Monitoring physiological changes in haloarchaeal cell during virus release

The slow rate of adsorption and non-synchronous release of some archaeal viruses have hindered more thorough analyses of the mechanisms of archaeal virus release. To address this deficit, we utilized four viruses that infect Haloarcula hispanica that represent the four virion morphotypes currently known for halophilic euryarchaeal viruses: (1) icosahedral internal membrane-containing SH1; (2) icosahedral tailed HHTV-1; (3) spindle-shaped His1; and (4) pleomorphic His2. To discern the events occurring as the progeny viruses exit, we monitored culture turbidity, as well as viable cell and progeny virus counts of infected and uninfected cultures. In addition to these traditional metrics, we measured three parameters associated with membrane integrity: the binding of the lipophilic anion phenyldicarbaundecaborane, oxygen consumption, and both intra- and extra-cellular ATP levels.Peer reviewe

The bacterial cell envelope as delimiter of anti-infective bioavailability - An in vitro permeation model of the Gram-negative bacterial inner membrane.

Gram-negative bacteria possess a unique and complex cell envelope, composed of an inner and outer membrane separated by an intermediate cell wall-containing periplasm. This tripartite structure acts intrinsically as a significant biological barrier, often limiting the permeation of anti-infectives, and so preventing such drugs from reaching their target. Furthermore, identification of the specific permeation-limiting envelope component proves difficult in the case of many anti-infectives, due to the challenges associated with isolation of individual cell envelope structures in bacterial culture. The development of an in vitro permeation model of the Gram-negative inner membrane, prepared by repeated coating of physiologically-relevant phospholipids on Transwell(®) filter inserts, is therefore reported, as a first step in the development of an overall cell envelope model. Characterization and permeability investigations of model compounds as well as anti-infectives confirmed the suitability of the model for quantitative and kinetically-resolved permeability assessment, and additionally confirmed the importance of employing bacteria-specific base materials for more accurate mimicking of the inner membrane lipid composition - both advantages compared to the majority of existing in vitro approaches. Additional incorporation of further elements of the Gram-negative bacterial cell envelope could ultimately facilitate model application as a screening tool in anti-infective drug discovery or formulation development

The ζ Toxin Induces a Set of Protective Responses and Dormancy

The ζε module consists of a labile antitoxin protein, ε, which in dimer form (ε2) interferes with the action of the long-living monomeric ζ phosphotransferase toxin through protein complex formation. Toxin ζ, which inhibits cell wall biosynthesis and may be bactericide in nature, at or near physiological concentrations induces reversible cessation of Bacillus subtilis proliferation (protective dormancy) by targeting essential metabolic functions followed by propidium iodide (PI) staining in a fraction (20–30%) of the population and selects a subpopulation of cells that exhibit non-inheritable tolerance (1–5×10−5). Early after induction ζ toxin alters the expression of ∼78 genes, with the up-regulation of relA among them. RelA contributes to enforce toxin-induced dormancy. At later times, free active ζ decreases synthesis of macromolecules and releases intracellular K+. We propose that ζ toxin induces reversible protective dormancy and permeation to PI, and expression of ε2 antitoxin reverses these effects. At later times, toxin expression is followed by death of a small fraction (∼10%) of PI stained cells that exited earlier or did not enter into the dormant state. Recovery from stress leads to de novo synthesis of ε2 antitoxin, which blocks ATP binding by ζ toxin, thereby inhibiting its phosphotransferase activity

