- Email: [email protected]
A Single Transcription Factor Drives Toxoplasma gondii Differentiation
Hughes, C.E., Coody, T.K., Jeong, M.-Y., Berg, J.A., Winge, D.R., and Hughes, A.L. (2020). Cysteine Toxicity Drives Age-Related Mitochondrial Decline by Altering Iron Homeostasis. Cell 180, this issue, 296–310. Kumar, N., Leonzino, M., Hancock-Cerutti, W., Horenkamp, F.A., Li, P., Lees, J.A., Wheeler, H., Reinisch, K.M., and De Camilli, P. (2018). VPS13A and VPS13C are lipid transport proteins differen-
tially localized at ER contact sites. J. Cell Biol. 217, 3625–3639. Pagliarini, D.J., and Rutter, J. (2013). Hallmarks of a new era in mitochondrial biochemistry. Genes Dev. 27, 2615–2627. Perera, R.M., and Zoncu, R. (2016). The Lysosome as a Regulatory Hub. Annu. Rev. Cell Dev. Biol. 32, 223–253.
Sun, N., Youle, R.J., and Finkel, T. (2016). The Mitochondrial Basis of Aging. Mol. Cell 61, 654–666. Yambire, K.F., Rostosky, C., Watanabe, T., Pacheu-Grau, D., Torres-Odio, S., Sanchez-Guerrero, A., Senderovich, O., Meyron-Holtz, E.G., Milosevic, I., Frahm, J., et al. (2019). Impaired lysosomal acidification triggers iron deficiency and inflammation in vivo. eLife 8, 8.
A Single Transcription Factor Drives Toxoplasma gondii Differentiation Joshua A. Kochanowsky1 and Anita A. Koshy1,2,3,* 1Department
of Immunobiology, University of Arizona, Tucson, Arizona, USA of Neurology, University of Arizona, Tucson, Arizona, USA 3Bio5 Institute, University of Arizona, Tucson, Arizona, USA *Correspondence: [email protected] https://doi.org/10.1016/j.cell.2019.12.038 2Department
Microbes that cause persistent infections (e.g., herpes viruses) do so by switching from fast-growing lytic states to slow-growing latent states. Waldman et al. have identified a single transcription factor that governs the switch between the lytic and latent forms of Toxoplasma gondii, a parasite that causes a persistent brain infection. To establish lifelong colonization of their host, persistent pathogens often undergo dramatic changes to alter their metabolic state, rate of replication, and immunogenicity. Toxoplasma gondii is an obligate intracellular protozoan parasite that establishes a lifelong infection in the brain and skeletal and cardiac muscle of many mammals, including humans (Remington and Cavanaugh, 1965). This lifelong persistence allows T. gondii to reactivate and cause life-threatening disease in those with acquired immune deficiencies (Porter and Sande, 1992). To persist, T. gondii switches from its fast-replicating form, the tachyzoite, to its slow-replicating and encysted form, the bradyzoite. In vivo, bradyzoite-filled cysts are primarily found in neurons and myocytes, consistent with in vitro data suggesting that T. gondii may have higher rates of spontaneous differentiation and encystment within these cell types (Ferreira da Silva et al., 2008, 2009; Lu¨der et al., 1999; Swierzy and Lu¨der, 2015). Differen-
tiation in other cell types, such as fibroblasts, can be induced in vitro by exogenous stressors such as alkaline pH, heat shock, small molecules (Compound 1), and nutrient starvation, suggesting that the tachyzoite-to-bradyzoite transition may be a general stress response (Lu¨der and Rahman, 2017) (Figure 1A). In this issue of Cell, Waldman et al. (2020) upend the current model of differentiation by identifying a single transcription factor that governs the switch between tachyzoites and bradyzoites (Figure 1B). The current model of differentiation is built upon prior work that defined a family of parasite transcription factors (ApiAP2s) that modulate differentiation. Some of the ApiAP2s (AP2IV-3) induce the expression of bradyzoite-associated genes while others (AP2IV-4 and AP2IX-9) act as repressors of bradyzoite gene expression (Hong et al., 2017; Radke et al., 2018). However, no single ApiAP2 transcription factor is capable of completely ablating differentiation, leading to a model in-
