RESEARCH NEWS & VIEWS MICROBIO LO GY
Fungus against the wall A compound derived from plant cell-wall material that is a waste product of biofuel manufacture has been found to have fungicidal properties: it interacts with a carbohydrate called β1,3 glucan, thus compromising the integrity of fungal cells. PA U L O ’ M A I L L E
F
ungal infections are a major problem in agriculture, and as the world’s population grows, a rising tide of resistance to antifungal agents is threatening global food security. This threat has led to an intensification in the use of fungicidal agrochemicals, which may themselves put human health at risk and damage the environment. New fungicidal agents are therefore desperately needed; and now it seems that help is on the way, but from unlikely sources. Writing in Proceedings of the National Academy of Sciences, Piotrowski and colleagues1 report the discovery of molecules in the waste products of biofuel manufacture that possess antifungal activity against plant pathogens. Dwindling oil reserves have spurred the development of technologies that use lignocellulosics — dry plant matter such as grass — as a renewable source of sugar to produce biofuels and bio-based chemicals. a
A key step in the process is the hydrolysis of polysaccharides in the plant cell wall, which liberates sugar monomers so they can be fermented by yeast2 (Fig. 1a). But other molecules are also liberated by hydrolysis, including diferulates, metabolites of the phenylpropanoid family of natural products. These molecules crosslink polysaccharides to each other, and to lignin polymers, to stiffen the plant cell wall, but the waste products stunt the growth of the yeast, inhibiting the fermentation process. Piotrowski and colleagues saw this problem as an opportunity for ‘bioprospecting’, reasoning that these molecules might be an untapped resource of antifungal agents. The authors screened diferulates for antifungal activity by testing the molecules’ ability to inhibit growth of a fungus, the budding yeast Saccharomyces cerevisiae. They identified two active compounds that inhibited growth, the most potent of which they dubbed poacic acid because of its prevalence in grasses
Plant cell wall
Yeast cell Sugar monomers
Diferulates Bioprocessing
Hydrolysis
Biofuel
Polysaccharide
b Poacic acid
Yeast cell wall
β1,3 Glucan
Abnormal shape
Leaky cell
Figure 1 | Waste of a fungicide. a, Grasses of the Poaceae family have cell walls fortified with polysaccharides that are crosslinked with metabolites called diferulates, which act as an anchor for the formation of lignin polymers (not shown). Bioprocessing of such grasses liberates the polysaccharides for hydrolysis to sugar monomers, from which yeast can produce biofuels and bio-based chemicals. The process also liberates diferulates such as poacic acid as waste products. b, Piotrowski et al.1 report that poacic acid has antifungal activity against yeast cells. The compound attacks the carbohydrate β1,3 glucan, which acts as a fortification in the yeast cell wall. As a result, cells become leaky and adopt abnormal shapes. 1 6 8 | NAT U R E | VO L 5 2 1 | 1 4 M AY 2 0 1 5
© 2015 Macmillan Publishers Limited. All rights reserved
