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. ... Read more

Novel phage defense systems featuring diverse enzymatic activities are continually being discovered. Among these, defense systems employing proteolytic enzymes have been identified, revealing a previously unrecognized enzymatic activity in phage defense. These protease-associated defense systems represent an untapped reservoir for new biotechnological tools and may serve as a springboard for the development of proteome editors. This review outlines recent advancements in the discovery and characterization of protease-containing defense systems, proposes methods for further exploration and investigation of protease activity, and considers the prospect of protease defense systems for modulating protein processing and cell fate. ... Read more

Jumbo phages protect their genomes from DNA-sensing bacterial defense systems by enclosing them within vesicles and nucleus-like compartments. Very little is known about defense systems specialized to counter these phages. Here, we show that AVAST type 5 (Avs5) systems, part of the signal transduction ATPases of numerous domains (STAND) superfamily, confer conserved immunity against jumbo phages. Using fluorescence microscopy and biotin proximity labeling, we demonstrate that Avs5 localizes to early infection vesicles, where it senses an essential, early-expressed phage protein named JADA (Jumbo phage Avs5 Defense Activator). Recognition of phage infection triggers the Sir2-like effector domain of Avs5 across three Avs5 clades, resulting in rapid NAD + hydrolysis, disruption of phage nucleus formation, and arrest of infection. These findings reveal a spatially coordinated bacterial immune strategy that targets an early vulnerability in jumbo phage infection. ... Read more

Cystic fibrosis is a genetic disorder that affects mucus clearance, particularly of the lungs. As a result, cystic fibrosis patients often experience infections from bacteria, which contribute to the disease progression. Pseudomonas aeruginosa is one of the most common opportunistic pathogens associated with cystic fibrosis. The presence of P. aeruginosa complicates the treatment due to its high antibiotic resistance. Thus, research is ongoing to treat these infections with bacterial viruses instead, known as bacteriophages. Notably, P. aeruginosa clinical strains possess a variety of phage defense mechanisms that may limit the effectiveness of phage therapy. In this study, we compared the defense system repertoire of P. aeruginosa strains isolated from cystic fibrosis patients with those from non-cystic fibrosis patients. Our findings reveal that P. aeruginosa strains isolated from cystic fibrosis patients have fewer phage defense mechanisms per strain than from non-cystic fibrosis patients, suggesting altered phage selection pressures in strains colonizing CF patient lungs. ... Read more

The presence of tRNAs in bacteriophage genomes has intrigued scientists since their discovery in the early 1960s, as phages were believed to rely on the host tRNAs for their translation. Over the years, a multitude of hypotheses have been explored, providing evidence that phages with different lifestyles utilize tRNAs in distinct ways. In recent years, several studies have provided evidence that phage tRNAs play a crucial role in evading phage defense systems. In this review we summarize the current state of the field of phage tRNAs, highlighting their diverse roles in phage infection. We also discuss the host response to phage tRNAs and the application of this knowledge to improve phage-based therapeutics to combat bacterial infections. ... Read more

The evolutionary arms race between bacteria and bacteriophages drives rapid evolution of bacterial defense mechanisms with scattered distribution across genomes. We hypothesized that this variability in bacterial defense systems leads to equally variable counter-defense repertoires in phage genomes. Examining the variable regions in Pseudomonas model phages of the Pbunavirus genus revealed five anti-defense genes, including one inhibiting Druantia type III named DadIII-1, another targeting Thoeris type III named TadIII-1, one inhibiting Zorya type I named ZadI-1, and two related broad defense inhibitors named Bdi1 and Bdi2 targeting four defenses. A typical Pbunavirus encodes up to five known anti-defense genes, some inhibiting four unrelated defense systems with distinct nucleic-acid-targeting mechanisms. Structural homologs of broad-acting Bdi1 and Bdi2 are encoded across diverse phage taxa infecting multiple bacterial hosts. These findings show that phages face a variety of bacterial defenses, driving them to evolve both specific and general strategies to overcome these barriers. ... Read more

Prokaryotes encode multiple distinct anti-phage defense systems in their genomes. However, the impact of carrying a multitude of defense systems on phage resistance remains unclear, especially in a clinical context. Using a collection of antibiotic-resistant clinical strains of Pseudomonas aeruginosa and a broad panel of phages, we demonstrate that defense systems contribute substantially to defining phage host range and that overall phage resistance scales with the number of defense systems in the bacterial genome. We show that many individual defense systems target specific phage genera and that defense systems with complementary phage specificities co-occur in P. aeruginosa genomes likely to provide benefits in phage-diverse environments. Overall, we show that phage-resistant phenotypes of P. aeruginosa with at least 19 phage defense systems exist in the populations of clinical, antibiotic-resistant P. aeruginosa strains. ... Read more

