- Email: [email protected]
Can the Microbiome Deliver? A Proof-of-Concept Engineered E. coli PKU Therapeutic
Cell Host & Microbe
Previews ushers in a new era of CDN and now CTN signaling, and it is going to be fascinating to see what music they make. ACKNOWLEDGMENTS C.M.W. acknowledges support from NIH grants GM109259, GM110444, and AI130554. We appreciate Nicolas Fernandez for comments on the manuscript.
€ndling, A. (2013). Cyclic diCorrigan, R.M., and Gru AMP: another second messenger enters the fray. Nat. Rev. Microbiol. 11, 513–524. Davies, B.W., Bogard, R.W., Young, T.S., and Mekalanos, J.J. (2012). Coordinated regulation of accessory genetic elements produces cyclic dinucleotides for V. cholerae virulence. Cell 149, 358–370. Margolis, S.R., Wilson, S.C., and Vance, R.E. (2017). Evolutionary origins of cGAS-STING signaling. Trends Immunol. 38, 733–743.
REFERENCES €ffer, D.E., Iyer, Burroughs, A.M., Zhang, D., Scha L.M., and Aravind, L. (2015). Comparative genomic analyses reveal a vast, novel network of nucleotide-centric systems in biological conflicts, immunity and signaling. Nucleic Acids Res. 43, 10633–10654.
McFarland, A.P., Luo, S., Ahmed-Qadri, F., Zuck, M., Thayer, E.F., Goo, Y.A., Hybiske, K., Tong, L., and Woodward, J.J. (2017). Sensing of bacterial cyclic dinucleotides by the oxidoreductase RECON promotes NF-kB activation and shapes a proinflammatory antibacterial state. Immunity 46, 433–445.
Nelson, J.W., and Breaker, R.R. (2017). The lost language of the RNA World. Sci. Signal. 10, 1–11. Ro¨mling, U., Galperin, M.Y., and Gomelsky, M. (2013). Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol. Mol. Biol. Rev. 77, 1–52. Severin, G.B., Ramliden, M.S., Hawver, L.A., Wang, K., Pell, M.E., Kieninger, A.K., Khataokar, A., O’Hara, B.J., Behrmann, L.V., Neiditch, M.B., et al. (2018). Direct activation of a phospholipase by cyclic GMP-AMP in El Tor Vibrio cholerae. Proc. Natl. Acad. Sci. USA 115, E6048–E6055. Whiteley, A.T., Eaglesham, J.B., de Oliveira Mann, C.C., Morehouse, B.R., Lowey, B., Nieminen, E.A., Danilchanka, O., King, D.S., Lee, A.S.Y., Mekalanos, J.J., and Kranzusch, P.J. (2019). Bacterial cGAS-like enzymes synthesize diverse nucleotide signals. Nature 567, 194–199.
