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Cooperation among Conflict: Prophages Protect Bacteria from Phagocytosis
Cell Host & Microbe
Previews Cooperation among Conflict: Prophages Protect Bacteria from Phagocytosis Brittany A. Leigh1,2,* 1Biological
Sciences, Vanderbilt University, Nashville, TN, USA Microbiome Initiative, Vanderbilt University, Nashville, TN, USA *Correspondence: [email protected] https://doi.org/10.1016/j.chom.2019.09.003 2Vanderbilt
Bacteriophages, viruses that infect bacteria, are the most abundant biological entities within the holobiont. In this issue of Cell Host & Microbe, Jahn et al. (2019) describe a group of phages that can suppress immune cell function in marine sponges using secreted ankyrin proteins. They call these phages Ankyphages. The holobiont is comprised of an animal and all of its associated microbes including bacteria, archaea, fungi, protists, and viruses. The smallest, yet most abundant members of the holobiont are bacteriophages, the viruses that infect bacteria. These tiny predators significantly structure the bacterial community through lysis and have the potential to serve as an arsenal of genetic diversity for colonizing microbes. Phages often maintain one of two life cycles: lytic or lysogenic. Lytic phages are free-living until they encounter their bacterial host, inject their DNA, and produce a number of virions that resultantly lyse the cell to release new phages into the environment and repeat the cycle. Lysogenic phages, on the other hand, integrate their genomes into the chromosome of the bacterial host cell as prophages. Here, they lie dormant until triggered by some environmental factor to excise and enter the lytic cycle. During integration, phages can introduce new genes to the bacterial host that alter their phenotype, a phenomenon known as lysogenic conversion. In healthy animal-associated communities, lysogeny often dominates (Minot et al., 2011). It seems counterintuitive that a bacterium would harbor a prophage, a ticking time bomb, within its genome, but prophages often confer fitness advantages to their bacterial hosts. For example, an E. coli prophage encodes a Shiga toxin that is induced by phage latent gene expression, enhancing the bacteria’s pathogenesis (Plunkett et al., 1999). Additionally, integration of a prophage prevents infection by other phages, both lytic and lysogenic, through superimmunity
exclusion, a phenomenon where an existing viral infection precludes the re-infection with the same or closely related virus. Most research has been conducted on these isolated pathogenic bacteria-phage systems while mutualistic bacteria-phage interactions, which are key in successful establishment of a holobiont, remain enigmatic due, in part, to their limited number in culture. Metagenomics, however, has changed the landscape. The ability to sequence all viral genomes within a community, independent of cultivation, allows for environmental characterization of both bacterial and phage diversity. Metagenomic studies have since uncovered that animals maintain species-specific bacterial and viral communities that harbor a vast diversity of genes necessary for host survival (Laffy et al., 2018). In this issue of Cell Host & Microbe, Jahn et al. (2019) use both viral metagenomics and immunoassays to characterize sponge phage communities in order to determine if phages interact with the animal host. Simple animal systems, such as sponges, provide a powerful tool to dissect the biology of phages in animal-associated communities. In particular, marine invertebrates are hotspots of microbial diversity and often maintain a relatively simple body plan and immune system. Through constant filtration of seawater, sponges are exposed to a continuous influx of bacteria and viruses, yet maintain species-specific signatures for both (Laffy et al., 2018). This tripartite interaction forms a stable holobiont. Interactions between bacteria and eukaryotic hosts as well as bacteria and phages have been relatively well-studied previously, but researchers here assess the
