Corrigendum to “Cytokinesis and cancer” [FEBS Lett. 584 (2010) 2652–2661]

Cytokinesis is the final stage of cell division during which the two daughter cells separate completely. Although less well understood than some of the earlier phases of the cell cycle, recent discoveries have shed light on the mechanisms that orchestrate this process, including cleavage furrow formation, midbody maturation and abscission. One of the reasons why research on cytokinesis has been attracting increasing attention is the concept that failure of this process in mammals is associated with carcinogenesis. In this minireview, we will discuss the possible links between cytokinesis and cancer, and highlight key mechanisms that connect these processes

Genetically modified bacteriophages

Phages or bacteriophages, viruses that infect and replicate inside bacteria, are the most abundant microorganisms on earth. The realization that antibiotic resistance poses a substantial risk to the world’s health and global economy is revitalizing phage therapy as a potential solution. The increasing ease by which phage genomes can be modified, owing to the influx of new technologies, has led to an expansion of their natural capabilities, and a reduced dependence on phage isolation from environmental sources. This review will discuss the way synthetic biology has accelerated the construction of genetically modified phages and will describe the wide range of their applications. It will further provide insight into the societal and economic benefits that derive from the use of recombinant phages in various sectors, from health to biodetection, biocontrol and the food industry

A Review of Using Mathematical Modeling to Improve Our Understanding of Bacteriophage, Bacteria, and Eukaryotic Interactions

Phage therapy, the therapeutic usage of viruses to treat bacterial infections, has many theoretical benefits in the ‘post antibiotic era’. Nevertheless, there are currently no approved mainstream phage therapies. One reason for this is a lack of understanding of the complex interactions between bacteriophage, bacteria and eukaryotic hosts. These three-component interactions are complex, with non-linear or synergistic relationships, anatomical barriers and genetic or phenotypic heterogeneity all leading to disparity between performance and efficacy in in vivo versus in vitro environments. Realistic computer or mathematical models of these complex environments are a potential route to improve the predictive power of in vitro studies and to streamline lab work. Here, we review the current status of mathematical modelling and highlight that data on mutational stochasticity, time delays and population densities could be critical in the development of realistic phage therapy models. With this in mind, we aim to inform and encourage the collaboration and sharing of knowledge and expertise between microbiologists and theoretical modellers, smoothing the road to regulatory approval and widespread use of phage therapy

Engineered K1F bacteriophages kill intracellular Escherichia coli K1 in human epithelial cells

Bacterial infections can be treated with bacteriophages that show great specificity towards their bacterial host and can be genetically modified for different applications. However, whether and how bacteriophages can kill intracellular bacteria in human cells remains elusive. Here, using CRISPR/Cas selection, we have engineered a fluorescent bacteriophage specific for E. coli K1, a nosocomial pathogen responsible for urinary tract infections, neonatal meningitis and sepsis. By confocal and live microscopy, we show that engineered bacteriophages K1F-GFP and E. coli EV36-RFP bacteria displaying the K1 capsule, enter human cells via phagocytosis. Importantly, we show that bacteriophage K1F-GFP efficiently kills intracellular E. coli EV36-RFP in T24 human urinary bladder epithelial cells. Finally, we provide evidence that bacteria and bacteriophages are degraded by LC3-associated phagocytosis and xenophagy

Bacteriophage K1F targets Escherichia coli K1 in cerebral endothelial cells and influences the barrier function

Bacterial neonatal meningitis results in high mortality and morbidity rates for those affected. Although improvements in diagnosis and treatment have led to a decline in mortality rates, morbidity rates have remained relatively unchanged. Bacterial resistance to antibiotics in this clinical setting further underlines the need for developing other technologies, such as phage therapy. We exploited an in vitro phage therapy model for studying bacterial neonatal meningitis based on Escherichia coli (E. coli) EV36, bacteriophage (phage) K1F and human cerebral microvascular endothelial cells (hCMECs). We show that phage K1F is phagocytosed and degraded by constitutive- and PAMP-dependent LC3-assisted phagocytosis and does not induce expression of inflammatory cytokines TNFα, IL-6, IL-8 or IFNβ. Additionally, we observed that phage K1F temporarily decreases the barrier resistance of hCMEC cultures, a property that influences the barrier permeability, which could facilitate the transition of immune cells across the endothelial vessel in vivo. Collectively, we demonstrate that phage K1F can infect intracellular E. coli EV36 within hCMECs without themselves eliciting an inflammatory or defensive response. This study illustrates the potential of phage therapy targeting infections such as bacterial neonatal meningitis and is an important step for the continued development of phage therapy targeting antibiotic-resistant bacterial infections generally

Microencapsulation of Enteric Bacteriophages in a pH-Responsive Solid Oral Dosage Formulation Using a Scalable Membrane Emulsification Process