Prokaryotes have evolved a multitude of defense systems to protect against phage predation. Some of these resemble eukaryotic genes involved in antiviral responses. Here, we set out to systematically project the current knowledge of eukaryotic-like antiviral defense systems onto prokaryotic genomes, using Pseudomonas aeruginosa as a model organism. Searching for phage defense systems related to innate antiviral genes from vertebrates and plants, we uncovered over 450 candidates. We validated six of these phage defense systems, including factors preventing viral attachment, R-loop-acting enzymes, the inflammasome, ubiquitin pathway, and pathogen recognition signaling. Collectively, these defense systems support the concept of deep evolutionary links and shared antiviral mechanisms between prokaryotes and eukaryotes. ... Read more

Serratia sp. ATCC 39006 is a Gram-negative bacterium that has been used to study the function of phage defences, such as CRISPR-Cas, and phage counter-defence mechanisms. To expand our phage collection to study the phage-host interaction with Serratia sp. ATCC 39006, we isolated the T4-like myovirus LC53 in Ōtepoti Dunedin, Aotearoa New Zealand. Morphological, phenotypic and genomic characterization revealed that LC53 is virulent and similar to other Serratia, Erwinia and Kosakonia phages belonging to the genus Winklervirus. Using a transposon mutant library, we identified the host ompW gene as essential for phage infection, suggesting that it encodes the phage receptor. The genome of LC53 encodes all the characteristic T4-like core proteins involved in phage DNA replication and generation of viral particles. Furthermore, our bioinformatic analysis suggests that the transcriptional organization of LC53 is similar to that of Escherichia coli phage T4. Importantly, LC53 encodes 18 tRNAs, which likely compensate for differences in GC content between phage and host genomes. Overall, this study describes a newly isolated phage infecting Serratia sp. ATCC 39006 that expands the diversity of phages available to study phage-host interactions. ... Read more

Transfer RNAs (tRNAs) in bacteriophage genomes are widespread across bacterial host genera, but their exact function has remained unclear for more than 50 years. Several hypotheses have been proposed, and the most widely accepted one is codon compensation, which suggests that phages encode tRNAs that supplement codons that are less frequently used by the host. Here, we combine several observations and propose a new hypothesis that phage-encoded tRNAs counteract the tRNA-depleting strategies of the host using enzymes such as VapC, PrrC, Colicin D, and Colicin E5 to defend from viral infection. Based on mutational patterns of anticodon loops of tRNAs encoded by phages, we predict that these tRNAs are insensitive to host tRNAses. For phage-encoded tRNAs targeted in the anticodon itself, we observe that phages typically avoid encoding these tRNAs, further supporting the hypothesis that phage tRNAs are selected to be insensitive to host anticodon nucleases. Altogether, our results support the hypothesis that phage-encoded tRNAs have evolved to be insensitive to host anticodon nucleases. ... Read more

The bacterial chromosome is spatially organized through protein-mediated compaction, supercoiling, and cell-boundary confinement. Structural Maintenance of Chromosomes (SMC) complexes are a major class of chromosome-organizing proteins present throughout all domains of life. Here, we study the role of the Escherichia coli SMC complex MukBEF in chromosome architecture and segregation. Using quantitative live-cell imaging of shape-manipulated cells, we show that MukBEF is crucial to preserve the toroidal topology of the Escherichia coli chromosome and that it is non-uniformly distributed along the chromosome: it prefers locations toward the origin and away from the terminus of replication, and it is unevenly distributed over the origin of replication along the two chromosome arms. Using an ATP hydrolysis-deficient MukB mutant, we confirm that MukBEF translocation along the chromosome is ATP-dependent, in contrast to its loading onto DNA. MukBEF and MatP are furthermore found to be essential for sister chromosome decatenation. We propose a model that explains how MukBEF, MatP, and their interacting partners organize the chromosome and contribute to sister segregation. The combination of bacterial cell-shape modification and quantitative fluorescence microscopy paves way to investigating chromosome-organization factors in vivo. ... Read more

Bacteriophages are an invaluable source of novel genetic diversity. Sequencing of phage genomes can reveal new proteins with potential uses as biotechnological and medical tools, and help unravel the diversity of biological mechanisms employed by phages to take over the host during viral infection. Aiming to expand the available collection of phage genomes, we have isolated, sequenced, and assembled the genome sequences of four phages that infect the clinical pathogen Klebsiella pneumoniae: vB_KpnP_FBKp16, vB_KpnP_FBKp27, vB_KpnM_FBKp34, and Jumbo phage vB_KpnM_FBKp24. The four phages show very low (0-13%) identity to genomic phage sequences deposited in the GenBank database. Three of the four phages encode tRNAs and have a GC content very dissimilar to that of the host. Importantly, the genome sequences of the phages reveal potentially novel DNA packaging mechanisms as well as distinct clades of tubulin spindle and nucleus shell proteins that some phages use to compartmentalize viral replication. Overall, this study contributes to uncovering previously unknown virus diversity, and provides novel candidates for phage therapy applications against antibiotic-resistant K. pneumoniae infections. ... Read more