The Macrophage Model Was Used To Study The Relationship Between The Melanin Layer Of Aspergillus Fumigatus Cell Wall And Pathogenicity

Aspergillus fumigatus is an important pathogen of the immunocompromised host causing pneumonia and invasive disseminated disease with high mortality.Melanin is a complex polymer,which is secreted outside or constitutes the structure of fungal cell wall.It is considered as an important virulence factor linked to fungal virulence.Six genes(pksP,arpl,arp2,ayg1,abr1,abr2),forming a gene cluster spanning 19 kb,were identified as involved in conidial pigment biosynthesis in A.fumigatus.In the previous study,the conidia deleted of pksP gene showed a smooth surface morphology and appeared to be less protected against killing by the host defence.When compared to the WT,it also showed reduced virulence in a murine animal model.However,few studies focused on the interaction of Aspergillus fumigatus DHN-melanin with the host and on the other genes involved in the synthesis of melanin.So pathogenic mechanism of melanin in the complex interplay with host still need further study.To explore the function of melanin in modulating host response in A.fumigatus,six A.fumigatus mutant strains were used as followed:Af293 and A1160(wild-type strains),CZ01,CZ02(two different white transformants which result from Agrobacterium tumefaciens-mediated transformation of Af293),CZ03(brown mutant results from insert mutation of abrl gene in background of A1160),CZ04(white mutant is the production of gene knockout from A1160).Since murine monocyte macrophage cell line RAW264.7 was able to phagocytose aspergillus condia,the author tested the difference of the phagocytosis and killing of our six color A..fumigatus conidia by RAW264.7.The results indicated the conidia CZ01 and CZ02 and CZ04 were more likely to be phagocytosed and killed by RAW264.7 compared with the green conidia Af293 and A1160 as well as the brown conidia CZ03,indicating melanin in conidial cell wall plays an important role for survival during phagocytosis process.For futher study,the author detected the hydrophobicity and adhesion of conidia in different strains.As a result,melanised conidia of Af293,A1160 and brown mutant CZ03 showen more hydrophobicity than that of pigment-less conidia CZ01,CZ02 and CZ04 strains.And the CZ02 and CZ04 conidia had a higher biofilm formation.Then we using the red probe lysosomal Lyso-Tracker RedDND-99 to labele lysosomal and Calcofluor White to labele conidia for detecting the each color conidia acidification in macrophages,and the results showed that Af293,A1160 and brown mutant CZ03 slightly,if any,inhibit the acidification of phagolysosomes,as compared to albino conidia CZ01,CZ02,CZ04.The author also measured cytokines by ELISA kits and Reactive Oxygen Species by the fluorescent probe DCFH-DA of RAW264.7 macrophages in the stimulation of different experimental strains,the results indicated that the conidia CZ01,CZ02,CZ04 lacking of dihydroxynaphthalene(DHN)-melanin produced a stronger inflammatory response and more reactive oxygen species.This paper focuseed on the detection of different pathogenicity of different color strains associatied with the synthesis of melanin by using our new cell model,and systematically explored the virulence differences in cell internal mechanisms.All the data we got showed Aspergillus fumigatus melanin played an impontant role in virulence when invaded their host,and also explored the role of melanin gene abrl during Aspergillus fumigatus invasion process,which may provide a new theoretical basis of researching the virulence of Aspergillus fumigatus.