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Flavodoxin-Like Proteins Protect Candida albicans from Oxidative Stress and Promote Virulence.

Li L, Naseem S, Sharma S, Konopka JB - PLoS Pathog. (2015)

Bottom Line: Quinone reductase activity confers important antioxidant effects because resistance to oxidation was restored in the quadruple mutant by expressing either Escherichia coli wrbA or mammalian NQO1, two distinct types of quinone reductases.These observations suggested that FLPs reduce ubiquinone (coenzyme Q), enabling it to serve as an antioxidant in the membrane.The potential of FLPs as novel targets for antifungal therapy is further underscored by their absence in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York, United States of America.

ABSTRACT
The fungal pathogen Candida albicans causes lethal systemic infections in humans. To better define how pathogens resist oxidative attack by the immune system, we examined a family of four Flavodoxin-Like Proteins (FLPs) in C. albicans. In agreement with previous studies showing that FLPs in bacteria and plants act as NAD(P)H quinone oxidoreductases, a C. albicans quadruple mutant lacking all four FLPs (pst1Δ, pst2Δ, pst3Δ, ycp4Δ) was more sensitive to benzoquinone. Interestingly, the quadruple mutant was also more sensitive to a variety of oxidants. Quinone reductase activity confers important antioxidant effects because resistance to oxidation was restored in the quadruple mutant by expressing either Escherichia coli wrbA or mammalian NQO1, two distinct types of quinone reductases. FLPs were detected at the plasma membrane in C. albicans, and the quadruple mutant was more sensitive to linolenic acid, a polyunsaturated fatty acid that can auto-oxidize and promote lipid peroxidation. These observations suggested that FLPs reduce ubiquinone (coenzyme Q), enabling it to serve as an antioxidant in the membrane. In support of this, a C. albicans coq3Δ mutant that fails to synthesize ubiquinone was also highly sensitive to oxidative stress. FLPs are critical for survival in the host, as the quadruple mutant was avirulent in a mouse model of systemic candidiasis under conditions where infection with wild type C. albicans was lethal. The quadruple mutant cells initially grew well in kidneys, the major site of C. albicans growth in mice, but then declined after the influx of neutrophils and by day 4 post-infection 33% of the mice cleared the infection. Thus, FLPs and ubiquinone are important new antioxidant mechanisms that are critical for fungal virulence. The potential of FLPs as novel targets for antifungal therapy is further underscored by their absence in mammalian cells.

No MeSH data available.


Related in: MedlinePlus

Heterologous expression of E. coli wrbA or rat NQO1 rescues the sensitivity of Δ/Δ/Δ/Δ mutant to oxidants.The distinct NAD(P)H quinone oxidoreductase genes wrbA and NQO1 were expressed in the Δ/Δ/Δ/Δ strain under control of the ADH1 promoter. A control strain was also constructed in a similar manner that expressed GFP. The cells were then incubated with 0.5 mM linolenic acid (LNA) for 6 h at 37˚C and then (A) assayed for viable CFUs or (B) assayed for TBARS as an indicator of lipid peroxidation. Some cells in panel A were also incubated with oleic acid (OA) as a control. (C) Dilutions of cells were spotted onto different agar plates containing synthetic medium and the indicated oxidant, and then incubated at 37°C for 2 d. The plates contained H2O2, tert-butyl hydroperoxide (TBHP), cumene hydroperoxide (CHP), menadione (MND), monounsaturated oleic acid (OA), and polyunsaturated linolenic acid (LNA). Strains used included the wild type control strain LLF100, Δ/Δ/Δ/Δ strain LLF060, the complemented strain LLF079, and the Δ/Δ/Δ/Δ strain in which E. coli wrbA (LLF074), rat NQO1 (LLF076) or GFP (LLF080) was expressed under control of the constitutive ADH1 promoter. Error bars indicate SE. *, p <0.05 by ANOVA.
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ppat.1005147.g004: Heterologous expression of E. coli wrbA or rat NQO1 rescues the sensitivity of Δ/Δ/Δ/Δ mutant to oxidants.The distinct NAD(P)H quinone oxidoreductase genes wrbA and NQO1 were expressed in the Δ/Δ/Δ/Δ strain under control of the ADH1 promoter. A control strain was also constructed in a similar manner that expressed GFP. The cells were then incubated with 0.5 mM linolenic acid (LNA) for 6 h at 37˚C and then (A) assayed for viable CFUs or (B) assayed for TBARS as an indicator of lipid peroxidation. Some cells in panel A were also incubated with oleic acid (OA) as a control. (C) Dilutions of cells were spotted onto different agar plates containing synthetic medium and the indicated oxidant, and then incubated at 37°C for 2 d. The plates contained H2O2, tert-butyl hydroperoxide (TBHP), cumene hydroperoxide (CHP), menadione (MND), monounsaturated oleic acid (OA), and polyunsaturated linolenic acid (LNA). Strains used included the wild type control strain LLF100, Δ/Δ/Δ/Δ strain LLF060, the complemented strain LLF079, and the Δ/Δ/Δ/Δ strain in which E. coli wrbA (LLF074), rat NQO1 (LLF076) or GFP (LLF080) was expressed under control of the constitutive ADH1 promoter. Error bars indicate SE. *, p <0.05 by ANOVA.