216 Cell 180, January 23, 2020 ª 2019 Elsevier Inc.
volving many regulators that influence the tachyzoite-bradyzoite switch but no single master regulator. In contrast to this model, Waldman et al. (2020) identify a single gene, bradyzoite-formation deficient 1 (BFD1), that is both necessary and sufficient for differentiation in vitro. To find this master regulator, Waldman and colleagues utilized alkaline pHinduced differentiation in conjunction with a CRISPR/Cas9 screen targeting genes with nucleic acid or DNA binding domains and parasites expressing a stage-specific bradyzoite fluorescent reporter. Parasites that lack BFD1 fail to encyst under several stress conditions, and transgenic parasites that constitutively express a stabilized BFD1 protein show high levels of spontaneous differentiation in unstressed conditions. Consistent with BFD1 being a transcription factor that regulates bradyzoite differentiation, BFD1 binds to the promoter regions of many bradyzoite-specific genes as well as to the AP2IX-9 promoter (Figure 1B).
Figure 1. A Single Transcription Factor Regulates T. gondii Bradyzoite Differentiation In response to a wide variety of host cell stressors, fast-replicating tachyzoites convert to slow-growing bradyzoites. (A) The tachyzoite-to-bradyzoite transition is characterized by morphological changes of the parasite that include narrowing of the parasite body and movement of the nucleus toward the parasite posterior. Additionally, a cyst wall (black circle) composed of parasite proteins is formed underneath the parasitophorous vacuole. (B) Waldman et al. (2020) identified a single transcription factor, bradyzoite-formation deficient 1 (BFD1), which governs this tachyzoite-bradyzoite switch. BFD1 mRNA levels are equivalent in both parasite stages, but BFD1 protein is only observed under stress conditions. BFD1 binds to the promoter of bradyzoite-specific genes, and depletion of BFD1 ablates the ability of parasites to differentiate to bradyzoites.
The identification of BFD1 as a master regulator of bradyzoite differentiation has several important implications. For instance, it will enable the elucidation of a core bradyzoite transcriptional program. Such potential is highlighted by the Waldman et al. (2020) finding that many T. gondii genes upregulated under alkaline stress are not influenced by BFD1 expression and, thus, may not be specific to the bradyzoite stage but rather a byproduct of the response to alkaline stress. The identification of BFD1 may also have implications for the treatment and control of T. gondii, a microbe for which there is no human vaccine. The inability of BFD1-deficient parasites to form cysts makes it an intriguing candidate for a live attenuated vaccine because, in theory, it should generate immune memory but be unable to chronically persist in the vaccinated host. Despite these exciting implications, many questions remain. Though Waldman et al. (2020) observe that parasites that lack BFD1 fail to produce brain cysts in vivo, it remains unclear whether this lack stems from a failure of parasites to encyst after reaching the brain or from parasites never reaching the brain. The latter possibility would suggest that BFD1 may play a yet-to-be-determined role in parasite dissemination or tachy-
zoite growth in vivo. A role for BFD1 in tachyzoites might explain why BFD1 transcripts are present in tachyzoites, though appreciable levels of the protein are not detected by immunofluorescence. In addition, even with the recognition of the primary role of BFD1 in bradyzoite differentiation, the field still lacks any understanding of how parasites sense host cellular stress and the ensuing specific molecular triggers that initiate differentiation. The observation by Waldman et al. (2020) that BFD1 mRNA levels are constant across different stages, but BFD1 protein levels increase under stress conditions, suggests translational regulation likely plays a role in moving from sensing stress to initiating BFD1-dependent differentiation. Finally, while BFD1 may be the master regulator, how BFD1 and the ApiAP2s work in conjunction to form the overall differentiation program remains unclear. In summary, future work involving BFD1 has the potential to (1) elucidate a core bradyzoite transcriptional program that has remained elusive, (2) aid in identifying the molecular mechanisms that drive differentiation in vivo, and (3) enable the development of an effective human vaccine against T. gondii. Ultimately, the discovery of BFD1 marks an important new era in our understanding of the tachyzoite-bradyzoite transition.