Can the Microbiome Deliver? A Proof-of-Concept Engineered E. coli PKU Therapeutic Christopher J. Alteri1,* 1Department of Natural Sciences, University of Michigan Dearborn, Dearborn, MI 48128, USA *Correspondence: [email protected] https://doi.org/10.1016/j.chom.2019.03.015
Phenylketonuria (PKU) is a rare genetic disorder that causes phenylalanine toxicity in the brain. Two studies, Crook et al. (2019), in this issue of Cell Host & Microbe, and Isabella et al. (2018), employ synthetic biology to develop a live bacterial therapeutic to treat PKU and potentially other metabolic diseases. The interaction between diet, the microbiome, and human health has been the focus of intense basic research over the past decade. An explosion of high-profile studies have implicated the human intestinal microbiome in a multitude of maladies, from neurologic disorders (Griffiths and Mazmanian, 2018; Sampson et al., 2016) to obesity (Turnbaugh et al., 2006) and even lung cancer (Jin et al., 2019). As microbiome research moves toward mechanistic studies, synthetic biology has combined with what can be reasonably described as a historical probiotic, Escherichia coli Nissle (EcN) 1917, to deliver on the promise that intentional directed manipulation of the intestinal microbiome can treat human disease (Ruder et al., 2011). As the name implies, EcN has been used in humans since its isolation in 1917 (Sonnenborn, 2016), and as with
most E. coli, EcN is also highly tractable. These traits have led to the current interest among synthetic biologists in using EcN as a chassis for the development of engineered bacterial therapeutics. Phenylketonuria (PKU) is an autosomal recessive genetic disorder in humans caused by a defect in the gene encoding phenylalanine hydroxylase. In patients with PKU, the absence of this function leads to phenylalanine (Phe) accumulation in the brain, resulting in cognitive and emotional problems. Thus, PKU represents a specific target whereby the gut microbiome could be exploited to facilitate degradation of Phe from the diet that would otherwise be toxic in these patients. It has been shown in the Gram-positive Clostridium genus that manipulation of an endogenous bacterial metabolic pathway that uses aromatic amino acids such as
Phe as substrates affects levels of metabolites in the serum of gnotobiotic mice (Dodd et al., 2017). More recently, Isabella and colleagues have taken a translational approach to reduce serum Phe levels with clinical trials in mind by using EcN as a biotherapeutic agent suitable for the treatment of PKU (Isabella et al., 2018). In order to achieve Phe degradation in EcN, Isabella and colleagues first engineered two pathways into EcN (Isabella et al., 2018). One Phe degradation pathway was provided by inserting the gene sltA encoding Phe ammonia lyase into the EcN chromosome. Since sltA encodes a cytosolic enzyme, the strain also had to be engineered to express pheP, which encodes a Phe transporter. The advantage of using SltA is its lack of oxygen dependence. In addition, a second pathway was engineered into EcN, by insertion of Proteus mirabilis pma, which
Cell Host & Microbe 25, April 10, 2019 ª 2019 Elsevier Inc. 473
Cell Host & Microbe
Previews encodes L-amino acid deaminase and has greater Phe degradation capability than SltA but is dependent on oxygen. This engineered system provides a mechanism to deliver Phe degradation capability at dosing (oxygen present) and in the low-oxygen environment of the human gut. Since constitutive expression of these pathways retards the growth of EcN, the sltA and pheP genes were placed under the control of the FNR promoter, which is active under anaerobic conditions, and pma was placed under the control of the arabinose promoter AraC (Isabella et al., 2018). Ultimately, use of the dual Phe degradation pathway EcN in non-human primates led to increased levels of trans-cinnamate and hippuric acid, both evidence for Phe degradation by the SltA pathway (Isabella et al., 2018). While the study indirectly attempted to deal with genome stability by introducing multiple copies pheP and sltA into EcN, little was done to directly assess genome stability or to understand how EcN colonizes and maintains population density in the intestinal tract. In this issue of Cell Host & Microbe, Crook et al. demonstrate up to 50% reduction in serum Phe levels using various engineered EcN strains in the mouse model of PKU. This study also finds that their engineered EcN chassis, which encodes Phe ammonia lyase activity from Arabidopsis thaliana, was stable in vivo (Crook et al., 2019). Crook et al. also investigated how transit through the gut, microbiota complexity, antibiotic pressure, and engineered genetics affect EcN during longterm colonization of the murine intestinal tract (Crook et al., 2019). This study specifically examined whole-genome sequences of EcN in vivo adapted isolates containing metagenomic libraries to select for genetic elements that enhance degradation of complex polysaccharides, a role that is normally provided to E. coli by intestinal anaerobes (Porter and Martens, 2017). The metagenomic libraries were prepared from DNA isolated from
