Defence co-operation in bacteria and anti-defence strategies in phages

Abstract

In the face of the antimicrobial resistance crisis, bacteriophages (phages), natural predators of bacteria, have emerged as a promising alternative or complement to antibiotics. Achieving effective bacterial suppression through phage therapy requires a comprehensive understanding of how bacteria develop phage resistance, which can guide the rational design of such treatments. Bacterial defence systems, essential mechanisms by which bacteria resist phage infection, have also been repurposed into powerful gene editing tools, such as Restriction-Modification systems and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated proteins (Cas) systems. Over the past decade, more than 150 defence systems have been discovered, though the mechanisms of many remain unknown. To identify novel bacterial defence systems and study their interactions, I utilised the Prokaryotic Antiviral Defence LOCator (PADLOC), a tool I originally helped develop to identify and study the distribution of defence systems in prokaryotic genomes. In this thesis, I used PADLOC specifically to discover new systems using a guilt-by-embedding approach and to investigate patterns of defence systems co-occurrence. This approach led to the discovery of 145 candidate defence systems, of which I experimentally tested 34, with 24 exhibiting anti-phage activity. These findings highlight the effectiveness of this method for uncovering functional systems. Analysis of bacterial genomes using PADLOC revealed that bacteria typically harbour six to eight defence systems, raising questions about whether these systems operate independently or form functional networks. By analysing 26,362 Escherichia coli genomes, I identified 118 combinations of co-occurring systems. Experimental validation of three such pairs demonstrated, for the first time, synergistic anti-phage activity, including the interaction of Tmn with Gabija, Septu, and PrrC, which significantly enhanced bacterial fitness in evolutionary experiments. These findings provide the first experimental evidence supporting the pan-immunity model, which considers the population-level dynamics of defence systems. Building on this, I explored the molecular mechanisms of two membrane-associated defence systems: Tmn and Kiwa. Tmn was shown to sense two distinct phage proteins and halt phage replication through reversible plasmolysis, a process also observed in plants. The role of Tmn in synergy with other systems underscores its importance in bacterial immunity. Meanwhile, Kiwa detects mechanical changes in the membrane caused by phage adsorption or plasmid conjugation and binds invading DNA preventing replication and late transcription. Unlike Tmn, Kiwa cooperates with RecBCD to overcome phage ani-defence strategies, illustrating a different form of functional interaction. These findings reveal the diverse mechanisms by which bacterial defence systems interact with one another and their environments, highlighting their adaptability and complexity in responding to phage threats.

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

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