ACKNOWLEDGMENTS A.A.K. is funded by the National Institutes of Health (NS095994 and AI147756).
REFERENCES Ferreira da Silva, Mda.F., Barbosa, H.S., Gross, U., and Lu¨der, C.G. (2008). Stressrelated and spontaneous stage differentiation of Toxoplasma gondii. Mol. Biosyst. 4, 824–834. Ferreira-da-Silva, Mda.F., Taka´cs, A.C., Barbosa, H.S., Gross, U., and Lu¨der, C.G. (2009). Primary skeletal muscle cells trigger spontaneous Toxoplasma gondii tachyzoite-to-bradyzoite conversion at higher rates than fibroblasts. Int. J. Med. Microbiol. 299, 381–388. Hong, D.P., Radke, J.B., and White, M.W. (2017). Opposing Transcriptional mechanisms regulate Toxoplasma development. mSphere 2, e00347–16. Lu¨der, C.G.K., and Rahman, T. (2017). Impact of the host on Toxoplasma stage differentiation. Microb. Cell 4, 203–211. Lu¨der, C.G., Giraldo-Vela´squez, M., Sendtner, M., and Gross, U. (1999). Toxoplasma gondii in primary rat CNS cells: differential contribution of neurons, astrocytes, and microglial cells for the intracerebral development and stage differentiation. Exp. Parasitol. 93, 23–32. Porter, S.B., and Sande, M.A. (1992). Toxoplasmosis of the central nervous system in the acquired immunodeficiency syndrome. N. Engl. J. Med. 327, 1643–1648. Radke, J.B., Worth, D., Hong, D., Huang, S., Sullivan, W.J., Jr., Wilson, E.H., and White, M.W.
Cell 180, January 23, 2020 217
(2018). Transcriptional repression by ApiAP2 factors is central to chronic toxoplasmosis. PLoS Pathog. 14, e1007035. Remington, J.S., and Cavanaugh, E.N. (1965). Isolation of the encysted form of Toxoplasma gon-
dii from human skeletal muscle and brain. N. Engl. J. Med. 273, 1308–1310.
wards the bradyzoite stage. Cell. Microbiol. 17, 2–17.
Swierzy, I.J., and Lu¨der, C.G.K. (2015). Withdrawal of skeletal muscle cells from cell cycle progression triggers differentiation of Toxoplasma gondii to-
Waldman, B.S., Schwarz, D., Wadsworth, M.H., II, Saeij, J.P., Shalek, A.K., and Lourido, S. (2020). Identification of a master regulator of differentiation in Toxoplasma. Cell 180, this issue, 359–372.
Bacteriophage Prevents Alcoholic Liver Disease lu,1,2 Jing Xue,1,2 and Mirko Trajkovski1,2,* Melis C¸olakog 1Department of Cell Physiology and Metabolism, Centre Me ´ dical Universitaire (CMU), Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland 2Diabetes Center, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland *Correspondence: [email protected] https://doi.org/10.1016/j.cell.2019.12.034
Alcoholic hepatitis is a severe alcohol-associated liver disease with minimal treatment options. A recent study by Duan et al. uncovers that the exotoxin-secreting gut bacterium Enterococcus faecalis is a critical contributor to alcoholic hepatitis. This bacterium can now be eliminated with a bacteriophage, suggesting a new way to treat this life-threatening disease. Like other viruses, bacteriophages infect host cells to reproduce. Some bacteriophages can only reproduce via a lytic life cycle, in which they burst and kill their host cells, bacteria. In a recent study published in the November 21, 2019 issue of Nature, Schnabl and colleagues (Duan et al., 2019) identified a bacteriophage that could be used to treat alcoholic hepatitis by killing a bacterium that seem to be a major, previously underestimated, causal factor for this liver disease. Alcoholic hepatitis is the most severe form of alcohol-related liver disease with up to a 40% mortality rate and has recently emerged as the most common reason for liver transplant in the United States (Rehm et al., 2014) (Lee et al., 2019). To identify a gut microbiota signature linked to alcoholic hepatitis, Duan et al. (2019) compared patients with this disease to subjects with alcohol use disorder or healthy controls, finding an over 2,700-fold increase in the bacterium Enterococcus faecalis in the alcoholic hepatitis group. Thirty percent of the E. faecalis strains had genes encoding an exotoxin called cytolysin. The mortality of the patients correlated with the cytolysin presence in their stool samples; as
