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Sample records for active bacterial cells

  1. Isolation of biologically active nanomaterial (inclusion bodies) from bacterial cells

    PubMed Central

    2010-01-01

    Background In recent years bacterial inclusion bodies (IBs) were recognised as highly pure deposits of active proteins inside bacterial cells. Such active nanoparticles are very interesting for further downstream protein isolation, as well as for many other applications in nanomedicine, cosmetic, chemical and pharmaceutical industry. To prepare large quantities of a high quality product, the whole bioprocess has to be optimised. This includes not only the cultivation of the bacterial culture, but also the isolation step itself, which can be of critical importance for the production process. To determine the most appropriate method for the isolation of biologically active nanoparticles, three methods for bacterial cell disruption were analyzed. Results In this study, enzymatic lysis and two mechanical methods, high-pressure homogenization and sonication, were compared. During enzymatic lysis the enzyme lysozyme was found to attach to the surface of IBs, and it could not be removed by simple washing. As this represents an additional impurity in the engineered nanoparticles, we concluded that enzymatic lysis is not the most suitable method for IBs isolation. During sonication proteins are released (lost) from the surface of IBs and thus the surface of IBs appears more porous when compared to the other two methods. We also found that the acoustic output power needed to isolate the IBs from bacterial cells actually damages proteins structures, thereby causing a reduction in biological activity. High-pressure homogenization also caused some damage to IBs, however the protein loss from the IBs was negligible. Furthermore, homogenization had no side-effects on protein biological activity. Conclusions The study shows that among the three methods tested, homogenization is the most appropriate method for the isolation of active nanoparticles from bacterial cells. PMID:20831775

  2. Enhanced Efflux Activity Facilitates Drug Tolerance in Dormant Bacterial Cells

    PubMed Central

    Pu, Yingying; Zhao, Zhilun; Li, Yingxing; Zou, Jin; Ma, Qi; Zhao, Yanna; Ke, Yuehua; Zhu, Yun; Chen, Huiyi; Baker, Matthew A.B.; Ge, Hao; Sun, Yujie; Xie, Xiaoliang Sunney; Bai, Fan

    2016-01-01

    Summary Natural variations in gene expression provide a mechanism for multiple phenotypes to arise in an isogenic bacterial population. In particular, a sub-group termed persisters show high tolerance to antibiotics. Previously, their formation has been attributed to cell dormancy. Here we demonstrate that bacterial persisters, under β-lactam antibiotic treatment, show less cytoplasmic drug accumulation as a result of enhanced efflux activity. Consistently, a number of multi-drug efflux genes, particularly the central component TolC, show higher expression in persisters. Time-lapse imaging and mutagenesis studies further establish a positive correlation between tolC expression and bacterial persistence. The key role of efflux systems, among multiple biological pathways involved in persister formation, indicates that persisters implement a positive defense against antibiotics prior to a passive defense via dormancy. Finally, efflux inhibitors and antibiotics together effectively attenuate persister formation, suggesting a combination strategy to target drug tolerance. PMID:27105118

  3. Prodigiosin inhibits motility and activates bacterial cell death revealing molecular biomarkers of programmed cell death.

    PubMed

    Darshan, N; Manonmani, H K

    2016-12-01

    The antimicrobial activity of prodigiosin from Serratia nematodiphila darsh1, a bacterial pigment was tested against few food borne bacterial pathogens Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli. The mode of action of prodigiosin was studied. Prodigiosin induced bactericidal activity indicating a stereotypical set of biochemical and morphological feature of Programmed cell death (PCD). PCD involves DNA fragmentation, generation of ROS, and expression of a protein with caspase-like substrate specificity in bacterial cells. Prodigiosin was observed to be internalized into bacterial cells and was localized predominantly in the membrane and the nuclear fraction, thus, facilitating intracellular trafficking and then binding of prodigiosin to the bacterial DNA. Corresponding to an increasing concentration of prodigiosin, the level of certain proteases were observed to increase in bacteria studied, thus initiating the onset of PCD. Prodigiosin at a sub-inhibitory concentration inhibits motility of pathogens. Our observations indicated that prodigiosin could be a promising antibacterial agent and could be used in the prevention of bacterial infections. PMID:27460563

  4. Bacterial Manipulation of NK Cell Regulatory Activity Increases Susceptibility to Listeria monocytogenes Infection

    PubMed Central

    Guthrie, Brandon S.; Schmidt, Rebecca L.; Jamieson, Amanda; Merkel, Patricia; Knight, Vijaya; Cole, Caroline M.; Raulet, David H.; Lenz, Laurel L.

