Phage immunity and evasion

Abstract

Bacteria are under constant predation by their viruses, also known as bacteriophages or phages. To protect themselves against phages, bacteria evolved a multitude of phage defense mechanisms. Some of these phage defense mechanisms, such as CRISPR-Cas systems, have been known since 2007. More recently, it has become evident that the repertoire of phage defense systems of bacteria is far more diverse and numerous than previously thought. This finding not only reshaped our understanding of phage defense systems but also raised the question of how phages can still successfully infect these complex immune responses. In this dissertation, we investigate this complex interplay between phages and their hosts, specifically looking at the importance of phage defense systems in phage resistance, approaches for discovering additional phage defenses, and how phages evade these phage defense systems.

In the first chapter, we provide a brief overview of the current understanding of bacterial immune systems and how phages evade these systems. We begin by describing how phages infect bacteria, and how bacteria respond to this infection. We then provide an overview of the known strategies that phages use to neutralize the host response, which in response prompted bacteria to evolve new strategies. Over time, creating a complex interplay between phage defense systems and evasion strategies of the phage.
In the second chapter, we show that bacterial species Pseudomonas aeruginosa has a large and numerous repertoires of phage defense systems. We demonstrate that these phage defenses the number of phage defense systems per strain correlates with the broadness of its resistance against a wide range of phages.
In the third chapter, we aimed to uncover previously unknown phage defense systems by searching for gene clusters that are associated with a higher resistance to phage infection. To achieve this, we assessed the infection ability of our Pseudomonas phages across our P. aeruginosa collection and conducted a genome-wide-association study. We identified one gene-cluster to be significantly associated with an increased phage resistance, corresponding to a R2-type pyocin. These pyocins are remnants of ancient phages that have been domesticated by bacteria to lyse nearby cells. How these R2-type pyocin may convey phage defense remains unknown.
In the fourth chapter, we look for homologs of eukaryotic viral defense systems in bacteria to uncover previously unknown phage defenses. We demonstrate that these homologs provide protection against phages using P. aeruginosa as a model organism. These bacterial phage defense systems resemble eukaryotic viral defense mechanisms in several ways, including preventing viral attachment, R-loop-acting enzymes, the inflammasome, the ubiquitin pathway, and the pathogen recognition signalling.
In the fifth chapter, we search for previously unknown phage defense systems by capitalizing on the observation that phage defense systems often exhibit high degrees of modularity, with sensing, signal transmission, and effector enzymes frequently being exchanged among phage defense gene clusters. By searching for gene clusters with defense-associated genes or functional domains, we uncovered several new phage defense systems.
In the sixth chapter, we observe that the prevalence of phage defense systems of P. aeruginosa strains from cystic fibrosis lung patients is reduced compared to P. aeruginosa strains from patients with other lung conditions, suggesting that cystic fibrosis-associated strains are more susceptible to phages. This observation provides a promising perspective for treating P. aeruginosa infections in these patients.
In the seventh chapter, we set-out to identify additional evasion strategies that phages use to evade phage defense systems. In this chapter, we focussed on phage genes that were located within the highly variable genomic regions of Pbunaviruses, a Pseudomonas phage family, and testing their ability to inhibit bacterial phage defense systems. Using this approach, we discovered several genes that were able to prevent the host immune response from acting, including those both broad and specific inhibitors. We showed that these genes are prevalent among a large variety of phage taxa.
In the eight chapter, we provide a hypothesis that offers a new perspective on a long-standing mystery: why phages encode their own tRNAs. An observation that has intrigued the phage field since its discovery in the early 1950s. We suggest that these phage tRNAs serve as an evasion strategy against phage defense systems that deplete host tRNAs, which would otherwise inhibit the ability of the phage to translate its genes and prevent phage propagation. Supporting our hypothesis, we observe that phage tRNAs have mutations that render these insensitive to tRNA targeting phage defenses.
In the nineth chapter, we review the current state of the phage tRNA field by highlighting their diverse roles in phage infection. We discuss their multifunctional roles for temperate, as well as the role of phage tRNAs for virulent phages, where they primarily serve to replenish the depleted tRNA pool of the host. Additionally, we highlight currently known phage defense systems that convey phage protection by depleting the host tRNA pool. We conclude the review by discussing the multiple layers of tRNA-targeting phage defenses, not all of which act by depleting tRNAs, since some also act on tRNA maturation steps and incorporation during translation.
In the tenth chapter, we present a general summary of the thesis and discuss the implications of these insights. Many questions remain, including: What are the biological laws that seem to govern the composition of the phage defense repertoire? And how can such a relatively small entity overcome the defenses of a much larger host? We also discuss several conceptual considerations, such as: Are we studying phage defense systems in the appropriate biological context? The discussion concludes by providing a perspective on the future of the field.

In conclusion, this dissertation investigates phage defense systems and evasion strategies. It provides insights into the cumulative role of Pseudomonas phage defense systems that facilitate a broad resistance to phages, it describes several methods to further uncover the bacterial immune system and provides new perspectives on how phages circumvent these defenses.