450 Cell Host & Microbe 26, October 9, 2019 ª 2019 Elsevier Inc.
potential for phage interactions with the eukaryotic host. Using metagenomics, purified viruses were sequenced from 32 sponge samples and 4 seawater samples, revealing a plethora of previously undescribed viruses specifically associated with sponges. Not only are sponges a repository of yet unidentified bacteria and their associated compounds, but they also harbor phages (and phage-associated proteins). Here, phages were grouped into four categories: generalists (prevalent in more than one sponge or seawater sample), specialists (prevalent in only one sample), individualists (present in only one individual but in both tissues), and intermediates (does not fall into above groups). Surprisingly, the individualist category was the most represented viral group, highlighting the individual variation even among members of the same species. As has been reported in humans (Moreno-Gallego et al., 2019), phages are often individual specific, providing a ‘‘viral fingerprint’’ that can be differentiated among even the most closely related entities. Bacterial communities that are specifically associated with animals often have factors that enhance this association. Phages seem to be no different. Auxiliary ankyrin repeats (ANKs) were found in 12/ 32 sponge viromes and were completely absent in all seawater samples. ANKs are one of the most abundant classes of protein-protein interactors and are widespread in all domains of life (Jernigan and Bordenstein, 2014). However, reports of ANKs in phages are strikingly rare, with only one previously reported in phage WO of the obligate intracellular bacteria Wolbachia (Wu et al., 2004). The authors
Cell Host & Microbe
Previews
Figure 1. Bacteria and Ankyphage Work Together to Enhance Colonization (A) Colonizing bacteria without an integrated Ankyphage are more often phagocytosed by host macrophages than those bacteria with an integrated Ankyphage. (B) Ankyphage-infected bacterial cells secrete an ankyrin protein (ANKp) that functions to inhibit phagocytosis. These Ankyphages are also present as viral particles, suggesting they have the ability to lyse the bacterial cell in which they reside.
name these sponge-associated phages harboring these ANKs ‘‘Ankyphages,’’ perhaps the best name for any phage group to date. These Ankyphages fall into the intermediate viral group and are in the top 75th percentile of most abundant viruses for some sponges sampled in the study. The identified ankyrin proteins did not maintain a transmembrane domain but instead N-terminal signal peptides that suggest it is secreted out of a bacterial cell infected with an Anky-prophage. Utilizing E. coli expression systems, the secretion of one ankyrin protein (ANKp) was indeed validated. Why then would a bacterium infected with an Anky-prophage secrete this protein that is involved in eukaryotic protein interactions? To functionally evaluate ANKp, Jahn et al. exposed E. coli to murine macrophages pre-incubated with ANKp and measured a higher survivability of the bacteria when ANKp was present. This same pattern also occurred when the E. coli was itself expressing
ANKp. Gentamycin protection and MTS assays determined that increased survivability of the bacteria was a result of decreased phagocytosis by the macrophages and not cytotoxicity of the cells (Figure 1). Additionally, the macrophages exhibited a reduction in the production of pro-inflammatory cytokines in response to ANKp. Lastly, homology searches uncovered Ankyphages in numerous host-associated environments, including human oral cavity, gut, and stomach. Altogether, these results highlight a widely distributed phage-encoded ankyrin protein that can actively suppress host immune cell function. Following the discovery of a phage-encoded immunomodulatory protein, several fundamental questions remain. How are these Ankyphages acquiring these proteins? Phages are masters at gene exchange, but the ANKps may be acquired directly from the eukaryotic host or from the bacterial chromosome as a result of integration. Comparisons
of all homologous ANKps found across all domains of life may provide insight into the modes of this lateral gene transfer. Second, is there a natural trigger for expression of ANKp? Integration of the prophage into the bacterial chromosome can cause expression of phage encoded proteins such as the Shiga toxin in E. coli, but in a stable long-term symbiosis, is secretion of ANKp constantly occurring or only during initial colonization? Lastly, is there host specificity to the ANKps? Microbiomes can be highly distinguishable between species, and phage communities are unique to individuals. What diversity of ANKp, if any, is present within a population, and is their immunomodulatory capacity similar across the animal kingdom? Sponge ANKp suppression of murine macrophages suggests broad potential impacts on an array of animal hosts. Future research into the biology of phage-encoded eukaryotic-interacting proteins will be key to understanding the establishment and maintenance of the holobiont Cell Host & Microbe 26, October 9, 2019 451
Cell Host & Microbe
Previews and finally drawing a line connecting phages and animals in this dynamic tripartite symbiosis. ACKNOWLEDGMENTS B.A.L. is supported by the National Institutes of Health Ruth L. Kirchstein Postdoctoral Individual National Research Service Award 1F32AI140694-01A1.