of the Poaceae plant family. Importantly, poacic acid had comparable antifungal activity to two widely used agricultural fungicides. Next, Piotrowski et al. investigated how poacic acid exerts its effect, by challenging a pooled mixture of approximately 4,000 yeast strains with the compound. Each strain carried a different gene deletion that was marked by a unique molecular barcode — a short sequence of DNA that does not affect the cell’s function but can be used to identify the strain. After exposing the cells to poacic acid, the authors took a head count with next-generation DNA sequencing, measuring the frequency of each barcode in the population to determine the relative sensitivity or resistance of each strain to the fungicide3. The result was clear: deletion of genes involved in cell-wall organization conferred poacic-acid sensitivity, suggesting that the compound affects the yeast cell wall, thus mirroring the mechanism of many known antifungal compounds4. The authors then tested poacic acid in combination with two antifungal compounds that compromise cellular integrity5 and that are normally used to treat human fungal infections: fluconazole, which targets the biosynthesis of ergosterol, an essential component of the fungal cell membrane; and capsofungin, which inhibits6 the biosynthesis of β1,3 glucan, a polysaccharide that acts as both a barrier and structural support in the fungal cell wall. Perhaps not unexpectedly, poacic acid acted in synergy with these compounds, indicating that each drug strikes the same or related pathways, albeit through different targets7. To further investigate how poacic acid alters the cell wall, Piotrowski et al. used highdimensional microscopy to study yeast cells treated with poacic acid. The neck between the yeast cell and the daughter that buds off it was unusually wide in treated cells, and the degree of morphological variability in the cell population was higher than normal — similar changes to those caused by other drugs that disrupt the cell wall8 (Fig. 1b). Poacic acid also caused rapid influx of propidium iodide dye into the cell, consistent with cell-wall disruption. In a stroke of resourcefulness, the authors used the natural fluorescence of poacic acid to study its location within the cell. The cell’s surface lit up under the microscope, suggesting that poacic acid interacts directly with the β1,3 glucan layer. The researchers then incubated poacic acid with purified fungal β1,3 glucan — the glucan particles began to fluoresce, confirming the interaction. Given these promising results, the researchers next tested the potency of poacic acid against key agricultural fungal pathogens. Preliminary studies showed that poacic acid inhibited the fungi Sclerotinia sclerotiorum and Alternaria solani in a dose-dependent manner and protected soya-bean leaves from the oomycete (fungus-like) pathogen Phytophthora sojae, suggesting that poacic
NEWS & VIEWS RESEARCH
Paul O’Maille is at the John Innes Centre and Institute of Food Research, Norwich NR4 7UH, UK. e-mail: paul.o’[email protected] 1. Piotrowski, J. S. et al. Proc. Natl Acad. Sci. USA 112, E1490–E1497 (2015). 2. Sun, Y. & Cheng, J. Bioresour. Technol. 83, 1–11 (2002). 3. Parsons, A. B. et al. Cell 126, 611–625 (2006). 4. Latgé, J.-P. Mol. Microbiol. 66, 279–290 (2007). 5. Kiraz, N. et al. Antimicrob. Agents Chemother. 54, 2244–2247 (2010). 6. Kurtz, M. B. & Douglas, C. M. J. Med. Vet. Mycol. 35, 79–86 (1997). 7. Cokol, M. et al. Mol. Syst. Biol. 7, 544 (2011). 8. Okada, H., Ohnuki, S., Roncero, C., Konopka, J. B. & Ohya, Y. Mol. Biol. Cell 25, 222–233 (2014). 9. Aliferis, K. A. & Jabaji, S. Pestic. Biochem. Physiol. 100, 105–117 (2011). 10. Bouhifd, M., Hartung, T., Hogberg, H. T., Kleensang, A. & Zhao, L. J. Appl. Toxicol. 33, 1365–1383 (2013).
