news & views METABOLISM
Pathogens love the poison
Itaconate is a metabolite secreted by activated macrophages that inhibits pathogen growth. Some pathogens use itaconate degradation enzymes to promote their survival and infectivity, highlighting metabolic pathways to be considered in host-pathogen interactions.
Stéphane Ménage & Ina Attrée
326
In this issue, Sasikaran et al.6 identified enzymes capable of using itaconate as a sole carbon source in two human pathogens, Yersinia pestis, a causative agent of ‘black death’, and Pseudomonas aeruginosa, a major human opportunistic pathogen. They propose that pathogens may use these enzymes as a new defense strategy against the burst of itaconate secretion by macrophages. Indeed, the Y. pestis genes encoding itaconate-degradative enzymes were previously shown to be mandatory for pathogen survival in macrophages and were thus named rip (‘required for intracellular proliferation’)7. The rip genes (ripA, ripB and ripC) and their homologs in P. aeruginosa (PA0882, PA0878 and PA0883) respectively encode a CoA transferase (Ict), a specific enoyl-CoA hydratase acting as an itaconyl-CoA hydratase that is also capable of isomerization and subsequent hydration (Ich) and a citramomalyl-CoA lyase (Ccl) (Fig. 1). The enzyme activities and specificities were determined in vitro using purified recombinant proteins and were shown to harbor different properties
depending on the gene origin. Notably, although P. aeruginosa Ict is highly specific to itaconate and succinyl-CoA compared to Y. pestis Ict, which may use different substrates, Ich and Ccl were found to be substantially more efficient for itaconate metabolism in P. aeruginosa than in Y. pestis. Whether or not these differences have consequences in pathogen survival in macrophages in vivo is completely unknown. Notably, itaconate degradation enzymes are encoded by three- and six-gene operons in Y. pestis and P. aeruginosa subgroups, respectively (http://www.pseudomonas.com/8), and the conserved synteny further suggests their functional relationship. However, the exact function of the three additional genes in P. aeruginosa is still speculative. Deciphering metabolic specificities of enzymes encoded by these genes is necessary to obtain the global view of itaconate degradation and utilization pathways. Moreover, beside the fact that the rip genes have been found to be required for pathogenicity, direct evidence for a role of the P. aeruginosa operon in infectivity is still missing.
O OH
CoAS
O HO
O
Ich
Ich
OH O
Ict
Ich Ccl
Ccl Ich
Ict
Ccl Ccl
Ict Ich
Ccl Ccl
Ccl
O
Ict
Ccl Ict
O HO
OH
HO
Ccl Ict Ccl Ich
Ccl
O
OH
Ict
O
Ccl O
Ict CoAS
OH
HO
O
Ccl
Ich Ict
Ict Ict
O
O
OH
HO O
Marina Corral Spence
npg
© 2014 Nature America, Inc. All rights reserved.
B
acteria thrive in diverse environments, and the pressure to adapt is constant. In the context of host-pathogen interactions, bacteria confront a highly aggressive host environment that is activated to eliminate the foreign agent. Neutrophils and macrophages, cells of the innate immune system, are at the first line of defense against infecting bacteria. They use a panoply of different strategies to combat colonization and tissue invasion by the pathogen. Pathogens do not take these onslaughts lightly: they have evolved antihost strategies that permit successful host invasion and establishment of infection1. Neutrophils and macrophages secrete toxic products, such as antimicrobial peptides, cytokines, reactive oxygen species and nitric oxide, that are well known to participate in inflammation and pathogen elimination2. Recently, Michelucci et al.3 discovered that immunoresponsive gene 1 (irg1), which is highly expressed in macrophages during activation, encodes an enzyme producing the metabolite itaconic acid (methylenesuccinic acid). This metabolite was recently identified as the product of a specific decarboxylation reaction of cis-aconitate, an intermediate of the Krebs cycle in mammalian cells4. However, the molecule has potent antimicrobial activity, as evaluated by changes in cell growth of Mycobacterium tuberculosis and Salmonella enterica in vitro and within macrophages3. The mechanistic explanation for this seemingly contradictory activity lies in itaconate’s inhibitory effects on cytosolic isocitrate lyase, an essential enzyme of the glyoxylate cycle, a key pathway for pathogen survival in cells. Interestingly, some bacteria are able to grow in the presence of itaconate, which is also produced by Aspergillus spp. and other fungi; this topic was of particular interest in the 1970s and has been more recently as itaconate synthesis by different microorganisms has potential applications in the industrial production of plastics, resins, paints and similar products5.