REFERENCES Jahn, M.T., Arkhipova, K., Market, S.M., Stigloher, C., Lachnit, T., Pita, L., Kupczok, A., Ribes, M., Stengel, S.T., Rosenstiel, P., et al. (2019). A marine sponge symbiont phage protein aids in eukaryote
452 Cell Host & Microbe 26, October 9, 2019
immune evasion. Cell Host Microbe 26, this issue, 542–550. Jernigan, K.K., and Bordenstein, S.R. (2014). Ankyrin domains across the Tree of Life. PeerJ 2, e264. Laffy, P.W., Wood-Charlson, E.M., Turaev, D., Jutz, S., Pascelli, C., Botte´, E.S., Bell, S.C., Peirce, T.E., Weynberg, K.D., van Oppen, M.J.H., et al. (2018). Reef invertebrate viromics: diversity, host specificity and functional capacity. Environ. Microbiol. 20, 2125–2141. Minot, S., Sinha, R., Chen, J., Li, H., Keilbaugh, S.A., Wu, G.D., Lewis, J.D., and Bushman, F.D. (2011). The human gut virome: inter-individual variation and dynamic response to diet. Genome Res. 21, 1616–1625.
Moreno-Gallego, J.L., Chou, S.P., Di Rienzi, S.C., Goodrich, J.K., Spector, T.D., Bell, J.T., Youngblut, N.D., Hewson, I., Reyes, A., and Ley, R.E. (2019). Virome diversity correlates with intestinal microbiome diversity in adult monozygotic twins. Cell Host Microbe 25, 261–272.e5. Plunkett, G., 3rd, Rose, D.J., Durfee, T.J., and Blattner, F.R. (1999). Sequence of Shiga toxin 2 phage 933W from Escherichia coli O157:H7: Shiga toxin as a phage late-gene product. J. Bacteriol. 181, 1767–1778. Wu, M., Sun, L.V., Vamathevan, J., Riegler, M., Deboy, R., Brownlie, J.C., McGraw, E.A., Martin, W., Esser, C., Ahmadinejad, N., et al. (2004). Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements. PLoS Biol. 2, E69.
Previews Cooperation among Conflict: Prophages Protect Bacteria from Phagocytosis Brittany A. Leigh1,2,* 1Biological
Sciences, Vanderbilt University, Nashville, TN, USA Microbiome Initiative, Vanderbilt University, Nashville, TN, USA *Correspondence: [email protected] https://doi.org/10.1016/j.chom.2019.09.003 2Vanderbilt
Bacteriophages, viruses that infect bacteria, are the most abundant biological entities within the holobiont. In this issue of Cell Host & Microbe, Jahn et al. (2019) describe a group of phages that can suppress immune cell function in marine sponges using secreted ankyrin proteins. They call these phages Ankyphages. The holobiont is comprised of an animal and all of its associated microbes including bacteria, archaea, fungi, protists, and viruses. The smallest, yet most abundant members of the holobiont are bacteriophages, the viruses that infect bacteria. These tiny predators significantly structure the bacterial community through lysis and have the potential to serve as an arsenal of genetic diversity for colonizing microbes. Phages often maintain one of two life cycles: lytic or lysogenic. Lytic phages are free-living until they encounter their bacterial host, inject their DNA, and produce a number of virions that resultantly lyse the cell to release new phages into the environment and repeat the cycle. Lysogenic phages, on the other hand, integrate their genomes into the chromosome of the bacterial host cell as prophages. Here, they lie dormant until triggered by some environmental factor to excise and enter the lytic cycle. During integration, phages can introduce new genes to the bacterial host that alter their phenotype, a phenomenon known as lysogenic conversion. In healthy animal-associated communities, lysogeny often dominates (Minot et al., 2011). It seems counterintuitive that a bacterium would harbor a prophage, a ticking time bomb, within its genome, but prophages often confer fitness advantages to their bacterial hosts. For example, an E. coli prophage encodes a Shiga toxin that is induced by phage latent gene expression, enhancing the bacteria’s pathogenesis (Plunkett et al., 1999). Additionally, integration of a prophage prevents infection by other phages, both lytic and lysogenic, through superimmunity
