Control of bacterial anti-phage signaling by a WYL domain transcription factor

Bacteria use diverse immune systems to defend themselves from ubiquitous viruses termed bacteriophages (phages). Many anti-phage systems function by abortive infection to kill a phage-infected cell, raising the question of how these systems are regulated to avoid activation and cell killing outside the context of infection. Here, we identify a transcription factor associated with the widespread CBASS bacterial immune system, that we term CapW. CapW forms a homodimer and binds a palindromic DNA sequence in the CBASS promoter region. Two crystal structures of CapW reveal how the protein switches from a DNA binding-competent state to a ligand-bound state that cannot bind DNA due to misalignment of dimer-related DNA binding domains. We show that CapW strongly represses CBASS gene expression in uninfected cells, and that CapW disruption likely results in toxicity due to uncontrolled CBASS expression. Our results parallel recent findings with BrxR, a transcription factor associated with the BREX anti-phage system, and suggest that CapW and BrxR are the founding members of a family of universal anti-phage signaling proteins.

A short prokaryotic argonaute cooperates with membrane effector to confer antiviral defense

SummaryArgonaute (Ago) proteins are widespread nucleic acid-guided enzymes that recognize targets through complementary base pairing. While in eukaryotes Agos are involved in RNA silencing, the functions of prokaryotic Agos (pAgos) remain largely unknown. In particular, a clade of truncated and catalytically inactive pAgos (short pAgos) lacks characterization. Here, we reveal that a short pAgo protein in Sulfolobus islandicus, together with its two genetically associated proteins, Aga1 and Aga2, provide robust antiviral protection via abortive infection. Aga2 is a membrane-associated toxic effector that binds anionic phospholipids via a basic pocket, which is essential for its cell killing ability. Ago and Aga1 form a stable complex that exhibits RNA-directed nucleic acid recognition ability and directly interacts with Aga2, pointing to an immune sensing mechanism. Together, our results highlight the cooperation between pAgos and their widespread associated proteins, suggesting an uncharted diversity of pAgo-derived immune systems that await to be discovered.

UG/Abi: a highly diverse family of prokaryotic reverse transcriptases associated with defense functions

Reverse transcriptases (RTs) are enzymes capable of synthesizing DNA using RNA as a template. Within the last few years, a burst of research has led to the discovery of novel prokaryotic RTs with diverse antiviral properties, such as DRTs (Defense-associated RTs), which belong to the so-called group of unknown RTs (UG) and are closely related to the Abortive Infection system (Abi) RTs. In this work, we performed a systematic analysis of UG and Abi RTs, increasing the number of UG/Abi members up to 42 highly diverse groups, most of which are predicted to be functionally associated with other gene(s) or domain(s). Based on this information, we classified these systems into three major classes. In addition, we reveal that most of these groups are associated with defense functions and/or mobile genetic elements, and demonstrate the antiphage role of four novel groups. Besides, we highlight the presence of one of these systems in novel families of human gut viruses infecting members of the Bacteroidetes and Firmicutes phyla. This work lays the foundation for a comprehensive and unified understanding of these highly diverse RTs with enormous biotechnological potential.

Cell wall deficiency as an escape mechanism from phage infection

The cell wall plays a central role in protecting bacteria from some environmental stresses, but not against all. In fact, in some cases, an elaborate cell envelope may even render the cell more vulnerable. For example, it contains molecules or complexes that bacteriophages recognize as the first step of host invasion, such as proteins and sugars, or cell appendages such as pili or flagella. In order to counteract phages, bacteria have evolved multiple escape mechanisms, such as restriction-modification, abortive infection, CRISPR/Cas systems or phage inhibitors. In this perspective review, we present the hypothesis that bacteria may have additional means to escape phage attack. Some bacteria are known to be able to shed their cell wall in response to environmental stresses, yielding cells that transiently lack a cell wall. In this wall-less state, the bacteria may be temporarily protected against phages, since they lack the essential entities that are necessary for phage binding and infection. Given that cell wall deficiency can be triggered by clinically administered antibiotics, phage escape could be an unwanted consequence that limits the use of phage therapy for treating stubborn infections.