Figure 1 | Bacterial defense against itaconate. An enzyme trio identified in pathogenic bacteria (Ict, Ich and Ccl) may represent a unique protection barrier against itaconic acid, a metabolite secreted by activated macrophages. nature chemical biology | VOL 10 | MAY 2014 | www.nature.com/naturechemicalbiology
npg
© 2014 Nature America, Inc. All rights reserved.
news & views Even though the whole genomes of major bacterial pathogens were sequenced more than a decade ago, still about 20% of predicted proteins harbor so-called domains of unknown function9. Some of these domains belong to proteins that are essential for bacterial lifestyle, revealing the need to explore their biological function in more detail. Assigning roles to the rip genes, as achieved by Sasikaran et al.6, clearly contributes to the general understanding of bacterial biology and opens new perspectives in the host-pathogen field of research. Notably, on the basis of these recent findings, one can speculate that bacterial success in invading tissue may be also correlated to the fine balance between itaconate production by the macrophages and the itaconate degradation pathway orchestrated by the pathogens. This postulate opens up the debate about the possibility of using the itaconate degradation pathway as a target for antibacterial treatment, presenting an alternative to
classical antibiotic therapies. However, going in this direction seems to be premature. Targeting bacterial enzymes with small chemicals without the complete knowledge of homologous host enzymes and their specificities may provoke undesirable side effects on host side. Indeed, searching for similar enzymes in mammals revealed that the itaconate degradation pathway relies solely on the presence of the Ccl enzyme, thus leaving the degradation metabolites and eventually other players, such as other less specific CoA transferases or carboxylic CoA synthases, to be identified. In conclusion, deciphering all of the intricate metabolic pathways related both to the pathogen and the host will be an exciting challenge in the future to define new antibacterial targets. ■ Stéphane Ménage is at the Chemistry and Biology of Metals Laboratory, UMR5249, CNRS; Université Grenoble Alpes, Grenoble, France; and Commissariat à l’énergie atomique et aux énergies alternatives, l’Institut de recherches en technologies
et sciences pour le vivant (iRTSV), Grenoble, France. Ina Attrée is at Bacterial Pathogenicity and Cellular Responses, ERL5261, CNRS; Biologie du cancer et de l’infection (UMR-S1036), INSERM, Grenoble, France; Université Grenoble Alpes, Grenoble, France; and CEA, iRTSV, Grenoble, France. e-mail: [email protected] References
1. Sarantis, H. & Grinstein, S. Cell Host Microbe 12, 419–431 (2012). 2. Laskin, D.L., Sunil, V.R., Gardner, C.R. & Laskin, J.D. Annu. Rev. Pharmacol. Toxicol. 51, 267–288 (2011). 3. Michelucci, A. et al. Proc. Natl. Acad. Sci. USA 110, 7820–7825 (2013). 4. Strelko, C.L. et al. J. Am. Chem. Soc. 133, 16386–16389 (2011). 5. Steiger, M.G., Blumhoff, M.L., Mattanovich, D. & Sauer, M. Front. Microbiol. 4, 23 (2013). 6. Sasikaran, J., Ziemski, M., Zadora, P.K., Fleig, A. & Berg, I.A. Nat. Chem. Biol. 10, 371–377 (2014). 7. Pujol, C., Grabenstein, J.P., Perry, R.D. & Bliska, J.B. Proc. Natl. Acad. Sci. USA 102, 12909–12914 (2005). 8. Winsor, G.L. et al. Nucleic Acids Res. 39, D596–D600 (2011). 9. Goodacre, N.F., Gerloff, D.L. & Uetz, P. MBio. 5, e00744–e00713 (2013).
Competing financial interests
The authors declare no competing financial interests.
PLURIPOTENCY
Citrullination unravels stem cells
Maintenance of the pluripotent stem cell state is regulated by the post-translational modification of histones. The discovery that citrullination of the linker histone H1 is critical to this process represents a new role for the protein arginine deiminases in development.