exclusion, a phenomenon where an existing viral infection precludes the re-infection with the same or closely related virus. Most research has been conducted on these isolated pathogenic bacteria-phage systems while mutualistic bacteria-phage interactions, which are key in successful establishment of a holobiont, remain enigmatic due, in part, to their limited number in culture. Metagenomics, however, has changed the landscape. The ability to sequence all viral genomes within a community, independent of cultivation, allows for environmental characterization of both bacterial and phage diversity. Metagenomic studies have since uncovered that animals maintain species-specific bacterial and viral communities that harbor a vast diversity of genes necessary for host survival (Laffy et al., 2018). In this issue of Cell Host & Microbe, Jahn et al. (2019) use both viral metagenomics and immunoassays to characterize sponge phage communities in order to determine if phages interact with the animal host. Simple animal systems, such as sponges, provide a powerful tool to dissect the biology of phages in animal-associated communities. In particular, marine invertebrates are hotspots of microbial diversity and often maintain a relatively simple body plan and immune system. Through constant filtration of seawater, sponges are exposed to a continuous influx of bacteria and viruses, yet maintain species-specific signatures for both (Laffy et al., 2018). This tripartite interaction forms a stable holobiont. Interactions between bacteria and eukaryotic hosts as well as bacteria and phages have been relatively well-studied previously, but researchers here assess the
450 Cell Host & Microbe 26, October 9, 2019 ª 2019 Elsevier Inc.
potential for phage interactions with the eukaryotic host. Using metagenomics, purified viruses were sequenced from 32 sponge samples and 4 seawater samples, revealing a plethora of previously undescribed viruses specifically associated with sponges. Not only are sponges a repository of yet unidentified bacteria and their associated compounds, but they also harbor phages (and phage-associated proteins). Here, phages were grouped into four categories: generalists (prevalent in more than one sponge or seawater sample), specialists (prevalent in only one sample), individualists (present in only one individual but in both tissues), and intermediates (does not fall into above groups). Surprisingly, the individualist category was the most represented viral group, highlighting the individual variation even among members of the same species. As has been reported in humans (Moreno-Gallego et al., 2019), phages are often individual specific, providing a ‘‘viral fingerprint’’ that can be differentiated among even the most closely related entities. Bacterial communities that are specifically associated with animals often have factors that enhance this association. Phages seem to be no different. Auxiliary ankyrin repeats (ANKs) were found in 12/ 32 sponge viromes and were completely absent in all seawater samples. ANKs are one of the most abundant classes of protein-protein interactors and are widespread in all domains of life (Jernigan and Bordenstein, 2014). However, reports of ANKs in phages are strikingly rare, with only one previously reported in phage WO of the obligate intracellular bacteria Wolbachia (Wu et al., 2004). The authors
Cell Host & Microbe
Previews
Figure 1. Bacteria and Ankyphage Work Together to Enhance Colonization (A) Colonizing bacteria without an integrated Ankyphage are more often phagocytosed by host macrophages than those bacteria with an integrated Ankyphage. (B) Ankyphage-infected bacterial cells secrete an ankyrin protein (ANKp) that functions to inhibit phagocytosis. These Ankyphages are also present as viral particles, suggesting they have the ability to lyse the bacterial cell in which they reside.
