Identification and characterization of an anti-oxidative stress-associated mutant of Aspergillus fumigatus transformed by Agrobacterium tumefaciens

Abstract

Aspergillus fumigatus is one of the most common opportunistic pathogenic fungi, surviving in various environmental conditions. Maintenance of the redox homeostasis of the fungus relies upon the well-organized regulation between reactive oxygen species generated by immune cells or its own organelles, and the activated anti-oxidative stress mechanism. To investigate such a mechanism, the present study obtained a number of randomly-inserted mutants of A. fumigatus, mediated by Agrobacterium tumefaciens. In addition, a high throughput hydrogen peroxide screening system was established to examine ~1,000 mutants. A total of 100 mutants exhibited changes in hydrogen peroxide sensitivity, among which a significant increase in sensitivity was observed in the AFM2658 mutant. Further investigations of the mutant were also performed, in which the sequence of this mutant was characterized using thermal asymmetric interlaced-polymerase chain reaction. This revealed that the insertion site was located on chromosome 2 afu1_92, and the 96 bp sequence was knocked out, which partially comprised a sequence localized between the integral membrane protein coding region and the helix-loop-helix transcription factor coding region. A decrease in the levels of anti-oxidative stress-associated mRNAs were observed, and an increase in reactive oxygen species were detected using fluorescence. The results of the present study demonstrated that this sequence may have a protective role in A. fumigatus in the presence of oxidative stress.

Identification of glutathione metabolic genes from a dimorphic fungus Talaromyces marneffei and their gene expression patterns under different environmental conditions

“…Yet, the Sc GPX1 gene was induced by glucose starvation while the Sc GPX2 gene was induced by oxidative stress 44 . In A. fumigatus , the hyr1 gene was moderately induced upon H 2 O 2 treatment (fold-change < 2, p -value < 0.05; 45 . In C. albicans , only the GPX31 gene was highly induced upon exposure to oxidative stress 43 .…”

The sat1 Gene Is Required for the Growth and Virulence of the Human Pathogenic Fungus Aspergillus fumigatus

“…We constructed the T-DNA insertion mutant library according to a previously described method involving ATMT ( 12 ). The T-DNA flanking sequences were identified by touchdown TAIL-PCR ( 13 ), and the amplified fragments were sequenced by Comate Bioscience Co., Ltd. (Jilin, China).…”

“…To search for genes regulating A. fumigatus growth and analyze their contribution to virulence, Agrobacterium tumefaciens -mediated transformation (ATMT) was used to construct a transfer DNA (T-DNA) random insertion mutation library ( 12 ). The mutant of AFM2954 with severe growth defect was obtained by screening the library for colonies with altered morphologies.…”

Contribution of NADPH-cytochrome P450 Reductase to Azole Resistance in Fusarium oxysporum

“…The F. oxysporum T-DNA insertion mutants were generated as previously described ( Fan et al, 2016 ). Briefly, fungal spores (1×10 4 CFU/ml) were mixed with an equal volume (1ml) of AgrN cells (OD 600nm =0.8).…”