Regulation of anti-phage defense mechanisms by using cinnamaldehyde as a quorum sensing inhibitor.

Background: Multidrug-resistant bacteria and the shortage of new antibiotics constitute a serious health problem. This problem has led to increased interest in the use of bacteriophages, which have great potential as antimicrobial agents but also carry the risk of inducing resistance. The objective of the present study was to minimize the development of phage resistance in Klebsiella pneumoniae strains by inhibiting quorum sensing (QS) and thus demonstrate the role of QS in regulating defense mechanisms. Results: Cinnamaldehyde (CAD) was added to K. pneumoniae cultures to inhibit QS and thus demonstrate the role of the signaling system in regulating the anti-phage defense mechanism. The QS inhibitory activity of CAD in K. pneumoniae was confirmed by a reduction in the quantitative expression of the lsrB gene (AI-2 pathway) and by proteomic analysis. The infection assays showed that the phage was able to infect a previously resistant K. pneumoniae strain in the cultures to which CAD was added. The results were confirmed using proteomic analysis. Thus, anti-phage defense-related proteins from different systems, such as cyclic oligonucleotide-based bacterial anti-phage signaling systems (CBASS), restriction-modification (R-M) systems, clustered regularly interspaced short palindromic repeat-Cas (CRISPR-Cas) system, and bacteriophage control infection (BCI), were present in the cultures with phage but not in the cultures with phage and CAD. When the QS and anti-phage defense systems were inhibited by the combined treatment, proteins related to phage infection and proliferation, such as the tail fiber protein, the cell division protein DamX, and the outer membrane channel protein TolC, were detected. Conclusion: Inhibition of QS reduces phage resistance in K. pneumoniae, resulting in the infection of a previously resistant strain by phage, with a significant increase in phage proliferation and a significant reduction in bacterial growth. QS inhibitors could be considered for therapeutic application by including them in phage cocktails or in phage-antibiotic combinations to enhance synergistic effects and reduce the emergence of antimicrobial resistance.

Retron-Eco1 assembles NAD+-hydrolyzing filaments that provide immunity against bacteriophages.

Retrons are toxin-antitoxin systems protecting bacteria against bacteriophages via abortive infection. The Retron-Eco1 antitoxin is formed by a reverse transcriptase (RT) and a non-coding RNA (ncRNA)/multi-copy single-stranded DNA (msDNA) hybrid that neutralizes an uncharacterized toxic effector. Yet, the molecular mechanisms underlying phage defense remain unknown. Here, we show that the N-glycosidase effector, which belongs to the STIR superfamily, hydrolyzes NAD+ during infection. Cryoelectron microscopy (cryo-EM) analysis shows that the msDNA stabilizes a filament that cages the effector in a low-activity state in which ADPr, a NAD+ hydrolysis product, is covalently linked to the catalytic E106 residue. Mutations shortening the msDNA induce filament disassembly and the effector's toxicity, underscoring the msDNA role in immunity. Furthermore, we discovered a phage-encoded Retron-Eco1 inhibitor (U56) that binds ADPr, highlighting the intricate interplay between retron systems and phage evolution. Our work outlines the structural basis of Retron-Eco1 defense, uncovering ADPr's pivotal role in immunity.

RNA-based regulation in bacteria-phage interactions.

Interactions of bacteria with their viruses named bacteriophages or phages shape the bacterial genome evolution and contribute to the diversity of phages. RNAs have emerged as key components of several anti-phage defense systems in bacteria including CRISPR-Cas, toxin-antitoxin and abortive infection. Frequent association with mobile genetic elements and interplay between different anti-phage defense systems are largely discussed. Newly discovered defense systems such as retrons and CBASS include RNA components. RNAs also perform their well-recognized regulatory roles in crossroad of phage-bacteria regulatory networks. Both regulatory and defensive function can be sometimes attributed to the same RNA molecules including CRISPR RNAs. This review presents the recent advances on the role of RNAs in the bacteria-phage interactions with a particular focus on clostridial species including an important human pathogen, Clostridioides difficile.