    2016-01-01

    Natural killer (NK) cells produce interferon (IFN)-γ and thus have been suggested to promote type I immunity during bacterial infections. Yet, Listeria monocytogenes (Lm) and some other pathogens encode proteins that cause increased NK cell activation. Here, we show that stimulation of NK cell activation increases susceptibility during Lm infection despite and independent from robust NK cell production of IFNγ. The increased susceptibility correlated with IL-10 production by responding NK cells. NK cells produced IL-10 as their IFNγ production waned and the Lm virulence protein p60 promoted induction of IL-10 production by mouse and human NK cells. NK cells consequently exerted regulatory effects to suppress accumulation and activation of inflammatory myeloid cells. Our results reveal new dimensions of the role played by NK cells during Lm infection and demonstrate the ability of this bacterial pathogen to exploit the induction of regulatory NK cell activity to increase host susceptibility. PMID:27295349

  5. Bacterial Manipulation of NK Cell Regulatory Activity Increases Susceptibility to Listeria monocytogenes Infection.

    PubMed

    Clark, Sarah E; Filak, Holly C; Guthrie, Brandon S; Schmidt, Rebecca L; Jamieson, Amanda; Merkel, Patricia; Knight, Vijaya; Cole, Caroline M; Raulet, David H; Lenz, Laurel L

    2016-06-01

    Natural killer (NK) cells produce interferon (IFN)-γ and thus have been suggested to promote type I immunity during bacterial infections. Yet, Listeria monocytogenes (Lm) and some other pathogens encode proteins that cause increased NK cell activation. Here, we show that stimulation of NK cell activation increases susceptibility during Lm infection despite and independent from robust NK cell production of IFNγ. The increased susceptibility correlated with IL-10 production by responding NK cells. NK cells produced IL-10 as their IFNγ production waned and the Lm virulence protein p60 promoted induction of IL-10 production by mouse and human NK cells. NK cells consequently exerted regulatory effects to suppress accumulation and activation of inflammatory myeloid cells. Our results reveal new dimensions of the role played by NK cells during Lm infection and demonstrate the ability of this bacterial pathogen to exploit the induction of regulatory NK cell activity to increase host susceptibility. PMID:27295349

  6. Transcriptional activity around bacterial cell death reveals molecular biomarkers for cell viability

    PubMed Central

    Kort, Remco; Keijser, Bart J; Caspers, Martien PM; Schuren, Frank H; Montijn, Roy

    2008-01-01

    Background In bacteriology, the ability to grow in selective media and to form colonies on nutrient agar plates is routinely used as a retrospective criterion for the detection of living bacteria. However, the utilization of indicators for bacterial viability-such as the presence of specific transcripts or membrane integrity-would overcome bias introduced by cultivation and reduces the time span of analysis from initiation to read out. Therefore, we investigated the correlation between transcriptional activity, membrane integrity and cultivation-based viability in the Gram-positive model bacterium Bacillus subtilis. Results We present microbiological, cytological and molecular analyses of the physiological response to lethal heat stress under accurately defined conditions through systematic sampling of bacteria from a single culture exposed to gradually increasing temperatures. We identified a coherent transcriptional program including known heat shock responses as well as the rapid expression of a small number of sporulation and competence genes, the latter only known to be active in the stationary growth phase. Conclusion The observed coordinated gene expression continued even after cell death, in other words after all bacteria permanently lost their ability to reproduce. Transcription of a very limited number of genes correlated with cell viability under the applied killing regime. The transcripts of the expressed genes in living bacteria – but silent in dead bacteria-include those of essential genes encoding chaperones of the protein folding machinery and can serve as molecular biomarkers for bacterial cell viability. PMID:19061518

  7. Are the actively respiring cells (CTC+) those responsible for bacterial production in aquatic environments?

    PubMed

    Servais, P; Agogué, H; Courties, C; Joux, F; Lebaron, P

    2001-04-01

    The 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) staining method is commonly and increasingly used to detect and to enumerate actively respiring cells (CTC+ cells) in aquatic systems. However, this method remains controversial since some authors promote this technique while others pointed out several drawbacks of the method. Using flow cytometry (FCM), we showed that CTC staining kinetics vary greatly from one sample to another. Therefore, there is no universal staining protocol that can be applied to aquatic bacterial communities. Furthermore, using (3)H-leucine incorporation, it was shown that the CTC dye has a rapid toxic effect on bacterial cells by inhibiting protein synthesis, a key physiological function. The coupling of radioactive labelling with cell sorting by FCM suggested that CTC+ cells contribute to less than 60% of the whole bacterial activity determined at the community level. From these results, it is clearly demonstrated that the CTC method is not valid to detect active bacteria, i.e. cells responsible for bacterial production. PMID:11295456

  8. Evidence from a carbohydrate incorporation assay for direct activation of bone marrow myelopoietic precursor cells by bacterial cell wall constitutents.

    PubMed Central

    Monner, D A; Gmeiner, J; Mühlradt, P F

    1981-01-01

    The stimulation of incorporation of [3H]galactose into membrane glycoconjugates, measured in a precipitation test, was used as a criterion for activation of bone marrow cells. In this assay, purified bacterial lipopolysaccharide, lipoprotein, and murein monomer and dimer fragments all activated rat bone marrow cells in vitro. The response was dose dependent, followed a defined time course, and was not serum dependent. O-Acetylated murein dimer fragments from Proteus mirabilis were much less active than their unsubstituted counterparts, indicating a structural specificity for murein activation. Removal of adherent and phagocytizing cells from the marrow suspensions did not alter these results. The labeled, activated cells constituted a distinct population of buoyant density 1.064 to 1.069 g/cm3 when centrifuged on a continuous gradient of Percoll. Enrichment of the target cell population was achieved by a combination of adherent cell removal and discontinuous density gradient centrifugation to remove granulocytes and erythropoietic cells. It was concluded that a population of myelopoietic precursors could be activated by direct contact with bacterial cell wall constituents. The stimulation of galactose incorporation was not coupled to active deoxyribonucleic acid synthesis in the marrow cells. Thus, the activation was interpreted as an induction of differentiation rather than a mitotic event. PMID:7014467

  9. Bacterial cell wall components as immunomodulators--II. The bacterial cell wall extract OM-85 BV as unspecific activator, immunogen and adjuvant in mice.