Entrée de bactériophage SPP1 dans la cellule hôte

Les quatre étapes principales d'infection des bactéries par leurs virus sont (i) la reconnaissance spécifique de la cellule hôte et l'entrée du génome dans le cytoplasme,(ii) la réplication du génome viral, (iii) l'assemblage des particules virales, et (iv) leur relâchement, menant dans la plupart des cas à la lyse de la cellule. Bien que la description des étapes individuelles du cycle viral a été relativement bien établie, les détails de comment d'ADN viral chemine du virion jusqu au cytoplasme de la bactérie hôte et de comment l'environnement cellulaire participe au processus restent mal compris.La première étape de l infection est la reconnaissance d un récepteur à la surface de la bactérie hôte par la machinerie d adsorption du phage. Les barrières que l agent infectieux doit franchir par la suite sont la membrane externe de la bactérie Gram-negative, la paroi cellulaire et la membrane cytoplasmique. Ceci implique une dégradation localisée de la paroi et le cheminement de l ADN à travers un pore dans la membrane. L ADN linéaire se circularise normalement dans le cytoplasme et il est répliqué par la suite. On a utilisé le bactériophage SPP1 qui infecte la bactérie Gram-positive Bacillus subtilis comme modèle d étude pour disséquer ces différentes étapes clés pour le démarrage de l infection virale. Dans ce travail de thèse les conditions d infection et d acquisition de données pour suivre en temps réel la dépolarisation de la membrane cellulaire de B. subtilis lors de l infection par SPP1 ont été mis au point. Il est montré que le démarrage de l infection déclenche une dépolarisation très rapide de la membrane cytoplasmique.Le potentiel de membrane n est plus rétablit pendant toute la durée du cycle d'infection. Ce changement du potentiel de membrane au début de l infection dépend de la présence du récepteur YueB. L amplitude de la dépolarisation dépend du nombre de particules virales infectieuses présentes et de la concentration du récepteur YueB à la surface de la bactérie hôte. L interaction du phage avec le récepteur YueB conduit à l interaction irréversible et à l'éjection de l ADN de SPP1. Pour établir si c est l interaction avec YueB ou le début de l entrée de l ADN qui conduit à la dépolarisation de la membrane on a utilisé des phages SPP1 éclates par EDTA qui adsorbent normalement à B. subtilis mais qui n avaient plus leur ADN. Les résultats obtenus ont montré que la dépolarisation requiert l interaction du virus intacte avec le récepteur YueB. Des concentrations sous-millimolaire de Ca2+ sont nécessaires et suffisantes pour SPP1 liaison réversible à l'enveloppe d'hôte et donc de déclencher la dépolarisation.La cinétique d entrée de l ADN du bactériophage SPP1 dans la bactérie Bacillus subtilis a été suivie en temps réel par microscopie de fluorescence. On a mis au point une méthode de microscopie pour visualiser des particules virales marquées avec des quantum dots ce qui permit de démontrer que ces particules se fixent préférentiellement aux pôles des bacilli. L immuno-marquage du récepteur de SPP1,la protéine YueB, a montré que celle-ci a une organisation ponctuée à la surface de B.subtilis et se concentre particulièrement aux extrémités de la bactérie. Cette localisation particulière du phage sur la surface de la cellule hôte corrèle avec l observation que l ADN viral rentre dans le cytoplasme (<2 min) et se réplique dans des foci situés dans la plupart des cas à proximité des pôles de B. subtilis. L étude spatio-temporelle de l interaction de SPP1 avec son hôte Gram-positive montre que le virus cible des régions spécifiques de la bactérie pour son entrée et pour sa réplication. Transfert d'ADN dans le cytoplasme dépend des concentrations millimolaires de Ca2+. Un modèle décrivant les événements précoces de l'infection bactériophage SPP1 est présenté.The four main steps of bacterial viruses (bacteriophages) lytic infection are (i) specific recognition and genome entry into the host bacterium, (ii) replication of the viral genome, (iii) assembly of viral particles, and (iv) their release, leading in most cases to cell lysis. Although the course of individual steps of the viral infection cycle has been relatively well established, the details of how viral DNA transits from the virion to the host cytoplasm and of how the cellular environment catalyzes and possibly organizes the entire process remain poorly understood.Tailed bacteriophages are by far the most abundant viruses that infect Eubacteria. The first event in their infection is recognition of a receptor on the surface of host bacterium by the phage adsorption machinery. The barriers that the infectious particle overcomes subsequently are the cell wall and the cytoplasmic membrane of bacteria. This implies a localized degradation of the wall and the flow of its double stranded DNA (dsDNA) through a hydrophilic pore in the membrane. The lineards DNA molecule is most frequently circularized in the cytoplasm followed by its replication. In this study we used bacteriophage SPP1 that infects the Gram-positive bacterium Bacillus subtilis as a model system to dissect the different steps leading to transfer of the phage genome from the viral capsid to the host cell cytoplasm.normally to B. subtilis but do not trigger depolarization of the CM. Attachment of intact SPP1 particles is thus required for phage-induced depolarization.The beginning of B. subtilis infection by bacteriophage SPP1 was followed inspace and time. The position of SPP1 binding at the cell surface was imaged by fluorescence microscopy using virus particles labeled with "quantum dots". We found that SPP1 reversible adsorption occurs preferentially at the cell poles. This initial binding facilitates irreversible adsorption to the SPP1 phage receptor protein YueB,which is encoded by a putative type VII secretion system gene cluster.Immunostaining and YueB GFP fusion showed that the phage receptor protein YueB is found over the entire cell surface. It concentrates at the bacterial poles too,and displays a punctate distribution over the sidewalls. The dynamics of SPP1 DNA entry and replication was visualised in real time by assaying specific binding of a fluorescent protein to tandem sequences present in the SPP1 genome. During infection, most of the infecting phages DNA entered and replicated near the bacterial poles in a defined focus. Therefore, SPP1 assembles a replication factory at a specific location in the host cell cytoplasm. DNA delivery to the cytoplasm depends on millimolar concentrations of Ca2+ allowing uncoupling it from the precedent steps of SPP1 adsorption to the cell envelope and CM depolarization that require only micromolar amounts of this divalent cation. A model describing the early events of bacteriophage SPP1 infection is presented.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Influence of the Metabolic Activity of Microorganisms on Disinfection Efficiency of the Visible Light and P25 TiO2 Photocatalyst