EVO LU TI O N
Steps on the road to eukaryotes A new archaeal phylum represents the closest known relatives of eukaryotes, the group encompassing all organisms that have nucleated cells. The discovery holds promise for a better understanding of eukaryotic origins. See Article p.173 T. M A R T I N E M B L E Y & T O M A . W I L L I A M S
T
here are many competing hypotheses1 for how eukaryotic cells, which contain a nucleus and other membrane-bound organelles, evolved from their prokaryotic ancestors, whose cells lack a nucleus. But testing these theories has been difficult owing to a lack of known intermediate stages in the prokaryote-to-eukaryote transition. On page 173 of this issue, Spang et al.2 describe a prokaryotic lineage that is more closely related to eukaryotes than any yet sampled and that shares with eukaryotes several genes previously thought to define aspects of eukaryotic biology. This technically outstanding paper has far-reaching implications for how we view early eukaryotic evolution, including our own deep ancestry. In most textbooks the cellular world is divided into three domains3: the eukaryotes (Eukarya) and two distinct prokaryotic groups, the Bacteria and the Archaea. In the classical three-domains tree, the eukaryotes are separated from a common prokaryotic ancestor that they share with Archaea by a long
branch that has been variously interpreted as representing a long period of time with unsampled diversity, a high rate of evolution in the eukaryotic ancestor, or the extinction of intermediate forms. Surveys of environmental microbial diversity using the tools of molecular biology have sought to populate this long branch, but have so far failed to identify any fundamentally new eukaryotic groups. In the three-domains tree, the eukaryotes appear fully formed, with almost all of the cellular complexity that we associate with modern eukaryotes already in place1. The three-domains tree is the most visible image depicting the diversity of cellular life, but it has not gone unchallenged. An alternative two-domains tree, in which the eukaryotic lineage originated within the archaeal domain, has gathered support from recent phylogenetic analyses4–6 and is now arguably the favoured hypothesis. In this tree, the eukaryotes are related to a diverse group of Archaea called the TACK superphylum7. Thus, unlike the threedomains tree, the two-domains tree includes an explicit prediction about where we should look for closer relatives of the eukaryotic Most recent common ancestor of eukaryotes
Most recent common ancestor of eukaryotes and Archaea • • • •
• Nucleus • Mitochondrion • Membrane-bound organelles
Cytoskeleton Membrane remodelling Ubiquitin modification Endocytosis and/or phagocytosis
Protists Plants Prokaryotes
Eukaryotes
Algae Fungi
Eukaryotes
acid has broad-spectrum activity. Although the ability of poacic acid to target β1,3 glucan and to disrupt cell walls in yeast is clear, more work is needed to establish the extent to which these effects are mirrored in agricultural pathogens and to gain deeper insights into the biochemical mechanism of action of poacic acid. Metabolomics (the study of metabolites within an organism, cell or tissue) provides a powerful tool with which to examine specific changes in the cellular biochemicals induced by drug action, and it has been successfully used to study pesticides9. When applied to poacic acid and other antifungals, metabolomics may help us to define the specific enzymatic targets that underlie fungicidal action. This could lead to the discovery of synergistic drug pairings and might help to explain why some fungicides are toxic to humans10. Piotrowski and colleagues’ demonstration of synergistic effects between poacic acid and human antifungal drugs is promising, suggesting that poacic acid and derivatives might have roles in medicine. But although poacic acid is probably also synergistic with agricultural fungicides, this remains to be examined and will be a crucial part of future studies. Finally, before poacic acid can be translated into a marketready fungicide there are still major hurdles to be overcome, including testing its effectiveness in field trials, efficacy against more-diverse pathogens and persistence in the environment, and establishing whether it is safe for humans. Although much work is still needed, this study is a timely illustration of how the right biological techniques, combined with resourcefulness — in this case discovering fungicides in waste biomass — can be used to gain insights into drug action. The work also shows how understanding drug mechanisms can help to identify synergistic drug pairings. This strategy holds great potential for rationally designed combination therapies that could extend the potency and lifetimes of existing fungicides. On its own, in combination with other fungicides or as a lead compound for further development, poacic acid is a potentially valuable new weapon in our armamentarium of antifungals. ■