    PubMed

    Bessler, W G; Huber, M; Baier, W

    1997-01-01

    The bacterial extract Broncho-Vaxom used for the prevention and treatment of recurrent respiratory tract infections is an immunomodulator in vitro and in vivo, as determined in a murine model. The extract acts, on the one hand, as macrophage activator and polyclonal B-lymphocyte stimulant. On the other hand, after repeated intraperitoneal or oral immunizations, the extract is immunogenic, inducing serum IgG binding to the bacterial strains used for the preparation of the extract. On bacteria, the sera recognize the cell wall components porin, lipoprotein/lipopeptide and murein. The bacterial extract also exhibits adjuvant properties when applied in mixture with antigens, such as TNP-BSA or an influenza vaccine preparation. The unspecific and the immunospecific stimulatory effect of the extract as well as its adjuvant properties could be of importance for understanding its therapeutic effect. PMID:9637353

  10. Human-restricted bacterial pathogens block shedding of epithelial cells by stimulating integrin activation.

    PubMed

    Muenzner, Petra; Bachmann, Verena; Zimmermann, Wolfgang; Hentschel, Jochen; Hauck, Christof R

    2010-09-01

    Colonization of mucosal surfaces is the key initial step in most bacterial infections. One mechanism protecting the mucosa is the rapid shedding of epithelial cells, also termed exfoliation, but it is unclear how pathogens counteract this process. We found that carcinoembryonic antigen (CEA)-binding bacteria colonized the urogenital tract of CEA transgenic mice, but not of wild-type mice, by suppressing exfoliation of mucosal cells. CEA binding triggered de novo expression of the transforming growth factor receptor CD105, changing focal adhesion composition and activating beta1 integrins. This manipulation of integrin inside-out signaling promotes efficient mucosal colonization and represents a potential target to prevent or cure bacterial infections. PMID:20813953

  11. T-cell activation or tolerization: the Yin and Yang of bacterial superantigens.

    PubMed

    Sähr, Aline; Förmer, Sandra; Hildebrand, Dagmar; Heeg, Klaus

    2015-01-01

    Bacterial superantigens (SAg) are exotoxins from pathogens which interact with innate and adaptive immune cells. The paradox that SAgs cause activation and inactivation/anergy of T-cells was soon recognized. The structural and molecular events following SAg binding to antigen presenting cells (APCs) followed by crosslinking of T-cell receptors were characterized in detail. Activation, cytokine burst and T-cell anergy have been described in vitro and in vivo. Later it became clear that SAg-induced T-cell anergy is in part caused by SAg-dependent activation of T-regulatory cells (Tregs). Although the main focus of analyses was laid on T-cells, it was also shown that SAg binding to MHC class II molecules on APCs induces a signal, which leads to activation and secretion of pro-inflammatory cytokines. Accordingly APCs are mandatory for T-cell activation. So far it is not known, whether APCs play a role during SAg-triggered activation of Tregs. We therefore tested whether in SAg (Streptococcal pyrogenic exotoxin A) -treated APCs an anti-inflammatory program is triggered in addition. We show here that not only the anti-inflammatory cytokine IL-10 and the co-inhibitory surface molecule PD-L1 (CD274) but also inhibitory effector systems like indoleamine 2,3-dioxygenase (IDO) or intracellular negative feedback loops (suppressor of cytokine signaling molecules, SOCS) are induced by SAgs. Moreover, cyclosporine A completely prevented induction of this program. We therefore propose that APCs triggered by SAgs play a key role in T-cell activation as well as inactivation and induction of Treg cells. PMID:26539181

  12. Targeting Bacterial Cell Wall Peptidoglycan Synthesis by Inhibition of Glycosyltransferase Activity.

    PubMed

    Mesleh, Michael F; Rajaratnam, Premraj; Conrad, Mary; Chandrasekaran, Vasu; Liu, Christopher M; Pandya, Bhaumik A; Hwang, You Seok; Rye, Peter T; Muldoon, Craig; Becker, Bernd; Zuegg, Johannes; Meutermans, Wim; Moy, Terence I

