| Aspergillus fumigatus is a saprophytic fungus that is ubiquitously distributed in a wide range of environments and spreads by producing conidia.It is an important opportunistic pathogenic fungus,second only to Candida albicans in systemic fungal infection,which can cause invasive aspergillosis(IA)in immunocompromised people,has become an important lethal fungus.In healthy people,innate immunity can effectively clear inhaled conidia,but in immunocompromised people,the conidia of A.fumigatus can adhere to lung epithelial cells and germinate to hyphae,which can invade host tissues and blood vessels and cause IA.Due to limited antifungal drugs,lack of fungal vaccines,increasing immune-compromised populations,and the emergence of fungal resistance,IA is increased year by year,with a lethality as high as 50%～90%.Although some improvement has been made in A.fumigatus,the pathogenic mechanism remains largely unknown.There are still a large number of non-coding regions and unannotated proteins with unknown functions in the A.fumigatus genome;however,the functions of only a few percent of annotated proteins have been verified by experiments.The T-DNA random insertion mutation can be used to annotate the functions of non-coding regions,hypothetical proteins,and new functions of annotated proteins.Regulated fungal growth is essential for disease development and progression.Studies suggest that there are some associations between pathogenicity and the growth of fungus.Studies have shown that abnormal growth and development,including changes in colony morphology,hyphal growth,and spore production,may exhibit altered pathogenicity of fungal mutants.Knockout of key genes of the TCA cycle in C.albicans resulted in defective hyphal growth.Knockout of cell wall integrity signaling pathway and calcineurin-related genes in A.fumigatus resulted in defective colony growth and reduced pathogenicity.Overexpression of brlA,which is related to conidia production affects hyphae growth and pathogenicity of A.fumigatus.Thus,screening for genes that regulate fungal growth and development may obtain pathogenic factors,annotate functions of genes,help to explore the pathogenic mechanisms and lead to the identification of potential therapeutic targets for IA.The contents of this study can be summarized as follows:(1)Screening and analysis of abnormal growth and development mutants of A.fumigatusTo search for genes regulating A.fumigatus growth and analyze their contribution to pathogenicity,Agrobacterium tumefaciens-mediated transformation(ATMT)was used to construct a transfer DNA(T-DNA)random insertion mutation library.By screening 3042 mutants,40 mutants with defective growth and development were obtained.The colony morphology changes included smaller colonies,albino,increased aerial hyphae,wrinkled appearance,or irregular edge.T-DNA flanking sequences of mutants were identified by touchdown TAIL-PCR and identified the genes related to growth and development.It has laid a solid foundation for the functional analysis of the genes related to the growth and development of A.fumigatus and the exploration of the pathogenic mechanism.Mutant AFM3725 had defective colony growth and T-DNA disruption of the noncoding region located on chromosome 6.Sequence analysis showing that the noncoding region was completely consistent with the sequences of other A.fumigatus isolates.The pathogenicity of the mutant was analyzed by C.elegans and mouse infection models,and the results showed that compared with WT,AFM3725 infected animals had lower mortality,less fungal load,and attenuated histopathological damage in the lungs of mice,showed that deletion of the noncoding region in AFM3725 resulted in reduced pathogenicity of A.fumigatus.The mutant AFM2954 was severely defective in colony growth,and the T-DNA interrupted sat1,encoding the putative intracellular protein transporter Sat1.The amino acid sequence analysis showed that Sat1 is conserved in common filamentous pathogenic fungi,and its function has not been reported.The effect of sat1 on the growth of A.fumigatus was further verified by constructing a targeted knockout strainΔsat1 and a compensatory strain sat1 C.Microscopic observation showed that the sat1 deficient strains had many growth defects,including impaired hyphal polarity growth,abnormal apical vesicle,and decreased sporulation.The effect of sat1 on the pathogenicity of A.fumigatus was analyzed by the animal models,and the results showed that sat1 deletion reduced mortality in infected animals and attenuated fungal burden and histopathological damage in the lungs of mice,indicating that sat1 deletion reduces the pathogenicity of A.fumigatus.(2)The sat1 affects the mitochondrial function of A.fumigatusRNA-seq analysis showed that the differentially expressed genes related to the TCA(Tricarboxylic Acid)cycle,oxidative phosphorylation,and cell wall synthesis were significantly enriched in the sat1 mutant.The reduction of mitochondrial membrane potential,ATP levels,and down-regulation of the TCA cycle and oxidative phosphorylation-related genes in sat1-deficient strains indicated that sat1 deletion affected A.fumigatus mitochondrial function.Furthermore,the increase in phosphatidylserine exposure and DNA fragmentation and the upregulation of casA and casB,which related to apoptosis of A.fumigatus indicated that sat1 deletion further induced apoptosis of A.fumigatus.(3)The sat1 is involved in cell wall synthesis-related processesCell wall analysis showed that the production of protoplasts in the sat1-deficient strains were reduced,the resistance to cell wall perturbing agents was increased,the cell wall was thickened,and the content of cell wall components such as β-1,3-glucan was increased,indicating that sat1 affects the synthesis of A.fumigatus cell wall.A.fumigatus MpkA is a key factor in the cell wall integrity signaling pathway.Western Blot results showed that the loss of sat1 increased the phosphorylation of MpkA,indicating that sat1 may affect the synthesis of A.fumigatus cell wall by regulating the cell wall integrity signaling pathway.The β-1,3-glucan synthase affects the synthesis ofβ-1,3-glucan,which is a target of echinocandin antifungal drugs.In this study,the drug susceptibility of strains to caspofungin was examined,and the results showed that the sat1 deleted strains had reduced drug susceptibility to caspofungin.The results of cell experiments showed that sat1 deficient strains increased the adhesion rate to lung epithelial cells and polyethylene planes but the damage to lung epithelial cells was reduced.In summary,the T-DNA random insertion mutation library of A.fumigatus was screened,some mutants with abnormal growth and development were obtained,and located some genes related to growth and development of A.fumigatus.Further analysis was carried out on the two mutants with significantly abnormal growth and development.The mutant AFM3725 T-DNA disrupted a non-coding region,which affects growth,development,and pathogenicity.The mutant AFM2954 T-DNA interrupted the sat1 gene with an unknown function.The results of the study revealed that sat1 affects the growth,development,and pathogenicity of A.fumigatus by regulating mitochondrial function and cell wall synthesis.This study will lay a foundation for the study of growth,development,and pathogenic mechanism of A.fumigatus,and provide a theoretical basis for the development of IA therapeutic targets. |
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