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Cyclic Nucleotide Signaling: A Second Messenger of Death
Cell Host & Microbe
Previews Cyclic Nucleotide Signaling: A Second Messenger of Death Nina Molin Høyland-Kroghsbo1,* 1Department of Veterinary and Animal Sciences, University of Copenhagen, DK-1870 Frederiksberg, Denmark *Correspondence: [email protected] https://doi.org/10.1016/j.chom.2019.10.017
All life forms rely on defenses to fight off viruses that prey on them. Intriguingly, bacteria often develop the blueprints for these. Recently published in Nature, Cohen et al. (2019) uncover a widespread bacterial anti-viral defense system, which may be the evolutionary precursor to the metazoan cGAS-STING immune pathway. Second messengers are signaling molecules that regulate cellular behaviors across all domains of life. In bacteria, cyclic nucleotide second messengers regulate bacterial activities including virulence, biofilm formation, morphogenesis, and motility. The cyclic di-nucleotide second messenger cyclic GMP-AMP (cGAMP) was discovered in the current pandemic El Tor biotype of Vibrio cholerae, where it is synthesized by DncV (Davies et al., 2012). cGAMP activates the phospholipase CapV, which in turn degrades the V. cholerae cell membrane causing growth inhibition (Severin et al., 2018). Recently, DncV homologs were shown to be widespread across diverse bacterial phyla. They synthesize a broad range of cyclic di- and tri-nucleotides, suggesting diverse regulatory roles (Whiteley et al., 2019). Recently published in Nature, Cohen, Melamed et al. uncover a new role for bacterial cGAMP signaling, as a messenger of bacteriophage (phage) virus infection (Cohen et al., 2019). Finding that dncV homologs are often located within ‘‘defense islands,’’ encoding known anti-phage defense systems such as the CRISPR-Cas adaptive immune system and restriction-modification systems, Cohen, Melamed et al. investigated whether DncV (denoted as bacterial cGAS in the Nature article) plays a role in protection against phages. They cloned the four gene capV-dncV operon that includes two additional conserved genes, from V. cholerae and a homologous Escherichia coli system into an E. coli strain lacking the system. DncV and the phospholipase CapV conferred protection against select double-stranded DNA phages, serving as a minimal anti-phage
defense. Protection against other phages required one or both additional genes in the operon, encoding an E1/E2 domain protein with homology to metazoan ubiquitin transfer and ligase enzymes and a JAB deubiquitinase. The authors call this anti-phage defense system CBASS, for cyclic-oligonucleotide-based anti-phage signaling system. In metazoans, the DncV homolog cGAS patrols the cytosol for double-stranded DNA, a sign of infection. cGAS sensing of double-stranded DNA outside the nucleus triggers cGAMP synthesis and cGAMP activates the STING inflammatory pathway (reviewed in Xia et al., 2016). Likewise, Cohen, Melamed et al. showed that approximately halfway through the phage lytic cycle, cGAMP was synthesized and the phospholipase activity of CapV increased. As the membrane-degrading CapV appeared to be the central effector of the CBASS antiphage defense, Cohen, Melamed et al. hypothesized that CBASS could rely on a mechanism called abortive infection in which altruistic suicide eliminates the infected cell prior to phage-mediated killing, thereby sparing the bacterial population from infection by phage progeny (Figure 1) (reviewed in Dy et al., 2014). Indeed, when a small fraction of bacteria was infected by phage, CBASS eliminated the infected cells, minimizing phage spread. By contrast, when the majority of the population was infected, CBASS induced massive suicide prior to phage completion of its replication cycle, a hallmark feature of abortive infection. Interestingly, the CBASS system did not appear to protect against plasmid DNA. Thus, how phage-dependent activation of DncV occurs remains to be
defined. Potentially, the specificity of CBASS to phage DNA could allow cells to avoid autoimmunity. The timing of DncV activation suggests that CBASS possibly detects particular phage DNA replication or packaging intermediates. If so, CBASS would not be activated in a bacterial lysogen where the phage genome is stably integrated in the bacterial chromosome but could lead to abortive infection in response to prophage induction. Cohen, Melamed et al. expanded their analysis to include the dncV homologs recently discovered in 10% of sequenced bacterial genomes (Whiteley et al., 2019). In agreement with previous findings (Burroughs et al., 2015), some of the effector genes contained domains known from other phage defense mechanisms, endonuclease or transmembrane helical domains, suggesting that these systems comprise widespread CBASS defenses. A predicted CBASS system of the latter type was confirmed to confer phage resistance. Remarkably, in a class of two-gene CBASS systems, the predicted effector gene contained an N-terminal TIR domain and a C-terminal STING domain, an arrangement with striking similarity to the simple cGAS-STING system found in the oyster Crassostrea gigas and the annelid worm Capitella teleta. Whether the CBASS TIR/STING domain enzymes induce abortive infection, or if they serve other immune functions, remains to be discovered. Cohen, Melamed et al. propose that these STING-based CBASS systems could represent the ancient evolutionary origin of the metazoan cGAS-STING immune pathway. In metazoans, STING activation leads to release of type I interferon, which alerts
Cell Host & Microbe 26, November 13, 2019 ª 2019 Elsevier Inc. 567
Cell Host & Microbe
Previews
Figure 1. The CBASS Anti-phage Defense System A phage infects a bacterium. The di-nucleotide cyclase DncV senses the infection and synthesizes the second messenger cGAMP, which in turn activates the phospholipase CapV. CapV degrades the bacterial membrane, causing altruistic suicide prior to succumbing by the phage infection. This mechanism, which serves to prevent the spread of phage progeny to neighboring cells, is known as abortive infection.
