Discovery of antiphage systems and the study of their cooperation to stop viral attacks

Abstract

Bacteria possess a diverse array of innate immune mechanisms that protect them from invading genetic elements, such as bacteriophages (phages). In recent years, the discovery of over 152 distinct defence systems, and more expected to emerge, has broadened our understanding of bacteria-phage interactions. However, at the start of this doctorate, there was an absence of computational tools to systematically identify defence systems in prokaryotic genomes. Furthermore, many defence systems remain unexplored and lack functional understanding. Therefore, in this thesis, I present the development of a computational tool for the identification of defence systems named the Prokaryotic Antiviral Defence LOCator (PADLOC). PADLOC was subsequently used to identify defence-associated genes, leading to the identification of putative defence subtypes and the discovery of a new system named Hma. Several candidates were experimentally verified by me for anti-phage functionality, overall indicating that PADLOC can identify new defence systems. Additionally, I investigated the uncharacterised Kiwa system, which is associated with membrane defence mechanisms. I provide mechanistic insights into Kiwa, revealing that the transmembrane protein KwaA detects phage infection by sensing inhibition of the host RNA polymerase, leading to the release of the DNA-binding effector KwaB. I also discovered that anti-RecBCD proteins antagonise Kiwa, and consequently, discuss the coexistence of RecBCD and Kiwa in the cell, emphasizing their crucial roles in ensuring protection and highlighting the complexity of bacterial immunity. To gain further insights into Kiwa, I employed comparative genomics on closely related Kiwa-resistant phages resulting in the identification of several putative anti-Kiwa genes. The observation that closely related phages display diverse Kiwa susceptibility underscores the importance of defence systems in determining phage resistance. Finally, I discuss the molecular characterisation of a novel Restriction-modification-like system, named Ronin, which induces cell death upon activation by phage anti-RM proteins. Using single-molecule super-resolution microscopy, I present evidence that Ronin localises at the cellular membrane upon activation by the RM inhibitor ArdA, suggesting that its toxicity is exerted through a membrane function. Ronin employs a strategy reminiscent of eukaryotic immune systems, further highlighting the conservation of immune strategies across domains of life. Overall, my thesis describes the development of an accessible tool for the identification of defence systems and expands on our current knowledge of bacterial antiphage mechanisms. The advancement in our understanding of phage-host interactions significantly contributes to the wider scientific community for the development of effective phage therapies and applications in biotechnology.

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Identifiers

ORCID for Thomas Christopher Todeschini: orcid.org/0009-0000-1648-8029

ORCID for Franklin Nobrega: orcid.org/0000-0002-8238-1083

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