Animals Lokiarchaeota
TACK Archaea
Figure 1 | Lokiarchaeota are the closest known prokaryotic relatives of eukaryotes. Phylogenetic trees presented by Spang et al.2 place eukaryotes within the Lokiarchaeota — a new group of Archaea described by the authors. The genomes of these Archaea contain more eukaryotic-like genes than other known Archaea. This finding implies that some of the defining features of eukaryotes — including a cytoskeleton, membrane remodelling, ubiquitin modification and the capacity for endocytosis and/or phagocytosis — might have already evolved in the last common ancestor of eukaryotes and Archaea. Spang and colleagues’ findings suggest that it is likely that other new lineages will be found to further close the evolutionary gap between Archaea and eukaryotes, increasing the precision with which we can identify when key cellular innovations such as the nucleus, mitochondrion and endoplasmic reticulum first evolved. 1 4 M AY 2 0 1 5 | VO L 5 2 1 | NAT U R E | 1 6 9
© 2015 Macmillan Publishers Limited. All rights reserved
Fungus against the wall A compound derived from plant cell-wall material that is a waste product of biofuel manufacture has been found to have fungicidal properties: it interacts with a carbohydrate called β1,3 glucan, thus compromising the integrity of fungal cells. PA U L O ’ M A I L L E
F
ungal infections are a major problem in agriculture, and as the world’s population grows, a rising tide of resistance to antifungal agents is threatening global food security. This threat has led to an intensification in the use of fungicidal agrochemicals, which may themselves put human health at risk and damage the environment. New fungicidal agents are therefore desperately needed; and now it seems that help is on the way, but from unlikely sources. Writing in Proceedings of the National Academy of Sciences, Piotrowski and colleagues1 report the discovery of molecules in the waste products of biofuel manufacture that possess antifungal activity against plant pathogens. Dwindling oil reserves have spurred the development of technologies that use lignocellulosics — dry plant matter such as grass — as a renewable source of sugar to produce biofuels and bio-based chemicals. a
A key step in the process is the hydrolysis of polysaccharides in the plant cell wall, which liberates sugar monomers so they can be fermented by yeast2 (Fig. 1a). But other molecules are also liberated by hydrolysis, including diferulates, metabolites of the phenylpropanoid family of natural products. These molecules crosslink polysaccharides to each other, and to lignin polymers, to stiffen the plant cell wall, but the waste products stunt the growth of the yeast, inhibiting the fermentation process. Piotrowski and colleagues saw this problem as an opportunity for ‘bioprospecting’, reasoning that these molecules might be an untapped resource of antifungal agents. The authors screened diferulates for antifungal activity by testing the molecules’ ability to inhibit growth of a fungus, the budding yeast Saccharomyces cerevisiae. They identified two active compounds that inhibited growth, the most potent of which they dubbed poacic acid because of its prevalence in grasses
Plant cell wall
Yeast cell Sugar monomers
Diferulates Bioprocessing
Hydrolysis
Biofuel
Polysaccharide
b Poacic acid
Yeast cell wall
β1,3 Glucan
Abnormal shape
Leaky cell
Figure 1 | Waste of a fungicide. a, Grasses of the Poaceae family have cell walls fortified with polysaccharides that are crosslinked with metabolites called diferulates, which act as an anchor for the formation of lignin polymers (not shown). Bioprocessing of such grasses liberates the polysaccharides for hydrolysis to sugar monomers, from which yeast can produce biofuels and bio-based chemicals. The process also liberates diferulates such as poacic acid as waste products. b, Piotrowski et al.1 report that poacic acid has antifungal activity against yeast cells. The compound attacks the carbohydrate β1,3 glucan, which acts as a fortification in the yeast cell wall. As a result, cells become leaky and adopt abnormal shapes. 1 6 8 | NAT U R E | VO L 5 2 1 | 1 4 M AY 2 0 1 5
© 2015 Macmillan Publishers Limited. All rights reserved
