Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Effects of metal ions and hydrogen peroxide on the phenotype of yeast hom6Δ mutant N.M. Tun, B.R. Lennon, P.J. O’Doherty, A.J. Johnson, G. Petersingham, T.D. Bailey, C. Kersaitis and M.J. Wu School of Science and Health, University of Western Sydney, Penrith, NSW, Australia
Significance and Impact of the Study: This study focuses on the yeast strain which lacks homoserine dehydrogenase encoded by HOM6 gene in aspartate metabolism. The HOM6-deletion mutant (hom6D) was analysed in the context of varying environmental parameters such as metal ions and oxidants, under anaerobic and aerobic conditions. We demonstrated that both manganese and hydrogen peroxide can promote the growth of hom6D, with the latter exerting such effect only under anaerobic condition. The findings are relevant to the research areas of ageing and anti-fungal drug development. It highlights the importance of interactions between gene expression and environmental factors as well as culture conditions.
Keywords aspartate pathway, HOM6 deletion, hydrogen peroxide, metal ions, Saccharomyces cerevisiae. Correspondence Ming J. Wu, School of Science and Health, University of Western Sydney, Locked Bag 1797, Penrith, NSW 2751, Australia. E-mail: [email protected] 2014/0946: received 11 May 2014, revised 3 September 2014 and accepted 25 September 2014 doi:10.1111/lam.12336
Abstract HOM6 is a major gene in the aspartate pathway which leads to biosynthesis of threonine and methionine. The phenotypes of the gene deletion mutant (hom6Δ) in a variety of cultural conditions have previously provided meaningful insights into the biological roles of HOM6 and its upstream intermediate metabolites. Here, we conducted a survey on a spectrum of metal ions for their effect on the aspartate pathway and broader sulphur metabolism. We show that manganese (Mn2+) promoted the growth of hom6Δ under both anaerobic and aerobic conditions. Unexpectedly, 4 mmol l 1 hydrogen peroxide (H2O2), a dose normally causing temporary cell growth arrest, enhanced the growth of hom6Δ under the anaerobic condition only, while it had no effect on the wild type strain BY4743. We propose that Mn2+ and H2O2 promote the growth of hom6Δ by reducing the accumulation of the toxic intermediate metabolite—aspartate b-semialdehyde, via directing the aspartate pathway to the central sugar metabolism–tricarboxylic acid cycle.
Introduction Homoserine dehydrogenase, encoded by HOM6 gene in Saccharomyces cerevisiae, is an enzyme in the aspartate metabolic pathway which catalyses the reduction of aspartic b-semialdehyde to homoserine in a number of organisms (DeLaBarre et al. 2000). It is required for the biosynthesis of threonine and methionine. The multi-step pathway from aspartate to homoserine and threonine is unique for fungal species, therefore presenting itself as an ideal target for drug development against fungal infections in human hosts (Jacques et al. 2001). HOM6 is the focus of this study due to our overall interest in oxidative stress, because it is part of the sulphur amino acid 20
biosynthesis pathway and consequently involved in antioxidant response. Deletion of HOM6 results in reduced growth when compared to the wild type (McGary et al. 2007). The phenotype of hom6D has also been associated with a significant increase in longevity of yeast cells (Burtner et al. 2011). Hence, there appears to be a trade-off taking place within hom6D between proliferation and longevity. As mentioned, the studies on HOM6 have been conducted in the context of antifungal drug development. It was found that deletion of HOM6 led to accumulation of aspartate b-semialdehyde, which consequently resulted in the inhibition of fungal cell growth (Arevalo-Rodrıguez et al. 2004). From a perspective of
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metabolism, we assume that reagents which could stimulate the growth of hom6D should shed light on a molecular mechanism in regulation of aspartate metabolism. In this study, we examined the hom6D mutant under the treatment of various metal ions, which might activate an alternative pathway for the removal of the toxic intermediate-aspartate b-semialdehyde or directly interact with it. The underlying rationale for such an approach is as follows: HOM6-encoded homoserine dehydrogenase is part of the aspartate pathway in antioxidant response, and in turn, the antioxidant response is modulated by metal ions (Wu et al. 2011b, 2012; O’Doherty et al. 2013). Indeed, the addition of minor levels of one metal ion can exert fundamental effects on cell physiology and defence. For example, trace amounts of Fe2+ can lead to lipid oxidation and stimulation of autocatalytic lipid peroxidation reactions (Zimniak 2011). Elevation of brain magnesium levels can enhance learning and memory (Slutsky et al. 2010). Mukhopadhyay and Linstedt (2012) found that manganese can block intracellular trafficking of Shiga toxin and protect against Shiga toxicoses. Zn2+, Fe2+/3+ and Mn2+ are heavily involved in cellular metabolism. Mn2+ is an essential trace element with multiple biological roles such as being a cofactor for manganese superoxide dismutase, and a high level of Mn2+ can increase the antioxidant response (Aguirre and Culotta 2012). Consequently, a range of metal ions was tested in this study for their effects on the phenotype of hom6Δ including Mn2+, Zn2+, Cu2+, Fe2+/3+, Ca2+, Mg2+, K+, Co2+, Cd2+, Ga3+ and Al3+. Exposure to hydrogen peroxide, which is known to trigger an adaptive response in yeast and higher eukaryotes, may also affect the growth of hom6Δ. Godon et al. (1998) demonstrated the ability of yeast to alter metabolic pathways under H2O2 stress. The cellular responses that are known to be altered by H2O2 include heat shock, antioxidant defence, protein translational apparatus and carbohydrate metabolism. While HOM6 and HOM2 protein expression was observed to remain constant under H2O2 exposure (Godon et al. 1998), expression of numerous other proteins can be varied by the oxidant. Therefore, a nonlethal dose of H2O2 may influence the phenotype of hom6D with respect to its growth. In this report, we screened a cohort of metal ions on hom6Δ, as well as four other related deletion mutants (hom3Δ, hom2Δ, th1Δ and th2Δ). The effect of Mn2+ and H2O2 on hom6Δ under both aerobic and anaerobic conditions was then further investigated. A molecular mechanism for the positive effect of Mn2+ and H2O2 on the mutant is finally proposed.