the surrounding tissues of infection (Xia et al., 2016), raising the question of whether CBASS-induced second messengers can transmit the information regarding infection to neighboring bacteria, and if so, whether stimulation of other phage defense mechanisms occurs. It is known that cyclic oligoadenylate second messengers activate type III CRISPRCas phage defenses (Kazlauskiene et al., 2017; Niewoehner et al., 2017). Perhaps a subset of the second messengers synthesized by DncV homologs enhance type III CRISPR-Cas activity. Ring nucleases keep type III CRISPR-Cas activity in check by degrading cyclic oligoadenylate (Athukoralage et al., 2018). It would be interesting if phages hijacked ring nu-
cleases to counteract CBASS, possibly explaining why CBASS does not confer universal protection against phage. As Cohen, Melamed et al. point out, the roles of the CBASS accessory E1/E2 ubiquitin transfer/ligase and JAB domain proteins remain to be solved. One hypothesis is that they function as a defense against phage-encoded anti-CBASS systems. The findings by Cohen, Melamed et al. highlight the importance of cyclic dinucleotide second messengers in regulating bacterial responses to environmental challenges, now including phage attack. The discovery of widespread CBASS abortive infection as a mechanism of phage defense offers the opportunity to turn these suicidal systems against harmful bacteria that harbor them. Specifically, development of targeted CBASS activators to kill CBASS-positive pathogens could be pursued, or alternatively, synthetic CBASS systems could be delivered via phages or other nano-vehicles as therapies. ACKNOWLEDGMENTS The author was supported by the Lundbeck Foundation (Grant R264-2017-3936). The author thanks Hanne Ingmer and Bonnie L. Bassler for helpful comments.
REFERENCES Athukoralage, J.S., Rouillon, C., Graham, S., €schow, S., and White, M.F. (2018). Ring nucleGru ases deactivate type III CRISPR ribonucleases by degrading cyclic oligoadenylate. Nature 562, 277–280.
568 Cell Host & Microbe 26, November 13, 2019
€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. Cohen, D., Melamed, S., Millman, A., Shulman, G., Oppenheimer-Shaanan, Y., Kacen, A., Doron, S., Amitai, G., and Sorek, R. (2019). Cyclic GMP-AMP signalling protects bacteria against viral infection. Nature. Published online September 18, 2019. https://doi.org/10.1038/s41586-019-1605-5. 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. Dy, R.L., Richter, C., Salmond, G.P., and Fineran, P.C. (2014). Remarkable mechanisms in microbes to resist phage infections. Annu. Rev. Virol. 1, 307–331. Kazlauskiene, M., Kostiuk, G., Venclovas, C., Tamulaitis, G., and Siksnys, V. (2017). A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems. Science 357, 605–609. Niewoehner, O., Garcia-Doval, C., Rostøl, J.T., Berk, C., Schwede, F., Bigler, L., Hall, J., Marraffini, L.A., and Jinek, M. (2017). Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers. Nature 548, 543–548. 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. Xia, P., Wang, S., Gao, P., Gao, G., and Fan, Z. (2016). DNA sensor cGAS-mediated immune recognition. Protein Cell 7, 777–791.