OLD family nuclease function across diverse anti-phage defense systems.

Bacteriophages constitute a ubiquitous threat to bacteria, and bacteria have evolved numerous anti-phage defense systems to protect themselves. These systems include well-studied phenomena such as restriction endonucleases and CRISPR, while emerging studies have identified many new anti-phage defense systems whose mechanisms are unknown or poorly understood. Some of these systems involve overcoming lysogenization defect (OLD) nucleases, a family of proteins comprising an ABC ATPase domain linked to a Toprim nuclease domain. Despite being discovered over 50 years ago, OLD nuclease function remained mysterious until recent biochemical, structural, and bioinformatic studies revealed that OLD nucleases protect bacteria by functioning in diverse anti-phage defense systems including the Gabija system and retrons. In this review we will highlight recent discoveries in OLD protein function and their involvement in multiple discrete anti-phage defense systems.

Mycobacterial phage TM4 requires a eukaryotic-like Ser/Thr protein kinase to silence and escape anti-phage immunity.

In eukaryotic cells, serine/threonine protein kinases (StpKs) play important roles in limiting viral infections. StpKs are commonly activated upon infections, inhibiting the expression of genes central for viral replication. Here, we report that a eukaryotic-like StpK7 encoded by MSMEG_1200 in M. smegmatis is required for mycobacteriophage TM4 to escape bacterial defense. stpK7 is located within a gene island, MSMEG_1191-MSMEG_1200, containing multiple anti-phage genes resembling the BREX (bacteriophage exclusion) phage-resistance system. StpK7 negatively regulates the expression of this gene island. Following phage TM4 infection, StpK7 is induced, directly phosphorylating the transcriptional regulator MSMEG_1198 and inhibiting its positive regulatory activity, thus reducing the expression of multiple downstream genes in the BREX-like gene island. Further analysis showed that genes within this anti-phage island critically regulate mycobacterial lipid hemostasis and phage adsorption. Collectively, this work characterizes a regulatory network driven by StpK7, which is utilized by phage TM4 to escape from the host defense against mycobacteria.

Viral sponges sequester nucleotide signals to inactivate immunity.

Bacteria synthesize specialized nucleotide signals to control anti-phage defense. Two papers - by Huiting et al. and Jenson et al. - now reveal that bacteriophages encode protein 'sponges' that sequester cyclic oligonucleotide immune signals and inactivate host antiviral immunity.

Molecular basis of RADAR anti-phage supramolecular assemblies.

Adenosine-to-inosine RNA editing has been proposed to be involved in a bacterial anti-phage defense system called RADAR. RADAR contains an adenosine triphosphatase (RdrA) and an adenosine deaminase (RdrB). Here, we report cryo-EM structures of RdrA, RdrB, and currently identified RdrA-RdrB complexes in the presence or absence of RNA and ATP. RdrB assembles into a dodecameric cage with catalytic pockets facing outward, while RdrA adopts both autoinhibited tetradecameric and activation-competent heptameric rings. Structural and functional data suggest a model in which RNA is loaded through the bottom section of the RdrA ring and translocated along its inner channel, a process likely coupled with ATP-binding status. Intriguingly, up to twelve RdrA rings can dock one RdrB cage with precise alignments between deaminase catalytic pockets and RNA-translocation channels, indicative of enzymatic coupling of RNA translocation and deamination. Our data uncover an interesting mechanism of enzymatic coupling and anti-phage defense through supramolecular assemblies.

A Glimpse at the Anti-Phage Defenses Landscape in the Foodborne Pathogen Salmonella enterica subsp. enterica serovar Typhimurium.