Mentions: To confirm whether quinone reductase activity is important to promote resistance to oxidative stress in C. albicans, the Δ/Δ/Δ/Δ mutant was engineered to express two distinct types of NAD(P)H quinone oxidoreductases: rat NQO1 and E. coli wrbA. NQO1 and wrbA were selected because their proteins have been well-studied biochemically [21, 39, 40]. These genes were expressed under the control of the strong ADH1 promoter. As a control, cells were also engineered to express GFP in a similar manner. Incubation of the cells in the presence of 0.5 mM linolenic acid for 6 h showed that expression of either wrbA or NQO1 rescued the viability of the Δ/Δ/Δ/Δ mutant (Fig 4A). In contrast, the Δ/Δ/Δ/Δ mutant or the Δ/Δ/Δ/Δ mutant that expressed only GFP showed a significant drop in CFUs. Similarly, expression of wrbA or NQO1, but not GFP, diminished lipid peroxidation in cells that were exposed to linolenic acid (Fig 4B). Growth assays on agar plates also showed that wrbA and NQO1 could complement the increased sensitivity of the Δ/Δ/Δ/Δ mutant to H2O2, tert-butyl hydroperoxide, cumene hydroperoxide and menadione (Fig 4C). The ability of two distinct quinone reductases to complement the Δ/Δ/Δ/Δ mutant phenotype demonstrates that this activity plays a key antioxidant role in C. albicans.


Flavodoxin-Like Proteins Protect Candida albicans from Oxidative Stress and Promote Virulence.

Li L, Naseem S, Sharma S, Konopka JB - PLoS Pathog. (2015)

Heterologous expression of E. coli wrbA or rat NQO1 rescues the sensitivity of Δ/Δ/Δ/Δ mutant to oxidants.The distinct NAD(P)H quinone oxidoreductase genes wrbA and NQO1 were expressed in the Δ/Δ/Δ/Δ strain under control of the ADH1 promoter. A control strain was also constructed in a similar manner that expressed GFP. The cells were then incubated with 0.5 mM linolenic acid (LNA) for 6 h at 37˚C and then (A) assayed for viable CFUs or (B) assayed for TBARS as an indicator of lipid peroxidation. Some cells in panel A were also incubated with oleic acid (OA) as a control. (C) Dilutions of cells were spotted onto different agar plates containing synthetic medium and the indicated oxidant, and then incubated at 37°C for 2 d. The plates contained H2O2, tert-butyl hydroperoxide (TBHP), cumene hydroperoxide (CHP), menadione (MND), monounsaturated oleic acid (OA), and polyunsaturated linolenic acid (LNA). Strains used included the wild type control strain LLF100, Δ/Δ/Δ/Δ strain LLF060, the complemented strain LLF079, and the Δ/Δ/Δ/Δ strain in which E. coli wrbA (LLF074), rat NQO1 (LLF076) or GFP (LLF080) was expressed under control of the constitutive ADH1 promoter. Error bars indicate SE. *, p <0.05 by ANOVA.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4556627&req=5

