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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

Differential sensitivity to oxidants for C. albicans strains expressing a single FLP gene or the analogous NQO1 gene.Strains expressing individual C. albicans FLP genes under control of their native promoter, or the E. coli wrbA or rat NQO1 gene under control of the ADH1 promoter were created by introducing the corresponding genes into the Δ/Δ/Δ/Δ mutant strain LLF054. (A) Serial 10-fold dilutions of cells were spotted onto agar plates containing synthetic medium and the indicated chemical. Plates were incubated at 37°C for 48 h and then photographed. (B) Summary of mutant phenotypes shown in panel A. A brighter yellow color indicates better cell growth of under the specified condition. Black indicates no growth detected. The values were determined from the relative extent of growth detected in two to three independent spot assays as shown in panel A. (C) Deletion mutant strains lacking a single FLP gene were spotted onto medium containing the indicated quinones. Note that the pst3Δ strain was more sensitive. Strains used included the wild type control strain LLF100, Δ/Δ/Δ/Δ strain LLF060, and the complemented strain LLF079. Also, the Δ/Δ/Δ/Δ strain engineered to express E. coli wrbA (strain LLF074), rat NQO1 (strain LLF076), GFP (strain LLF080), PST1 (LLF064), PST2 (LLF081), PST3 (LLF066) or YCP4 (LLF082). Single mutant deletion strains used in panel C were pst1Δ (LLF052), pst2Δ (LLF059), pst3Δ (LLF036), and ycp4Δ (LLF037).
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ppat.1005147.g005: Differential sensitivity to oxidants for C. albicans strains expressing a single FLP gene or the analogous NQO1 gene.Strains expressing individual C. albicans FLP genes under control of their native promoter, or the E. coli wrbA or rat NQO1 gene under control of the ADH1 promoter were created by introducing the corresponding genes into the Δ/Δ/Δ/Δ mutant strain LLF054. (A) Serial 10-fold dilutions of cells were spotted onto agar plates containing synthetic medium and the indicated chemical. Plates were incubated at 37°C for 48 h and then photographed. (B) Summary of mutant phenotypes shown in panel A. A brighter yellow color indicates better cell growth of under the specified condition. Black indicates no growth detected. The values were determined from the relative extent of growth detected in two to three independent spot assays as shown in panel A. (C) Deletion mutant strains lacking a single FLP gene were spotted onto medium containing the indicated quinones. Note that the pst3Δ strain was more sensitive. Strains used included the wild type control strain LLF100, Δ/Δ/Δ/Δ strain LLF060, and the complemented strain LLF079. Also, the Δ/Δ/Δ/Δ strain engineered to express E. coli wrbA (strain LLF074), rat NQO1 (strain LLF076), GFP (strain LLF080), PST1 (LLF064), PST2 (LLF081), PST3 (LLF066) or YCP4 (LLF082). Single mutant deletion strains used in panel C were pst1Δ (LLF052), pst2Δ (LLF059), pst3Δ (LLF036), and ycp4Δ (LLF037).

Mentions: The properties of the different quinone reductase homologues were examined by expressing individual genes in the Δ/Δ/Δ/Δ mutant. The C. albicans genes were reintroduced under control of their native promoters, whereas wrbA and NQO1 were controlled by the ADH1 promoter. Growth assays were performed to test the ability of cells carrying only one quinone reductase gene to resist different quinones and oxidants. All of the different quinone reductases were able to promote resistance to H2O2, tert-butyl hydroperoxide, and linolenic acid (Fig 5A). However, some of the strains had differential ability to resist cumene hydroperoxide and the small molecule quinones: p-benzoquinone and menadione (Fig 5A and summarized in Fig 5B).


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

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

Differential sensitivity to oxidants for C. albicans strains expressing a single FLP gene or the analogous NQO1 gene.Strains expressing individual C. albicans FLP genes under control of their native promoter, or the E. coli wrbA or rat NQO1 gene under control of the ADH1 promoter were created by introducing the corresponding genes into the Δ/Δ/Δ/Δ mutant strain LLF054. (A) Serial 10-fold dilutions of cells were spotted onto agar plates containing synthetic medium and the indicated chemical. Plates were incubated at 37°C for 48 h and then photographed. (B) Summary of mutant phenotypes shown in panel A. A brighter yellow color indicates better cell growth of under the specified condition. Black indicates no growth detected. The values were determined from the relative extent of growth detected in two to three independent spot assays as shown in panel A. (C) Deletion mutant strains lacking a single FLP gene were spotted onto medium containing the indicated quinones. Note that the pst3Δ strain was more sensitive. Strains used included the wild type control strain LLF100, Δ/Δ/Δ/Δ strain LLF060, and the complemented strain LLF079. Also, the Δ/Δ/Δ/Δ strain engineered to express E. coli wrbA (strain LLF074), rat NQO1 (strain LLF076), GFP (strain LLF080), PST1 (LLF064), PST2 (LLF081), PST3 (LLF066) or YCP4 (LLF082). Single mutant deletion strains used in panel C were pst1Δ (LLF052), pst2Δ (LLF059), pst3Δ (LLF036), and ycp4Δ (LLF037).
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ppat.1005147.g005: Differential sensitivity to oxidants for C. albicans strains expressing a single FLP gene or the analogous NQO1 gene.Strains expressing individual C. albicans FLP genes under control of their native promoter, or the E. coli wrbA or rat NQO1 gene under control of the ADH1 promoter were created by introducing the corresponding genes into the Δ/Δ/Δ/Δ mutant strain LLF054. (A) Serial 10-fold dilutions of cells were spotted onto agar plates containing synthetic medium and the indicated chemical. Plates were incubated at 37°C for 48 h and then photographed. (B) Summary of mutant phenotypes shown in panel A. A brighter yellow color indicates better cell growth of under the specified condition. Black indicates no growth detected. The values were determined from the relative extent of growth detected in two to three independent spot assays as shown in panel A. (C) Deletion mutant strains lacking a single FLP gene were spotted onto medium containing the indicated quinones. Note that the pst3Δ strain was more sensitive. Strains used included the wild type control strain LLF100, Δ/Δ/Δ/Δ strain LLF060, and the complemented strain LLF079. Also, the Δ/Δ/Δ/Δ strain engineered to express E. coli wrbA (strain LLF074), rat NQO1 (strain LLF076), GFP (strain LLF080), PST1 (LLF064), PST2 (LLF081), PST3 (LLF066) or YCP4 (LLF082). Single mutant deletion strains used in panel C were pst1Δ (LLF052), pst2Δ (LLF059), pst3Δ (LLF036), and ycp4Δ (LLF037).
Mentions: The properties of the different quinone reductase homologues were examined by expressing individual genes in the Δ/Δ/Δ/Δ mutant. The C. albicans genes were reintroduced under control of their native promoters, whereas wrbA and NQO1 were controlled by the ADH1 promoter. Growth assays were performed to test the ability of cells carrying only one quinone reductase gene to resist different quinones and oxidants. All of the different quinone reductases were able to promote resistance to H2O2, tert-butyl hydroperoxide, and linolenic acid (Fig 5A). However, some of the strains had differential ability to resist cumene hydroperoxide and the small molecule quinones: p-benzoquinone and menadione (Fig 5A and summarized in Fig 5B).

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