    2016-02-01

    Synthesis of bacterial cell wall peptidoglycan requires glycosyltransferase enzymes that transfer the disaccharide-peptide from lipid II onto the growing glycan chain. The polymerization of the glycan chain precedes cross-linking by penicillin-binding proteins and is essential for growth for key bacterial pathogens. As such, bacterial cell wall glycosyltransferases are an attractive target for antibiotic drug discovery. However, significant challenges to the development of inhibitors for these targets include the development of suitable assays and chemical matter that is suited to the nature of the binding site. We developed glycosyltransferase enzymatic activity and binding assays using the natural products moenomycin and vancomycin as model inhibitors. In addition, we designed a library of disaccharide compounds based on the minimum moenomycin fragment with peptidoglycan glycosyltransferase inhibitory activity and based on a more drug-like and synthetically versatile disaccharide building block. A subset of these disaccharide compounds bound and inhibited the glycosyltransferase enzymes, and these compounds could serve as chemical entry points for antibiotic development. PMID:26358369

  13. Fluorescence-Activated Cell Sorting of Live Versus Dead Bacterial Cells and Spores

    NASA Technical Reports Server (NTRS)

    Bernardini, James N.; LaDuc, Myron T.; Diamond, Rochelle; Verceles, Josh

    2012-01-01

    This innovation is a coupled fluorescence-activated cell sorting (FACS) and fluorescent staining technology for purifying (removing cells from sampling matrices), separating (based on size, density, morphology, and live versus dead), and concentrating cells (spores, prokaryotic, eukaryotic) from an environmental sample.

  14. Chronic bacterial infection activates autoreactive B cells and induces isotype switching and autoantigen-driven mutations.

    PubMed

    Jung, Sophie; Schickel, Jean-Nicolas; Kern, Aurélie; Knapp, Anne-Marie; Eftekhari, Pierre; Da Silva, Sylvia; Jaulhac, Benoît; Brink, Robert; Soulas-Sprauel, Pauline; Pasquali, Jean-Louis; Martin, Thierry; Korganow, Anne-Sophie

    2016-01-01

    The links between infections and the development of B-cell-mediated autoimmune diseases are still unclear. In particular, it has been suggested that infection-induced stimulation of innate immune sensors can engage low affinity autoreactive B lymphocytes to mature and produce mutated IgG pathogenic autoantibodies. To test this hypothesis, we established a new knock-in mouse model in which autoreactive B cells could be committed to an affinity maturation process. We show that a chronic bacterial infection allows the activation of such B cells and the production of nonmutated IgM autoantibodies. Moreover, in the constitutive presence of their soluble antigen, some autoreactive clones are able to acquire a germinal center phenotype, to induce Aicda gene expression and to introduce somatic mutations in the IgG heavy chain variable region on amino acids forming direct contacts with the autoantigen. Paradoxically, only lower affinity variants are detected, which strongly suggests that higher affinity autoantibodies secreting B cells are counterselected. For the first time, we demonstrate in vivo that a noncross-reactive infectious agent can activate and induce autoreactive B cells to isotype switching and autoantigen-driven mutations, but on a nonautoimmune background, tolerance mechanisms prevent the formation of consequently dangerous autoimmunity. PMID:26474536

  15. Bacterial Cell Wall Components

    NASA Astrophysics Data System (ADS)

    Ginsberg, Cynthia; Brown, Stephanie; Walker, Suzanne

    Bacterial cell-surface polysaccharides cells are surrounded by a variety of cell-surface structures that allow them to thrive in extreme environments. Components of the cell envelope and extracellular matrix are responsible for providing the cells with structural support, mediating intercellular communication, allowing the cells to move or to adhere to surfaces, protecting the cells from attack by antibiotics or the immune system, and facilitating the uptake of nutrients. Some of the most important cell wall components are polysaccharide structures. This review discusses the occurrence, structure, function, and biosynthesis of the most prevalent bacterial cell surface polysaccharides: peptidoglycan, lipopolysaccharide, arabinogalactan, and lipoarabinomannan, and capsular and extracellular polysaccharides. The roles of these polysaccharides in medicine, both as drug targets and as therapeutic agents, are also described.

  16. A 17-mer Membrane-Active MSI-78 Derivative with Improved Selectivity toward Bacterial Cells.

    PubMed

    Monteiro, Claudia; Pinheiro, Marina; Fernandes, Mariana; Maia, Sílvia; Seabra, Catarina L; Ferreira-da-Silva, Frederico; Reis, Salette; Gomes, Paula; Martins, M Cristina L

    2015-08-01

    Antimicrobial peptides are widely recognized as an excellent alternative to conventional antibiotics. MSI-78, a highly effective and broad spectrum AMP, is one of the most promising AMPs for clinical application. In this study, we have designed shorter derivatives of MSI-78 with the aim of improving selectivity while maintaining antimicrobial activity. Shorter 17-mer derivatives were created by truncating MSI-78 at the N- and/or C-termini, while spanning MSI-78 sequence. Despite the truncations made, we found a 17-mer peptide, MSI-78(4-20) (KFLKKAKKFGKAFVKIL), which was demonstrated to be as effective as MSI-78 against the Gram-positive Staphylococcus strains tested and the Gram-negative Pseudomonas aeruginosa. This shorter derivative is more selective toward bacterial cells as it was less toxic to erythrocytes than MSI-78, representing an improved version of the lead peptide. Biophysical studies support a mechanism of action for MSI-78(4-20) based on the disruption of the bacterial membrane permeability barrier, which in turn leads to loss of membrane integrity and ultimately to cell death. These features point to a mechanism of action similar to the one described for the lead peptide MSI-78. PMID:26066462