Daniel J Slade, Venkataraman Subramanian & Paul R Thompson
T
he protein arginine deiminases (PADs) 1, 2, 3, 4 and 6 convert protein-encoded arginines into citrulline through a calcium-dependent reaction called citrullination or deimination. PAD expression and activity is upregulated in inflammatory diseases and cancer1,2, and multiple isozymes represent attractive therapeutic targets. Despite their disease relevance, the cellular roles of the PADs remain poorly understood. Their best-characterized role is as histone-modifying enzymes that regulate gene transcription. For example, PAD2 and PAD4 citrullinate histones H3 and H4, and these modifications are correlated with either the repression or activation of genes under the control of the estrogen receptor and p53 (refs. 3–5). Histone citrullination affects chromatin structure, as citrullination of histone H3 leads to the expulsion of heterochromatin protein 1α from the chromatin, thereby creating an ‘open’ state that promotes
gene transcription6. The PAD4-catalyzed citrullination of histones H1 and H3 in neutrophils leads to massive chromatin decondensation and expulsion of DNA to form neutrophil extracellular traps7, a pro-inflammatory form of cell death that is aberrantly increased in numerous inflammatory diseases2. Adding to the role of the PADs in histone biology, Christophorou et al.8 report that PAD4 citrullination of histone H1 promotes its dissociation from DNA, thereby creating an open chromatin architecture that is necessary for stem cell pluripotency during early embryogenesis. Pluripotent stem cells are ‘master’ cells that differentiate into any cell lineage and can either be isolated as embryonic stem (ES) cells or genetically reprogrammed through the reversion of differentiated cells into induced pluripotent (iPS) cells. Reprogramming of iPS cells is initiated by the upregulation of pluripotency gene expression due to formation of an open
nature chemical biology | VOL 10 | MAY 2014 | www.nature.com/naturechemicalbiology
chromatin structure around these genes. This process involves modifications of the proteins that constitute the core histone octamer as well as histone H1, which directly binds nucleosome-bound DNA and maintains a properly compacted state (Fig. 1). Given the ability of the PADs to modulate the chromatin architecture in neutrophils, Christophorou et al.8 questioned whether PAD4 has a role in ES and iPS cells. Initial experiments performed with mouse ES cells (ES Oct4-GIP) and committed neural stem cells (NSO4G) showed that PAD4 was only expressed in ES cells. Upon reprogramming into iPS cells, NSO4G cells express PAD4, and, remarkably, this expression highly correlates with levels of Nanog, an essential stem cell transcription factor, as well as the expression of a subset of other known pluripotency genes, including Klf2, Tcl1, Tcfap2c and Kit. Nanog seems to induce PAD4 activity because the levels of citrullinated H3 are reduced in its absence. 327
Pathogens love the poison
Itaconate is a metabolite secreted by activated macrophages that inhibits pathogen growth. Some pathogens use itaconate degradation enzymes to promote their survival and infectivity, highlighting metabolic pathways to be considered in host-pathogen interactions.
Stéphane Ménage & Ina Attrée
326
In this issue, Sasikaran et al.6 identified enzymes capable of using itaconate as a sole carbon source in two human pathogens, Yersinia pestis, a causative agent of ‘black death’, and Pseudomonas aeruginosa, a major human opportunistic pathogen. They propose that pathogens may use these enzymes as a new defense strategy against the burst of itaconate secretion by macrophages. Indeed, the Y. pestis genes encoding itaconate-degradative enzymes were previously shown to be mandatory for pathogen survival in macrophages and were thus named rip (‘required for intracellular proliferation’)7. The rip genes (ripA, ripB and ripC) and their homologs in P. aeruginosa (PA0882, PA0878 and PA0883) respectively encode a CoA transferase (Ict), a specific enoyl-CoA hydratase acting as an itaconyl-CoA hydratase that is also capable of isomerization and subsequent hydration (Ich) and a citramomalyl-CoA lyase (Ccl) (Fig. 1). The enzyme activities and specificities were determined in vitro using purified recombinant proteins and were shown to harbor different properties
depending on the gene origin. Notably, although P. aeruginosa Ict is highly specific to itaconate and succinyl-CoA compared to Y. pestis Ict, which may use different substrates, Ich and Ccl were found to be substantially more efficient for itaconate metabolism in P. aeruginosa than in Y. pestis. Whether or not these differences have consequences in pathogen survival in macrophages in vivo is completely unknown. Notably, itaconate degradation enzymes are encoded by three- and six-gene operons in Y. pestis and P. aeruginosa subgroups, respectively (http://www.pseudomonas.com/8), and the conserved synteny further suggests their functional relationship. However, the exact function of the three additional genes in P. aeruginosa is still speculative. Deciphering metabolic specificities of enzymes encoded by these genes is necessary to obtain the global view of itaconate degradation and utilization pathways. Moreover, beside the fact that the rip genes have been found to be required for pathogenicity, direct evidence for a role of the P. aeruginosa operon in infectivity is still missing.