Metals and oxidants on hom6D
Results and discussion The phenotype of a gene deletion mutant can reveal not only the genetic and biological function of the gene, but also the intricacies between the gene function and environmental parameters, if screened under different environmental conditions. Here, we firstly showed that Mn2+ can enhance hom6D growth under anaerobic condition. A schematic description of the aspartate pathway is given in Fig. 1a. The effect of metal ions (Fe3+, Zn2+, Mn2+, K+, Mg2+, Fe2+, Cu2+, Ca2+, Al3+, Co2+, Cd2+ and Ga3+) on deletion mutants (hom3Δ, hom2Δ, hom6Δ, thr1Δ and thr4Δ) is shown in Fig. 1b,c. The phenotypic growth of mutants was measured at OD600 under anaerobic condition, and fold change for each treatment was calculated by dividing the final reading T24 by the initial measurement T0. The fold changes range from 0 to 11 and are expressed in green and red for low and high growth, respectively. hom6Δ showed very low growth in the control medium alone and following exposure to various metals, except Mn2 + . Clearly, Mn2+ promoted the growth of hom6Δ, from 34 fold in the control medium to 87 fold under 8 mmol l 1 Mn2+. In contrast to hom6Δ, the growth of hom2Δ, hom3Δ, thr1Δ and thr4Δ showed the same or similar pattern as the parent strain, BY4743. hom6Δ was further investigated under the treatment with Mn2+, hydrogen peroxide (H2O2) and their combination. Both Mn2+ and H2O2 enhanced the growth of hom6D under anaerobic conditions. Under anaerobic conditions in medium only, hom6D exhibited 335 fold of growth and BY4743 showed 902 fold (Fig. 2). The results further confirmed the slow growth of hom6D mutant. There was, however, a significant increase in the growth of hom6D under 4 mmol l 1 H2O2 alone, 8 mmol l 1 Mn2+ alone, and the combination of 8 mmol l 1 Mn2+ and 4 mmol l 1 H2O2, when compared to the hom6D control (P value < 001) (Fig. 2a). The mutant grew approx. 78 fold under the treatment of 4 mmol l 1 H2O2. The same effect was observed with 8 mmol l 1 Mn2+, while a 93 fold increase of growth was observed with the combined treatment. In the experiment, diamide was employed as a control oxidant, and no significant effect on the mutant’s growth at 16 mmol l 1 diamide was observed while it became toxic at 32 mmol l 1. This demonstrates that the marked enhancement of the mutant’s growth by H2O2 is intrinsic to H2O2, not due to a general oxidative effect. For the wild type BY4743 (Fig. 2b), 4 mmol l 1 H2O2 treatment had no significant effect on growth. The treatment with 8 mmol l 1 Mn2+ resulted in a 737 fold growth, significantly less than the medium only control (P value < 001). The fold increase for 8 mmol l 1 Mn2+
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Figure 1 Aspartate pathway and the phenotypes of its gene deletion mutants following exposure to various metal ions. (a) The metabolic steps from aspartate to homoserine and threonine. (b) The effect of each metal ion on hom6Δ and the other deletion mutants. The averages of growth folds for the mutants and BY4743 were analysed and visualized by means of the hierarchical clustering program MULTIEXPERIMENT VIEWER. Each average was calculated from three replicates. And these fold changes are tabulated in (c).
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Figure 2 Effects of Mn2+ and H2O2 on the growth of hom6D and BY4743 under anaerobic condition. The growth of hom6D and BY4743 was measured spectrophotometrically as OD600 nm in 96-well plates after 24-h anaerobic incubation at 30°C. The average of fold changes and standard deviation for each treatment were calculated from 12 replicates. (a) hom6D treated with H2O2, MnCl2, H2O2 plus MnCl2, 16 mmol l 1 diamide, 32 mmol l 1 diamide, 32 mmol l 1 diamide plus MnCl2; (b) BY4743 treated with H2O2, Mn2+, H2O2 plus Mn2+, 16 mmol l 1 diamide, 32 mmol l 1 diamide, 32 mmol l 1 diamide plus Mn2+. ** denotes statistical significance (P < 001).