Bacteriophages, which specifically infect and kill bacteria, are currently used as additives to control pathogens such as Salmonella in human food (PhageGuard S®) or animal feed (SalmoFREE®, Bafasal®). Indeed, salmonellosis is among the most important zoonotic foodborne illnesses. The presence of anti-phage defenses protecting bacteria against phage infection could impair phage applications aiming at reducing the burden of foodborne pathogens such as Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) to the food industry. In this study, the landscape of S. Typhimurium anti-phage defenses was bioinformatically investigated in publicly available genomes using the webserver PADLOC. The primary anti-phage systems identified in S. Typhimurium use nucleic acid degradation and abortive infection mechanisms. Reference systems were identified on an integrative and conjugative element, a transposon, a putative integrative and mobilizable element, and prophages. Additionally, the mobile genetic elements (MGEs) containing a subset of anti-phage systems were found in the Salmonella enterica species. Lastly, the MGEs alone were also identified in the Enterobacteriaceae family. The presented diversity assessment of the anti-phage defenses and investigation of their dissemination through MGEs in S. Typhimurium constitute a first step towards the design of preventive measures against the spread of phage resistance that may hinder phage applications.

An expanded arsenal of immune systems that protect bacteria from phages.

Bacterial anti-phage systems are frequently clustered in microbial genomes, forming defense islands. This property enabled the recent discovery of multiple defense systems based on their genomic co-localization with known systems, but the full arsenal of anti-phage mechanisms remains unknown. We report the discovery of 21 defense systems that protect bacteria from phages, based on computational genomic analyses and phage-infection experiments. We identified multiple systems with domains involved in eukaryotic antiviral immunity, including those homologous to the ubiquitin-like ISG15 protein, dynamin-like domains, and SEFIR domains, and show their participation in bacterial defenses. Additional systems include domains predicted to manipulate DNA and RNA molecules, alongside toxin-antitoxin systems shown here to function in anti-phage defense. These systems are widely distributed in microbial genomes, and in some bacteria, they form a considerable fraction of the immune arsenal. Our data substantially expand the inventory of defense systems utilized by bacteria to counteract phage infection.

The evolution of a counter-defense mechanism in a virus constrains its host range.

Bacteria use diverse immunity mechanisms to defend themselves against their viral predators, bacteriophages. In turn, phages can acquire counter-defense systems, but it remains unclear how such mechanisms arise and what factors constrain viral evolution. Here, we experimentally evolved T4 phage to overcome a phage-defensive toxin-antitoxin system, toxIN, in Escherichia coli. Through recombination, T4 rapidly acquires segmental amplifications of a previously uncharacterized gene, now named tifA, encoding an inhibitor of the toxin, ToxN. These amplifications subsequently drive large deletions elsewhere in T4's genome to maintain a genome size compatible with capsid packaging. The deleted regions include accessory genes that help T4 overcome defense systems in alternative hosts. Thus, our results reveal a trade-off in viral evolution; the emergence of one counter-defense mechanism can lead to loss of other such mechanisms, thereby constraining host range. We propose that the accessory genomes of viruses reflect the integrated evolutionary history of the hosts they infected.

Toxin-Antitoxin Systems as Phage Defense Elements.

Toxin-antitoxin (TA) systems are ubiquitous genetic elements in bacteria that consist of a growth-inhibiting toxin and its cognate antitoxin. These systems are prevalent in bacterial chromosomes, plasmids, and phage genomes, but individual systems are not highly conserved, even among closely related strains. The biological functions of TA systems have been controversial and enigmatic, although a handful of these systems have been shown to defend bacteria against their viral predators, bacteriophages. Additionally, their patterns of conservation-ubiquitous, but rapidly acquired and lost from genomes-as well as the co-occurrence of some TA systems with known phage defense elements are suggestive of a broader role in mediating phage defense. Here, we review the existing evidence for phage defense mediated by TA systems, highlighting how toxins are activated by phage infection and how toxins disrupt phage replication. We also discuss phage-encoded systems that counteract TA systems, underscoring the ongoing coevolutionary battle between bacteria and phage. We anticipate that TA systems will continue to emerge as central players in the innate immunity of bacteria against phage.

Deploying Viruses against Phytobacteria: Potential Use of Phage Cocktails as a Multifaceted Approach to Combat Resistant Bacterial Plant Pathogens.