O OH
CoAS
O HO
O
Ich
Ich
OH O
Ict
Ich Ccl
Ccl Ich
Ict
Ccl Ccl
Ict Ich
Ccl Ccl
Ccl
O
Ict
Ccl Ict
O HO
OH
HO
Ccl Ict Ccl Ich
Ccl
O
OH
Ict
O
Ccl O
Ict CoAS
OH
HO
O
Ccl
Ich Ict
Ict Ict
O
O
OH
HO O
Marina Corral Spence
npg
© 2014 Nature America, Inc. All rights reserved.
B
acteria thrive in diverse environments, and the pressure to adapt is constant. In the context of host-pathogen interactions, bacteria confront a highly aggressive host environment that is activated to eliminate the foreign agent. Neutrophils and macrophages, cells of the innate immune system, are at the first line of defense against infecting bacteria. They use a panoply of different strategies to combat colonization and tissue invasion by the pathogen. Pathogens do not take these onslaughts lightly: they have evolved antihost strategies that permit successful host invasion and establishment of infection1. Neutrophils and macrophages secrete toxic products, such as antimicrobial peptides, cytokines, reactive oxygen species and nitric oxide, that are well known to participate in inflammation and pathogen elimination2. Recently, Michelucci et al.3 discovered that immunoresponsive gene 1 (irg1), which is highly expressed in macrophages during activation, encodes an enzyme producing the metabolite itaconic acid (methylenesuccinic acid). This metabolite was recently identified as the product of a specific decarboxylation reaction of cis-aconitate, an intermediate of the Krebs cycle in mammalian cells4. However, the molecule has potent antimicrobial activity, as evaluated by changes in cell growth of Mycobacterium tuberculosis and Salmonella enterica in vitro and within macrophages3. The mechanistic explanation for this seemingly contradictory activity lies in itaconate’s inhibitory effects on cytosolic isocitrate lyase, an essential enzyme of the glyoxylate cycle, a key pathway for pathogen survival in cells. Interestingly, some bacteria are able to grow in the presence of itaconate, which is also produced by Aspergillus spp. and other fungi; this topic was of particular interest in the 1970s and has been more recently as itaconate synthesis by different microorganisms has potential applications in the industrial production of plastics, resins, paints and similar products5.
Figure 1 | Bacterial defense against itaconate. An enzyme trio identified in pathogenic bacteria (Ict, Ich and Ccl) may represent a unique protection barrier against itaconic acid, a metabolite secreted by activated macrophages. nature chemical biology | VOL 10 | MAY 2014 | www.nature.com/naturechemicalbiology
npg
© 2014 Nature America, Inc. All rights reserved.
news & views Even though the whole genomes of major bacterial pathogens were sequenced more than a decade ago, still about 20% of predicted proteins harbor so-called domains of unknown function9. Some of these domains belong to proteins that are essential for bacterial lifestyle, revealing the need to explore their biological function in more detail. Assigning roles to the rip genes, as achieved by Sasikaran et al.6, clearly contributes to the general understanding of bacterial biology and opens new perspectives in the host-pathogen field of research. Notably, on the basis of these recent findings, one can speculate that bacterial success in invading tissue may be also correlated to the fine balance between itaconate production by the macrophages and the itaconate degradation pathway orchestrated by the pathogens. This postulate opens up the debate about the possibility of using the itaconate degradation pathway as a target for antibacterial treatment, presenting an alternative to
classical antibiotic therapies. However, going in this direction seems to be premature. Targeting bacterial enzymes with small chemicals without the complete knowledge of homologous host enzymes and their specificities may provoke undesirable side effects on host side. Indeed, searching for similar enzymes in mammals revealed that the itaconate degradation pathway relies solely on the presence of the Ccl enzyme, thus leaving the degradation metabolites and eventually other players, such as other less specific CoA transferases or carboxylic CoA synthases, to be identified. In conclusion, deciphering all of the intricate metabolic pathways related both to the pathogen and the host will be an exciting challenge in the future to define new antibacterial targets. ■ Stéphane Ménage is at the Chemistry and Biology of Metals Laboratory, UMR5249, CNRS; Université Grenoble Alpes, Grenoble, France; and Commissariat à l’énergie atomique et aux énergies alternatives, l’Institut de recherches en technologies