Microbial Arsenal of Antiviral Defenses. Part II

Bacteriophages or phages are viruses that infect bacterial cells (for the scope of this review we will also consider viruses that infect Archaea). The constant threat of phage infection is a major force that shapes evolution of microbial genomes. To withstand infection, bacteria had evolved numerous strategies to avoid recognition by phages or to directly interfere with phage propagation inside the cell. Classical molecular biology and genetic engineering had been deeply intertwined with the study of phages and host defenses. Nowadays, owing to the rise of phage therapy, broad application of CRISPR-Cas technologies, and development of bioinformatics approaches that facilitate discovery of new systems, phage biology experiences a revival. This review describes variety of strategies employed by microbes to counter phage infection. In the first part defense associated with cell surface, roles of small molecules, and innate immunity systems relying on DNA modification were discussed. The second part focuses on adaptive immunity systems, abortive infection mechanisms, defenses associated with mobile genetic elements, and novel systems discovered in recent years through metagenomic mining.

Monocytes and macrophages, targets of SARS-CoV-2: the clue for Covid-19 immunoparalysis

Background Covid-19 clinical expression is pleiomorphic, severity is related to age and comorbidities such as diabetes and hypertension, and pathophysiology involves aberrant immune activation and lymphopenia. We wondered if the myeloid compartment was affected during Covid-19 and if monocytes and macrophages could be infected by SARS-CoV-2. Methods Monocytes and monocyte-derived macrophages from Covid-19 patients and controls were infected with SARS-CoV-2, and extensively investigated with immunofluorescence, viral RNA extraction and quantification, total RNA extraction followed by reverse transcription and q-PCR using specific primers, supernatant cytokines (IL-10, TNF-α, IL-1β, IFN-β, TGF-β1 and IL-6), flow cytometry. The effect of M1- versus M2-type or no polarization prior to infection was assessed. Results SARS-CoV-2 efficiently infected monocytes and MDMs but their infection is abortive. Infection was associated with immunoregulatory cytokines secretion and the induction of a macrophagic specific transcriptional program characterized by the upregulation of M2-type molecules. In vitro polarization did not account for permissivity to SARS-CoV-2, since M1- and M2-type MDMs were similarly infected. In Covid-19 patients, monocytes exhibited lower counts affecting all subsets, decreased expression of HLA-DR, and increased expression of CD163, irrespective of severity. Conclusion SARS-CoV-2 drives monocytes and macrophages to induce host immunoparalysis for the benefit of Covid-19 progression.

Prophage-encoded hotspots of bacterial immune systems

The arms race between bacteria and phages led to the emergence of a variety of genetic systems used by bacteria to defend against viral infection, some of which were repurposed as powerful biotechnological tools. While numerous defense systems have been identified in genomic regions termed defense islands, it is believed that many more remain to be discovered. Here, we show that P2- like prophages and their P4-like satellites have genomic hotspots that represent a significant source of novel anti-phage systems. We validate the defense activity of 14 systems spanning various protein domains and describe PARIS, an abortive infection system triggered by a phage-encoded anti-restriction protein. Immunity hotspots are present across prophages of distant bacterial species, highlighting their biological importance in the competition between bacteria and phages.

Antitoxin ε Reverses Toxin ζ-Facilitated Ampicillin Dormants

Toxin-antitoxin (TA) modules are ubiquitous in bacteria, but their biological importance in stress adaptation remains a matter of debate. The inactive ζ-ε2-ζ TA complex is composed of one labile ε2 antitoxin dimer flanked by two stable ζ toxin monomers. Free toxin ζ reduces the ATP and GTP levels, increases the (p)ppGpp and c-di-AMP pool, inactivates a fraction of uridine diphosphate-N-acetylglucosamine, and induces reversible dormancy. A small subpopulation, however, survives toxin action. Here, employing a genetic orthogonal control of ζ and ε levels, the fate of bacteriophage SPP1 infection was analyzed. Toxin ζ induces an active slow-growth state that halts SPP1 amplification, but it re-starts after antitoxin expression rather than promoting abortive infection. Toxin ζ-induced and toxin-facilitated ampicillin (Amp) dormants have been revisited. Transient toxin ζ expression causes a metabolic heterogeneity that induces toxin and Amp dormancy over a long window of time rather than cell persistence. Antitoxin ε expression, by reversing ζ activities, facilitates the exit of Amp-induced dormancy both in rec+ and recA cells. Our findings argue that an unexploited target to fight against antibiotic persistence is to disrupt toxin-antitoxin interactions.