Previews Cyclic Nucleotide Signaling: A Second Messenger of Death Nina Molin Høyland-Kroghsbo1,* 1Department of Veterinary and Animal Sciences, University of Copenhagen, DK-1870 Frederiksberg, Denmark *Correspondence: [email protected] https://doi.org/10.1016/j.chom.2019.10.017
All life forms rely on defenses to fight off viruses that prey on them. Intriguingly, bacteria often develop the blueprints for these. Recently published in Nature, Cohen et al. (2019) uncover a widespread bacterial anti-viral defense system, which may be the evolutionary precursor to the metazoan cGAS-STING immune pathway. Second messengers are signaling molecules that regulate cellular behaviors across all domains of life. In bacteria, cyclic nucleotide second messengers regulate bacterial activities including virulence, biofilm formation, morphogenesis, and motility. The cyclic di-nucleotide second messenger cyclic GMP-AMP (cGAMP) was discovered in the current pandemic El Tor biotype of Vibrio cholerae, where it is synthesized by DncV (Davies et al., 2012). cGAMP activates the phospholipase CapV, which in turn degrades the V. cholerae cell membrane causing growth inhibition (Severin et al., 2018). Recently, DncV homologs were shown to be widespread across diverse bacterial phyla. They synthesize a broad range of cyclic di- and tri-nucleotides, suggesting diverse regulatory roles (Whiteley et al., 2019). Recently published in Nature, Cohen, Melamed et al. uncover a new role for bacterial cGAMP signaling, as a messenger of bacteriophage (phage) virus infection (Cohen et al., 2019). Finding that dncV homologs are often located within ‘‘defense islands,’’ encoding known anti-phage defense systems such as the CRISPR-Cas adaptive immune system and restriction-modification systems, Cohen, Melamed et al. investigated whether DncV (denoted as bacterial cGAS in the Nature article) plays a role in protection against phages. They cloned the four gene capV-dncV operon that includes two additional conserved genes, from V. cholerae and a homologous Escherichia coli system into an E. coli strain lacking the system. DncV and the phospholipase CapV conferred protection against select double-stranded DNA phages, serving as a minimal anti-phage
defense. Protection against other phages required one or both additional genes in the operon, encoding an E1/E2 domain protein with homology to metazoan ubiquitin transfer and ligase enzymes and a JAB deubiquitinase. The authors call this anti-phage defense system CBASS, for cyclic-oligonucleotide-based anti-phage signaling system. In metazoans, the DncV homolog cGAS patrols the cytosol for double-stranded DNA, a sign of infection. cGAS sensing of double-stranded DNA outside the nucleus triggers cGAMP synthesis and cGAMP activates the STING inflammatory pathway (reviewed in Xia et al., 2016). Likewise, Cohen, Melamed et al. showed that approximately halfway through the phage lytic cycle, cGAMP was synthesized and the phospholipase activity of CapV increased. As the membrane-degrading CapV appeared to be the central effector of the CBASS antiphage defense, Cohen, Melamed et al. hypothesized that CBASS could rely on a mechanism called abortive infection in which altruistic suicide eliminates the infected cell prior to phage-mediated killing, thereby sparing the bacterial population from infection by phage progeny (Figure 1) (reviewed in Dy et al., 2014). Indeed, when a small fraction of bacteria was infected by phage, CBASS eliminated the infected cells, minimizing phage spread. By contrast, when the majority of the population was infected, CBASS induced massive suicide prior to phage completion of its replication cycle, a hallmark feature of abortive infection. Interestingly, the CBASS system did not appear to protect against plasmid DNA. Thus, how phage-dependent activation of DncV occurs remains to be
defined. Potentially, the specificity of CBASS to phage DNA could allow cells to avoid autoimmunity. The timing of DncV activation suggests that CBASS possibly detects particular phage DNA replication or packaging intermediates. If so, CBASS would not be activated in a bacterial lysogen where the phage genome is stably integrated in the bacterial chromosome but could lead to abortive infection in response to prophage induction. Cohen, Melamed et al. expanded their analysis to include the dncV homologs recently discovered in 10% of sequenced bacterial genomes (Whiteley et al., 2019). In agreement with previous findings (Burroughs et al., 2015), some of the effector genes contained domains known from other phage defense mechanisms, endonuclease or transmembrane helical domains, suggesting that these systems comprise widespread CBASS defenses. A predicted CBASS system of the latter type was confirmed to confer phage resistance. Remarkably, in a class of two-gene CBASS systems, the predicted effector gene contained an N-terminal TIR domain and a C-terminal STING domain, an arrangement with striking similarity to the simple cGAS-STING system found in the oyster Crassostrea gigas and the annelid worm Capitella teleta. Whether the CBASS TIR/STING domain enzymes induce abortive infection, or if they serve other immune functions, remains to be discovered. Cohen, Melamed et al. propose that these STING-based CBASS systems could represent the ancient evolutionary origin of the metazoan cGAS-STING immune pathway. In metazoans, STING activation leads to release of type I interferon, which alerts
Cell Host & Microbe 26, November 13, 2019 ª 2019 Elsevier Inc. 567
Cell Host & Microbe
Previews
Figure 1. The CBASS Anti-phage Defense System A phage infects a bacterium. The di-nucleotide cyclase DncV senses the infection and synthesizes the second messenger cGAMP, which in turn activates the phospholipase CapV. CapV degrades the bacterial membrane, causing altruistic suicide prior to succumbing by the phage infection. This mechanism, which serves to prevent the spread of phage progeny to neighboring cells, is known as abortive infection.
