Virus satellites drive viral evolution and ecology

Virus satellites are widespread subcellular entities, present both in eukaryotic and in prokaryotic cells. Their modus vivendi involves parasitism of the life cycle of their inducing helper viruses, which assures their transmission to a new host. However, the evolutionary and ecological implications of satellites on helper viruses remain unclear. Here, using staphylococcal pathogenicity islands (SaPIs) as a model of virus satellites, we experimentally show that helper viruses rapidly evolve resistance to their virus satellites, preventing SaPI proliferation, and SaPIs in turn can readily evolve to overcome phage resistance. Genomic analyses of both these experimentally evolved strains as well as naturally occurring bacteriophages suggest that the SaPIs drive the coexistence of multiple alleles of the phage-coded SaPI inducing genes, as well as sometimes selecting for the absence of the SaPI depressing genes. We report similar (accidental) evolution of resistance to SaPIs in laboratory phages used for Staphylococcus aureus typing and also obtain the same qualitative results in both experimental evolution and phylogenetic studies of Enterococcus faecalis phages and their satellites viruses. In summary, our results suggest that helper and satellite viruses undergo rapid coevolution, which is likely to play a key role in the evolution and ecology of the viruses as well as their prokaryotic hosts

Prácticas de laboratorio interdisciplinares de alto nivel científico con alumnos de diferentes grados universitarios guiados por WebQuest AICLE

[ES] Cada vez resulta más importante la colaboración entre expertos de diferentes áreas científicas multidisciplinares. En este trabajo, se han realizado prácticas de laboratorio agrupando alumnos de cuatro grados universitarios del área de biomedicina: Biotecnología, Ciencias del Mar, Veterinaria, Odontología y un grado impartido en inglés: Dentistry. Las asignaturas, que participaron en el estudio fueron: Biorreactores, Cultivos Celulares, Microbiología Marina, Microbiología Veterinaria, Microbiología de Odontología y Microbiology de Dentistry. Se abordó el tema de las síntesis química y por impresión 3D de biomateriales, su caracterización antimicrobiana por tres métodos complementarios (difusión en agar, contacto y formación de biofilm en biorreactor) y repoblación por cultivo con células madre adultas. Se diseñó una WebQuest con las instrucciones, laboratorio virtual y guías de prácticas en formato digital. Con motivo de llevar a cabo un Aprendizaje Integrado de Contenido y de Lenguas Extranjeras (AICLE), la WebQuest fue diseñada en inglés y los participantes realizaron una exposición en inglés al finalizar la experiencia. Las prácticas fueron realizadas en los laboratorios de la Universidad Católica de Valencia y en el Centro de Investigación Príncipe Felipe. Este procedimiento fue evaluado mediante un cuestionario de 14 preguntas, y mediante dos rúbricas para las memorias y exposiciones.[EN] Collaboration between experts from different scientific areas is becoming more and more important. Thus, in this work, transversal laboratory sessions have been carried out by students from four different university bachelor’s degrees in the area of biomedicine: Biotechnology, Marine Sciences, Veterinary, Dentistry and a degree taught in English: Dentistry. The subjects that participated in the study were: bioreactors, cell cultures, marine microbiology, veterinary microbiology and dentistry microbiology. Working teams addressing a scientific topic such as chemical synthesis and 3D printing of biomaterials, their antimicrobial characterization by three complementary methods (diffusion in agar, contact and biofilm formation in bioreactor) and repopulation by adult stem cell culture. A WebQuest was designed with the instructions, virtual laboratory and laboratory sessions guides in digital format. In order to carry out a Content and Language Integrated Learning (CLIL), the WebQuest was designed in English and the participants made a presentation in English at the end of this experience. The laboratory sessions were carried out in the laboratories of the Catholic University of Valencia and in the Príncipe Felipe Research Center. This procedure was evaluated through a questionnaire of 14 questions, and by means of two rubrics used for the reports and expositions.Serrano-Aroca, Á.; Frígols, B.; Martí, M.; Ingresa-Capaccioni, S.; Moreno-Manzano, V. (2019). Prácticas de laboratorio interdisciplinares de alto nivel científico con alumnos de diferentes grados universitarios guiados por WebQuest AICLE. En IN-RED 2019. V Congreso de Innovación Educativa y Docencia en Red. Editorial Universitat Politècnica de València. 141-155. https://doi.org/10.4995/INRED2019.2019.10365OCS14115

