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Abstract Background Transition from planktonic cells to biofilm is mediated by production of adhesion factors, such as extracellular polysaccharides (EPS), and modulated by complex regulatory networks that, in addition to controlling production of adhesion factors, redirect bacterial cell metabolism to the biofilm mode. Results Deletion of the pnp gene, encoding polynucleotide phosphorylase, an RNA processing enzyme and a component of the RNA degradosome, results in increased biofilm formation in Escherichia coli. This effect is particularly pronounced in the E. coli strain C-1a, in which deletion of the pnp gene leads to strong cell aggregation in liquid medium. Cell aggregation is dependent on the EPS poly-N-acetylglucosamine (PNAG), thus suggesting negative regulation of the PNAG biosynthetic operon pgaABCD by PNPase. Indeed, pgaABCD transcript levels are higher in the pnp mutant. Negative control of pgaABCD expression by PNPase takes place at mRNA stability level and involves the 5’-untranslated region of the pgaABCD transcript, which serves as a cis-element regulating pgaABCD transcript stability and translatability. Conclusions Our results demonstrate that PNPase is necessary to maintain bacterial cells in the planktonic mode through down-regulation of pgaABCD expression and PNAG production.

In 1952, Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Over 70 years later, our understanding of bacteriophage structure has grown dramatically, mainly thanks to the cryogenic electron microscopy revolution. In stark contrast, phage genome delivery in prokaryotes remains poorly understood, mainly due to the inherent challenge of studying such a transient and complex process. Here, we review the current literature on viral genome delivery across bacterial cell surfaces. We focus on icosahedral bacterial viruses that we arbitrarily sort into three groups based on the presence and size of a tail apparatus. We inventory the building blocks implicated in genome delivery and critically analyze putative mechanisms of genome ejection. Bacteriophage genome delivery into bacteria is a topic of growing interest, given the renaissance of phage therapy in Western medicine as a therapeutic alternative to face the antibiotic resistance crisis.

DEV is an obligatory lytic Pseudomonas phage of the N4-like genus, recently reclassified as Schitoviridae. The DEV genome encodes 91 ORFs, including a 3398 amino acid virion-associated RNA polymerase (vRNAP). Here, we describe the complete architecture of DEV, determined using a combination of cryo-electron microscopy localized reconstruction, biochemical methods, and genetic knockouts. We built de novo structures of all capsid factors and tail components involved in host attachment. We demonstrate that DEV long tail fibers are essential for infection of Pseudomonas aeruginosa but dispensable for infecting mutants with a truncated lipopolysaccharide devoid of the O-antigen. We determine that DEV vRNAP is part of a three-gene operon conserved in 191 Schitoviridae genomes. We propose these three proteins are ejected into the host to form a genome ejection motor spanning the cell envelope. We posit that the design principles of the DEV ejection apparatus are conserved in all Schitoviridae., (© 2024. The Author(s).)

Coronary artery aneurysms represent a rare pathology (0.2-4.9% of patients undergoing coronary angiography) that may reach considerable size. The clinical presentation is various, manifesting as acute coronary syndrome or, conversely, remaining silent lifelong. We here report the case of an incidental finding by transthoracic echocardiography of a paracardiac mass of considerable size in a patient with vasculopathy that underwent a Bentall procedure for acute aortic dissection 18 years earlier. On thoracic computed tomography angiography, a 62 mm-sized giant aneurysm located in the proximal right coronary artery was evidenced. The optimal treatment of patients affected by coronary artery aneurysms remains debated; therefore, the therapeutic strategy should be individualized considering the etiology, clinical presentation, anatomical characteristics and concomitant presence of obstructive coronary artery disease.

The rise of bacteria resistant to the antibiotics currently in use (multiple drug-resistant, MDR) is a serious problem for patients affected by infections. This situation is even more worrying in the case of chronic bacterial infections, such as those caused by Pseudomonas aeruginosa (Pa), in patients with cystic fibrosis (CF). As an alternative to antibiotic treatments, the use of bacteriophages (phages) to fight bacterial infections has gained increasing interest in the last few years. Phages are viruses that specifically infect and multiply within the bacteria without infecting eukaryotic cells. It is well assumed that phage therapy has a high bacterial specificity, which, unlike antibiotics, should limit the damage to the endogenous microbiome. In addition, phages can kill antibiotic-resistant bacteria and perform self-amplification at the site of the infection.The protocol detailed in this chapter describes how the antimicrobial effect of phages can be studied in vivo in the zebrafish (Danio rerio) model infected with Pa. The same procedure can be applied to test the effectiveness of several different phages killing other bacterial species and for the rapid preclinical testing of phages to be used as personalized medicine., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Importance: In this work, we identified the putative receptors of 16 Pseudomonas phages and evaluated how resistance to phages recognizing different bacterial receptors may affect the virulence. Our findings are relevant for the implementation of phage therapy of Pseudomonas aeruginosa infections, which are difficult to treat with antibiotics. Overall, our results highlight the need to modify natural phages to enlarge the repertoire of receptors exploited by therapeutic phages and suggest that phages using the PAO1-type T4P as receptor may have limited value for the therapy of the cystic fibrosis infection., Competing Interests: The authors declare no conflict of interest.