  17. Complement activation and cytokine response by BioProtein, a bacterial single cell protein.

    PubMed

    Sikkeland, L I B; Thorgersen, E B; Haug, T; Mollnes, T E

    2007-04-01

    The bacterial single cell protein (BSCP), BioProtein, is dried bacterial mass derived from fermentation of the gram negative bacteria Methylococcus capsulatus, used for animal and fish feed. Workers in this industry suffer frequently from pulmonary and systemic symptoms which may be induced by an inflammatory reaction. The aim of the present study was to examine the effect of BSCP on inflammation in vitro as evaluated by complement activation and cytokine production. Human serum was incubated with BSCP and complement activation products specific for all pathways were detected by enzyme-linked immunosorbent assay (ELISA). Human whole blood anti-coagulated with lepirudin was incubated with BSCP and a panel of 27 biological mediators was measured using multiplex technology. BSCP induced a dose-dependent complement activation as revealed by a pronounced increase in alternative and terminal pathway activation (fivefold and 20-fold, respectively) at doses from 1 microg BSCP/ml serum and a similar, but less extensive (two- to fourfold) increase in activation of the lectin and classical pathways at doses from 100 and 1000 microg BSCP/ml serum, respectively. Similarly, BSCP induced a dose-dependent production of a number of cytokines, chemokines and growth factors in human whole blood. At doses as low as 0 x 05-0 x 5 microg BSCP/ml blood a substantial increase was seen for tumour necrosis factor (TNF)-alpha, interleukin (IL)-1-beta, IL-6, interferon (IFN)-gamma, IL-8, monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, IL-4, IL-9, IL-17, IL-1Ra, granulocyte-colony-stimulating factor (G-CSF) and vascular endothelial growth factor (VEGF). Thus, BSCP induced a substantial activation of all three initial complement pathways as well as a pronounced cytokine response in vitro, indicating a potent inflammatory property of this agent. PMID:17302729

  18. Activation of natural killer cells and cytokine production in man by bacterial extracts.

    PubMed

    Wybran, J; Libin, M; Schandene, L

    1989-01-01

    Broncho-Vaxon (OM-85 BV) is a bacterial extract of eight bacterias usually involved in the respiratory tract infections. Since Broncho-Vaxom is clinically active in decreasing the incidence of such infections, its immunological effect was investigated, in vitro, using peripheral blood mononuclear cells (PBMC). The experimental data indicate that Broncho-Vaxom can modulate various immune functions. It was shown, using a radioimmunoassay for these cytokines, that Broncho-Vaxom will spontaneously enhance TNF alpha and IL-2 production whereas it has no action on IF gamma production. However, when the PBMC are stimulated with PHA, an increased production for IF gamma, TNF alpha and IL-2 was observed suggesting that, under appropriate conditions, Broncho-Vaxom enhances the production of these cytokines. In other experiments, Broncho-Vaxom was shown to markedly increase the natural killer activity of PBMC. All these results demonstrate that Broncho-Vaxom is an immunomodulator affecting multiple immunological mechanisms including the activation of natural killer cells, of monocytes and of T cells through direct mechanisms or through the cytokine cascade. PMID:2503554

  19. Effect of Bacterial Cell Moisture on the Sporicidal Activity of β-Propiolactone Vapor

    PubMed Central

    Hoffman, Robert K.

    1968-01-01

    The activity of a vapor-phase disinfectant is usually expressed in terms of the atmospheric relative humidity (RH). This study shows that, in β-propiolactone (BPL) vapor disinfection, the important factor is really the moisture content and location of water in the cell, and not necessarily the atmospheric RH. Previous studies revealed that only about 50% of the bacterial spores equilibrated to 45% RH were killed when exposed to the same RH to BPL vapor. On the other hand, all the spores equilibrated to and then exposed at 75% RH to BPL were readily killed. The present study shows that spores equilibrated to 98% RH are readily killed by BPL at 45% RH, but only 99% of the spores equilibrated to 75% RH are killed by BPL at 45% RH. Also, in order to be killed, desiccated spores must be exposed to BPL at higher humidities than would be required if the spores had not been previously desiccated. PMID:4967453

  20. Bacterial capsular polysaccharide prevents the onset of asthma through T-cell activation.

    PubMed

    Johnson, Jenny L; Jones, Mark B; Cobb, Brian A

    2015-04-01

    Over the last four decades, increases in the incidence of immune-mediated diseases in the Western world have been linked to changes in microbial exposure. It is becoming increasingly clear that the normal microbiota in the gut can profoundly alter susceptibility to a wide range of diseases, such as asthma, in which immune homeostasis is disrupted, yet the mechanisms governing this microbial influence remains poorly defined. In this study, we show that gastrointestinal exposure to PSA, a capsular polysaccharide derived from the commensal bacterium Bacteroides fragilis, significantly limits susceptibility to the induction of experimental asthma. We report that direct treatment of mice with PSA generates protection from asthma, and this effect can be given to a naïve recipient by adoptive transfer of CD4(+) T cells from PSA-exposed mice. Remarkably, we found that these PSA-induced T cells are not canonical FoxP3(+) regulatory T cells, but that they potently inhibit both Th1 and Th2 models of asthma in an IL-10-dependent fashion. These findings reveal that bacterial polysaccharides link the microbiota with the peripheral immune system by activating CD4(+)Foxp3(-) T cells upon exposure in the gut, and they facilitate resistance to unnecessary inflammatory responses via the production of IL-10. PMID:25347992