the surrounding tissues of infection (Xia et al., 2016), raising the question of whether CBASS-induced second messengers can transmit the information regarding infection to neighboring bacteria, and if so, whether stimulation of other phage defense mechanisms occurs. It is known that cyclic oligoadenylate second messengers activate type III CRISPRCas phage defenses (Kazlauskiene et al., 2017; Niewoehner et al., 2017). Perhaps a subset of the second messengers synthesized by DncV homologs enhance type III CRISPR-Cas activity. Ring nucleases keep type III CRISPR-Cas activity in check by degrading cyclic oligoadenylate (Athukoralage et al., 2018). It would be interesting if phages hijacked ring nu-
cleases to counteract CBASS, possibly explaining why CBASS does not confer universal protection against phage. As Cohen, Melamed et al. point out, the roles of the CBASS accessory E1/E2 ubiquitin transfer/ligase and JAB domain proteins remain to be solved. One hypothesis is that they function as a defense against phage-encoded anti-CBASS systems. The findings by Cohen, Melamed et al. highlight the importance of cyclic dinucleotide second messengers in regulating bacterial responses to environmental challenges, now including phage attack. The discovery of widespread CBASS abortive infection as a mechanism of phage defense offers the opportunity to turn these suicidal systems against harmful bacteria that harbor them. Specifically, development of targeted CBASS activators to kill CBASS-positive pathogens could be pursued, or alternatively, synthetic CBASS systems could be delivered via phages or other nano-vehicles as therapies. ACKNOWLEDGMENTS The author was supported by the Lundbeck Foundation (Grant R264-2017-3936). The author thanks Hanne Ingmer and Bonnie L. Bassler for helpful comments.
REFERENCES Athukoralage, J.S., Rouillon, C., Graham, S., €schow, S., and White, M.F. (2018). Ring nucleGru ases deactivate type III CRISPR ribonucleases by degrading cyclic oligoadenylate. Nature 562, 277–280.
568 Cell Host & Microbe 26, November 13, 2019
€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. Cohen, D., Melamed, S., Millman, A., Shulman, G., Oppenheimer-Shaanan, Y., Kacen, A., Doron, S., Amitai, G., and Sorek, R. (2019). Cyclic GMP-AMP signalling protects bacteria against viral infection. Nature. Published online September 18, 2019. https://doi.org/10.1038/s41586-019-1605-5. 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. Dy, R.L., Richter, C., Salmond, G.P., and Fineran, P.C. (2014). Remarkable mechanisms in microbes to resist phage infections. Annu. Rev. Virol. 1, 307–331. Kazlauskiene, M., Kostiuk, G., Venclovas, C., Tamulaitis, G., and Siksnys, V. (2017). A cyclic oligonucleotide signaling pathway in type III CRISPR-Cas systems. Science 357, 605–609. Niewoehner, O., Garcia-Doval, C., Rostøl, J.T., Berk, C., Schwede, F., Bigler, L., Hall, J., Marraffini, L.A., and Jinek, M. (2017). Type III CRISPR-Cas systems produce cyclic oligoadenylate second messengers. Nature 548, 543–548. 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. Xia, P., Wang, S., Gao, P., Gao, G., and Fan, Z. (2016). DNA sensor cGAS-mediated immune recognition. Protein Cell 7, 777–791.