Periodic Reporting for period 4 - PhageResist (Beyond CRISPR: Systematic characterization of novel anti-phage defense systems in the microbial pan-genome)
Okres sprawozdawczy: 2021-01-01 do 2021-12-31
To survive in the face of perpetual phage attacks, bacteria and archaea have developed a variety of anti-phage defense systems, commonly known as the microbial “immune system”. The purpose of the project was to systematically discover and characterize new, yet unknown, defense mechanisms used by bacteria to defend themselves against viruses that infect them.
This project is important to society in several aspects. First, bacterial defense systems play a major role in shaping the evolution of both phage and bacterial genomes, and it is hence crucial to understand these systems if we want to understand the processes of consequences of microbial genome evolution. Second, In the past, the discovery of new defense systems contributed not only to our appreciation of the arms race between bacteria and phage, but also provided important molecular tools and proved invaluable for biotechnological utilization. Defense systems, including the CRISPR-Cas systems and multiple abortive infection systems, are being used in the dairy industry to protect cheese- and yogurt-producing bacteria against detrimental phage attacks. Moreover, deep mechanistic understanding of the mode of action of individual systems lead to revolutions in molecular biology, as demonstrated by the utilization of restriction enzymes for genetic engineering, and now the adaptation of the CRISPR-Cas system into a powerful genome editing tool.
Our overall goal was to discover, and then decipher the mechanism, of new defense systems that protect bacteria from viral infection. Over the course of the project we discovered more than 15 new defense systems, and solved the mechanism of action to many of them
This project is important to society in several aspects. First, bacterial defense systems play a major role in shaping the evolution of both phage and bacterial genomes, and it is hence crucial to understand these systems if we want to understand the processes of consequences of microbial genome evolution. Second, In the past, the discovery of new defense systems contributed not only to our appreciation of the arms race between bacteria and phage, but also provided important molecular tools and proved invaluable for biotechnological utilization. Defense systems, including the CRISPR-Cas systems and multiple abortive infection systems, are being used in the dairy industry to protect cheese- and yogurt-producing bacteria against detrimental phage attacks. Moreover, deep mechanistic understanding of the mode of action of individual systems lead to revolutions in molecular biology, as demonstrated by the utilization of restriction enzymes for genetic engineering, and now the adaptation of the CRISPR-Cas system into a powerful genome editing tool.
Our overall goal was to discover, and then decipher the mechanism, of new defense systems that protect bacteria from viral infection. Over the course of the project we discovered more than 15 new defense systems, and solved the mechanism of action to many of them
The following studies have been performed as part of the current project:
We have developed a pilot computational genomics algorithm that enables the discovery of yet-unknown immune defense systems in microbial genomes. As a pilot run, we used this algorithm to reveal and study a new 5-gene defense systems that we denoted “DISARM” (after the acronym – Defense Island System Associated with Restriction and Modification). We further studied the predicted system and showed experimentally that it indeed confers resistance to a wide array of phages under multiple conditions. Our relevant results were summarized in the following paper:
Ofir G, Melamed S, Sberro H, Mukamel Z, Silverman S, Yaakov G, Doron S, Sorek R. “DISARM is a widespread bacterial defence system with broad anti-phage activities.” Nature Microbiology, 3(1):90-98 (2018).
With the success of the pilot project, we moved to a larger scale project that used massive genomic analyses of defense islands in ~50,000 microbial genomes. This involved algorithm refinement, application of mathematical methodologies from the field of graph models, and extended statistical analyses of the results. On the experimental side, we have set up a pipeline of phage infection assays that involves two model organisms (Escherichia coli and Bacillus subtilis) and 16 different phages the represent different viral families. The overall endeavor has led to the discovery of 10 completely new defense systems that are widespread in microbes and shown to strongly protect against foreign DNA invasion. The results of this study were published in the following paper:
Doron S, Melamed S, Ofir G, Leavitt A, Lopatina A, Keren M, Amitai G, Sorek R. “Systematic discovery of antiphage defense systems in the microbial pangenome.“ Science, 359(6379), pii: eaar4120 (2018).
We then extended the efforts to discover additional new defense systems, and studied their mechanisms of action. This resulted in the discovery of systems that use small molecules to mediate immune signals, systems that produce antiviral molecules, and systems that utilize reverse-transcribed non-coding RNAs for their defensive capacity. The results were described in a series of publications:
Cohen D, Melamed S, Millman A, Shulman G, Oppenheimer-Shaanan Y, Kacen A, Doron S, Amitai G, Sorek R. Cyclic GMP-AMP signaling protects bacteria against viral infection. Nature, 574(7780):691-695 (2019).
Millman A, Melamed S, Amitai G, Sorek R. Diversity and classification of cyclic-oligonucleotide-based anti-phage signalling systems. Nature Microbiology, 5(12):1608-1615 (2020).
Morehouse B, Govande AA, Millman A, Keszei A, Lowey B, Ofir G, Shao S, Sorek R, Kranzusch PJ. STING cyclic dinucleotide sensing originated in bacteria. Nature, 586(7829):429-433 (2020).
Bernheim A, Millman A, Ofir G, Meitav G, Avraham C, Shomar H, Rosenberg MM, Tal N, Melamed S, Amitai G, Sorek R. Prokaryotic viperins produce diverse antiviral molecules. Nature, 589(7840):120-124 (2021).