A scalable low-shear membrane emulsification process was used to produce microencapsulated Escherichia coli-phages in a solid oral dosage form. Uniform pH-responsive composite microparticles (mean size ~100 µm) composed of Eudragit® S100 and alginate were produced. The internal microstructure of the gelled microcapsules was studied using ion-milling and imaging, which showed that the microparticles had a solid internal core. The microencapsulation process significantly protected phages upon prolonged exposure to a simulated gastric acidic environment. Encapsulated phages that had been pre-exposed to simulated gastric acid were added to actively growing bacterial cells using in vitro cell cultures and were found to be effective in killing E. coli. Encapsulated phages were also shown to be effective in killing actively growing E. coli in the presence of human epithelial cells. Confocal microscopy images showed that the morphology of encapsulated phage-treated epithelial cells was considerably better than controls without phage treatment. The encapsulated phages were stable during refrigerated storage over a four-week period. The process of membrane emulsification is highly scalable and is a promising route to produce industrial quantities of pH-responsive oral solid dosage forms suitable for delivering high titres of viable phages to the gastrointestinal tract

Investigating bacteriophages targeting the opportunistic pathogen Acinetobacter baumannii

The multi-drug resistance of the opportunistic pathogen Acinetobacter baumannii is of growing concern, with many clinical isolates proving to be resistant to last resort as well as front line antibiotic treatments. The use of bacteriophages is an attractive alternative to controlling and treating this emerging nosocomial pathogen. In this study, we have investigated bacteriophages collected from hospital wastewater in Thailand and we have explored their activity against clinical isolates of A. baumannii. Bacteriophage vB_AbaM_PhT2 showed 28% host range against 150 multidrug resistant (MDR) isolates and whole genome sequencing did not detect any known virulence factors or antibiotic resistance genes. Purified vB_AbaM_PhT2 samples had endotoxin levels below those recommended for preclinical trials and were not shown to be directly cytotoxic to human cell lines in vitro. The treatment of human brain and bladder cell lines grown in the presence of A. baumannii with this bacteriophage released significantly less lactate dehydrogenase compared to samples with no bacteriophage treatment, indicating that vB_AbaM_PhT2 can protect from A. baumannii induced cellular damage. Our results have also indicated that there is synergy between this bacteriophage and the end line antibiotic colistin. We therefore propose bacteriophage vB_AbaM_PhT2 as a good candidate for future research and for its potential development into a surface antimicrobial for use in hospitals. View Full-Tex

Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain

p62 has been proposed to mark ubiquitinated protein bodies for autophagic degradation. We report that the Drosophila melanogaster p62 orthologue, Ref(2)P, is a regulator of protein aggregation in the adult brain. We demonstrate that Ref(2)P localizes to age-induced protein aggregates as well as to aggregates caused by reduced autophagic or proteasomal activity. A similar localization to protein aggregates is also observed in D. melanogaster models of human neurodegenerative diseases. Although atg8a autophagy mutant flies show accumulation of ubiquitin- and Ref(2)P-positive protein aggregates, this is abrogated in atg8a/ref(2)P double mutants. Both the multimerization and ubiquitin binding domains of Ref(2)P are required for aggregate formation in vivo. Our findings reveal a major role for Ref(2)P in the formation of ubiquitin-positive protein aggregates both under physiological conditions and when normal protein turnover is inhibited

Anionic synthietic polymers prevent bacteriophage infection

Bioprocessing and biotechnology exploit microorganisms (such as bacteria) for the production of chemicals, biologics, therapies, and food. A major unmet challenge is that bacteriophage (phage) contamination compromises products and necessitates shut-downs and extensive decontamination using nonspecific disinfectants. Here we demonstrate that poly(acrylic acid) prevents phage-induced killing of bacterial hosts, prevents phage replication, and that induction of recombinant protein expression is not affected by the presence of the polymer. Poly(acrylic acid) was more active than poly(methacrylic acid), and poly(styrenesulfonate) had no activity showing the importance of the carboxylic acids. Initial evidence supported a virustatic, not virucidal, mechanism of action. This simple, low-cost, mass-produced additive offers a practical, scalable, and easy to implement solution to reduce phage contamination

Polymer-mediated cryopreservation of bacteriophages

Bacteriophages (phages, bacteria-specific viruses) have biotechnological and therapeutic potential. To apply phages as pure or heterogeneous mixtures, it is essential to have a robust mechanism for transport and storage, with different phages having very different stability profiles across storage conditions. For many biologics, cryopreservation is employed for long-term storage and cryoprotectants are essential to mitigate cold-induced damage. Here, we report that poly(ethylene glycol) can be used to protect phages from cold damage, functioning at just 10 mg·mL–1 (∼1 wt %) and outperforms glycerol in many cases, which is a currently used cryoprotectant. Protection is afforded at both −20 and −80 °C, the two most common temperatures for frozen storage in laboratory settings. Crucially, the concentration of the polymer required leads to frozen solutions at −20 °C, unlike 50% glycerol (which results in liquid solutions). Post-thaw recoveries close to 100% plaque-forming units were achieved even after 2 weeks of storage with this method and kill assays against their bacterial host confirmed the lytic function of the phages. Initial experiments with other hydrophilic polymers also showed cryoprotection, but at this stage, the exact mechanism of this protection cannot be concluded but does show that water-soluble polymers offer an alternative tool for phage storage. Ice recrystallization inhibiting polymers (poly(vinyl alcohol)) were found to provide no additional protection, in contrast to their ability to protect proteins and microorganisms which are damaged by recrystallization. PEG’s low cost, solubility, well-established low toxicity/immunogenicity, and that it is fit for human consumption at the concentrations used make it ideal to help translate new approaches for phage therapy