Polyribonucleotide phosphorylase (PNPase) is a phosphorolytic RNA exonuclease highly conserved throughout evolution. In Escherichia coli, PNPase controls complex phenotypic traits like biofilm formation and growth at low temperature. In human cells, PNPase is located in mitochondria, where it is implicated in the RNA import from the cytoplasm, the mitochondrial RNA degradation and the processing of R-loops, namely stable RNA-DNA hybrids displacing a DNA strand. In this work, we show that the human PNPase (hPNPase) expressed in E. coli causes oxidative stress, SOS response activation and R-loops accumulation. Hundreds of E. coli RNAs are stabilized in presence of hPNPase, whereas only few transcripts are destabilized. Moreover, phenotypic traits typical of E. coli strains lacking PNPase are strengthened in presence of the human enzyme. We discuss the hypothesis that hPNPase expressed in E. coli may bind, but not degrade, the RNA, in agreement with previous in vitro data showing that phosphate concentrations in the range of those found in the bacterial cytoplasm and, more relevant, in the mitochondria, inhibit its activity., (© 2023. The Author(s).)

E217 is a Pseudomonas phage used in an experimental cocktail to eradicate cystic fibrosis-associated Pseudomonas aeruginosa. Here, we describe the structure of the whole E217 virion before and after DNA ejection at 3.1 Å and 4.5 Å resolution, respectively, determined using cryogenic electron microscopy (cryo-EM). We identify and build de novo structures for 19 unique E217 gene products, resolve the tail genome-ejection machine in both extended and contracted states, and decipher the complete architecture of the baseplate formed by 66 polypeptide chains. We also determine that E217 recognizes the host O-antigen as a receptor, and we resolve the N-terminal portion of the O-antigen-binding tail fiber. We propose that E217 design principles presented in this paper are conserved across PB1-like Myoviridae phages of the Pbunavirus genus that encode a ~1.4 MDa baseplate, dramatically smaller than the coliphage T4., (© 2023. The Author(s).)

The chloroplast proteome is a dynamic mosaic of plastid- and nuclear-encoded proteins. Plastid protein homeostasis is maintained through the balance between de novo synthesis and proteolysis. Intracellular communication pathways, including the plastid-to-nucleus signalling and the protein homeostasis machinery, made of stromal chaperones and proteases, shape chloroplast proteome based on developmental and physiological needs. However, the maintenance of fully functional chloroplasts is costly and under specific stress conditions the degradation of damaged chloroplasts is essential to the maintenance of a healthy population of photosynthesising organelles while promoting nutrient redistribution to sink tissues. In this work, we have addressed this complex regulatory chloroplast-quality-control pathway by modulating the expression of two nuclear genes encoding plastid ribosomal proteins PRPS1 and PRPL4. By transcriptomics, proteomics and transmission electron microscopy analyses, we show that the increased expression of PRPS1 gene leads to chloroplast degradation and early flowering, as an escape strategy from stress. On the contrary, the overaccumulation of PRPL4 protein is kept under control by increasing the amount of plastid chaperones and components of the unfolded protein response (cpUPR) regulatory mechanism. This study advances our understanding of molecular mechanisms underlying chloroplast retrograde communication and provides new insights into cellular responses to impaired plastid protein homeostasis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Tadini et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

