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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

The Δ/Δ/Δ/Δ mutant strain is more sensitive to linolenic acid-induced cell death and lipid peroxidation.(A) C. albicans strains were incubated with 0.5 mM linolenic acid (LNA) at 37°C for the indicated time (hours), and then dilutions of cells were plated to determine the viable colony forming units (CFU). (B) Cells were incubated with 0.5 LNA for the different times and then thiobarbituric acid reactive substance (TBARS) assays were carried out to detect malondialdehyde (MDA), a byproduct of lipid peroxidation. (C) Cells were exposed to different concentrations of LNA for 6 h, and then CFUs were determined. (D) TBARS assays to detect lipid peroxidation in cells treated with different concentrations of LNA for 6 h. (E) CFU analysis and (F) TBARS assays of the indicated FLP mutant strains. Note that in contrast to the Δ/Δ/Δ/Δ quadruple mutant strain LLF060, the single, double and triple FLP deletion mutant strains did not display increased sensitivity to LNA. Error bars indicate SE. * = p<0.05, ** = p<0.01, *** = p< 0.001 by ANOVA. Strains used included the wild type strain LLF100, Δ/Δ/Δ/Δ strain LLF060, and the complemented strain LLF079 in which one copy of each FLP gene was introduced into the Δ/Δ/Δ/Δ strain.
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ppat.1005147.g002: The Δ/Δ/Δ/Δ mutant strain is more sensitive to linolenic acid-induced cell death and lipid peroxidation.(A) C. albicans strains were incubated with 0.5 mM linolenic acid (LNA) at 37°C for the indicated time (hours), and then dilutions of cells were plated to determine the viable colony forming units (CFU). (B) Cells were incubated with 0.5 LNA for the different times and then thiobarbituric acid reactive substance (TBARS) assays were carried out to detect malondialdehyde (MDA), a byproduct of lipid peroxidation. (C) Cells were exposed to different concentrations of LNA for 6 h, and then CFUs were determined. (D) TBARS assays to detect lipid peroxidation in cells treated with different concentrations of LNA for 6 h. (E) CFU analysis and (F) TBARS assays of the indicated FLP mutant strains. Note that in contrast to the Δ/Δ/Δ/Δ quadruple mutant strain LLF060, the single, double and triple FLP deletion mutant strains did not display increased sensitivity to LNA. Error bars indicate SE. * = p<0.05, ** = p<0.01, *** = p< 0.001 by ANOVA. Strains used included the wild type strain LLF100, Δ/Δ/Δ/Δ strain LLF060, and the complemented strain LLF079 in which one copy of each FLP gene was introduced into the Δ/Δ/Δ/Δ strain.

Mentions: The effects of linolenic acid on C. albicans were analyzed further in quantitative assays. A time course of cell death was assayed by incubating cells for different times in the presence of 0.5 mM linolenic acid followed by plating dilutions on agar medium to determine the viable colony forming units (CFUs). The results confirmed the spotting assays. The Δ/Δ/Δ/Δ mutant showed a significant trend toward decreased viability by 6–8 h that was not observed for the wild-type control or complemented strains (Fig 2A). Analysis of the dose-response to incubation with linolenic acid for 6 h revealed a loss of viability starting at 0.25 mM that became more significant at 0.5 and 1.0 mM (Fig 2C). In contrast, the cells remained viable after incubation in the monounsaturated oleic acid (S2 Fig).


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

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

The Δ/Δ/Δ/Δ mutant strain is more sensitive to linolenic acid-induced cell death and lipid peroxidation.(A) C. albicans strains were incubated with 0.5 mM linolenic acid (LNA) at 37°C for the indicated time (hours), and then dilutions of cells were plated to determine the viable colony forming units (CFU). (B) Cells were incubated with 0.5 LNA for the different times and then thiobarbituric acid reactive substance (TBARS) assays were carried out to detect malondialdehyde (MDA), a byproduct of lipid peroxidation. (C) Cells were exposed to different concentrations of LNA for 6 h, and then CFUs were determined. (D) TBARS assays to detect lipid peroxidation in cells treated with different concentrations of LNA for 6 h. (E) CFU analysis and (F) TBARS assays of the indicated FLP mutant strains. Note that in contrast to the Δ/Δ/Δ/Δ quadruple mutant strain LLF060, the single, double and triple FLP deletion mutant strains did not display increased sensitivity to LNA. Error bars indicate SE. * = p<0.05, ** = p<0.01, *** = p< 0.001 by ANOVA. Strains used included the wild type strain LLF100, Δ/Δ/Δ/Δ strain LLF060, and the complemented strain LLF079 in which one copy of each FLP gene was introduced into the Δ/Δ/Δ/Δ strain.
© Copyright Policy
Related In: Results  -  Collection

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ppat.1005147.g002: The Δ/Δ/Δ/Δ mutant strain is more sensitive to linolenic acid-induced cell death and lipid peroxidation.(A) C. albicans strains were incubated with 0.5 mM linolenic acid (LNA) at 37°C for the indicated time (hours), and then dilutions of cells were plated to determine the viable colony forming units (CFU). (B) Cells were incubated with 0.5 LNA for the different times and then thiobarbituric acid reactive substance (TBARS) assays were carried out to detect malondialdehyde (MDA), a byproduct of lipid peroxidation. (C) Cells were exposed to different concentrations of LNA for 6 h, and then CFUs were determined. (D) TBARS assays to detect lipid peroxidation in cells treated with different concentrations of LNA for 6 h. (E) CFU analysis and (F) TBARS assays of the indicated FLP mutant strains. Note that in contrast to the Δ/Δ/Δ/Δ quadruple mutant strain LLF060, the single, double and triple FLP deletion mutant strains did not display increased sensitivity to LNA. Error bars indicate SE. * = p<0.05, ** = p<0.01, *** = p< 0.001 by ANOVA. Strains used included the wild type strain LLF100, Δ/Δ/Δ/Δ strain LLF060, and the complemented strain LLF079 in which one copy of each FLP gene was introduced into the Δ/Δ/Δ/Δ strain.
Mentions: The effects of linolenic acid on C. albicans were analyzed further in quantitative assays. A time course of cell death was assayed by incubating cells for different times in the presence of 0.5 mM linolenic acid followed by plating dilutions on agar medium to determine the viable colony forming units (CFUs). The results confirmed the spotting assays. The Δ/Δ/Δ/Δ mutant showed a significant trend toward decreased viability by 6–8 h that was not observed for the wild-type control or complemented strains (Fig 2A). Analysis of the dose-response to incubation with linolenic acid for 6 h revealed a loss of viability starting at 0.25 mM that became more significant at 0.5 and 1.0 mM (Fig 2C). In contrast, the cells remained viable after incubation in the monounsaturated oleic acid (S2 Fig).

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