name these sponge-associated phages harboring these ANKs ‘‘Ankyphages,’’ perhaps the best name for any phage group to date. These Ankyphages fall into the intermediate viral group and are in the top 75th percentile of most abundant viruses for some sponges sampled in the study. The identified ankyrin proteins did not maintain a transmembrane domain but instead N-terminal signal peptides that suggest it is secreted out of a bacterial cell infected with an Anky-prophage. Utilizing E. coli expression systems, the secretion of one ankyrin protein (ANKp) was indeed validated. Why then would a bacterium infected with an Anky-prophage secrete this protein that is involved in eukaryotic protein interactions? To functionally evaluate ANKp, Jahn et al. exposed E. coli to murine macrophages pre-incubated with ANKp and measured a higher survivability of the bacteria when ANKp was present. This same pattern also occurred when the E. coli was itself expressing
ANKp. Gentamycin protection and MTS assays determined that increased survivability of the bacteria was a result of decreased phagocytosis by the macrophages and not cytotoxicity of the cells (Figure 1). Additionally, the macrophages exhibited a reduction in the production of pro-inflammatory cytokines in response to ANKp. Lastly, homology searches uncovered Ankyphages in numerous host-associated environments, including human oral cavity, gut, and stomach. Altogether, these results highlight a widely distributed phage-encoded ankyrin protein that can actively suppress host immune cell function. Following the discovery of a phage-encoded immunomodulatory protein, several fundamental questions remain. How are these Ankyphages acquiring these proteins? Phages are masters at gene exchange, but the ANKps may be acquired directly from the eukaryotic host or from the bacterial chromosome as a result of integration. Comparisons
of all homologous ANKps found across all domains of life may provide insight into the modes of this lateral gene transfer. Second, is there a natural trigger for expression of ANKp? Integration of the prophage into the bacterial chromosome can cause expression of phage encoded proteins such as the Shiga toxin in E. coli, but in a stable long-term symbiosis, is secretion of ANKp constantly occurring or only during initial colonization? Lastly, is there host specificity to the ANKps? Microbiomes can be highly distinguishable between species, and phage communities are unique to individuals. What diversity of ANKp, if any, is present within a population, and is their immunomodulatory capacity similar across the animal kingdom? Sponge ANKp suppression of murine macrophages suggests broad potential impacts on an array of animal hosts. Future research into the biology of phage-encoded eukaryotic-interacting proteins will be key to understanding the establishment and maintenance of the holobiont Cell Host & Microbe 26, October 9, 2019 451
Cell Host & Microbe
Previews and finally drawing a line connecting phages and animals in this dynamic tripartite symbiosis. ACKNOWLEDGMENTS B.A.L. is supported by the National Institutes of Health Ruth L. Kirchstein Postdoctoral Individual National Research Service Award 1F32AI140694-01A1.
REFERENCES Jahn, M.T., Arkhipova, K., Market, S.M., Stigloher, C., Lachnit, T., Pita, L., Kupczok, A., Ribes, M., Stengel, S.T., Rosenstiel, P., et al. (2019). A marine sponge symbiont phage protein aids in eukaryote
452 Cell Host & Microbe 26, October 9, 2019
immune evasion. Cell Host Microbe 26, this issue, 542–550. Jernigan, K.K., and Bordenstein, S.R. (2014). Ankyrin domains across the Tree of Life. PeerJ 2, e264. Laffy, P.W., Wood-Charlson, E.M., Turaev, D., Jutz, S., Pascelli, C., Botte´, E.S., Bell, S.C., Peirce, T.E., Weynberg, K.D., van Oppen, M.J.H., et al. (2018). Reef invertebrate viromics: diversity, host specificity and functional capacity. Environ. Microbiol. 20, 2125–2141. Minot, S., Sinha, R., Chen, J., Li, H., Keilbaugh, S.A., Wu, G.D., Lewis, J.D., and Bushman, F.D. (2011). The human gut virome: inter-individual variation and dynamic response to diet. Genome Res. 21, 1616–1625.
Moreno-Gallego, J.L., Chou, S.P., Di Rienzi, S.C., Goodrich, J.K., Spector, T.D., Bell, J.T., Youngblut, N.D., Hewson, I., Reyes, A., and Ley, R.E. (2019). Virome diversity correlates with intestinal microbiome diversity in adult monozygotic twins. Cell Host Microbe 25, 261–272.e5. Plunkett, G., 3rd, Rose, D.J., Durfee, T.J., and Blattner, F.R. (1999). Sequence of Shiga toxin 2 phage 933W from Escherichia coli O157:H7: Shiga toxin as a phage late-gene product. J. Bacteriol. 181, 1767–1778. Wu, M., Sun, L.V., Vamathevan, J., Riegler, M., Deboy, R., Brownlie, J.C., McGraw, E.A., Martin, W., Esser, C., Ahmadinejad, N., et al. (2004). Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements. PLoS Biol. 2, E69.