, hyphal dry weight and biofilm formation, but significantly suppressed the invasion of A. fumigatus into A549 epithelial cells without affecting conidial adhesion to epithelial cells. The suppressed invasion was fully rescued in the presence of 20μM phosphatidic acid (PA), the PLD product. And there was no differences in invasion ability between Apld and pldc strains in the presence of 100μM PA. Phagocytosis of A. fumigatus conidia by J774 cell was not affected by the absence of the pld gene. Pre-treatment of conidia with 1-butanol and a specific PLD inhibitor decreased the invasion of A. fumigatus into A549 epithelial cells, but there was no effect on phagocytosis by J774 macrophages. Finally, loss of the pld gene attenuated the virulence of A. fumigatus in mice immunosuppressed with hydrocortisone acetate, but not with cyclophosphamide.Conclusions:Invasion ability of A. fumigatus△pld into A549 epithelial cells and virulence in mice immunosuppressed with hydrocortisone acetate are decreased. Phospholipase D is one of important virulence factor for A. fumigatus.