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

Fan,Zhongqi; Yu,Huimei; Guo,Qi; He,Dan; Xue,Baiji; Xie,Xiangli; Yokoyama,Koji; Wang,Li

doi:10.3892/mmr.2016.4839

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

Authors:
- Zhongqi Fan
- Huimei Yu
- Qi Guo
- Dan He
- Baiji Xue
- Xiangli Xie
- Koji Yokoyama
- Li Wang
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Affiliations: Department of Pathogenobiology, Jilin University Mycology Research Center, Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun, Jilin 130021, P.R. China, Medical Mycology Research Center, Chiba University, Chiba 260‑8673, Japan
Published online on: February 1, 2016 https://doi.org/10.3892/mmr.2016.4839
Pages: 2367-2376
Copyright: © Fan et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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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.

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1	Singh N and Paterson DL: Aspergillus infections in transplant recipients. Clin Microbiol Rev. 18:44–69. 2005. View Article : Google Scholar : PubMed/NCBI
2	Steinbach WJ, Marr KA, Anaissie EJ, Azie N, Quan SP, Meier-Kriesche HU, Apewokin S and Horn DL: Clinical epidemiology of 960 patients with invasive aspergillosis from the PATH Alliance registry. J Infect. 65:453–464. 2012. View Article : Google Scholar : PubMed/NCBI
3	Horn F, Heinekamp T, Kniemeyer O, Pollmächer J, Valiante V and Brakhage AA: Systems biology of fungal infection. Front Microbiol. 3:1082012. View Article : Google Scholar : PubMed/NCBI
4	Nierman WC, Pain A, Anderson MJ, Wortman JR, Kim HS, Arroyo J, Berriman M, Abe K, Archer DB, Bermejo C, et al: Genomic sequence of the pathogenic and allergenic filamentous fungus Aspergillus fumigatus. Nature. 438:1151–1156. 2005. View Article : Google Scholar : PubMed/NCBI
5	Hartmann T, Sasse C, Schedler A, Hasenberg M, Gunzer M and Krappmann S: Shaping the fungal adaptome-stress responses of Aspergillus fumigatus. Int J Med Microbiol. 301:408–416. 2011. View Article : Google Scholar : PubMed/NCBI
6	Segal BH and Romani LR: Invasive aspergillosis in chronic granulomatous disease. Med Mycol. 47(Suppl 1): S282–S290. 2009. View Article : Google Scholar : PubMed/NCBI
7	Boyle KB, Stephens LR and Hawkins PT: Activation of the neutrophil NADPH oxidase by Aspergillus fumigatus. Ann NY Acad Sci. 1273:68–73. 2012. View Article : Google Scholar : PubMed/NCBI
8	Lambou K, Lamarre C, Beau R, Dufour N and Latge JP: Functional analysis of the superoxide dismutase family in Aspergillus fumigatus. Mol Microbiol. 75:910–923. 2010. View Article : Google Scholar : PubMed/NCBI
9	Burns C, Geraghty R, Neville C, Murphy A, Kavanagh K and Doyle S: Identification, cloning and functional expression of three glutathione transferase genes from Aspergillus fumigatus. Fungal Genet Biol. 42:319–327. 2005. View Article : Google Scholar : PubMed/NCBI
10	Shibuya K, Paris S, Ando T, Nakayama H, Hatori T and Latge JP: Catalases of Aspergillus fumigatus and inflammation in aspergillosis. Nihon Ishinkin Gakkai Zasshi. 47:249–255. 2006. View Article : Google Scholar : PubMed/NCBI
11	Lamarre C, Ibrahim-Granet O, Du C, Calderone R and Latgé JP: Characterization of the SKN7 ortholog of Aspergillus fumigatus. Fungal Genet Biol. 44:682–690. 2007. View Article : Google Scholar : PubMed/NCBI