many as 89% of the subjects that were cytolysin-positive died within 180 days after admission, mostly owing to liver failure or related complications. To examine the causative effects of the cytolytic E. faecalis on the alcoholic liver disease, the authors turned to mice. Feeding the animals ethanol together with the cytolytic E. faecalis led to higher rates of liver injury, steatosis, inflammation, and shorter survival rates compared to the cytolytic E. faecalis PBS-fed mice or ethanol-fed non-cytolytic E. faecalisgavaged controls. Consistent with these findings, transplanting stool microbiota from human subjects with alcoholic hepatitis that contained the cytolytic E. faecalis to high alcohol-fed germ-free mice (mice without microbiota of their own) led to liver damage and liver cell death. In contrast, germ-free mice colonized with microbiota from cytolysin-negative patients showed no major signs of liver damage, despite the high alcohol feeding. In search of the mechanism by which the cytolytic E. faecalis causes liver damage, the authors tested the effect of cytolysin on primary hepatocytes. Cytolysin led to cell death in a dose-dependant manner; however, incubation of the hepa-
218 Cell 180, January 23, 2020 ª 2019 Elsevier Inc.
tocytes with ethanol together with the cytolysin did not further increase the cell death, suggesting that the direct effect of cytolysin on the hepatocytes is independent of alcohol. Moreover, cytolysin was only detectable in livers of mice chronically fed ethanol together with the cytolytic E. faecalis administration but not in those of non-cytolytic E. faecalisand ethanol-supplemented mice or of mice given cytolytic E. faecalis but without alcohol. These results indicated that alcohol enables the cytolytic E. faecalis to reach the liver by affecting the intestinal permeability. Indeed, there was evidence of ethanol-induced alterations in the gut barrier, which was not affected by gavaging the mice with the cytolytic or with the non-cytolytic E. faecalis. The treatment options for alcoholic hepatitis are limited. In the context of the findings above, the authors decided to uncover specific ways for targeting the cytolytic E. faecalis by using bacteriophages. The advantage of these viral particles over antibiotics is primarily their specificity for a single host bacteria (although some phages have a broad host range) (Kortright et al., 2019) and that some E. faecalis strains exhibit
tially localized at ER contact sites. J. Cell Biol. 217, 3625–3639. Pagliarini, D.J., and Rutter, J. (2013). Hallmarks of a new era in mitochondrial biochemistry. Genes Dev. 27, 2615–2627. Perera, R.M., and Zoncu, R. (2016). The Lysosome as a Regulatory Hub. Annu. Rev. Cell Dev. Biol. 32, 223–253.
Sun, N., Youle, R.J., and Finkel, T. (2016). The Mitochondrial Basis of Aging. Mol. Cell 61, 654–666. Yambire, K.F., Rostosky, C., Watanabe, T., Pacheu-Grau, D., Torres-Odio, S., Sanchez-Guerrero, A., Senderovich, O., Meyron-Holtz, E.G., Milosevic, I., Frahm, J., et al. (2019). Impaired lysosomal acidification triggers iron deficiency and inflammation in vivo. eLife 8, 8.