Carbon Nanofibers in Pure Form and in Calcium Alginate Composites Films: New Cost-Effective Antibacterial Biomaterials against the Life-Threatening Multidrug-Resistant Staphylococcus epidermidis

Due to the current global health problem of antibiotic resistant recently announced by the World Health Organization, there is an urgent necessity of looking for new alternative antibacterial materials able to treat and impede multidrug-resistant infections which are cost-effective and non-toxic for human beings. In this regard, carbon nanofibers (CNFs) possess currently much lower cost than other carbon nanomaterials, such as graphene oxide, and exhibit excellent chemical, mechanical and electric properties. Furthermore, here, the first report on the antibacterial activity of CNFs was demonstrated. Thus, these nanomaterials, in pure form or incorporated in a minuscule amount into calcium alginate composite films to reduce production costs as much as possible, showed to be new weapons against a globally spreading multidrug-resistant pathogen, the methicillin-resistant Staphylococcus epidermidis (MRSE). This Gram-positive bacterium is becoming one of the most dangerous pathogens, due to its abundance on skin. In this study, these hollow filamentous materials, in direct contact with cells and loaded in the low-cost calcium alginate composite films, showed no cytotoxicity for human keratinocyte HaCaT cells, which render them very promising for biomedical applications. The CNFs used in this work were characterized by Raman spectroscopy and observed by high-resolution transmission electron with energy-disperse X-ray spectroscopy

Carbon Nanomaterials and LED Irradiation as Antibacterial Strategies against Gram-Positive Multidrug-Resistant Pathogens

Background: Due to current antibiotic resistance worldwide, there is an urgent need to find new alternative antibacterial approaches capable of dealing with multidrug-resistant pathogens. Most recent studies have demonstrated the antibacterial activity and non-cytotoxicity of carbon nanomaterials such as graphene oxide (GO) and carbon nanofibers (CNFs). On the other hand, light-emitting diodes (LEDs) have shown great potential in a wide range of biomedical applications. Methods: We investigated a nanotechnological strategy consisting of GO or CNFs combined with light-emitting diod (LED) irradiation as novel nanoweapons against two clinically relevant Gram-positive multidrug-resistant pathogens: methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus epidermidis (MRSE). The cytotoxicity of GO and CNFs was studied in the presence of human keratinocyte HaCaT cells. Results: GO or CNFs exhibited no cytotoxicity and high antibacterial activity in direct contact with MRSE and MRSA cells. Furthermore, when GO or CNFs were illuminated with LED light, the MRSE and MRSA cells lost viability. The rate of decrease in colony forming units from 0 to 3 h, measured per mL, increased to 98.5 ± 1.6% and 95.8 ± 1.4% for GO and 99.5 ± 0.6% and 99.7 ± 0.2% for CNFs. Conclusions: This combined antimicrobial approach opens up many biomedical research opportunities and provides an enhanced strategy for the prevention and treatment of Gram-positive multidrug-resistant infections

Virus Satellites Drive Viral Evolution and Ecology

19 páginas, 4 figuras, 4 tablasVirus satellites are widespread subcellular entities, present both in eukaryotic and in prokaryotic cells. Their modus vivendi involves parasitism of the life cycle of their inducing helper viruses, which assures their transmission to a new host. However, the evolutionary and ecological implications of satellites on helper viruses remain unclear. Here, using staphylococcal pathogenicity islands (SaPIs) as a model of virus satellites, we experimentally show that helper viruses rapidly evolve resistance to their virus satellites, preventing SaPI proliferation, and SaPIs in turn can readily evolve to overcome phage resistance. Genomic analyses of both these experimentally evolved strains as well as naturally occurring bacteriophages suggest that the SaPIs drive the coexistence of multiple alleles of the phage-coded SaPI inducing genes, as well as sometimes selecting for the absence of the SaPI depressing genes. We report similar (accidental) evolution of resistance to SaPIs in laboratory phages used for Staphylococcus aureus typing and also obtain the same qualitative results in both experimental evolution and phylogenetic studies of Enterococcus faecalis phages and their satellites viruses. In summary, our results suggest that helper and satellite viruses undergo rapid coevolution, which is likely to play a key role in the evolution and ecology of the viruses as well as their prokaryotic hostsThis work was supported by grants Consolider-Ingenio CSD2009-00006, BIO2011- 30503-C02-01, ERAnet-Pathogenomics PIM2010EPA-00606 and MR/M003876/1 from the Medical Research Council (UK) to JRP, BIO2010-15424 to AM, and BFU2012-30805 to SFE, all from the Spanish Ministerio de Economía y Competitividad (MINECO), and grant R01AI022159 to RPN and JRP, from the USA National Institutes of Health. AB acknowledges support from the Royal Society, NERC, BBSRC and AXA research fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscriptPeer reviewe