12	Lessing F, Kniemeyer O, Wozniok I, Loeffler J, Kurzai O, Haertl A and Brakhage AA: The Aspergillus fumigatus transcriptional regulator AfYap1 represents the major regulator for defense against reactive oxygen intermediates but is dispensable for pathogenicity in an intranasal mouse infection model. Eukaryot Cell. 6:2290–2302. 2007. View Article : Google Scholar : PubMed/NCBI
13	Frealle E, Aliouat-Denis CM, Delhaes L, Hot D and Dei-Cas E: Transcriptomic insights into the oxidative response of stress-exposed Aspergillus fumigatus. Curr Pharm Des. 19:3713–3737. 2013. View Article : Google Scholar : PubMed/NCBI
14	Wiedner SD, Burnum KE, Pederson LM, Anderson LN, Fortuin S, Chauvigné-Hines LM, Shukla AK, Ansong C, Panisko EA, Smith RD and Wright AT: Multiplexed activity-based protein profiling of the human pathogen Aspergillus fumigatus reveals large functional changes upon exposure to human serum. J Biol Chem. 287:33447–33459. 2012. View Article : Google Scholar : PubMed/NCBI
15	Weld RJ, Plummer KM, Carpenter MA and Ridgway HJ: Approaches to functional genomics in filamentous fungi. Cell Res. 16:31–44. 2006. View Article : Google Scholar : PubMed/NCBI
16	Zhang Y, Li G, He D, Yu B, Yokoyama K and Wang L: Efficient insertional mutagenesis system for the dimorphic pathogenic fungus Sporothrix schenckii using Agrobacterium tumefaciens. J Microbiol Methods. 84:418–422. 2011. View Article : Google Scholar : PubMed/NCBI
17	Lazo GR, Stein PA and Ludwig RA: A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Biotechnology (NY). 9:963–967. 1991. View Article : Google Scholar
18	Chen XL, Yang J and Peng YL: Large-scale insertional mutagenesis in Magnaporthe oryzae by Agrobacterium tumefaciens-mediated transformation. Methods Mol Biol. 722:213–224. 2011. View Article : Google Scholar : PubMed/NCBI
19	Mullins ED, Chen X, Romaine P, Raina R, Geiser DM and Kang S: Agrobacterium-mediated transformation of Fusarium oxysporum: An efficient tool for insertional mutagenesis and gene transfer. Phytopathology. 91:173–180. 2001. View Article : Google Scholar
20	Michielse CB, Hooykaas PJ, van den Hondel CA and Ram AF: Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori. Nat Protoc. 3:1671–1678. 2008. View Article : Google Scholar : PubMed/NCBI
21	Valiante V, Heinekamp T, Jain R, Härtl A and Brakhage AA: The mitogen-activated protein kinase MpkA of Aspergillus fumigatus regulates cell wall signaling and oxidative stress response. Fungal Genet Biol. 45:618–627. 2008. View Article : Google Scholar
22	Penttilä M, Nevalainen H, Rättö M, Salminen E and Knowles J: A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene. 61:155–164. 1987. View Article : Google Scholar : PubMed/NCBI
23	Canton E, Espinel-Ingroff A and Pemán J: Trends in antifungal susceptibility testing using CLSI reference and commercial methods. Expert Rev Anti Infect Ther. 7:107–119. 2009. View Article : Google Scholar : PubMed/NCBI
24	Morano KA, Grant CM and Moye-Rowley WS: The response to heat shock and oxidative stress in Saccharomyces cerevisiae. Genetics. 190:1157–1195. 2012. View Article : Google Scholar : PubMed/NCBI
25	Sugui JA, Chang YC and Kwon-Chung KJ: Agrobacterium tumefaciens-mediated transformation of Aspergillus fumigatus: An efficient tool for insertional mutagenesis and targeted gene disruption. Appl Environ Microbiol. 71:1798–1802. 2005. View Article : Google Scholar : PubMed/NCBI
26	Brakhage AA, Bruns S, Thywissen A, Zipfel PF and Behnsen J: Interaction of phagocytes with filamentous fungi. Curr Opin Microbiol. 13:409–415. 2010. View Article : Google Scholar : PubMed/NCBI