474 Cell Host & Microbe 25, April 10, 2019
the feces of healthy human infants and adult females and introduced into EcN. Interestingly, following long-term colonization in mice, it was found that genes encoding a glycoside hydrolase family 32 (GH32) enzyme and other enzymes involved in carbohydrate utilization were selected for (Crook et al., 2019). Subsequently, it was found that GH32 containing EcN was capable of cross-feeding EcN lacking GH32 for growth on longchain polysaccharides. These findings indicated that the primary selective pressure for EcN is carbon source limitation, but this can be overcome by other taxa presumably encoding GH32 enzymatic activity. Next, Crook et al. looked at withingenome changes that occur in the EcN chromosome during colonization of the intestine with varying levels of microbiota complexity under three different dietary conditions. The major change that occurred in the EcN chromosome was mutation of genes encoding gluconate utilization, specifically gntT, which encodes a gluconate transporter. EcN strains with defects in gluconate utilization demonstrated superior growth on mucin as compared to wild-type EcN. This finding indicates that inhibition of gluconate utilization is advantageous when mucin is the primary carbon source. The studies by Isabella et al. (2018) and Crook et al. (2019) both demonstrate the potential for a live biotherapeutic to treat the genetic disorder PKU. Isabella and colleagues brought a significant conceptual advance by developing the two pathways for Phe degradation with optimization for therapeutic dosing and translating their engineered EcN strain for clinical trials (Isabella et al., 2018). On the other hand, Crook and colleagues brought a significant advance by elucidating selective pressures that EcN is under during intestinal colonization under a variety of microbiomes and dietary conditions, which is apropos given the diverse healthy and dysbiotic microbiomes that such engineered EcN chassis would likely be used.
Given that, the work by Isabella et al. (2018) and Crook et al. (2019) represent powerful synthetic biology proof-ofconcept studies that deliver on the promising notion that manipulation of the gut microbiome can treat metabolic disorders. REFERENCES Crook, N., Ferreiro, A., Gasparrini, A.J., Pesesky, M.W., Gibson, M.K., Wang, B., Sun, X., Condiotte, Z., Dobrowolski, S., Peterson, D., and Dantas, G. (2019). Adaptive strategies of the candidate probiotic E. coli Nissle in the mammalian gut. Cell Host Microbe 25, this issue, 499–512. Dodd, D., Spitzer, M.H., Van Treuren, W., Merrill, B.D., Hryckowian, A.J., Higginbottom, S.K., Le, A., Cowan, T.M., Nolan, G.P., Fischbach, M.A., and Sonnenburg, J.L. (2017). A gut bacterial pathway metabolizes aromatic amino acids into nine circulating metabolites. Nature 551, 648–652. Griffiths, J.A., and Mazmanian, S.K. (2018). Emerging evidence linking the gut microbiome to neurologic disorders. Genome Med. 10, 98. Isabella, V.M., Ha, B.N., Castillo, M.J., Lubkowicz, D.J., Rowe, S.E., Millet, Y.A., Anderson, C.L., Li, N., Fisher, A.B., West, K.A., et al. (2018). Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria. Nat. Biotechnol. 36, 857–864. Jin, C., Lagoudas, G.K., Zhao, C., Bullman, S., Bhutkar, A., Hu, B., Ameh, S., Sandel, D., Liang, X.S., Mazzilli, S., et al. (2019). Commensal microbiota promote lung cancer development via gd T cells. Cell 176, 998–1013.e16. Porter, N.T., and Martens, E.C. (2017). The critical roles of polysaccharides in gut microbial ecology and physiology. Annu. Rev. Microbiol. 71, 349–369. Ruder, W.C., Lu, T., and Collins, J.J. (2011). Synthetic biology moving into the clinic. Science 333, 1248–1252. Sampson, T.R., Debelius, J.W., Thron, T., Janssen, S., Shastri, G.G., Ilhan, Z.E., Challis, C., Schretter, C.E., Rocha, S., Gradinaru, V., et al. (2016). Gut microbiota regulate motor deficits and neuroinflammation in a model of Parkinson’s disease. Cell 167, 1469–1480.e12. Sonnenborn, U. (2016). Escherichia coli strain Nissle 1917-from bench to bedside and back: history of a special Escherichia coli strain with probiotic properties. FEMS Microbiol. Lett. 363, fnw212. Turnbaugh, P.J., Ley, R.E., Mahowald, M.A., Magrini, V., Mardis, E.R., and Gordon, J.I. (2006). An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444, 1027–1031.