  1. Whole‐cell bacterial bioreporter for actively searching and sensing of alkanes and oil spills

    PubMed Central

    Zhang, Dayi; He, Yi; Wang, Yun; Wang, Hui; Wu, Lin; Aries, Eric; Huang, Wei E.

    2012-01-01

    Summary Acinetobacter baylyi ADP1 was found to tolerate seawater and have a special ability of adhering to an oil–water interface of 10–80 µm emulsified mineral and crude oil droplets. These properties make ADP1 an ideal bacterial chassis for constructing bioreporters that are able to actively search and sense oil spill in water and soils. Acinetobacter baylyi bioreporter ADPWH_alk was developed and applied to the detection of alkanes and alkenes in water, seawater and soils. Bioreporter ADPWH_alk was able to detect a broad range of alkanes and alkenes with carbon chain length from C7 to C36. So far, ADPWH_alk is the only bioreporter that is able to detect alkane with carbon chain length greater than C18. This bioreporter responded to the alkanes in about 30 min and it was independent to the cell growth phase because of two point mutations in alkM promoter recognized by alkane regulatory protein ALKR. ADPWH_alk was applied to detect mineral oil, Brent, Chestnut and Sirri crude oils in water and seawater in the range 0.1–100 mg l−1, showing that the bioreporter oil detection was semi‐quantitative. This study demonstrates that ADPWH_alk is a rapid, sensitive and semi‐quantitative bioreporter that can be useful for environmental monitoring and assessment of oil spills in seawater and soils. PMID:21951420

  2. Defective disposal of immune complexes and polyclonal B cell activation persist long after exposure to bacterial lipopolysaccharide in mice

    SciTech Connect

    Granholm, N.A.; Cavallo, T. )

    1989-11-01

    Patients with systemic lupus erythematosus experience clinical exacerbation during superimposed bacterial infection. Previous studies in mice indicated that heightened immune phenomena during exposure to bacterial lipopolysaccharide (LPS) appear to be related, in part, to polyclonal B cell activation, to abnormal disposal of immune complexes (IC), and to increased localization of IC in tissues. To investigate whether such effects were reversible, we administered bacterial LPS to C57BL/6 mice for 5 weeks. Control mice received vehicle alone. We then discontinued LPS, and 6 weeks later LPS and control mice were challenged with a subsaturating dose of radiolabeled IC; the removal of IC from the circulation, their localization in the liver, spleen, and kidney were determined. In comparison to values in control mice, in mice previously exposed to LPS, serologic features of polyclonal B cell activation persisted; liver uptake of pathogenic IC (greater than Ag2Ab2) was normal, but removal of small size IC (less than or equal to Ag2Ab2) from the circulation was delayed; localization of IC in the kidneys was enhanced, and pathologic proteinuria developed. The effects of repeated exposure to bacterial LPS are partially reversible, but they last long after LPS is discontinued and may contribute to altered disposal of IC, enhanced organ localization of IC, and organ dysfunction.

  3. Nitrate reducing bacterial activity in concrete cells of nuclear waste disposal

    NASA Astrophysics Data System (ADS)

    Alquier, M.; Kassim, C.; Bertron, A.; Rafrafi, Y.; Sablayrolles, C.; Albrecht, A.; Erable, B.

    2013-07-01

    Leaching experiments of solid matrices (bitumen and cement pastes) have been first implemented to define the physicochemical conditions that microorganisms are likely to meet at the bitumen-concrete interface (see the paper of Bertron et al.). Of course, as might be suspected, the cement matrix imposes highly alkaline pH conditions (10 bacterial strains led us to select Halomonas desiderata as a model bacterium capable of catalyzing the reaction of nitrate reduction in these extreme conditions of pH. The denitrifying activity of Halomonas desiderata was quantified in batch bioreactor in the presence of solid matrices and / or leachate from bitumen and cement matrices. Denitrification was relatively fast in the presence of cement matrix (<100 hours) and 2 to 3 times slower in the presence of bituminous matrix. Overall, the presence of solid cement promoted the kinetics of denitrification. The observation of solid surfaces at the end of the experiment revealed the presence of a biofilm of Halomonas desiderata on the cement paste surface. These attached bacteria showed a denitrifying activity comparable to planktonic bacterial culture. On the other side, no colonization of bitumen could be highlighted as either by SEM or epifluorescence microscopy. Now, we are currently developing a continuous experimental bioreactor which should allow us a more rational understanding of the bitumen-cement-microbe interactions.