The beneficial photocatalytic properties of UV light activated TiO2 powder are well-known and have been demonstrated with various pollutants and pathogens. However, traditionally observed photocatalytic activity of visible light activated pristine TiO2 is insignificant but there are a few studies which have reported that under some specific conditions commercially available TiO2 powder could at least partially disinfect microorganisms even under visible light. To better understand this phenomenon, in the current study we focused on bacteria response to the treatment by visible light and P25 TiO2 powder. More specifically, we analyzed the relationship between the bacteria viability, outer membrane permeability, metabolism, and its capacity to generate intracellular reactive oxygen species. During the study we assayed the viability of treated bacteria by the spread plate technique and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction method. Changes in bacterial outer membrane permeability were determined by measuring the fluorescence of N-phenyl-1-naphthylamine (NPN). To detect intracellular reactive oxygen species formation, the fluorescence of dichlorodihydrofluorescein diacetate (DCFH-DA) was assayed. Results of our study indicated that TiO2 and wide spectrum visible light irradiation damaged the integrity of the outer membrane and caused oxidative stress in the metabolizing bacteria. When favorable conditions were created, these effects added up and unexpectedly high bacterial inactivation was achieved

Evaluation of the efficiency of synthesized efflux pump inhibitors on salmonella enterica ser. typhimurium cells

Multidrug efflux pump inhibitors have a great potential as pharmacological agents that increase the drug susceptibility of bacterial pathogens. Our study was focused on the synthesis and evaluation of the efficiency of resistance\u2013nodulation\u2013division (RND) family efflux pump inhibitors. The efficiency of these inhibitors was investigated on Salmonella enterica ser. typhimurium cells using tetraphenylphosphonium (TPP+) and ethidium cations as the efflux pump substrates. Results of our study indicated that efficiency of the inhibitors depends on the cell outer membrane permeability and method of the assay used. Temperature of the incubation medium and a solvent of the inhibitor used have only minor effect on results of the assay

C-TiO₂+Ni and ZnO+Ni Magnetic Photocatalyst Powder Synthesis by Reactive Magnetron Sputtering Technique and Their Application for Bacteria Inactivation

In the current study, a bi-layered magnetic photocatalyst powder consisting of a Ni layer on one side and carbon-doped TiO2 or ZnO photocatalyst layers on the other side was synthesized by magnetron sputtering technique. SEM, XRD, and XPS analysis of powders revealed that the photocatalytic TiO2 layer had a mixed anatase-rutile structure, was doped by carbon to approximately 3 at. % and had a fraction of Ti(III) oxide. Meanwhile, the ZnO layer was crystalized in a wurtzite structure and had a considerable number of intrinsic defects, which are useful for visible light photocatalysis. The activity of magnetic photocatalyst powder was tested by photocatalytic bleaching of dyes, as well as performing photocatalytic inactivation of Salmonella bacteria under UV and visible light irradiation. It was observed, that C-TiO2+Ni magnetic photocatalyst had relatively high and stable activity under both light sources (for five consecutive cycles dye degradation reached approximately 95%), but ZnO+Ni was generally lacking in activity and stability (over five cycles under UV and visible light, dye degradation fell from approximately 60% to 55% and from 90% to 70%, respectively). Photocatalytic treatment of bacteria also provided mixed results. On one hand, in all tests bacteria were not inactivated completely. However, on the other hand, their susceptibility to antibiotics increased significantly

TiO2 Application for the Photocatalytical Inactivation of S. enterica, E. coli and M. luteus Bacteria Mixtures

Water contamination by various bacteria, viruses and other pathogens is a great threat to human health. Amongst other Advanced Oxidation Processes TiO2 photocatalysis is considered as one of the most efficient treatment for the polluted wastewater disinfection. Usually, the wastewater produced by higher risk objects, such as hospitals, implicates diverse contaminants, but efficiency of most of the Advanced Oxidation Processes is tested by using only single pathogens and information on inactivation of bacteria mixtures is still limited. In this study, photocatalytical inactivation of three commonly found bacterial pathogens (gram-positive (Micrococcus luteus) and gram-negative (Salmonella enterica, Escherichia coli)) was investigated. Efficiency of traditional photocatalytic disinfection process using single bacterial pathogens was compared to the one observed for their mixtures. The impact of photocatalytical process parameters and treatment time on bacteria disinfection efficiency was studied. Photocatalytic disinfection efficiency testing with bacteria mixtures revealed, that in the presence of TiO2 photocatalyst and UV irradiation tested gram-positive cells were inactivated slower than gram-negative cells. Another important finding was that an overall photocatalytic disinfection efficiency of bacteria mixtures is not a straight forward sum of inactivation rates of individually tested pathogens but has a strong relationship to the properties of their competitive growth