Gene Disruption in Scedosporium aurantiacum: Proof of Concept with the Disruption of SODC Gene Encoding a Cytosolic Cu,Zn-Superoxide Dismutase

Abstract

Scedosporium species are opportunistic pathogens responsible for a large variety of infections in humans. An increasing occurrence was observed in patients with underlying conditions such as immunosuppression or cystic fibrosis. Indeed, the genus Scedosporium ranks the second among the filamentous fungi colonizing the respiratory tracts of the CF patients. To date, there is very scarce information on the pathogenic mechanisms, at least in part because of the limited genetic tools available. In the present study, we successfully developed an efficient transformation and targeted gene disruption approach on the species Scedosporium aurantiacum. The disruption cassette was constructed using double-joint PCR procedure, and resistance to hygromycin B as the selection marker. This proof of concept was performed on the functional gene SODC encoding the Cu,Zn-superoxide dismutase. Disruption of the SODC gene improved susceptibility of the fungus to oxidative stress. This technical advance should open new research areas and help to better understand the biology of Scedosporium species.

Synthesis of the Hydroxamate Siderophore Nα-Methylcoprogen B in Scedosporium apiospermum Is Mediated by sidD Ortholog and Is Required for Virulence

“…Previously, Pateau et al (2018) prepared from S. aurantiacum defective strains for a cytosolic Cu, Zn-superoxide dismutase A B…”

“…Monospore isolates of transformants growing in the presence of 20 µg/mL phleomycin or 50 µg/mL hygromycin B were subcultured, and their genotype was analyzed by Southern blot as previously described (Pateau et al, 2018) to confirm the integration of the disruption cassette at the target locus. Genomic DNA was extracted and digested overnight with the appropriate restriction enzyme (EcoRI for analysis of the Dku70 mutant, and SacI for the double mutant).…”

Fungal Respiratory Infections in Cystic Fibrosis (CF): Recent Progress and Future Research Agenda

“…Host response of fungal diseases in CF patients [21] 7. Fungal virulence mechanisms [22][23][24][25][26][27] large study involved 243 CF patients from seven centers in France over a 15-month period. The investigators found that about half of the patients presented with at least one fungal species, while 37% were co-colonized with two species.…”

“…The genetic manipulations of fungal pathogens have yielded valuable insights into their virulence mechanisms. This experimental approach is now possible with the disruption of Cu/Zn-superoxide dismutase (SOD) gene in S. aurantiacum [23]. The research group led by Sandrine Giraud used dominant selection marker for the construction of the SOD disruption mutants and confirmed increased susceptibility to oxidative stress by the menadione sensitivity test [23].…”

“…This experimental approach is now possible with the disruption of Cu/Zn-superoxide dismutase (SOD) gene in S. aurantiacum [23]. The research group led by Sandrine Giraud used dominant selection marker for the construction of the SOD disruption mutants and confirmed increased susceptibility to oxidative stress by the menadione sensitivity test [23]. This proof-of-concept study is likely to lead to future in-depth experiments on the precise mechanisms of host-pathogen interactions in disease caused by S. aurantiacum and other Scedosporium species.…”