  4. Evaluating bacterial activity from cell-specific ribosomal RNA content measured with oligonucleotide probes

    SciTech Connect

    Kemp, P.F.; Lee, S.; LaRoche, J.

    1992-10-01

    We describe a procedure for measuring the cell-specific quantity of ribosomal RNA (rRNA) and DNA in order to evaluate the frequency distribution of activity among cells. The procedure is inherently quantitative, does not require sample incubation and potentially can be taxon-specific. Fluorescently-labelled oligonucleotide probes are hybridized to the complementary 16S rRNA sequences in preserved, intact cells. The resulting cell fluorescence is proportional to cellular rRNA content and can be measured with a microscope-mounted photometer system, by image analysis, or by flow cytometry. Similarly, DNA content is measured as fluorescence of cells stained with the DNA specific fluorochrome DAPI. These are either prepared as separate samples for purposes of enumeration and DNA measurements, or are dual-labelled cells which are also hybridized with oligonucleotide probes.

  5. Evaluating bacterial activity from cell-specific ribosomal RNA content measured with oligonucleotide probes

    SciTech Connect

    Kemp, P.F.; Lee, S.; LaRoche, J.

    1992-01-01

    We describe a procedure for measuring the cell-specific quantity of ribosomal RNA (rRNA) and DNA in order to evaluate the frequency distribution of activity among cells. The procedure is inherently quantitative, does not require sample incubation and potentially can be taxon-specific. Fluorescently-labelled oligonucleotide probes are hybridized to the complementary 16S rRNA sequences in preserved, intact cells. The resulting cell fluorescence is proportional to cellular rRNA content and can be measured with a microscope-mounted photometer system, by image analysis, or by flow cytometry. Similarly, DNA content is measured as fluorescence of cells stained with the DNA specific fluorochrome DAPI. These are either prepared as separate samples for purposes of enumeration and DNA measurements, or are dual-labelled cells which are also hybridized with oligonucleotide probes.

  6. Helminth-Induced Interleukin-4 Abrogates Invariant Natural Killer T Cell Activation-Associated Clearance of Bacterial Infection

    PubMed Central

    Hsieh, Yi-Ju; Fu, Chi-Ling

    2014-01-01

    Helminth infections affect 1 billion people worldwide and render these individuals susceptible to bacterial coinfection through incompletely understood mechanisms. This includes urinary tract coinfection by bacteria and Schistosoma haematobium worms, the etiologic agent of urogenital schistosomiasis. To study the mechanisms of S. haematobium-bacterial urinary tract coinfections, we combined the first tractable model of urogenital schistosomiasis with an established mouse model of bacterial urinary tract infection (UTI). A single bladder exposure to S. haematobium eggs triggers interleukin-4 (IL-4) production and makes BALB/c mice susceptible to bacterial UTI when they are otherwise resistant. Ablation of IL-4 receptor alpha (IL-4Rα) signaling restored the baseline resistance of BALB/c mice to bacterial UTI despite prior exposure to S. haematobium eggs. Interestingly, numbers of NKT cells were decreased in coexposed versus bacterially monoinfected bladders. Given that schistosome-induced, non-natural killer T (NKT) cell leukocyte infiltration may dilute NKT cell numbers in the bladders of coexposed mice without exerting a specific functional effect on these cells, we next examined NKT cell biology on a per-cell basis. Invariant NKT (iNKT) cells from coexposed mice expressed less gamma interferon (IFN-γ) per cell than did those from mice with UTI alone. Moreover, coexposure resulted in lower CD1d expression in bladder antigen-presenting cells (APC) than did bacterial UTI alone in an IL-4Rα-dependent fashion. Finally, coexposed mice were protected from prolonged bacterial infection by administration of α-galactosylceramide, an iNKT cell agonist. Our findings point to a previously unappreciated role for helminth-induced IL-4 in impairment of iNKT cell-mediated clearance of bacterial coexposure. PMID:24643536

  7. Efficient Gene Editing in Pluripotent Stem Cells by Bacterial Injection of Transcription Activator-Like Effector Nuclease Proteins

    PubMed Central

    Jia, Jingyue; Bai, Fang; Jin, Yongxin; Santostefano, Katherine E.; Ha, Un-Hwan; Wu, Donghai