As part of our efforts aimed to reveal new mechanisms of defense in bacteria, we initiated a study that was aimed to test whether bacteria can alert other bacteria once they have been infected by a virus. Although this study did not verify the hypothesis that bacteria can communicate to alert each other from viruses, in unexpectedly revealed that viruses can communicate, during infection, to coordinate their infection dynamics. We showed that some viruses can use this kind of molecular communication to “decide” whether to replicate or to become dormant in the bacterium they infect. These discoveries were published in the following papers:
Erez Z, Steinberger-Levy I, Shamir M, Doron S, Stokar-Avihail A, Peleg Y, Melamed S, Leavitt A, Savidor A, Albeck S, Amitai G, Sorek R. “Communication between viruses guides lysis-lysogeny decisions.” Nature, 541(7638):488-493 (2017).
Stokar-Avihail A, Tal N, Erez Z, Lopatina A, Sorek R. Widespread utilization of peptide communication in phages infecting soil and pathogenic bacteria. Cell Host & Microbe, 25:746-755 (2019).
Furthermore, we have written a number of review articles to summarize the progress in the field:
Lopatina A, Tal N, Sorek R. Abortive Infection: Bacterial Suicide as an Antiviral Immune Strategy. Annual Review of Virology, 7:371-384 (2020).
Bernheim A, Sorek R. The pan-immune system of bacteria: antiviral defence as a community resource. Nature Reviews Microbiology, 18(2):113-119 (2020).
We have developed a pilot computational genomics algorithm that enables the discovery of yet-unknown immune defense systems in microbial genomes. As a pilot run, we used this algorithm to reveal and study a new 5-gene defense systems that we denoted “DISARM” (after the acronym – Defense Island System Associated with Restriction and Modification). We further studied the predicted system and showed experimentally that it indeed confers resistance to a wide array of phages under multiple conditions. Our relevant results were summarized in the following paper:
Ofir G, Melamed S, Sberro H, Mukamel Z, Silverman S, Yaakov G, Doron S, Sorek R. “DISARM is a widespread bacterial defence system with broad anti-phage activities.” Nature Microbiology, 3(1):90-98 (2018).
With the success of the pilot project, we moved to a larger scale project that used massive genomic analyses of defense islands in ~50,000 microbial genomes. This involved algorithm refinement, application of mathematical methodologies from the field of graph models, and extended statistical analyses of the results. On the experimental side, we have set up a pipeline of phage infection assays that involves two model organisms (Escherichia coli and Bacillus subtilis) and 16 different phages the represent different viral families. The overall endeavor has led to the discovery of 10 completely new defense systems that are widespread in microbes and shown to strongly protect against foreign DNA invasion. The results of this study were published in the following paper:
Doron S, Melamed S, Ofir G, Leavitt A, Lopatina A, Keren M, Amitai G, Sorek R. “Systematic discovery of antiphage defense systems in the microbial pangenome.“ Science, 359(6379), pii: eaar4120 (2018).
We then extended the efforts to discover additional new defense systems, and studied their mechanisms of action. This resulted in the discovery of systems that use small molecules to mediate immune signals, systems that produce antiviral molecules, and systems that utilize reverse-transcribed non-coding RNAs for their defensive capacity. The results were described in a series of publications:
Cohen D, Melamed S, Millman A, Shulman G, Oppenheimer-Shaanan Y, Kacen A, Doron S, Amitai G, Sorek R. Cyclic GMP-AMP signaling protects bacteria against viral infection. Nature, 574(7780):691-695 (2019).
Millman A, Melamed S, Amitai G, Sorek R. Diversity and classification of cyclic-oligonucleotide-based anti-phage signalling systems. Nature Microbiology, 5(12):1608-1615 (2020).
Morehouse B, Govande AA, Millman A, Keszei A, Lowey B, Ofir G, Shao S, Sorek R, Kranzusch PJ. STING cyclic dinucleotide sensing originated in bacteria. Nature, 586(7829):429-433 (2020).
Bernheim A, Millman A, Ofir G, Meitav G, Avraham C, Shomar H, Rosenberg MM, Tal N, Melamed S, Amitai G, Sorek R. Prokaryotic viperins produce diverse antiviral molecules. Nature, 589(7840):120-124 (2021).
As part of our efforts aimed to reveal new mechanisms of defense in bacteria, we initiated a study that was aimed to test whether bacteria can alert other bacteria once they have been infected by a virus. Although this study did not verify the hypothesis that bacteria can communicate to alert each other from viruses, in unexpectedly revealed that viruses can communicate, during infection, to coordinate their infection dynamics. We showed that some viruses can use this kind of molecular communication to “decide” whether to replicate or to become dormant in the bacterium they infect. These discoveries were published in the following papers:
Erez Z, Steinberger-Levy I, Shamir M, Doron S, Stokar-Avihail A, Peleg Y, Melamed S, Leavitt A, Savidor A, Albeck S, Amitai G, Sorek R. “Communication between viruses guides lysis-lysogeny decisions.” Nature, 541(7638):488-493 (2017).
Stokar-Avihail A, Tal N, Erez Z, Lopatina A, Sorek R. Widespread utilization of peptide communication in phages infecting soil and pathogenic bacteria. Cell Host & Microbe, 25:746-755 (2019).
Furthermore, we have written a number of review articles to summarize the progress in the field:
Lopatina A, Tal N, Sorek R. Abortive Infection: Bacterial Suicide as an Antiviral Immune Strategy. Annual Review of Virology, 7:371-384 (2020).
Bernheim A, Sorek R. The pan-immune system of bacteria: antiviral defence as a community resource. Nature Reviews Microbiology, 18(2):113-119 (2020).
The major achievements of this project the discovery of 15 defense systems, understanding the mechanism of a subset of these systems, and the discovery that phages can communicate – represent progress beyond the state of the art. In addition to these discoveries, we have issued two patent applications that cover applicative avenues stemming from our discoveries.