A strategy that can be applied to the research of new molecules with antibacterial activity is to look for inhibitors of essential bacterial processes within large collections of chemically heterogeneous compounds. The implementation of this approach requires the development of assays aimed at the identification of molecules interfering with specific cell pathways that can also be used in high-throughput analysis of large chemical libraries. Here, we describe a fluorescence-based whole-cell assay in Escherichia coli devised to find inhibitors of the translation initiation pathway. Translation is a complex and essential mechanism. It involves numerous sub-steps performed by factors that are in many cases sufficiently dissimilar in bacterial and eukaryotic cells to be targetable with domain-specific drugs. As a matter of fact, translation has been proven as one of the few bacterial mechanisms pharmacologically tractable with specific antibiotics. The assay described in this updated chapter is tailored to the identification of molecules affecting the first stage of translation initiation, which is the most dissimilar step in bacteria versus mammals. The effect of the compounds under analysis is measured in living cells, thus allowing evaluation of their in vivo performance as inhibitors of translation initiation. Compared with other assays for antibacterials, the major advantages of this screen are its simplicity, high mechanism specificity, and amenability to scaling up to high-throughput analyses., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Pseudomonas phages are increasingly important biomedicines for phage therapy, but little is known about how these viruses package DNA. This paper explores the terminase subunits from the Myoviridae E217, a Pseudomonas-phage used in an experimental cocktail to eradicate P. aeruginosa in vitro and in animal models. We identified the large (TerL) and small (TerS) terminase subunits in two genes ∼58 kbs away from each other in the E217 genome. TerL presents a classical two-domain architecture, consisting of an N-terminal ATPase and C-terminal nuclease domain arranged into a bean-shaped tertiary structure. A 2.05 Å crystal structure of the C-terminal domain revealed an RNase H-like fold with two magnesium ions in the nuclease active site. Mutations in TerL residues involved in magnesium coordination had a dominant-negative effect on phage growth. However, the two ions identified in the active site were too far from each other to promote two-metal-ion catalysis, suggesting a conformational change is required for nuclease activity. We also determined a 3.38 Å cryo-EM reconstruction of E217 TerS that revealed a ring-like decamer, departing from the most common nonameric quaternary structure observed thus far. E217 TerS contains both N-terminal helix-turn-helix motifs enriched in basic residues and a central channel lined with basic residues large enough to accommodate double-stranded DNA. Overexpression of TerS caused a more than a 4-fold reduction of E217 burst size, suggesting a catalytic amount of the protein is required for packaging. Together, these data expand the molecular repertoire of viral terminase subunits to Pseudomonas-phages used for phage therapy., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Multi drug resistant (MDR) bacteria are insensitive to the most common antibiotics currently in use. The spread of antibiotic-resistant bacteria, if not contained, will represent the main cause of death for humanity in 2050. The situation is even more worrying when considering patients with chronic bacterial infections, such as those with Cystic Fibrosis (CF). The development of alternative approaches is essential and novel therapies that combine exogenous and host-mediated antimicrobial action are promising. In this work, we demonstrate that asymmetric phosphatidylserine/phosphatidic acid (PS/PA) liposomes administrated both in prophylactic and therapeutic treatments, induced a reduction in the bacterial burden both in wild-type and cftr -loss-of-function ( cftr -LOF) zebrafish embryos infected with Pseudomonas aeruginosa ( Pa ) PAO1 strain (PAO1). These effects are elicited through the enhancement of phagocytic activity of macrophages. Moreover, the combined use of liposomes and a phage-cocktail (CKΦ), already validated as a PAO1 "eater", improves the antimicrobial effects of single treatments, and it is effective also against CKΦ-resistant bacteria. We also address the translational potential of the research, by evaluating the safety of CKΦ and PS/PA liposomes administrations in in vitro model of human bronchial epithelial cells, carrying the homozygous F508del- CFTR mutation, and in THP-1 cells differentiated into a macrophage-like phenotype with pharmacologically inhibited CFTR. Our results open the way to the development of novel pharmacological formulations composed of both phages and liposomes to counteract more efficiently the infections caused by Pa or other bacteria, especially in patients with chronic infections such those with CF., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cafora, Poerio, Forti, Loberto, Pin, Bassi, Aureli, Briani, Pistocchi and Fraziano.)

Polynucleotide phosphorylase (PNPase) is a phosphorolytic RNA exonuclease highly conserved throughout evolution. Human PNPase (hPNPase) is located in mitochondria and is essential for mitochondrial function and homeostasis. Not surprisingly, mutations in the PNPT1 gene, encoding hPNPase, cause serious diseases. hPNPase has been implicated in a plethora of processes taking place in different cell compartments and involving other proteins, some of which physically interact with hPNPase. This paper reviews hPNPase RNA binding and catalytic activity in relation with the protein structure and in comparison, with the activity of bacterial PNPases. The functions ascribed to hPNPase in different cell compartments are discussed, highlighting the gaps that still need to be filled to understand the physiological role of this ancient protein in human cells.

Cystic Fibrosis (CF), one of the most frequent hereditary diseases due to mutations in the CFTR gene, causes mortality in humans mainly due to infection in the respiratory system. However, besides the massive inflammatory response triggered by chronic bacterial infections, a constitutive pro-inflammatory state associated with the most common CFTR mutations has been reported in paediatric cases before the onset of bacterial colonization. In previous works we isolated and characterized a mix of virulent bacteriophages (phage cocktail) able to efficiently counteract Pseudomonas aeruginosa infection in a zebrafish model with cftr loss-of-function (LOF), but also showing anti-inflammatory effects in zebrafish embryos not infected by bacteria. On these premises, in this work we demonstrated the anti-inflammatory role of the phage cocktail both in the wild-type (WT) and hyper-inflamed cftr LOF zebrafish embryos in terms of reduction of pro-inflammatory markers. We also dissect that only the virion proteinaceous components, but not the phage DNA, are responsible for the immune-modulatory effect and that this action is elicited through the activation of the Toll-like Receptor (TLR) pathway. In the cftr LOF zebrafish embryos, we demonstrated that phages injection significantly reduces neutrophil migration following acute inflammatory induction. The elucidation of the molecular interaction between phages and the cells of vertebrate immune system might open new possibility in their manipulation for therapeutic benefits especially in diseases such as cystic fibrosis, characterized by chronic infection and inflammation., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The funder has no role in data interpretations., (Copyright © 2020. Published by Elsevier B.V.)