Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - PhageResist (Beyond CRISPR: Systematic characterization of novel anti-phage defense systems in the microbial pan-genome)

Teaser

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 current project is to systematically discover and characterize new, yet...

Summary

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 current project is 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 is to discover, and then decipher the mechanism, of new defense systems that protect bacteria from viral infection. These, in turn, have the potential to become new and powerful molecular tools for biotechnology and biomedicine.

Work performed

Since the beginning of the project and until today, the following work has been performed:

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. Through methodologies involving genomics, molecular genetics, microscopy and phage infection techniques we begun to characterize the mechanism of action of the DISARM system. 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 systems discovered include ones that seem to have adopted components of the bacterial flagella and chromosome maintenance complexes and use these components for defensive capacities. Our data also showed that genes with Toll-interleukin receptor (TIR) domains are involved in bacterial defense against phages, providing evidence for a common, ancient ancestry of innate immunity components shared between animals, plants, and bacteria. 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).


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 paper:

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).

Final results

The three major achievements of this project so far – the discovery of DISARM, the discovery of additional 10 defense 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.

In the second half of the project, we will continue our studies as planned in the project proposal, in the following scientific fronts:

- We will study the mechanism of action of individual defense systems out of the ones that we discovered, including the mechanism of the BREX system that has been discovered earlier

- We will extend our searches of new defense systems also to additional genomes and metagenomes that have not been analyzed in the first round, including application of improved algorithms to detect new defense systems

- We will continue to study the phage communication system that we discovered as part of this project, with the aim of understanding how broad such phage communication is in nature, and what are the consequences for bacterial defense against communicating phages.

We anticipate that these studies would generate mechanistic knowledge on the new defense systems and phage communication systems, as well as lead to the discovery of yet additional defense systems that so far escaped discovery.