Letters in Applied Microbiology 60, 20--26 © 2014 The Society for Applied Microbiology
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Metals and oxidants on hom6D
plus 4 mmol l 1 H2O2 (984) was almost equal to 4 mmol l 1 H2O2 alone. Both 16 and 32 mmol l 1 diamide were toxic to BY4743. The promotion of hom6D growth by Mn2+ alone and H2O2 alone was then confirmed in scaled-up 2 ml cultures carried out in 5-ml tubes. The anaerobic condition was ensured by a layer of mineral oil over the medium in each tube. As shown in Fig. 3, both 4 mmol l 1 H2O2 and 8 mmol l 1 Mn2+ considerably enhanced the growth of hom6D in comparison to the control (~fivefold), while such a positive effect did not occur with BY4743. In fact, Mn2+ resulted in decreased growth, consistent with the finding in the 96-well plate. In terms of the combined treatment of H2O2 and Mn2+, it is shown that there is no synergistic effect between them. Under aerobic conditions, 4 mmol l 1 H2O2 was toxic to hom6D and the wild type (P value < 001) (Fig. 4). Growth was seen to be reduced approx. 3 and 2 fold,
respectively. However, the effects of Mn2+ on the mutant and wild type were consistent with the anaerobic condition, that is, it greatly enhanced the growth of hom6D and reduced the growth of BY4743 (P value < 001). These findings are intriguing, considering both of these agents are seemingly distant from aspartate metabolism. Data analysis, however, points to a possible underlying mechanism operational in yeast, and potentially other fungal species, under environmental variations. The opposite effects of H2O2 under anaerobic and aerobic conditions on the growth of hom6D demonstrate that oxygen plays an important role in the metabolism of the mutant. As the control oxidant, diamide displayed toxicity in BY4743 and the mutant as expected, this study highlights that the effect of H2O2 on hom6D is specific. A molecular mechanism must be in play for its positive effect on the growth of hom6D under anaerobic condition. Because both H2O2 and Mn2+ exerted significant
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Figure 3 Effects of Mn2+ and H2O2 on the growth of hom6D and BY4743 under anaerobic condition in scaled-up 2 ml cultures. Data represent the average of four replicates with standard deviation shown. ** denotes statistical significance (P < 001).
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positive effects on hom6D, their underlying mechanisms may be linked. The phenotypic changes induced by H2O2 and Mn2+, in terms of growth increase for hom6D, most likely reflect variations at a gene expression level. Numerous transcriptomic and proteomic data sets have been established for micro-organisms in response to H2O2. Both eukaryotic (e.g. S. cerevisiae) and prokaryotic (e.g. E. coli) organisms contain intrinsic defence enzymes, particularly catalase, to decompose hydrogen peroxide into oxygen and water. Their expression is highly upregulated upon exposure to the oxidant (Gasch et al. 2000; Zeller et al. 2005). Following the treatment of H2O2 in semi-anaerobic conditions or transition from an anaerobic to aerobic environment, citric acid cycle genes are markedly induced (de Groot et al. 2007). Also, from a systems biology point of view, the aspartate pathway is linked to central carbon metabolism via aspartate transaminases which convert aspartate to oxaloacetate, a component of the citric acid cycle (Jeffery et al. 1998; de Groot et al. 2007). On the basis of our findings, we propose the following mechanism as outlined in Fig. 5. Firstly, Mn2+ could work through aspartate transaminase, as it is a potential cofactor for this enzyme (Martins et al. 2002). Mn2+induced transaminase activity may increase oxaloacetate production from aspartate. This process would lead to a
(a) Without Mn2+ or H2O2
reduction of the toxic aspartate b-semialdehyde accumulation, hence the increased hom6D growth. Mn2+ could also exert an effect through decarboxylase (Reddi et al. 2009), which can reduce the level of aspartate. Hydrogen peroxide is normally associated with oxidative stress and cellular damage. In the case of hom6D under anaerobic condition, however, it seems to play a role in growth promotion. The proposed mechanism for this involves the reaction catalysed by catalase. That is, catalase may act on hydrogen peroxide to produce oxygen, the release of which would drive the metabolism of aspartate to oxaloacetate and the running of the citric acid cycle, resulting in the reduction of aspartate b-semialdehyde accumulation and promoting hom6D growth (Fig. 5). That Mn2+ enhances the growth of hom6Δ adds another row to the list of biological functions for manganese, re-enforcing its name of Greek origin, ‘magic’. The aspartate pathway is a target for drug development against fungal infection. Saccharomyces cerevisiae has long been used as a ‘surrogate’ for pathogenic fungi such as Candida albicans, because of their high genomic homology (Berman and Sudbery 2002). Three proteins for the first three catalytic steps of the aspartate pathway, encoded by HOM3, HOM2 and HOM6, are present in the Candida albicans proteome (Yin et al. 2004). This
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Figure 5 Proposed mechanism for the growth promotion of Mn2+ and H2O2 on hom6D. (a) Aspartate b-semialdehyde accumulation in hom6D. Such accumulation causes slow growth of the mutant. (b) Proposed mechanism for the growth promotion of Mn2+ and H2O2 on hom6D. The introduction of an excess of Mn2+, a cofactor of aspartate transaminase, may allow for the removal of the amino group in aspartate to produce oxaloacetate, an intermediate of the citric acid cycle. In this way, the Mn2+ is able to rescue the mutant by providing an exit pathway for aspartate. H2O2 reduction under anaerobic incubation is catalysed by the enzyme catalase in Saccharomyces cerevisiae. This reaction results in O2 in the cell which may feed into the electron transport chain, so stimulating the citric acid cycle and ultimately leading to reduction of aspartate bsemialdehyde, promoting the growth of hom6D.
