Antiphage small molecules produced by bacteria - beyond protein-mediated defenses.

Bacterial populations face the constant threat of viral predation exerted by bacteriophages ('phages'). In response, bacteria have evolved a wide range of defense mechanisms against phage challenges. Yet the vast majority of antiphage defense systems described until now are mediated by proteins or RNA complexes acting at the single-cell level. Here, we review small molecule-based defense strategies against phage infection, with a focus on the antiphage molecules described recently. Importantly, inhibition of phage infection by excreted small molecules has the potential to protect entire bacterial communities, highlighting the ecological significance of these antiphage strategies. Considering the immense repertoire of bacterial metabolites, we envision that the list of antiphage small molecules will be further expanded in the future.

[Bacterial immunity: Uncovering a new world]. / Immunité bactérienne : à la découverte d'un nouveau monde.

Viruses are parasites that infect all living organisms, and bacteria are no exception. To defend themselves against their viruses (phages), bacteria have developed numerous and sophisticated defense mechanisms, our understanding of which is rapidly growing. In the 2000s, only a handful of mechanisms were known and only two of them seemed to be found in most bacteria. In 2018, a new key method based on genome analysis revealed that there were likely many others. Indeed, over the past five years, more than 150 new mechanisms have been discovered. It is now estimated that there are probably thousands. This remarkable diversity, paralleled with the tremendous viral diversity, is evident both in terms of possible combinations of systems in bacterial genomes and in molecular mechanisms. One of the most surprising observations emerging from the exploration of this diversity is the discovery of striking similarities between certain bacterial defense systems and antiviral systems in humans, as well as plant (and eukaryotes in general) immune systems. Contrary to the previously accepted paradigm, organisms as diverse as fungi, plants, bacteria and humans share certain molecular strategies to fight viral infections, suggesting that an underestimated part of eukaryotic antiviral immunity could have evolved from bacterial antiviral defense systems.

Title: Immunité bactérienne : à la découverte d'un nouveau monde. Abstract: Les virus sont des parasites qui infectent tous les organismes vivants, et les bactéries n'y font pas exception. Pour se défendre contre leurs virus (les bactériophages ou phages), les bactéries se sont dotées d'un éventail de mécanismes élaborés, dont la découverte et la compréhension sont en pleine expansion. Dans les années 2000, seuls quelques systèmes de défense étaient connus et deux semblaient présents chez la plupart des bactéries. En 2018, une nouvelle méthode fondée sur l'analyse des génomes a révélé l'existence potentielle de nombreux autres. Plus de 150 nouveaux systèmes anti-phages ont été découverts au cours des cinq dernières années. On estime maintenant qu'il en existe probablement des milliers. Cette formidable diversité, qui est à mettre en parallèle avec la considérable diversité virale, s'exprime tant en termes de combinaisons de systèmes possibles dans les génomes bactériens que de mécanismes moléculaires. Une des observations les plus surprenantes qui émerge est la découverte de similarités entre certains systèmes de défense bactériens et des mécanismes antiviraux eucaryotes. Contrairement au paradigme jusqu'alors en place, des organismes aussi différents que des champignons, des plantes, des bactéries ou des êtres humains partagent certaines stratégies moléculaires pour combattre des infections virales, suggérant qu'une part sous-estimée de l'immunité antivirale eucaryote a directement évolué à partir des systèmes de défense bactériens.

Streptomyces development is involved in the efficient containment of viral infections.

The formation of plaques represents the hallmark of phage infection visualizing the clearance of the bacterial lawn in structured environments. In this study, we have addressed the impact of cellular development on phage infection in Streptomyces undergoing a complex developmental life cycle. Analysis of plaque dynamics revealed, after a period of plaque size enlargement, a significant regrowth of transiently phage-resistant Streptomyces mycelium into the lysis zone. Analysis of Streptomyces venezuelae mutant strains defective at different stages of cellular development indicated that this regrowth was dependent on the onset of the formation of aerial hyphae and spores at the infection interface. Mutants restricted to vegetative growth (ΔbldN) featured no significant constriction of plaque area. Fluorescence microscopy further confirmed the emergence of a distinct zone of cells/spores with reduced cell permeability towards propidium iodide staining at the plaque periphery. Mature mycelium was further shown to be significantly less susceptible to phage infection, which is less pronounced in strains defective in cellular development. Transcriptome analysis revealed the repression of cellular development at the early stages of phage infection probably facilitating efficient phage propagation. We further observed an induction of the chloramphenicol biosynthetic gene cluster highlighting phage infection as a trigger of cryptic metabolism in Streptomyces. Altogether, our study emphasizes cellular development and the emergence of transient phage resistance as an important layer of Streptomyces antiviral immunity.

Aminoglycoside Antibiotics Inhibit Phage Infection by Blocking an Early Step of the Infection Cycle.