of the Poaceae plant family. Importantly, poacic acid had comparable antifungal activity to two widely used agricultural fungicides. Next, Piotrowski et al. investigated how poacic acid exerts its effect, by challenging a pooled mixture of approximately 4,000 yeast strains with the compound. Each strain carried a different gene deletion that was marked by a unique molecular barcode — a short sequence of DNA that does not affect the cell’s function but can be used to identify the strain. After exposing the cells to poacic acid, the authors took a head count with next-generation DNA sequencing, measuring the frequency of each barcode in the population to determine the relative sensitivity or resistance of each strain to the fungicide3. The result was clear: deletion of genes involved in cell-wall organization conferred poacic-acid sensitivity, suggesting that the compound affects the yeast cell wall, thus mirroring the mechanism of many known antifungal compounds4. The authors then tested poacic acid in combination with two antifungal compounds that compromise cellular integrity5 and that are normally used to treat human fungal infections: fluconazole, which targets the biosynthesis of ergosterol, an essential component of the fungal cell membrane; and capsofungin, which inhibits6 the biosynthesis of β1,3 glucan, a polysaccharide that acts as both a barrier and structural support in the fungal cell wall. Perhaps not unexpectedly, poacic acid acted in synergy with these compounds, indicating that each drug strikes the same or related pathways, albeit through different targets7. To further investigate how poacic acid alters the cell wall, Piotrowski et al. used highdimensional microscopy to study yeast cells treated with poacic acid. The neck between the yeast cell and the daughter that buds off it was unusually wide in treated cells, and the degree of morphological variability in the cell population was higher than normal — similar changes to those caused by other drugs that disrupt the cell wall8 (Fig. 1b). Poacic acid also caused rapid influx of propidium iodide dye into the cell, consistent with cell-wall disruption. In a stroke of resourcefulness, the authors used the natural fluorescence of poacic acid to study its location within the cell. The cell’s surface lit up under the microscope, suggesting that poacic acid interacts directly with the β1,3 glucan layer. The researchers then incubated poacic acid with purified fungal β1,3 glucan — the glucan particles began to fluoresce, confirming the interaction. Given these promising results, the researchers next tested the potency of poacic acid against key agricultural fungal pathogens. Preliminary studies showed that poacic acid inhibited the fungi Sclerotinia sclerotiorum and Alternaria solani in a dose-dependent manner and protected soya-bean leaves from the oomycete (fungus-like) pathogen Phytophthora sojae, suggesting that poacic
NEWS & VIEWS RESEARCH
Paul O’Maille is at the John Innes Centre and Institute of Food Research, Norwich NR4 7UH, UK. e-mail: paul.o’[email protected] 1. Piotrowski, J. S. et al. Proc. Natl Acad. Sci. USA 112, E1490–E1497 (2015). 2. Sun, Y. & Cheng, J. Bioresour. Technol. 83, 1–11 (2002). 3. Parsons, A. B. et al. Cell 126, 611–625 (2006). 4. Latgé, J.-P. Mol. Microbiol. 66, 279–290 (2007). 5. Kiraz, N. et al. Antimicrob. Agents Chemother. 54, 2244–2247 (2010). 6. Kurtz, M. B. & Douglas, C. M. J. Med. Vet. Mycol. 35, 79–86 (1997). 7. Cokol, M. et al. Mol. Syst. Biol. 7, 544 (2011). 8. Okada, H., Ohnuki, S., Roncero, C., Konopka, J. B. & Ohya, Y. Mol. Biol. Cell 25, 222–233 (2014). 9. Aliferis, K. A. & Jabaji, S. Pestic. Biochem. Physiol. 100, 105–117 (2011). 10. Bouhifd, M., Hartung, T., Hogberg, H. T., Kleensang, A. & Zhao, L. J. Appl. Toxicol. 33, 1365–1383 (2013).
EVO LU TI O N
Steps on the road to eukaryotes A new archaeal phylum represents the closest known relatives of eukaryotes, the group encompassing all organisms that have nucleated cells. The discovery holds promise for a better understanding of eukaryotic origins. See Article p.173 T. M A R T I N E M B L E Y & T O M A . W I L L I A M S
T
here are many competing hypotheses1 for how eukaryotic cells, which contain a nucleus and other membrane-bound organelles, evolved from their prokaryotic ancestors, whose cells lack a nucleus. But testing these theories has been difficult owing to a lack of known intermediate stages in the prokaryote-to-eukaryote transition. On page 173 of this issue, Spang et al.2 describe a prokaryotic lineage that is more closely related to eukaryotes than any yet sampled and that shares with eukaryotes several genes previously thought to define aspects of eukaryotic biology. This technically outstanding paper has far-reaching implications for how we view early eukaryotic evolution, including our own deep ancestry. In most textbooks the cellular world is divided into three domains3: the eukaryotes (Eukarya) and two distinct prokaryotic groups, the Bacteria and the Archaea. In the classical three-domains tree, the eukaryotes are separated from a common prokaryotic ancestor that they share with Archaea by a long