A Single Transcription Factor Drives Toxoplasma gondii Differentiation Joshua A. Kochanowsky1 and Anita A. Koshy1,2,3,* 1Department
of Immunobiology, University of Arizona, Tucson, Arizona, USA of Neurology, University of Arizona, Tucson, Arizona, USA 3Bio5 Institute, University of Arizona, Tucson, Arizona, USA *Correspondence: [email protected] https://doi.org/10.1016/j.cell.2019.12.038 2Department
Microbes that cause persistent infections (e.g., herpes viruses) do so by switching from fast-growing lytic states to slow-growing latent states. Waldman et al. have identified a single transcription factor that governs the switch between the lytic and latent forms of Toxoplasma gondii, a parasite that causes a persistent brain infection. To establish lifelong colonization of their host, persistent pathogens often undergo dramatic changes to alter their metabolic state, rate of replication, and immunogenicity. Toxoplasma gondii is an obligate intracellular protozoan parasite that establishes a lifelong infection in the brain and skeletal and cardiac muscle of many mammals, including humans (Remington and Cavanaugh, 1965). This lifelong persistence allows T. gondii to reactivate and cause life-threatening disease in those with acquired immune deficiencies (Porter and Sande, 1992). To persist, T. gondii switches from its fast-replicating form, the tachyzoite, to its slow-replicating and encysted form, the bradyzoite. In vivo, bradyzoite-filled cysts are primarily found in neurons and myocytes, consistent with in vitro data suggesting that T. gondii may have higher rates of spontaneous differentiation and encystment within these cell types (Ferreira da Silva et al., 2008, 2009; Lu¨der et al., 1999; Swierzy and Lu¨der, 2015). Differen-
tiation in other cell types, such as fibroblasts, can be induced in vitro by exogenous stressors such as alkaline pH, heat shock, small molecules (Compound 1), and nutrient starvation, suggesting that the tachyzoite-to-bradyzoite transition may be a general stress response (Lu¨der and Rahman, 2017) (Figure 1A). In this issue of Cell, Waldman et al. (2020) upend the current model of differentiation by identifying a single transcription factor that governs the switch between tachyzoites and bradyzoites (Figure 1B). The current model of differentiation is built upon prior work that defined a family of parasite transcription factors (ApiAP2s) that modulate differentiation. Some of the ApiAP2s (AP2IV-3) induce the expression of bradyzoite-associated genes while others (AP2IV-4 and AP2IX-9) act as repressors of bradyzoite gene expression (Hong et al., 2017; Radke et al., 2018). However, no single ApiAP2 transcription factor is capable of completely ablating differentiation, leading to a model in-
216 Cell 180, January 23, 2020 ª 2019 Elsevier Inc.
volving many regulators that influence the tachyzoite-bradyzoite switch but no single master regulator. In contrast to this model, Waldman et al. (2020) identify a single gene, bradyzoite-formation deficient 1 (BFD1), that is both necessary and sufficient for differentiation in vitro. To find this master regulator, Waldman and colleagues utilized alkaline pHinduced differentiation in conjunction with a CRISPR/Cas9 screen targeting genes with nucleic acid or DNA binding domains and parasites expressing a stage-specific bradyzoite fluorescent reporter. Parasites that lack BFD1 fail to encyst under several stress conditions, and transgenic parasites that constitutively express a stabilized BFD1 protein show high levels of spontaneous differentiation in unstressed conditions. Consistent with BFD1 being a transcription factor that regulates bradyzoite differentiation, BFD1 binds to the promoter regions of many bradyzoite-specific genes as well as to the AP2IX-9 promoter (Figure 1B).
Figure 1. A Single Transcription Factor Regulates T. gondii Bradyzoite Differentiation In response to a wide variety of host cell stressors, fast-replicating tachyzoites convert to slow-growing bradyzoites. (A) The tachyzoite-to-bradyzoite transition is characterized by morphological changes of the parasite that include narrowing of the parasite body and movement of the nucleus toward the parasite posterior. Additionally, a cyst wall (black circle) composed of parasite proteins is formed underneath the parasitophorous vacuole. (B) Waldman et al. (2020) identified a single transcription factor, bradyzoite-formation deficient 1 (BFD1), which governs this tachyzoite-bradyzoite switch. BFD1 mRNA levels are equivalent in both parasite stages, but BFD1 protein is only observed under stress conditions. BFD1 binds to the promoter of bradyzoite-specific genes, and depletion of BFD1 ablates the ability of parasites to differentiate to bradyzoites.