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study suggests that limiting oxygen supply would reduce fungal growth, as oxygen derived from H2O2 enhanced the growth of hom6Δ. Therefore, delivering a given antifungal drug under anaerobic conditions for the infected tissue could be desirable. This study provides a perspective of systems biology. The central carbon metabolism is the centre of cellular metabolism under favourable or adverse environmental conditions. We show that the balance between high and low growth is dependent upon both genetic and environmental factors, as the gene deletion of HOM6 is responsible for reduced growth and agents such as manganese and hydrogen peroxide can stimulate the mutant’s metabolic machinery. It is further demonstrated that sometimes, unexpected findings like the positive effect of H2O2 under anaerobic condition, can shed light on the intricacy of metabolic networks. In conclusion, this study clearly found that the growth of yeast cells that lack a functioning homoserine dehydrogenase gene (HOM6) is promoted by the addition of Mn2+ and H2O2 under anaerobic condition, possibly due to the redirection of the aspartate metabolism to the tricarboxylic acid cycle. We are currently validating this molecular hypothesis through quantifying the gene expression of aspartate transaminase and catalase. Materials and methods
Metals and oxidants on hom6D
40, 80 mmol l 1), Al3+ (04, 08, 16 mmol l 1), Co2+ (004, 04, 40 mmol l 1), Cd2+ (5, 10, 20 lmol l 1) and Ga3+ (04, 08, 16 mmol l 1). Yeast cultures were diluted and inoculated into minimal medium in 96-well microtiter plates followed with addition of each metal ion. Absorbance (OD600) was measured initially (T0) using a 96-well plate reader and then again at 24 h (T24) after incubation at 30°C, under anaerobic conditions by placing the plates in a sealed box without shaking in an incubator. Phenotypic growth rates were calculated as fold change by dividing OD600 of T24 over that of T0. The screening was repeated thrice. After the initial screening of the metal ions, Mn2+ was chosen for further experiments with hom6Δ mutant. H2O2 was also assayed with the mutant. The concentration of 4 mmol l 1 H2O2 was chosen on the basis of previous studies (Wu et al. 2011a), which demonstrate that 4 mmol l 1 H2O2 causes temporary growth arrest of BY4743 while concentrations above 4 mmol l 1 are lethal. A combination of Mn2+ and the oxidant was also tested to see if there was any synergistic effect. Both aerobic and anaerobic conditions were used. The anaerobic environment in the culturing tubes was created by a layer of mineral oil. For the 96-well plates, anaerobic incubation was carried out in a sealed box without shaking inside an incubator. The aerobic condition was performed by shaking (150 rev min 1), unsealed, at 30°C.
Yeast strains and culture media
Bioinformatic and statistical analyses
Saccharomyces cerevisiae deletion mutant strains (hom3Δ, hom2Δ, hom6Δ, thr1Δ and thr4Δ) and their parent strain BY4743 were purchased from EUROSCARF. Yeast cultures were initiated by inoculating a single colony from its agar plate into minimal medium, as described in Wu et al. (2012).
Visualization of the metal ion screening data was obtained by means of the hierarchical clustering program, MULTIEXPERIMENT VIEWER (MeV v4.8, TIGR, Rockville, MD, USA) as previously described (Tun et al. 2013). The clustering process was carried out with Euclidean distance and the average clustering method. Data of hom6Δ under various treatments were statistically analysed with R statistical package, using one-way ANOVA (Analysis of Variance) and Tukey’s test to establish the significance between the means of treatments.
Screening the deletion mutants in the aspartate pathway Five deletion mutants (hom3Δ, hom2Δ, hom6Δ, thr1Δ and thr4Δ) and BY4743 were firstly screened with the following metal ions at nonlethal concentrations including Fe3+ (800 lmol l 1), Zn2+ (035 mmol l 1), Mn2+ (8 mmol l 1), K+ (20 mmol l 1), Mg2+ (80 mmol l 1), Fe2+ (400 lmol l 1), Cu2+ (4 lmol l 1), Ca2+ (40 mmol l 1), Al3+ (04 mmol l 1), Co2+ (4 mmol l 1), Cd2+ (5 lmol l 1) and Ga3+ (04 mmol l 1). Such nonlethal concentration for each ion was predetermined with BY4743 after titrating against a series of concentrations for each ion: Fe3+ (8, 80, 800 lmol l 1), Zn2+ (035, 07, 35 mmol l 1), Mn2+ (08, 16, 80 mmol l 1), K+ (20, 40, 80 mmol l 1), Mg2+ (20, 40, 80 mmol l 1), Fe2+ (4, 40, 400 lmol l 1), Cu2+ (4, 40, 400 lmol l 1), Ca2+ (20,
Conflict of interest The authors confirm that there is no conflict of interest to declare in connection with this manuscript. References Aguirre, J.D. and Culotta, V.C. (2012) Battles with iron: manganese in oxidative stress protection. J Biol Chem 287, 13541–13548. Arevalo-Rodrıguez, M., Pan, X., Boeke, J.D. and Heitman, J. (2004) FKBP12 controls aspartate pathway flux in
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ORIGINAL ARTICLE
Effects of metal ions and hydrogen peroxide on the phenotype of yeast hom6Δ mutant N.M. Tun, B.R. Lennon, P.J. O’Doherty, A.J. Johnson, G. Petersingham, T.D. Bailey, C. Kersaitis and M.J. Wu School of Science and Health, University of Western Sydney, Penrith, NSW, Australia
Significance and Impact of the Study: This study focuses on the yeast strain which lacks homoserine dehydrogenase encoded by HOM6 gene in aspartate metabolism. The HOM6-deletion mutant (hom6D) was analysed in the context of varying environmental parameters such as metal ions and oxidants, under anaerobic and aerobic conditions. We demonstrated that both manganese and hydrogen peroxide can promote the growth of hom6D, with the latter exerting such effect only under anaerobic condition. The findings are relevant to the research areas of ageing and anti-fungal drug development. It highlights the importance of interactions between gene expression and environmental factors as well as culture conditions.