Previews ushers in a new era of CDN and now CTN signaling, and it is going to be fascinating to see what music they make. ACKNOWLEDGMENTS C.M.W. acknowledges support from NIH grants GM109259, GM110444, and AI130554. We appreciate Nicolas Fernandez for comments on the manuscript.
€ndling, A. (2013). Cyclic diCorrigan, R.M., and Gru AMP: another second messenger enters the fray. Nat. Rev. Microbiol. 11, 513–524. Davies, B.W., Bogard, R.W., Young, T.S., and Mekalanos, J.J. (2012). Coordinated regulation of accessory genetic elements produces cyclic dinucleotides for V. cholerae virulence. Cell 149, 358–370. Margolis, S.R., Wilson, S.C., and Vance, R.E. (2017). Evolutionary origins of cGAS-STING signaling. Trends Immunol. 38, 733–743.
REFERENCES €ffer, D.E., Iyer, Burroughs, A.M., Zhang, D., Scha L.M., and Aravind, L. (2015). Comparative genomic analyses reveal a vast, novel network of nucleotide-centric systems in biological conflicts, immunity and signaling. Nucleic Acids Res. 43, 10633–10654.
McFarland, A.P., Luo, S., Ahmed-Qadri, F., Zuck, M., Thayer, E.F., Goo, Y.A., Hybiske, K., Tong, L., and Woodward, J.J. (2017). Sensing of bacterial cyclic dinucleotides by the oxidoreductase RECON promotes NF-kB activation and shapes a proinflammatory antibacterial state. Immunity 46, 433–445.
Nelson, J.W., and Breaker, R.R. (2017). The lost language of the RNA World. Sci. Signal. 10, 1–11. Ro¨mling, U., Galperin, M.Y., and Gomelsky, M. (2013). Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol. Mol. Biol. Rev. 77, 1–52. Severin, G.B., Ramliden, M.S., Hawver, L.A., Wang, K., Pell, M.E., Kieninger, A.K., Khataokar, A., O’Hara, B.J., Behrmann, L.V., Neiditch, M.B., et al. (2018). Direct activation of a phospholipase by cyclic GMP-AMP in El Tor Vibrio cholerae. Proc. Natl. Acad. Sci. USA 115, E6048–E6055. Whiteley, A.T., Eaglesham, J.B., de Oliveira Mann, C.C., Morehouse, B.R., Lowey, B., Nieminen, E.A., Danilchanka, O., King, D.S., Lee, A.S.Y., Mekalanos, J.J., and Kranzusch, P.J. (2019). Bacterial cGAS-like enzymes synthesize diverse nucleotide signals. Nature 567, 194–199.
Can the Microbiome Deliver? A Proof-of-Concept Engineered E. coli PKU Therapeutic Christopher J. Alteri1,* 1Department of Natural Sciences, University of Michigan Dearborn, Dearborn, MI 48128, USA *Correspondence: [email protected] https://doi.org/10.1016/j.chom.2019.03.015
Phenylketonuria (PKU) is a rare genetic disorder that causes phenylalanine toxicity in the brain. Two studies, Crook et al. (2019), in this issue of Cell Host & Microbe, and Isabella et al. (2018), employ synthetic biology to develop a live bacterial therapeutic to treat PKU and potentially other metabolic diseases. The interaction between diet, the microbiome, and human health has been the focus of intense basic research over the past decade. An explosion of high-profile studies have implicated the human intestinal microbiome in a multitude of maladies, from neurologic disorders (Griffiths and Mazmanian, 2018; Sampson et al., 2016) to obesity (Turnbaugh et al., 2006) and even lung cancer (Jin et al., 2019). As microbiome research moves toward mechanistic studies, synthetic biology has combined with what can be reasonably described as a historical probiotic, Escherichia coli Nissle (EcN) 1917, to deliver on the promise that intentional directed manipulation of the intestinal microbiome can treat human disease (Ruder et al., 2011). As the name implies, EcN has been used in humans since its isolation in 1917 (Sonnenborn, 2016), and as with
most E. coli, EcN is also highly tractable. These traits have led to the current interest among synthetic biologists in using EcN as a chassis for the development of engineered bacterial therapeutics. Phenylketonuria (PKU) is an autosomal recessive genetic disorder in humans caused by a defect in the gene encoding phenylalanine hydroxylase. In patients with PKU, the absence of this function leads to phenylalanine (Phe) accumulation in the brain, resulting in cognitive and emotional problems. Thus, PKU represents a specific target whereby the gut microbiome could be exploited to facilitate degradation of Phe from the diet that would otherwise be toxic in these patients. It has been shown in the Gram-positive Clostridium genus that manipulation of an endogenous bacterial metabolic pathway that uses aromatic amino acids such as