The identification of BFD1 as a master regulator of bradyzoite differentiation has several important implications. For instance, it will enable the elucidation of a core bradyzoite transcriptional program. Such potential is highlighted by the Waldman et al. (2020) finding that many T. gondii genes upregulated under alkaline stress are not influenced by BFD1 expression and, thus, may not be specific to the bradyzoite stage but rather a byproduct of the response to alkaline stress. The identification of BFD1 may also have implications for the treatment and control of T. gondii, a microbe for which there is no human vaccine. The inability of BFD1-deficient parasites to form cysts makes it an intriguing candidate for a live attenuated vaccine because, in theory, it should generate immune memory but be unable to chronically persist in the vaccinated host. Despite these exciting implications, many questions remain. Though Waldman et al. (2020) observe that parasites that lack BFD1 fail to produce brain cysts in vivo, it remains unclear whether this lack stems from a failure of parasites to encyst after reaching the brain or from parasites never reaching the brain. The latter possibility would suggest that BFD1 may play a yet-to-be-determined role in parasite dissemination or tachy-
zoite growth in vivo. A role for BFD1 in tachyzoites might explain why BFD1 transcripts are present in tachyzoites, though appreciable levels of the protein are not detected by immunofluorescence. In addition, even with the recognition of the primary role of BFD1 in bradyzoite differentiation, the field still lacks any understanding of how parasites sense host cellular stress and the ensuing specific molecular triggers that initiate differentiation. The observation by Waldman et al. (2020) that BFD1 mRNA levels are constant across different stages, but BFD1 protein levels increase under stress conditions, suggests translational regulation likely plays a role in moving from sensing stress to initiating BFD1-dependent differentiation. Finally, while BFD1 may be the master regulator, how BFD1 and the ApiAP2s work in conjunction to form the overall differentiation program remains unclear. In summary, future work involving BFD1 has the potential to (1) elucidate a core bradyzoite transcriptional program that has remained elusive, (2) aid in identifying the molecular mechanisms that drive differentiation in vivo, and (3) enable the development of an effective human vaccine against T. gondii. Ultimately, the discovery of BFD1 marks an important new era in our understanding of the tachyzoite-bradyzoite transition.
ACKNOWLEDGMENTS A.A.K. is funded by the National Institutes of Health (NS095994 and AI147756).
REFERENCES Ferreira da Silva, Mda.F., Barbosa, H.S., Gross, U., and Lu¨der, C.G. (2008). Stressrelated and spontaneous stage differentiation of Toxoplasma gondii. Mol. Biosyst. 4, 824–834. Ferreira-da-Silva, Mda.F., Taka´cs, A.C., Barbosa, H.S., Gross, U., and Lu¨der, C.G. (2009). Primary skeletal muscle cells trigger spontaneous Toxoplasma gondii tachyzoite-to-bradyzoite conversion at higher rates than fibroblasts. Int. J. Med. Microbiol. 299, 381–388. Hong, D.P., Radke, J.B., and White, M.W. (2017). Opposing Transcriptional mechanisms regulate Toxoplasma development. mSphere 2, e00347–16. Lu¨der, C.G.K., and Rahman, T. (2017). Impact of the host on Toxoplasma stage differentiation. Microb. Cell 4, 203–211. Lu¨der, C.G., Giraldo-Vela´squez, M., Sendtner, M., and Gross, U. (1999). Toxoplasma gondii in primary rat CNS cells: differential contribution of neurons, astrocytes, and microglial cells for the intracerebral development and stage differentiation. Exp. Parasitol. 93, 23–32. Porter, S.B., and Sande, M.A. (1992). Toxoplasmosis of the central nervous system in the acquired immunodeficiency syndrome. N. Engl. J. Med. 327, 1643–1648. Radke, J.B., Worth, D., Hong, D., Huang, S., Sullivan, W.J., Jr., Wilson, E.H., and White, M.W.
Cell 180, January 23, 2020 217
(2018). Transcriptional repression by ApiAP2 factors is central to chronic toxoplasmosis. PLoS Pathog. 14, e1007035. Remington, J.S., and Cavanaugh, E.N. (1965). Isolation of the encysted form of Toxoplasma gon-
dii from human skeletal muscle and brain. N. Engl. J. Med. 273, 1308–1310.
wards the bradyzoite stage. Cell. Microbiol. 17, 2–17.
Swierzy, I.J., and Lu¨der, C.G.K. (2015). Withdrawal of skeletal muscle cells from cell cycle progression triggers differentiation of Toxoplasma gondii to-
Waldman, B.S., Schwarz, D., Wadsworth, M.H., II, Saeij, J.P., Shalek, A.K., and Lourido, S. (2020). Identification of a master regulator of differentiation in Toxoplasma. Cell 180, this issue, 359–372.