et sciences pour le vivant (iRTSV), Grenoble, France. Ina Attrée is at Bacterial Pathogenicity and Cellular Responses, ERL5261, CNRS; Biologie du cancer et de l’infection (UMR-S1036), INSERM, Grenoble, France; Université Grenoble Alpes, Grenoble, France; and CEA, iRTSV, Grenoble, France. e-mail: [email protected] References
1. Sarantis, H. & Grinstein, S. Cell Host Microbe 12, 419–431 (2012). 2. Laskin, D.L., Sunil, V.R., Gardner, C.R. & Laskin, J.D. Annu. Rev. Pharmacol. Toxicol. 51, 267–288 (2011). 3. Michelucci, A. et al. Proc. Natl. Acad. Sci. USA 110, 7820–7825 (2013). 4. Strelko, C.L. et al. J. Am. Chem. Soc. 133, 16386–16389 (2011). 5. Steiger, M.G., Blumhoff, M.L., Mattanovich, D. & Sauer, M. Front. Microbiol. 4, 23 (2013). 6. Sasikaran, J., Ziemski, M., Zadora, P.K., Fleig, A. & Berg, I.A. Nat. Chem. Biol. 10, 371–377 (2014). 7. Pujol, C., Grabenstein, J.P., Perry, R.D. & Bliska, J.B. Proc. Natl. Acad. Sci. USA 102, 12909–12914 (2005). 8. Winsor, G.L. et al. Nucleic Acids Res. 39, D596–D600 (2011). 9. Goodacre, N.F., Gerloff, D.L. & Uetz, P. MBio. 5, e00744–e00713 (2013).
Competing financial interests
The authors declare no competing financial interests.
PLURIPOTENCY
Citrullination unravels stem cells
Maintenance of the pluripotent stem cell state is regulated by the post-translational modification of histones. The discovery that citrullination of the linker histone H1 is critical to this process represents a new role for the protein arginine deiminases in development.
Daniel J Slade, Venkataraman Subramanian & Paul R Thompson
T
he protein arginine deiminases (PADs) 1, 2, 3, 4 and 6 convert protein-encoded arginines into citrulline through a calcium-dependent reaction called citrullination or deimination. PAD expression and activity is upregulated in inflammatory diseases and cancer1,2, and multiple isozymes represent attractive therapeutic targets. Despite their disease relevance, the cellular roles of the PADs remain poorly understood. Their best-characterized role is as histone-modifying enzymes that regulate gene transcription. For example, PAD2 and PAD4 citrullinate histones H3 and H4, and these modifications are correlated with either the repression or activation of genes under the control of the estrogen receptor and p53 (refs. 3–5). Histone citrullination affects chromatin structure, as citrullination of histone H3 leads to the expulsion of heterochromatin protein 1α from the chromatin, thereby creating an ‘open’ state that promotes
gene transcription6. The PAD4-catalyzed citrullination of histones H1 and H3 in neutrophils leads to massive chromatin decondensation and expulsion of DNA to form neutrophil extracellular traps7, a pro-inflammatory form of cell death that is aberrantly increased in numerous inflammatory diseases2. Adding to the role of the PADs in histone biology, Christophorou et al.8 report that PAD4 citrullination of histone H1 promotes its dissociation from DNA, thereby creating an open chromatin architecture that is necessary for stem cell pluripotency during early embryogenesis. Pluripotent stem cells are ‘master’ cells that differentiate into any cell lineage and can either be isolated as embryonic stem (ES) cells or genetically reprogrammed through the reversion of differentiated cells into induced pluripotent (iPS) cells. Reprogramming of iPS cells is initiated by the upregulation of pluripotency gene expression due to formation of an open
nature chemical biology | VOL 10 | MAY 2014 | www.nature.com/naturechemicalbiology
chromatin structure around these genes. This process involves modifications of the proteins that constitute the core histone octamer as well as histone H1, which directly binds nucleosome-bound DNA and maintains a properly compacted state (Fig. 1). Given the ability of the PADs to modulate the chromatin architecture in neutrophils, Christophorou et al.8 questioned whether PAD4 has a role in ES and iPS cells. Initial experiments performed with mouse ES cells (ES Oct4-GIP) and committed neural stem cells (NSO4G) showed that PAD4 was only expressed in ES cells. Upon reprogramming into iPS cells, NSO4G cells express PAD4, and, remarkably, this expression highly correlates with levels of Nanog, an essential stem cell transcription factor, as well as the expression of a subset of other known pluripotency genes, including Klf2, Tcl1, Tcfap2c and Kit. Nanog seems to induce PAD4 activity because the levels of citrullinated H3 are reduced in its absence. 327