Plants in nature are under the persistent intimidation of severe microbial diseases, threatening a sustainable food production system. Plant-bacterial pathogens are a major concern in the contemporary era, resulting in reduced plant growth and productivity. Plant antibiotics and chemical-based bactericides have been extensively used to evade plant bacterial diseases. To counteract this pressure, bacteria have evolved an array of resistance mechanisms, including innate and adaptive immune systems. The emergence of resistant bacteria and detrimental consequences of antimicrobial compounds on the environment and human health, accentuates the development of an alternative disease evacuation strategy. The phage cocktail therapy is a multidimensional approach effectively employed for the biocontrol of diverse resistant bacterial infections without affecting the fauna and flora. Phages engage a diverse set of counter defense strategies to undermine wide-ranging anti-phage defense mechanisms of bacterial pathogens. Microbial ecology, evolution, and dynamics of the interactions between phage and plant-bacterial pathogens lead to the engineering of robust phage cocktail therapeutics for the mitigation of devastating phytobacterial diseases. In this review, we highlight the concrete and fundamental determinants in the development and application of phage cocktails and their underlying mechanism, combating resistant plant-bacterial pathogens. Additionally, we provide recent advances in the use of phage cocktail therapy against phytobacteria for the biocontrol of devastating plant diseases.

Genomic Analysis Unveils the Pervasiveness and Diversity of Prophages Infecting Erwinia Species.

Prophages are abundant elements integrated into bacterial genomes and contribute to inter-strain genetic variability and, in some cases, modulate the environmental behavior of bacteria, such as pathogen virulence. Here, we described prophage occurrence and diversity in publicly available Erwinia genome assemblies, a genus containing plant pathogens. Prophage-like sequences were identified and taxonomically classified. Sequence diversity was analyzed through intergenomic similarities. Furthermore, we searched for anti-phage defense systems in Erwinia spp., such as DISARM, BREX, and CRISPR-Cas systems, and identified the putative targets of CRISPR spacers. We identified 939 prophage-like sequences in 221 Erwinia spp. genome assemblies. Only 243 prophage-like sequences were classified, all belonging to the Caudoviricetes class. The set of putative Erwinia prophages was mostly unique since only three sequences showed more than 70% intergenomic similarities to known Erwinia phages. Overall, the number and type of CRISPR-Cas systems were conserved within Erwinia species, with many spacers directed to the putative prophages identified. This study increased the knowledge of the diversity and distribution of Erwinia prophages, contributing to the characterization of genetic and ecological factors influencing Erwinia spp. environmental fitness.

Eco-Evolutionary Effects of Bacterial Cooperation on Phage Therapy: An Unknown Risk?

If there is something we have learned from the antibiotic era, it is that indiscriminate use of a therapeutic agent without a clear understanding of its long-term evolutionary impact can have enormous health repercussions. This knowledge is particularly relevant when the therapeutic agents are remarkably adaptable and diverse biological entities capable of a plethora of interactions, most of which remain largely unexplored. Although phage therapy (PT) undoubtedly holds the potential to save lives, its current efficacy in case studies recalls the golden era of antibiotics, when these compounds were highly effective and the possibility of them becoming ineffective seemed remote. Safe PT schemes depend on our understanding of how phages interact with, and evolve in, highly complex environments. Here, we summarize and review emerging evidence in a commonly overlooked theme in PT: bacteria-phage interactions. In particular, we discuss the influence of quorum sensing (QS) on phage susceptibility, the consequent role of phages in modulating bacterial cooperation, and the potential implications of this relationship in PT, including how we can use this knowledge to inform PT strategies. We highlight that the influence of QS on phage susceptibility seems to be widespread but can have contrasting outcomes depending on the bacterial host, underscoring the need to thoroughly characterize this link in various bacterial models. Furthermore, we encourage researchers to exploit competition experiments, experimental evolution, and mathematical modeling to explore this relationship further in relevant infection models. Finally, we emphasize that long-term PT success requires research on phage ecology and evolution to inform the design of optimal therapeutic schemes.