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

Abstract

Fusarium species exhibit significant intrinsic resistance to most antifungal agents and fungicides, resulting in high mortality rates among immunocompromised patients. Consequently, a thorough characterization of the antifungal resistance mechanism is required for effective treatments and for preventing fungal infections and reducing antifungal resistance. In this study, an isolate of Fusarium oxysporum (wild-type) with broadly resistant to commonly antifungal agents was used to generate 1,450 T-DNA random insertion mutants via Agrobacterium tumefaciens-mediated transformation. Antifungal susceptibility test results revealed one mutant with increased sensitivity to azoles. Compared with the resistant wild-type, the mutant exhibited low MICs to KTZ, ITC, VRC, POS, and PCZ (0.125, 1, 0.06, 0.5, and 0.125μg/ml, respectively). The T-DNA insertion site of this mutant was characterized as involving two adjacent genes, one encoding a hypothetical protein with unknown function and the other encoding the NADPH-cytochrome P450 reductase, referred as CPR1. To confirm the involvement of these genes in the altered azole susceptibility, the independent deletion mutants were generated and the Cpr1 deletion mutant displayed the same phenotypes as the T-DNA random mutant. The deletion of Cpr1 significantly decreased ergosterol levels. Additionally, the expression of the downstream Cyp51 gene was affected, which likely contributed to the observed increased susceptibility to azoles. These findings verified the association between Cpr1 and azole susceptibility in F. oxysporum. Furthermore, this gene may be targeted to improve antifungal treatments.

Rapid Identification of Four Fusarium spp. Complex by High-Resolution Melting Curve Analysis and their Antifungal Susceptibility Profiles

“…Fusarium species are globally distributed lamentous ascomycete fungi that are frequently reported as plant pathogens and opportunistic human pathogens [13,14]. Species within Fusarium are causative agents of disease in a broad range of wild and cultivated plants.…”

“…While the prevalence of Fusarium infections of the pulmonary system is lower than that attributable to Aspergillus, its patient outcomes are generally poorer, with higher rates of morbidity and mortality [15,16]. This is considered to be resulted from the broad resistance of Fusarium species against commonly used antifungal agents [13,20,29]. Thus, in vitro evaluation of antifungal susceptibility of these strains provides signi cant guidance for appropriate antifungal drug selection in clinic.…”

“…are related to infections that cause fusariosis in humans [11,12]. The rate of human fungal infections increases with the upward trend of immunocompromised population, leading to an increase in the morbidity and mortality rates due to fusariosis [13][14][15][16][17]. This could due to the fact that most available antifungal drugs are ineffective against fusariosis [18].…”

Hypothetical protein FoDbp40 influences the growth and virulence of Fusarium oxysporum by regulating the expression of isocitrate lyase

“…Wild type F. oxysporum JLCC31768 and Agrobacterium tumefaciens AgrN (containing plasmid pXEN carrying neomycin and kanamycin resistance tags[ neo ]) were used to generate F. oxysporum mutants ( He et al, 2021 ). Wild type and AgrN ( Table 1 ) were preserved at and obtained from the Jilin University Mycology Research Center (Jilin, China).…”

“…ATMT of F. oxysporum was performed as described previously to obtain mutants with single-strand transferred DNA (T-DNA) inserts ( He et al, 2021 ). The DNA of randomly selected mutants containing the neo gene was isolated and amplified using DNA extraction kits (Beyotim, Shanghai, China) and specific neoF and neoR primers ( He et al, 2021 ). The products were then sequenced by Comate Bioscience Co., Ltd. (Jilin, China) to confirm whether T-DNA was inserted into the F. oxysporum genome.…”