Keywords aspartate pathway, HOM6 deletion, hydrogen peroxide, metal ions, Saccharomyces cerevisiae. Correspondence Ming J. Wu, School of Science and Health, University of Western Sydney, Locked Bag 1797, Penrith, NSW 2751, Australia. E-mail: [email protected] 2014/0946: received 11 May 2014, revised 3 September 2014 and accepted 25 September 2014 doi:10.1111/lam.12336
Abstract HOM6 is a major gene in the aspartate pathway which leads to biosynthesis of threonine and methionine. The phenotypes of the gene deletion mutant (hom6Δ) in a variety of cultural conditions have previously provided meaningful insights into the biological roles of HOM6 and its upstream intermediate metabolites. Here, we conducted a survey on a spectrum of metal ions for their effect on the aspartate pathway and broader sulphur metabolism. We show that manganese (Mn2+) promoted the growth of hom6Δ under both anaerobic and aerobic conditions. Unexpectedly, 4 mmol l 1 hydrogen peroxide (H2O2), a dose normally causing temporary cell growth arrest, enhanced the growth of hom6Δ under the anaerobic condition only, while it had no effect on the wild type strain BY4743. We propose that Mn2+ and H2O2 promote the growth of hom6Δ by reducing the accumulation of the toxic intermediate metabolite—aspartate b-semialdehyde, via directing the aspartate pathway to the central sugar metabolism–tricarboxylic acid cycle.
Introduction Homoserine dehydrogenase, encoded by HOM6 gene in Saccharomyces cerevisiae, is an enzyme in the aspartate metabolic pathway which catalyses the reduction of aspartic b-semialdehyde to homoserine in a number of organisms (DeLaBarre et al. 2000). It is required for the biosynthesis of threonine and methionine. The multi-step pathway from aspartate to homoserine and threonine is unique for fungal species, therefore presenting itself as an ideal target for drug development against fungal infections in human hosts (Jacques et al. 2001). HOM6 is the focus of this study due to our overall interest in oxidative stress, because it is part of the sulphur amino acid 20
biosynthesis pathway and consequently involved in antioxidant response. Deletion of HOM6 results in reduced growth when compared to the wild type (McGary et al. 2007). The phenotype of hom6D has also been associated with a significant increase in longevity of yeast cells (Burtner et al. 2011). Hence, there appears to be a trade-off taking place within hom6D between proliferation and longevity. As mentioned, the studies on HOM6 have been conducted in the context of antifungal drug development. It was found that deletion of HOM6 led to accumulation of aspartate b-semialdehyde, which consequently resulted in the inhibition of fungal cell growth (Arevalo-Rodrıguez et al. 2004). From a perspective of
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metabolism, we assume that reagents which could stimulate the growth of hom6D should shed light on a molecular mechanism in regulation of aspartate metabolism. In this study, we examined the hom6D mutant under the treatment of various metal ions, which might activate an alternative pathway for the removal of the toxic intermediate-aspartate b-semialdehyde or directly interact with it. The underlying rationale for such an approach is as follows: HOM6-encoded homoserine dehydrogenase is part of the aspartate pathway in antioxidant response, and in turn, the antioxidant response is modulated by metal ions (Wu et al. 2011b, 2012; O’Doherty et al. 2013). Indeed, the addition of minor levels of one metal ion can exert fundamental effects on cell physiology and defence. For example, trace amounts of Fe2+ can lead to lipid oxidation and stimulation of autocatalytic lipid peroxidation reactions (Zimniak 2011). Elevation of brain magnesium levels can enhance learning and memory (Slutsky et al. 2010). Mukhopadhyay and Linstedt (2012) found that manganese can block intracellular trafficking of Shiga toxin and protect against Shiga toxicoses. Zn2+, Fe2+/3+ and Mn2+ are heavily involved in cellular metabolism. Mn2+ is an essential trace element with multiple biological roles such as being a cofactor for manganese superoxide dismutase, and a high level of Mn2+ can increase the antioxidant response (Aguirre and Culotta 2012). Consequently, a range of metal ions was tested in this study for their effects on the phenotype of hom6Δ including Mn2+, Zn2+, Cu2+, Fe2+/3+, Ca2+, Mg2+, K+, Co2+, Cd2+, Ga3+ and Al3+. Exposure to hydrogen peroxide, which is known to trigger an adaptive response in yeast and higher eukaryotes, may also affect the growth of hom6Δ. Godon et al. (1998) demonstrated the ability of yeast to alter metabolic pathways under H2O2 stress. The cellular responses that are known to be altered by H2O2 include heat shock, antioxidant defence, protein translational apparatus and carbohydrate metabolism. While HOM6 and HOM2 protein expression was observed to remain constant under H2O2 exposure (Godon et al. 1998), expression of numerous other proteins can be varied by the oxidant. Therefore, a nonlethal dose of H2O2 may influence the phenotype of hom6D with respect to its growth. In this report, we screened a cohort of metal ions on hom6Δ, as well as four other related deletion mutants (hom3Δ, hom2Δ, th1Δ and th2Δ). The effect of Mn2+ and H2O2 on hom6Δ under both aerobic and anaerobic conditions was then further investigated. A molecular mechanism for the positive effect of Mn2+ and H2O2 on the mutant is finally proposed.