In response to viral predation, bacteria have evolved a wide range of defense mechanisms, which rely mostly on proteins acting at the cellular level. Here, we show that aminoglycosides, a well-known class of antibiotics produced by Streptomyces, are potent inhibitors of phage infection in widely divergent bacterial hosts. We demonstrate that aminoglycosides block an early step of the viral life cycle, prior to genome replication. Phage inhibition was also achieved using supernatants from natural aminoglycoside producers, indicating a broad physiological significance of the antiviral properties of aminoglycosides. Strikingly, we show that acetylation of the aminoglycoside antibiotic apramycin abolishes its antibacterial effect but retains its antiviral properties. Altogether, our study expands the knowledge of aminoglycoside functions, suggesting that aminoglycosides not only are used by their producers as toxic molecules against their bacterial competitors but also could provide protection against the threat of phage predation at the community level. IMPORTANCE Predation by phages is a major driver of bacterial evolution. As a result, elucidating antiphage strategies is crucial from both fundamental and therapeutic standpoints. While protein-mediated defense mechanisms, like restriction-modification systems or CRISPR/Cas, have been extensively studied, much less is known about the potential antiphage activity of small molecules. Focusing on the model bacteria Escherichia coli and Streptomyces venezuelae, our findings revealed significant antiphage properties of aminoglycosides, a major class of translation-targeting antibiotics produced by Streptomyces. Further, we demonstrate that supernatants from natural aminoglycoside producers protect bacteria from phage propagation, highlighting the physiological relevance of this inhibition. Suppression of phage infection by aminoglycosides did not result from the indirect inhibition of bacterial translation, suggesting a direct interaction between aminoglycosides and phage components. This work highlights the molecular versatility of aminoglycosides, which have evolved to efficiently block protein synthesis in bacterial competitors and provide protection against phages.

Phylogenetic Distribution of WhiB- and Lsr2-Type Regulators in Actinobacteriophage Genomes.

Viruses that infect different actinobacterial host species are known as actinobacteriophages. They are composed of highly divergent and mosaic genomes due to frequent gene exchange between their bacterial hosts and related viral species. This is also reflected by the adaptive incorporation of host transcription factors (TFs) into phage regulatory networks. Previous studies discovered Lsr2-type and WhiB-type regulators encoded by actinobacteriophage genomes. However, limited information is available about their distribution, evolution, and impact on host species. In this study, we computationally screened the distribution of known bacterial and phage TFs inside 2951 complete actinobacteriophage genomes and identified 13 different TF domains. Among those, WhiB, Lsr2, MerR, and Cro/CI-like proteins were widespread and found in more than 10% of the analyzed actinobacteriophage genomes. Neighboring genomic context analysis of the whiB and lsr2 loci showed group-specific conservation of gene synteny and potential involvement of these genes in diverse regulatory functions. Both genes were significantly enriched in temperate phages, and the Lsr2-encoding genomes featured an overall lower GC content. Phylogenetic analysis of WhiB and Lsr2 proteins showed the grouping of phage sequences within bacterial clades, suggesting gene acquisition by phages from their bacterial host species or by multiple, independent acquisition events. Overall, our study reports the global distribution of actinobacteriophage regulatory proteins and sheds light on their origin and evolution. IMPORTANCE Actinobacteriophages are viruses that infect bacterial species of the diverse phylum of Actinobacteria. Phages engage in a close relationship with their bacterial host. This is also reflected by the adoption of genetic material from their host and its incorporation into phage regulatory circuits. In this study, we systematically searched the genomes of actinobacteriophages for the presence of transcription factor domains. We show that proteins belonging to the regulator families of WhiB and Lsr2 belong to the most abundant regulatory proteins encoded by actinobacteriophages. Further phylogenetic analysis shed light on their origin and evolution. Altogether, this study provides an important basis for further experimental investigation of their role in the coordination of the phage life cycle and their interaction with the host regulatory network in this important bacterial phylum.

Correction: Hardy et al. Genome Sequence and Characterization of Five Bacteriophages Infecting Streptomyces coelicolor and Streptomyces venezuelae: Alderaan, Coruscant, Dagobah, Endor1 and Endor2. Viruses 2020, 12, 1065.

The authors wish to make the following corrections to this paper [...].

Genome Sequence of the Bacteriophage CL31 and Interaction with the Host Strain Corynebacterium glutamicum ATCC 13032.

In this study, we provide a comprehensive analysis of the genomic features of the phage CL31 and the infection dynamics with the biotechnologically relevant host strain Corynebacterium glutamicum ATCC 13032. Genome sequencing and annotation of CL31 revealed a 45-kbp genome composed of 72 open reading frames, mimicking the GC content of its host strain (54.4%). An ANI-based distance matrix showed the highest similarity of CL31 to the temperate corynephage Φ16. While the C. glutamicum ATCC 13032 wild type strain showed only mild propagation of CL31, a strain lacking the cglIR-cglIIR-cglIM restriction-modification system was efficiently infected by this phage. Interestingly, the prophage-free strain C. glutamicum MB001 featured an even accelerated amplification of CL31 compared to the ∆resmod strain suggesting a role of cryptic prophage elements in phage defense. Proteome analysis of purified phage particles and transcriptome analysis provide important insights into structural components of the phage and the response of C. glutamicum to CL31 infection. Isolation and sequencing of CL31-resistant strains revealed SNPs in genes involved in mycolic acid biosynthesis suggesting a role of this cell envelope component in phage adsorption. Altogether, these results provide an important basis for further investigation of phage-host interactions in this important biotechnological model organism.

Genome Sequence and Characterization of Five Bacteriophages Infecting Streptomyces Coelicolor and Streptomyces Venezuelae: Alderaan, Coruscant, Dagobah, Endor1 and Endor2.

Streptomyces are well-known antibiotic producers, also characterized by a complex morphological differentiation. Streptomyces, like all bacteria, are confronted with the constant threat of phage predation, which in turn shapes bacterial evolution. However, despite significant sequencing efforts recently, relatively few phages infecting Streptomyces have been characterized compared to other genera. Here, we present the isolation and characterization of five novel Streptomyces phages. All five phages belong to the Siphoviridae family, based on their morphology as determined by transmission electron microscopy. Genome sequencing and life style predictions suggested that four of them were temperate phages, while one had a lytic lifestyle. Moreover, one of the newly sequenced phages shows very little homology to already described phages, highlighting the still largely untapped viral diversity. Altogether, this study expands the number of characterized phages of Streptomyces and sheds light on phage evolution and phage-host dynamics in Streptomyces.