Bacteriophage Prevents Alcoholic Liver Disease lu,1,2 Jing Xue,1,2 and Mirko Trajkovski1,2,* Melis C¸olakog 1Department of Cell Physiology and Metabolism, Centre Me ´ dical Universitaire (CMU), Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland 2Diabetes Center, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland *Correspondence: [email protected] https://doi.org/10.1016/j.cell.2019.12.034
Alcoholic hepatitis is a severe alcohol-associated liver disease with minimal treatment options. A recent study by Duan et al. uncovers that the exotoxin-secreting gut bacterium Enterococcus faecalis is a critical contributor to alcoholic hepatitis. This bacterium can now be eliminated with a bacteriophage, suggesting a new way to treat this life-threatening disease. Like other viruses, bacteriophages infect host cells to reproduce. Some bacteriophages can only reproduce via a lytic life cycle, in which they burst and kill their host cells, bacteria. In a recent study published in the November 21, 2019 issue of Nature, Schnabl and colleagues (Duan et al., 2019) identified a bacteriophage that could be used to treat alcoholic hepatitis by killing a bacterium that seem to be a major, previously underestimated, causal factor for this liver disease. Alcoholic hepatitis is the most severe form of alcohol-related liver disease with up to a 40% mortality rate and has recently emerged as the most common reason for liver transplant in the United States (Rehm et al., 2014) (Lee et al., 2019). To identify a gut microbiota signature linked to alcoholic hepatitis, Duan et al. (2019) compared patients with this disease to subjects with alcohol use disorder or healthy controls, finding an over 2,700-fold increase in the bacterium Enterococcus faecalis in the alcoholic hepatitis group. Thirty percent of the E. faecalis strains had genes encoding an exotoxin called cytolysin. The mortality of the patients correlated with the cytolysin presence in their stool samples; as
many as 89% of the subjects that were cytolysin-positive died within 180 days after admission, mostly owing to liver failure or related complications. To examine the causative effects of the cytolytic E. faecalis on the alcoholic liver disease, the authors turned to mice. Feeding the animals ethanol together with the cytolytic E. faecalis led to higher rates of liver injury, steatosis, inflammation, and shorter survival rates compared to the cytolytic E. faecalis PBS-fed mice or ethanol-fed non-cytolytic E. faecalisgavaged controls. Consistent with these findings, transplanting stool microbiota from human subjects with alcoholic hepatitis that contained the cytolytic E. faecalis to high alcohol-fed germ-free mice (mice without microbiota of their own) led to liver damage and liver cell death. In contrast, germ-free mice colonized with microbiota from cytolysin-negative patients showed no major signs of liver damage, despite the high alcohol feeding. In search of the mechanism by which the cytolytic E. faecalis causes liver damage, the authors tested the effect of cytolysin on primary hepatocytes. Cytolysin led to cell death in a dose-dependant manner; however, incubation of the hepa-
218 Cell 180, January 23, 2020 ª 2019 Elsevier Inc.
tocytes with ethanol together with the cytolysin did not further increase the cell death, suggesting that the direct effect of cytolysin on the hepatocytes is independent of alcohol. Moreover, cytolysin was only detectable in livers of mice chronically fed ethanol together with the cytolytic E. faecalis administration but not in those of non-cytolytic E. faecalisand ethanol-supplemented mice or of mice given cytolytic E. faecalis but without alcohol. These results indicated that alcohol enables the cytolytic E. faecalis to reach the liver by affecting the intestinal permeability. Indeed, there was evidence of ethanol-induced alterations in the gut barrier, which was not affected by gavaging the mice with the cytolytic or with the non-cytolytic E. faecalis. The treatment options for alcoholic hepatitis are limited. In the context of the findings above, the authors decided to uncover specific ways for targeting the cytolytic E. faecalis by using bacteriophages. The advantage of these viral particles over antibiotics is primarily their specificity for a single host bacteria (although some phages have a broad host range) (Kortright et al., 2019) and that some E. faecalis strains exhibit