27	Moye-Rowley WS: Regulation of the transcriptional response to oxidative stress in fungi: Similarities and differences. Eukaryot Cell. 2:381–389. 2003. View Article : Google Scholar : PubMed/NCBI
28	Nikolaou E, Agrafioti I, Stumpf M, Quinn J, Stansfield I and Brown AJ: Phylogenetic diversity of stress signalling pathways in fungi. BMC Evol Biol. 9:442009. View Article : Google Scholar : PubMed/NCBI
29	Gasch AP, Spellman PT, Kao CM, Carmel-Harel O, Eisen MB, Storz G, Botstein D and Brown PO: Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell. 11:4241–4257. 2000. View Article : Google Scholar : PubMed/NCBI
30	Cuéllar-Cruz M, Briones-Martin-del-Campo M, Cañas-Villamar I, Montalvo-Arredondo J, Riego-Ruiz L, Castaño I and De Las Peñas A: High resistance to oxidative stress in the fungal pathogen Candida glabrata is mediated by a single catalase, Cta1p and is controlled by the transcription factors Yap1p, Skn7p, Msn2p and Msn4p. Eukaryot Cell. 7:814–825. 2008. View Article : Google Scholar
31	Nicholls S, Straffon M, Enjalbert B, Nantel A, Macaskill S, Whiteway M and Brown AJ: Msn2- and Msn4-like transcription factors play no obvious roles in the stress responses of the fungal pathogen Candida albicans. Eukaryot Cell. 3:1111–1123. 2004. View Article : Google Scholar : PubMed/NCBI
32	Winzeler EA, Shoemaker DD, Astromoff A, Liang H, Anderson K, Andre B, Bangham R, Benito R, Boeke JD, Bussey H, et al: Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science. 285:901–906. 1999. View Article : Google Scholar : PubMed/NCBI
33	Michielse CB, Hooykaas PJ, van den Hondel CA and Ram AF: Agrobacterium-mediated transformation as a tool for functional genomics in fungi. Curr Genet. 48:1–17. 2005. View Article : Google Scholar : PubMed/NCBI
34	Temple MD, Perrone GG and Dawes IW: Complex cellular responses to reactive oxygen species. Trends Cell Biol. 15:319–326. 2005. View Article : Google Scholar : PubMed/NCBI
35	Yan L, Li M, Cao Y, Gao P, Wang Y and Jiang Y: The alternative oxidase of Candida albicans causes reduced fluconazole susceptibility. J Antimicrob Chemother. 64:764–773. 2009. View Article : Google Scholar : PubMed/NCBI
36	Kontoyiannis DP: Modulation of fluconazole sensitivity by the interaction of mitochondria and erg3p in Saccharomyces cerevisiae. J Antimicrob Chemother. 46:191–197. 2000. View Article : Google Scholar : PubMed/NCBI
37	Xu XS, CY Z, QM L, HP D and W X: Study on the autofluorescence in fungi with laser scanning confocal microscope. Acta Laser Biology Sinica. 11:109–113. 2002.
38	Dreyer B, Morte A, Pérez-Gilabert M and Honrubia M: Autofluorescence detection of arbuscular mycorrhizal fungal structures in palm roots: An underestimated experimental method. Mycol Res. 110:887–897. 2006. View Article : Google Scholar : PubMed/NCBI
39	Zižka Z, Vêtrovský T and Gabriel J: Enhancement of autofluorescence of the brown-rot fungus Piptoporus betulinus by metal ions. Folia Microbiol (Praha). 55:625–628. 2010. View Article : Google Scholar
40	Mignone F, Gissi C, Liuni S and Pesole G: Untranslated regions of mRNAs. Genome Biol. 3:REVIEWS00042002. View Article : Google Scholar : PubMed/NCBI
41	Grillo G, Turi A, Licciulli F, Mignone F, Liuni S, Banfi S, Gennarino VA, Horner DS, Pavesi G, Picardi E and Pesole G: UTRdb and UTRsite (RELEASE 2010): A collection of sequences and regulatory motifs of the untranslated regions of eukaryotic mRNAs. Nucleic Acids Res. 38(Database Issue): D75–D80. 2010. View Article : Google Scholar :