Metals and oxidants on hom6D
Results and discussion The phenotype of a gene deletion mutant can reveal not only the genetic and biological function of the gene, but also the intricacies between the gene function and environmental parameters, if screened under different environmental conditions. Here, we firstly showed that Mn2+ can enhance hom6D growth under anaerobic condition. A schematic description of the aspartate pathway is given in Fig. 1a. The effect of metal ions (Fe3+, Zn2+, Mn2+, K+, Mg2+, Fe2+, Cu2+, Ca2+, Al3+, Co2+, Cd2+ and Ga3+) on deletion mutants (hom3Δ, hom2Δ, hom6Δ, thr1Δ and thr4Δ) is shown in Fig. 1b,c. The phenotypic growth of mutants was measured at OD600 under anaerobic condition, and fold change for each treatment was calculated by dividing the final reading T24 by the initial measurement T0. The fold changes range from 0 to 11 and are expressed in green and red for low and high growth, respectively. hom6Δ showed very low growth in the control medium alone and following exposure to various metals, except Mn2 + . Clearly, Mn2+ promoted the growth of hom6Δ, from 34 fold in the control medium to 87 fold under 8 mmol l 1 Mn2+. In contrast to hom6Δ, the growth of hom2Δ, hom3Δ, thr1Δ and thr4Δ showed the same or similar pattern as the parent strain, BY4743. hom6Δ was further investigated under the treatment with Mn2+, hydrogen peroxide (H2O2) and their combination. Both Mn2+ and H2O2 enhanced the growth of hom6D under anaerobic conditions. Under anaerobic conditions in medium only, hom6D exhibited 335 fold of growth and BY4743 showed 902 fold (Fig. 2). The results further confirmed the slow growth of hom6D mutant. There was, however, a significant increase in the growth of hom6D under 4 mmol l 1 H2O2 alone, 8 mmol l 1 Mn2+ alone, and the combination of 8 mmol l 1 Mn2+ and 4 mmol l 1 H2O2, when compared to the hom6D control (P value < 001) (Fig. 2a). The mutant grew approx. 78 fold under the treatment of 4 mmol l 1 H2O2. The same effect was observed with 8 mmol l 1 Mn2+, while a 93 fold increase of growth was observed with the combined treatment. In the experiment, diamide was employed as a control oxidant, and no significant effect on the mutant’s growth at 16 mmol l 1 diamide was observed while it became toxic at 32 mmol l 1. This demonstrates that the marked enhancement of the mutant’s growth by H2O2 is intrinsic to H2O2, not due to a general oxidative effect. For the wild type BY4743 (Fig. 2b), 4 mmol l 1 H2O2 treatment had no significant effect on growth. The treatment with 8 mmol l 1 Mn2+ resulted in a 737 fold growth, significantly less than the medium only control (P value < 001). The fold increase for 8 mmol l 1 Mn2+
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Figure 1 Aspartate pathway and the phenotypes of its gene deletion mutants following exposure to various metal ions. (a) The metabolic steps from aspartate to homoserine and threonine. (b) The effect of each metal ion on hom6Δ and the other deletion mutants. The averages of growth folds for the mutants and BY4743 were analysed and visualized by means of the hierarchical clustering program MULTIEXPERIMENT VIEWER. Each average was calculated from three replicates. And these fold changes are tabulated in (c).
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Figure 2 Effects of Mn2+ and H2O2 on the growth of hom6D and BY4743 under anaerobic condition. The growth of hom6D and BY4743 was measured spectrophotometrically as OD600 nm in 96-well plates after 24-h anaerobic incubation at 30°C. The average of fold changes and standard deviation for each treatment were calculated from 12 replicates. (a) hom6D treated with H2O2, MnCl2, H2O2 plus MnCl2, 16 mmol l 1 diamide, 32 mmol l 1 diamide, 32 mmol l 1 diamide plus MnCl2; (b) BY4743 treated with H2O2, Mn2+, H2O2 plus Mn2+, 16 mmol l 1 diamide, 32 mmol l 1 diamide, 32 mmol l 1 diamide plus Mn2+. ** denotes statistical significance (P < 001).