Phe as substrates affects levels of metabolites in the serum of gnotobiotic mice (Dodd et al., 2017). More recently, Isabella and colleagues have taken a translational approach to reduce serum Phe levels with clinical trials in mind by using EcN as a biotherapeutic agent suitable for the treatment of PKU (Isabella et al., 2018). In order to achieve Phe degradation in EcN, Isabella and colleagues first engineered two pathways into EcN (Isabella et al., 2018). One Phe degradation pathway was provided by inserting the gene sltA encoding Phe ammonia lyase into the EcN chromosome. Since sltA encodes a cytosolic enzyme, the strain also had to be engineered to express pheP, which encodes a Phe transporter. The advantage of using SltA is its lack of oxygen dependence. In addition, a second pathway was engineered into EcN, by insertion of Proteus mirabilis pma, which
Cell Host & Microbe 25, April 10, 2019 ª 2019 Elsevier Inc. 473
Cell Host & Microbe
Previews encodes L-amino acid deaminase and has greater Phe degradation capability than SltA but is dependent on oxygen. This engineered system provides a mechanism to deliver Phe degradation capability at dosing (oxygen present) and in the low-oxygen environment of the human gut. Since constitutive expression of these pathways retards the growth of EcN, the sltA and pheP genes were placed under the control of the FNR promoter, which is active under anaerobic conditions, and pma was placed under the control of the arabinose promoter AraC (Isabella et al., 2018). Ultimately, use of the dual Phe degradation pathway EcN in non-human primates led to increased levels of trans-cinnamate and hippuric acid, both evidence for Phe degradation by the SltA pathway (Isabella et al., 2018). While the study indirectly attempted to deal with genome stability by introducing multiple copies pheP and sltA into EcN, little was done to directly assess genome stability or to understand how EcN colonizes and maintains population density in the intestinal tract. In this issue of Cell Host & Microbe, Crook et al. demonstrate up to 50% reduction in serum Phe levels using various engineered EcN strains in the mouse model of PKU. This study also finds that their engineered EcN chassis, which encodes Phe ammonia lyase activity from Arabidopsis thaliana, was stable in vivo (Crook et al., 2019). Crook et al. also investigated how transit through the gut, microbiota complexity, antibiotic pressure, and engineered genetics affect EcN during longterm colonization of the murine intestinal tract (Crook et al., 2019). This study specifically examined whole-genome sequences of EcN in vivo adapted isolates containing metagenomic libraries to select for genetic elements that enhance degradation of complex polysaccharides, a role that is normally provided to E. coli by intestinal anaerobes (Porter and Martens, 2017). The metagenomic libraries were prepared from DNA isolated from
474 Cell Host & Microbe 25, April 10, 2019
the feces of healthy human infants and adult females and introduced into EcN. Interestingly, following long-term colonization in mice, it was found that genes encoding a glycoside hydrolase family 32 (GH32) enzyme and other enzymes involved in carbohydrate utilization were selected for (Crook et al., 2019). Subsequently, it was found that GH32 containing EcN was capable of cross-feeding EcN lacking GH32 for growth on longchain polysaccharides. These findings indicated that the primary selective pressure for EcN is carbon source limitation, but this can be overcome by other taxa presumably encoding GH32 enzymatic activity. Next, Crook et al. looked at withingenome changes that occur in the EcN chromosome during colonization of the intestine with varying levels of microbiota complexity under three different dietary conditions. The major change that occurred in the EcN chromosome was mutation of genes encoding gluconate utilization, specifically gntT, which encodes a gluconate transporter. EcN strains with defects in gluconate utilization demonstrated superior growth on mucin as compared to wild-type EcN. This finding indicates that inhibition of gluconate utilization is advantageous when mucin is the primary carbon source. The studies by Isabella et al. (2018) and Crook et al. (2019) both demonstrate the potential for a live biotherapeutic to treat the genetic disorder PKU. Isabella and colleagues brought a significant conceptual advance by developing the two pathways for Phe degradation with optimization for therapeutic dosing and translating their engineered EcN strain for clinical trials (Isabella et al., 2018). On the other hand, Crook and colleagues brought a significant advance by elucidating selective pressures that EcN is under during intestinal colonization under a variety of microbiomes and dietary conditions, which is apropos given the diverse healthy and dysbiotic microbiomes that such engineered EcN chassis would likely be used.