ppat.1005147.g004: Heterologous expression of E. coli wrbA or rat NQO1 rescues the sensitivity of Δ/Δ/Δ/Δ mutant to oxidants.The distinct NAD(P)H quinone oxidoreductase genes wrbA and NQO1 were expressed in the Δ/Δ/Δ/Δ strain under control of the ADH1 promoter. A control strain was also constructed in a similar manner that expressed GFP. The cells were then incubated with 0.5 mM linolenic acid (LNA) for 6 h at 37˚C and then (A) assayed for viable CFUs or (B) assayed for TBARS as an indicator of lipid peroxidation. Some cells in panel A were also incubated with oleic acid (OA) as a control. (C) Dilutions of cells were spotted onto different agar plates containing synthetic medium and the indicated oxidant, and then incubated at 37°C for 2 d. The plates contained H2O2, tert-butyl hydroperoxide (TBHP), cumene hydroperoxide (CHP), menadione (MND), monounsaturated oleic acid (OA), and polyunsaturated linolenic acid (LNA). Strains used included the wild type control strain LLF100, Δ/Δ/Δ/Δ strain LLF060, the complemented strain LLF079, and the Δ/Δ/Δ/Δ strain in which E. coli wrbA (LLF074), rat NQO1 (LLF076) or GFP (LLF080) was expressed under control of the constitutive ADH1 promoter. Error bars indicate SE. *, p <0.05 by ANOVA.
Mentions: To confirm whether quinone reductase activity is important to promote resistance to oxidative stress in C. albicans, the Δ/Δ/Δ/Δ mutant was engineered to express two distinct types of NAD(P)H quinone oxidoreductases: rat NQO1 and E. coli wrbA. NQO1 and wrbA were selected because their proteins have been well-studied biochemically [21, 39, 40]. These genes were expressed under the control of the strong ADH1 promoter. As a control, cells were also engineered to express GFP in a similar manner. Incubation of the cells in the presence of 0.5 mM linolenic acid for 6 h showed that expression of either wrbA or NQO1 rescued the viability of the Δ/Δ/Δ/Δ mutant (Fig 4A). In contrast, the Δ/Δ/Δ/Δ mutant or the Δ/Δ/Δ/Δ mutant that expressed only GFP showed a significant drop in CFUs. Similarly, expression of wrbA or NQO1, but not GFP, diminished lipid peroxidation in cells that were exposed to linolenic acid (Fig 4B). Growth assays on agar plates also showed that wrbA and NQO1 could complement the increased sensitivity of the Δ/Δ/Δ/Δ mutant to H2O2, tert-butyl hydroperoxide, cumene hydroperoxide and menadione (Fig 4C). The ability of two distinct quinone reductases to complement the Δ/Δ/Δ/Δ mutant phenotype demonstrates that this activity plays a key antioxidant role in C. albicans.

Bottom Line: Quinone reductase activity confers important antioxidant effects because resistance to oxidation was restored in the quadruple mutant by expressing either Escherichia coli wrbA or mammalian NQO1, two distinct types of quinone reductases.These observations suggested that FLPs reduce ubiquinone (coenzyme Q), enabling it to serve as an antioxidant in the membrane.The potential of FLPs as novel targets for antifungal therapy is further underscored by their absence in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York, United States of America.

ABSTRACT
The fungal pathogen Candida albicans causes lethal systemic infections in humans. To better define how pathogens resist oxidative attack by the immune system, we examined a family of four Flavodoxin-Like Proteins (FLPs) in C. albicans. In agreement with previous studies showing that FLPs in bacteria and plants act as NAD(P)H quinone oxidoreductases, a C. albicans quadruple mutant lacking all four FLPs (pst1Δ, pst2Δ, pst3Δ, ycp4Δ) was more sensitive to benzoquinone. Interestingly, the quadruple mutant was also more sensitive to a variety of oxidants. Quinone reductase activity confers important antioxidant effects because resistance to oxidation was restored in the quadruple mutant by expressing either Escherichia coli wrbA or mammalian NQO1, two distinct types of quinone reductases. FLPs were detected at the plasma membrane in C. albicans, and the quadruple mutant was more sensitive to linolenic acid, a polyunsaturated fatty acid that can auto-oxidize and promote lipid peroxidation. These observations suggested that FLPs reduce ubiquinone (coenzyme Q), enabling it to serve as an antioxidant in the membrane. In support of this, a C. albicans coq3Δ mutant that fails to synthesize ubiquinone was also highly sensitive to oxidative stress. FLPs are critical for survival in the host, as the quadruple mutant was avirulent in a mouse model of systemic candidiasis under conditions where infection with wild type C. albicans was lethal. The quadruple mutant cells initially grew well in kidneys, the major site of C. albicans growth in mice, but then declined after the influx of neutrophils and by day 4 post-infection 33% of the mice cleared the infection. Thus, FLPs and ubiquinone are important new antioxidant mechanisms that are critical for fungal virulence. The potential of FLPs as novel targets for antifungal therapy is further underscored by their absence in mammalian cells.

No MeSH data available.


Related in: MedlinePlus