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Metals and oxidants on hom6D
plus 4 mmol l 1 H2O2 (984) was almost equal to 4 mmol l 1 H2O2 alone. Both 16 and 32 mmol l 1 diamide were toxic to BY4743. The promotion of hom6D growth by Mn2+ alone and H2O2 alone was then confirmed in scaled-up 2 ml cultures carried out in 5-ml tubes. The anaerobic condition was ensured by a layer of mineral oil over the medium in each tube. As shown in Fig. 3, both 4 mmol l 1 H2O2 and 8 mmol l 1 Mn2+ considerably enhanced the growth of hom6D in comparison to the control (~fivefold), while such a positive effect did not occur with BY4743. In fact, Mn2+ resulted in decreased growth, consistent with the finding in the 96-well plate. In terms of the combined treatment of H2O2 and Mn2+, it is shown that there is no synergistic effect between them. Under aerobic conditions, 4 mmol l 1 H2O2 was toxic to hom6D and the wild type (P value < 001) (Fig. 4). Growth was seen to be reduced approx. 3 and 2 fold,
respectively. However, the effects of Mn2+ on the mutant and wild type were consistent with the anaerobic condition, that is, it greatly enhanced the growth of hom6D and reduced the growth of BY4743 (P value < 001). These findings are intriguing, considering both of these agents are seemingly distant from aspartate metabolism. Data analysis, however, points to a possible underlying mechanism operational in yeast, and potentially other fungal species, under environmental variations. The opposite effects of H2O2 under anaerobic and aerobic conditions on the growth of hom6D demonstrate that oxygen plays an important role in the metabolism of the mutant. As the control oxidant, diamide displayed toxicity in BY4743 and the mutant as expected, this study highlights that the effect of H2O2 on hom6D is specific. A molecular mechanism must be in play for its positive effect on the growth of hom6D under anaerobic condition. Because both H2O2 and Mn2+ exerted significant
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Figure 3 Effects of Mn2+ and H2O2 on the growth of hom6D and BY4743 under anaerobic condition in scaled-up 2 ml cultures. Data represent the average of four replicates with standard deviation shown. ** denotes statistical significance (P < 001).
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positive effects on hom6D, their underlying mechanisms may be linked. The phenotypic changes induced by H2O2 and Mn2+, in terms of growth increase for hom6D, most likely reflect variations at a gene expression level. Numerous transcriptomic and proteomic data sets have been established for micro-organisms in response to H2O2. Both eukaryotic (e.g. S. cerevisiae) and prokaryotic (e.g. E. coli) organisms contain intrinsic defence enzymes, particularly catalase, to decompose hydrogen peroxide into oxygen and water. Their expression is highly upregulated upon exposure to the oxidant (Gasch et al. 2000; Zeller et al. 2005). Following the treatment of H2O2 in semi-anaerobic conditions or transition from an anaerobic to aerobic environment, citric acid cycle genes are markedly induced (de Groot et al. 2007). Also, from a systems biology point of view, the aspartate pathway is linked to central carbon metabolism via aspartate transaminases which convert aspartate to oxaloacetate, a component of the citric acid cycle (Jeffery et al. 1998; de Groot et al. 2007). On the basis of our findings, we propose the following mechanism as outlined in Fig. 5. Firstly, Mn2+ could work through aspartate transaminase, as it is a potential cofactor for this enzyme (Martins et al. 2002). Mn2+induced transaminase activity may increase oxaloacetate production from aspartate. This process would lead to a
(a) Without Mn2+ or H2O2
reduction of the toxic aspartate b-semialdehyde accumulation, hence the increased hom6D growth. Mn2+ could also exert an effect through decarboxylase (Reddi et al. 2009), which can reduce the level of aspartate. Hydrogen peroxide is normally associated with oxidative stress and cellular damage. In the case of hom6D under anaerobic condition, however, it seems to play a role in growth promotion. The proposed mechanism for this involves the reaction catalysed by catalase. That is, catalase may act on hydrogen peroxide to produce oxygen, the release of which would drive the metabolism of aspartate to oxaloacetate and the running of the citric acid cycle, resulting in the reduction of aspartate b-semialdehyde accumulation and promoting hom6D growth (Fig. 5). That Mn2+ enhances the growth of hom6Δ adds another row to the list of biological functions for manganese, re-enforcing its name of Greek origin, ‘magic’. The aspartate pathway is a target for drug development against fungal infection. Saccharomyces cerevisiae has long been used as a ‘surrogate’ for pathogenic fungi such as Candida albicans, because of their high genomic homology (Berman and Sudbery 2002). Three proteins for the first three catalytic steps of the aspartate pathway, encoded by HOM3, HOM2 and HOM6, are present in the Candida albicans proteome (Yin et al. 2004). This
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Figure 5 Proposed mechanism for the growth promotion of Mn2+ and H2O2 on hom6D. (a) Aspartate b-semialdehyde accumulation in hom6D. Such accumulation causes slow growth of the mutant. (b) Proposed mechanism for the growth promotion of Mn2+ and H2O2 on hom6D. The introduction of an excess of Mn2+, a cofactor of aspartate transaminase, may allow for the removal of the amino group in aspartate to produce oxaloacetate, an intermediate of the citric acid cycle. In this way, the Mn2+ is able to rescue the mutant by providing an exit pathway for aspartate. H2O2 reduction under anaerobic incubation is catalysed by the enzyme catalase in Saccharomyces cerevisiae. This reaction results in O2 in the cell which may feed into the electron transport chain, so stimulating the citric acid cycle and ultimately leading to reduction of aspartate bsemialdehyde, promoting the growth of hom6D.