Given that, the work by Isabella et al. (2018) and Crook et al. (2019) represent powerful synthetic biology proof-ofconcept studies that deliver on the promising notion that manipulation of the gut microbiome can treat metabolic disorders. REFERENCES Crook, N., Ferreiro, A., Gasparrini, A.J., Pesesky, M.W., Gibson, M.K., Wang, B., Sun, X., Condiotte, Z., Dobrowolski, S., Peterson, D., and Dantas, G. (2019). Adaptive strategies of the candidate probiotic E. coli Nissle in the mammalian gut. Cell Host Microbe 25, this issue, 499–512. Dodd, D., Spitzer, M.H., Van Treuren, W., Merrill, B.D., Hryckowian, A.J., Higginbottom, S.K., Le, A., Cowan, T.M., Nolan, G.P., Fischbach, M.A., and Sonnenburg, J.L. (2017). A gut bacterial pathway metabolizes aromatic amino acids into nine circulating metabolites. Nature 551, 648–652. Griffiths, J.A., and Mazmanian, S.K. (2018). Emerging evidence linking the gut microbiome to neurologic disorders. Genome Med. 10, 98. Isabella, V.M., Ha, B.N., Castillo, M.J., Lubkowicz, D.J., Rowe, S.E., Millet, Y.A., Anderson, C.L., Li, N., Fisher, A.B., West, K.A., et al. (2018). Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria. Nat. Biotechnol. 36, 857–864. Jin, C., Lagoudas, G.K., Zhao, C., Bullman, S., Bhutkar, A., Hu, B., Ameh, S., Sandel, D., Liang, X.S., Mazzilli, S., et al. (2019). Commensal microbiota promote lung cancer development via gd T cells. Cell 176, 998–1013.e16. Porter, N.T., and Martens, E.C. (2017). The critical roles of polysaccharides in gut microbial ecology and physiology. Annu. Rev. Microbiol. 71, 349–369. Ruder, W.C., Lu, T., and Collins, J.J. (2011). Synthetic biology moving into the clinic. Science 333, 1248–1252. Sampson, T.R., Debelius, J.W., Thron, T., Janssen, S., Shastri, G.G., Ilhan, Z.E., Challis, C., Schretter, C.E., Rocha, S., Gradinaru, V., et al. (2016). Gut microbiota regulate motor deficits and neuroinflammation in a model of Parkinson’s disease. Cell 167, 1469–1480.e12. Sonnenborn, U. (2016). Escherichia coli strain Nissle 1917-from bench to bedside and back: history of a special Escherichia coli strain with probiotic properties. FEMS Microbiol. Lett. 363, fnw212. Turnbaugh, P.J., Ley, R.E., Mahowald, M.A., Magrini, V., Mardis, E.R., and Gordon, J.I. (2006). An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444, 1027–1031.