branch that has been variously interpreted as representing a long period of time with unsampled diversity, a high rate of evolution in the eukaryotic ancestor, or the extinction of intermediate forms. Surveys of environmental microbial diversity using the tools of molecular biology have sought to populate this long branch, but have so far failed to identify any fundamentally new eukaryotic groups. In the three-domains tree, the eukaryotes appear fully formed, with almost all of the cellular complexity that we associate with modern eukaryotes already in place1. The three-domains tree is the most visible image depicting the diversity of cellular life, but it has not gone unchallenged. An alternative two-domains tree, in which the eukaryotic lineage originated within the archaeal domain, has gathered support from recent phylogenetic analyses4–6 and is now arguably the favoured hypothesis. In this tree, the eukaryotes are related to a diverse group of Archaea called the TACK superphylum7. Thus, unlike the threedomains tree, the two-domains tree includes an explicit prediction about where we should look for closer relatives of the eukaryotic Most recent common ancestor of eukaryotes
Most recent common ancestor of eukaryotes and Archaea • • • •
• Nucleus • Mitochondrion • Membrane-bound organelles
Cytoskeleton Membrane remodelling Ubiquitin modification Endocytosis and/or phagocytosis
Protists Plants Prokaryotes
Eukaryotes
Algae Fungi
Eukaryotes
acid has broad-spectrum activity. Although the ability of poacic acid to target β1,3 glucan and to disrupt cell walls in yeast is clear, more work is needed to establish the extent to which these effects are mirrored in agricultural pathogens and to gain deeper insights into the biochemical mechanism of action of poacic acid. Metabolomics (the study of metabolites within an organism, cell or tissue) provides a powerful tool with which to examine specific changes in the cellular biochemicals induced by drug action, and it has been successfully used to study pesticides9. When applied to poacic acid and other antifungals, metabolomics may help us to define the specific enzymatic targets that underlie fungicidal action. This could lead to the discovery of synergistic drug pairings and might help to explain why some fungicides are toxic to humans10. Piotrowski and colleagues’ demonstration of synergistic effects between poacic acid and human antifungal drugs is promising, suggesting that poacic acid and derivatives might have roles in medicine. But although poacic acid is probably also synergistic with agricultural fungicides, this remains to be examined and will be a crucial part of future studies. Finally, before poacic acid can be translated into a marketready fungicide there are still major hurdles to be overcome, including testing its effectiveness in field trials, efficacy against more-diverse pathogens and persistence in the environment, and establishing whether it is safe for humans. Although much work is still needed, this study is a timely illustration of how the right biological techniques, combined with resourcefulness — in this case discovering fungicides in waste biomass — can be used to gain insights into drug action. The work also shows how understanding drug mechanisms can help to identify synergistic drug pairings. This strategy holds great potential for rationally designed combination therapies that could extend the potency and lifetimes of existing fungicides. On its own, in combination with other fungicides or as a lead compound for further development, poacic acid is a potentially valuable new weapon in our armamentarium of antifungals. ■
Animals Lokiarchaeota
TACK Archaea
Figure 1 | Lokiarchaeota are the closest known prokaryotic relatives of eukaryotes. Phylogenetic trees presented by Spang et al.2 place eukaryotes within the Lokiarchaeota — a new group of Archaea described by the authors. The genomes of these Archaea contain more eukaryotic-like genes than other known Archaea. This finding implies that some of the defining features of eukaryotes — including a cytoskeleton, membrane remodelling, ubiquitin modification and the capacity for endocytosis and/or phagocytosis — might have already evolved in the last common ancestor of eukaryotes and Archaea. Spang and colleagues’ findings suggest that it is likely that other new lineages will be found to further close the evolutionary gap between Archaea and eukaryotes, increasing the precision with which we can identify when key cellular innovations such as the nucleus, mitochondrion and endoplasmic reticulum first evolved. 1 4 M AY 2 0 1 5 | VO L 5 2 1 | NAT U R E | 1 6 9
© 2015 Macmillan Publishers Limited. All rights reserved
