Bacteria are under immense evolutionary pressure from their viral invaders-bacteriophages. Bacteria have evolved numerous immune mechanisms, both innate and adaptive, to cope with this pressure. The discovery and exploitation of CRISPR-Cas systems have stimulated a resurgence in the identification and characterization of anti-phage mechanisms. Bacteriophages use an extensive battery of counter-defence strategies to co-exist in the presence of these diverse phage defence mechanisms. Understanding the dynamics of the interactions between these microorganisms has implications for phage-based therapies, microbial ecology and evolution, and the development of new biotechnological tools. Here we review the spectrum of anti-phage systems and highlight their evasion by bacteriophages.

The arms race between bacteria and their phage foes.

(Ph)ighting Phages: How Bacteria Resist Their Parasites

Argonaute proteins constitute a highly diverse family of nucleic acid-guided proteins. They were first discovered in eukaryotes as key proteins in RNA interference systems, but homologous prokaryotic Argonaute proteins (pAgos) have also been found in archaea and bacteria. In this Progress article, we focus on long pAgo variants, a class of pAgos that are involved in nucleic acid-guided host defence against invading nucleic acids, and discuss the potential of pAgos in genome editing.

Prokaryotic Argonaute proteins: novel genome-editing tools?

Prokaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave complementary invading DNA. This activity can be repurposed for programmable DNA cleavage. However, currently characterized DNA-cleaving pAgos require elevated temperatures (≥65°C) for their activity, making them less suitable for applications that require moderate temperatures, such as genome editing. Here, we report the functional and structural characterization of the siDNA-guided DNA-targeting pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo displays a preference for siDNAs that have a deoxyadenosine at the 5'-end and thymidines at nucleotides 2-4. Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37°C). This study demonstrates that certain pAgos are capable of programmable DNA cleavage at moderate temperatures and thereby expands the scope of the potential pAgo-based applications.

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DNA-guided DNA cleavage at moderate temperatures by Clostridium butyricum Argonaute

Members of the conserved Argonaute protein family use small RNA guides to locate their mRNA targets and regulate gene expression and suppress mobile genetic elements in eukaryotes1,2. Argonautes are also present in many bacterial and archaeal species3–5. Unlike eukaryotic proteins, several prokaryotic Argonaute proteins use small DNA guides to cleave DNA, a process known as DNA interference6–10. However, the natural functions and targets of DNA interference are poorly understood, and the mechanisms of DNA guide generation and target discrimination remain unknown. Here we analyse the activity of a bacterial Argonaute nuclease from Clostridium butyricum (CbAgo) in vivo. We show that CbAgo targets multicopy genetic elements and suppresses the propagation of plasmids and infection by phages. CbAgo induces DNA interference between homologous sequences and triggers DNA degradation at double-strand breaks in the target DNA. The loading of CbAgo with locus-specific small DNA guides depends on both its intrinsic endonuclease activity and the cellular double-strand break repair machinery. A similar interaction was reported for the acquisition of new spacers during CRISPR adaptation, and prokaryotic genomes that encode Ago nucleases are enriched in CRISPR–Cas systems. These results identify molecular mechanisms that generate guides for DNA interference and suggest that the recognition of foreign nucleic acids by prokaryotic defence systems involves common principles. Argonaute protein from the bacterium C. butyricum targets multicopy genetic elements and functions in the suppression of plasmid and phage propagation, and there appears to be a DNA-mediated immunity pathway in prokaryotes.

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DNA targeting and interference by a bacterial Argonaute nuclease.

Prokaryotic Argonaute proteins acquire guide strands derived from invading or mobile genetic elements, via an unknown pathway, to direct guide-dependent cleavage of foreign DNA. Here, we report that Argonaute from the archaeal organism Methanocaldococcus jannaschii (MjAgo) possesses two modes of action: the canonical guide-dependent endonuclease activity and a non-guided DNA endonuclease activity. The latter allows MjAgo to process long double-stranded DNAs, including circular plasmid DNAs and genomic DNAs. Degradation of substrates in a guide-independent fashion primes MjAgo for subsequent rounds of DNA cleavage. Chromatinized genomic DNA is resistant to MjAgo degradation, and recombinant histones protect DNA from cleavage in vitro. Mutational analysis shows that key residues important for guide-dependent target processing are also involved in guide-independent MjAgo function. This is the first characterization of guide-independent cleavage activity for an Argonaute protein potentially serving as a guide biogenesis pathway in a prokaryotic system.

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Guide-independent DNA cleavage by archaeal Argonaute from Methanocaldococcus jannaschii.

Investigating interactions of glia cells and synapses during development and in adulthood is the focus of several research programmes which aim at understanding the neurobiology of brain physiological and pathological processes. Both glia-specific released and membrane-bound proteins play essential roles in the development, maintenance and functionality of synaptic connections. Alterations in synaptic contacts in specific brain areas are hallmarks of several brain diseases, such as major depressive disorder, autism spectrum disorder and schizophrenia. Thus, a deeper knowledge about putative astrocyte dysfunctions which might affect the synaptic compartment is warranted to improve treatment options. Here, we present the latest advances about the role of glia cells in orchestrating the arrangement of synapses and neuronal networks in physiological and pathological states. We specifically focus on the role of astrocytes in the phagocytosis of neuronal synapses as a novel mechanism which drives the refinement of neuronal circuits and might be affected in pathological conditions. Finally, we propose this astrocyte-dependent mechanism as a putative alternative target of pharmacological interventions for the treatment of brain disorders.

Luisa Egert

Papers

Guide-independent DNA cleavage by archaeal Argonaute from Methanocaldococcus jannaschii.

Astrocytic-neuronal crosstalk gets jammed: Alternative perspectives on the onset of neuropsychiatric disorders