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study suggests that limiting oxygen supply would reduce fungal growth, as oxygen derived from H2O2 enhanced the growth of hom6Δ. Therefore, delivering a given antifungal drug under anaerobic conditions for the infected tissue could be desirable. This study provides a perspective of systems biology. The central carbon metabolism is the centre of cellular metabolism under favourable or adverse environmental conditions. We show that the balance between high and low growth is dependent upon both genetic and environmental factors, as the gene deletion of HOM6 is responsible for reduced growth and agents such as manganese and hydrogen peroxide can stimulate the mutant’s metabolic machinery. It is further demonstrated that sometimes, unexpected findings like the positive effect of H2O2 under anaerobic condition, can shed light on the intricacy of metabolic networks. In conclusion, this study clearly found that the growth of yeast cells that lack a functioning homoserine dehydrogenase gene (HOM6) is promoted by the addition of Mn2+ and H2O2 under anaerobic condition, possibly due to the redirection of the aspartate metabolism to the tricarboxylic acid cycle. We are currently validating this molecular hypothesis through quantifying the gene expression of aspartate transaminase and catalase. Materials and methods
Metals and oxidants on hom6D
40, 80 mmol l 1), Al3+ (04, 08, 16 mmol l 1), Co2+ (004, 04, 40 mmol l 1), Cd2+ (5, 10, 20 lmol l 1) and Ga3+ (04, 08, 16 mmol l 1). Yeast cultures were diluted and inoculated into minimal medium in 96-well microtiter plates followed with addition of each metal ion. Absorbance (OD600) was measured initially (T0) using a 96-well plate reader and then again at 24 h (T24) after incubation at 30°C, under anaerobic conditions by placing the plates in a sealed box without shaking in an incubator. Phenotypic growth rates were calculated as fold change by dividing OD600 of T24 over that of T0. The screening was repeated thrice. After the initial screening of the metal ions, Mn2+ was chosen for further experiments with hom6Δ mutant. H2O2 was also assayed with the mutant. The concentration of 4 mmol l 1 H2O2 was chosen on the basis of previous studies (Wu et al. 2011a), which demonstrate that 4 mmol l 1 H2O2 causes temporary growth arrest of BY4743 while concentrations above 4 mmol l 1 are lethal. A combination of Mn2+ and the oxidant was also tested to see if there was any synergistic effect. Both aerobic and anaerobic conditions were used. The anaerobic environment in the culturing tubes was created by a layer of mineral oil. For the 96-well plates, anaerobic incubation was carried out in a sealed box without shaking inside an incubator. The aerobic condition was performed by shaking (150 rev min 1), unsealed, at 30°C.
Yeast strains and culture media
Bioinformatic and statistical analyses
Saccharomyces cerevisiae deletion mutant strains (hom3Δ, hom2Δ, hom6Δ, thr1Δ and thr4Δ) and their parent strain BY4743 were purchased from EUROSCARF. Yeast cultures were initiated by inoculating a single colony from its agar plate into minimal medium, as described in Wu et al. (2012).
Visualization of the metal ion screening data was obtained by means of the hierarchical clustering program, MULTIEXPERIMENT VIEWER (MeV v4.8, TIGR, Rockville, MD, USA) as previously described (Tun et al. 2013). The clustering process was carried out with Euclidean distance and the average clustering method. Data of hom6Δ under various treatments were statistically analysed with R statistical package, using one-way ANOVA (Analysis of Variance) and Tukey’s test to establish the significance between the means of treatments.
Screening the deletion mutants in the aspartate pathway Five deletion mutants (hom3Δ, hom2Δ, hom6Δ, thr1Δ and thr4Δ) and BY4743 were firstly screened with the following metal ions at nonlethal concentrations including Fe3+ (800 lmol l 1), Zn2+ (035 mmol l 1), Mn2+ (8 mmol l 1), K+ (20 mmol l 1), Mg2+ (80 mmol l 1), Fe2+ (400 lmol l 1), Cu2+ (4 lmol l 1), Ca2+ (40 mmol l 1), Al3+ (04 mmol l 1), Co2+ (4 mmol l 1), Cd2+ (5 lmol l 1) and Ga3+ (04 mmol l 1). Such nonlethal concentration for each ion was predetermined with BY4743 after titrating against a series of concentrations for each ion: Fe3+ (8, 80, 800 lmol l 1), Zn2+ (035, 07, 35 mmol l 1), Mn2+ (08, 16, 80 mmol l 1), K+ (20, 40, 80 mmol l 1), Mg2+ (20, 40, 80 mmol l 1), Fe2+ (4, 40, 400 lmol l 1), Cu2+ (4, 40, 400 lmol l 1), Ca2+ (20,
Conflict of interest The authors confirm that there is no conflict of interest to declare in connection with this manuscript. References Aguirre, J.D. and Culotta, V.C. (2012) Battles with iron: manganese in oxidative stress protection. J Biol Chem 287, 13541–13548. Arevalo-Rodrıguez, M., Pan, X., Boeke, J.D. and Heitman, J. (2004) FKBP12 controls aspartate pathway flux in
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