Graphene oxide in zinc alginate films: Antibacterial activity, cytotoxicity, zinc release, water sorption/diffusion, wettability and opacity.

Alginate is considered an exceptional biomaterial due to its hydrophilicity, biocompatibility, biodegradability, nontoxicity and low-cost in comparison with other biopolymers. We have recently demonstrated that the incorporation of 1% graphene oxide (GO) into alginate films crosslinked with Ca2+ cations provides antibacterial activity against Staphylococcus aureus and methicillin-resistant Staphylococcus epidermidis, and no cytotoxicity for human keratinocyte HaCaT cells. However, many other reports in literature have shown controversial results about the toxicity of GO demanding further investigation. Furthermore, the synergic effect of GO with other divalent cations with intrinsic antibacterial and cytotoxic activity such as Zn2+ has not been explored yet. Thus, here, two commercially available sodium alginates were characterised and utilized in the synthesis of zinc alginate films with GO following the same chemical route reported for the calcium alginate/GO composites. The results of this study showed that zinc release, water sorption/diffusion and wettability depended significantly on the type of alginate utilized. Furthermore, Zn2+ and GO produced alginate films with increased water diffusion, wettability and opacity. However, neither the combination of GO with Zn2+ nor the use of different types of sodium alginates modified the antibacterial activity and cytotoxicity of the zinc alginates against these Gram-positive pathogens and human cells respectively

Transfer of the coevolved SaPI islands by the evolved phage mutants^a.

<p>^aThe means of results from three independent experiments are presented. Variation was within 5% in all cases.</p><p>^bTransductants / ml of lysate, using RN4220 as recipient.</p><p>^cThis frequency is typical of transfer by generalized transduction and is not SaPI-specific.</p><p>Transfer of the coevolved SaPI islands by the evolved phage mutants<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005609#t004fn001" target="_blank">^a</a>.</p

SaPI interference in evolved phages.

<p>(A) SaPI interference with phage reproduction. Approximately 10⁸ bacteria were infected with 100 p.f.u. of phage 80α (upper panel) or an evolved 80α derivative carrying mutation in all three SaPI inducers (lower panel), plated on phage bottom agar, and incubated 24 h at 32°C. (B) Induction of SaPIbov1 (left) or SaPIbov2 (right) by evolved 80α phages carrying mutations in the <i>dut</i> or ORF15 genes, respectively. Samples from the different lysogenic strains were isolated 60 min after induction with mitomycin C, separated on agarose and blotted with a SaPIbov1- or SaPIbov2-specific probe. Upper band is ‘bulk’ DNA, including chromosomal, phage and replicating SaPI; lower band is SaPI linear monomers released from phage heads. (C) SaPIbov1 excision and replication after induction of cloned <i>dut</i> genes from different evolved phages. A non-lysogenic derivative of strain RN4220 carrying SaPIbov1 was complemented with plasmids expressing 3xFlag-tagged Dut proteins. One millilitre of each culture (optical density (OD)_540nm = 0.3) was collected and used to prepare standard minilysates, which were resolved on a 0.7% agarose gel, Southern blotted and probed for SaPIbov1 DNA. In these experiments, because no helper phage is present, the excised SaPI DNA appears as covalently closed circular molecules (CCC) rather than the linear monomers that are seen following helper-phage-mediated induction and packaging. The upper panel is a Southern blot probed for SaPIbov1 DNA; the lower panel is a western blot probed with antibody (Sigma) to the Flag tag carried by the proteins.</p