    2015-01-01

    The type III secretion system (T3SS) of Pseudomonas aeruginosa is a powerful tool for direct protein delivery into mammalian cells and has successfully been used to deliver various exogenous proteins into mammalian cells. In the present study, transcription activator-like effector nuclease (TALEN) proteins have been efficiently delivered using the P. aeruginosa T3SS into mouse embryonic stem cells (mESCs), human ESCs (hESCs), and human induced pluripotent stem cells (hiPSCs) for genome editing. This bacterial delivery system offers an alternative method of TALEN delivery that is highly efficient in cleavage of the chromosomal target and presumably safer by avoiding plasmid DNA introduction. We combined the method of bacterial T3SS-mediated TALEN protein injection and transfection of an oligonucleotide template to effectively generate precise genetic modifications in the stem cells. Initially, we efficiently edited a single-base in the gfp gene of a mESC line to silence green fluorescent protein (GFP) production. The resulting GFP-negative mESC was cloned from a single cell and subsequently mutated back to a GFP-positive mESC line. Using the same approach, the gfp gene was also effectively knocked out in hESCs. In addition, a defined single-base edition was effectively introduced into the X-chromosome-linked HPRT1 gene in hiPSCs, generating an in vitro model of Lesch-Nyhan syndrome. T3SS-mediated TALEN protein delivery provides a highly efficient alternative for introducing precise gene editing within pluripotent stem cells for the purpose of disease genotype-phenotype relationship studies and cellular replacement therapies. Significance The present study describes a novel and powerful tool for the delivery of the genome editing enzyme transcription activator-like effector nuclease (TALEN) directly into pluripotent stem cells (PSCs), achieving desired base changes on the genomes of PSCs with high efficiency. This novel approach uses bacteria as a protein delivery

  8. Efficacy of coating activated carbon with milk proteins to prevent binding of bacterial cells from foods for PCR detection.

    PubMed

    Opet, Nathan J; Levin, Robert E

    2013-08-01

    Foods contaminated with pathogens are common sources of illness. Currently, the most common and sensitive rapid detection method involves the PCR. However, food matrices are complex and contain inhibitors that limit the sensitivity of the PCR. The use of coated activated carbon can effectively facilitate the removal of PCR inhibitors without binding targeted bacterial cells from food samples. With the use of activated carbon coated with milk proteins, a cell recovery at pH 7.0 of 95.7%±2.0% was obtained, compared to control uncoated activated carbon, which yielded a cell recovery of only 1.1%±0.8%. In addition, the milk protein coated activated carbon was able to absorb similar amounts of soluble compounds as uncoated activated carbon, with the exception of bovine hemoglobin. This suggests that the use of milk proteins to coat activated carbon may therefore serve as a suitable replacement for bentonite in the coating of activated carbon, which has previously been used for the removal of PCR inhibitors from food. PMID:23680231

  9. Activity-dependent fluorescent labeling of bacterial cells expressing the TOL pathway

    SciTech Connect

    William K. Keener; Mary E. Watwood

    2005-01-01

    3-Ethynylbenzoate functions as an activity-dependent, fluorogenic and chromogenic probe for Pseudomonas putida mt-2, which is known to degrade toluene via conversion to benzoate, followed by meta ring fission of the intermediate, catechol. This direct physiological analysis allows the fluorescent labeling of cells whose toluene-degrading enzymes have been induced by an aromatic substrate.

  10. Efficient Gene Editing in Pluripotent Stem Cells by Bacterial Injection of Transcription Activator-Like Effector Nuclease Proteins.

    PubMed

    Jia, Jingyue; Bai, Fang; Jin, Yongxin; Santostefano, Katherine E; Ha, Un-Hwan; Wu, Donghai; Wu, Weihui; Terada, Naohiro; Jin, Shouguang

    2015-08-01

    The type III secretion system (T3SS) of Pseudomonas aeruginosa is a powerful tool for direct protein delivery into mammalian cells and has successfully been used to deliver various exogenous proteins into mammalian cells. In the present study, transcription activator-like effector nuclease (TALEN) proteins have been efficiently delivered using the P. aeruginosa T3SS into mouse embryonic stem cells (mESCs), human ESCs (hESCs), and human induced pluripotent stem cells (hiPSCs) for genome editing. This bacterial delivery system offers an alternative method of TALEN delivery that is highly efficient in cleavage of the chromosomal target and presumably safer by avoiding plasmid DNA introduction. We combined the method of bacterial T3SS-mediated TALEN protein injection and transfection of an oligonucleotide template to effectively generate precise genetic modifications in the stem cells. Initially, we efficiently edited a single-base in the gfp gene of a mESC line to silence green fluorescent protein (GFP) production. The resulting GFP-negative mESC was cloned from a single cell and subsequently mutated back to a GFP-positive mESC line. Using the same approach, the gfp gene was also effectively knocked out in hESCs. In addition, a defined single-base edition was effectively introduced into the X-chromosome-linked HPRT1 gene in hiPSCs, generating an in vitro model of Lesch-Nyhan syndrome. T3SS-mediated TALEN protein delivery provides a highly efficient alternative for introducing precise gene editing within pluripotent stem cells for the purpose of disease genotype-phenotype relationship studies and cellular replacement therapies. PMID:26062981

  11. Activation of natural killer cells and cytokine production in humans by bacterial extracts (OM-85 BV).

    PubMed

    Wybran, J; Libin, M; Schandene, L

    1990-01-01

    The influence of Broncho-Vaxom (BV) on different immune parameters was investigated in vitro on human peripheral blood mononuclear cells (PBMC). It was found that BV enhances the natural killer (NK) activity of PBMC and increases their spontaneous and phytohemagglutin (PHA)-induced production of tumor-necrosis factor--alpha and interleukin-2 as well as the PHA-stimulated production of interferon-gamma. These immunostimulating actions of BV on NK activity and cytokine production can contribute to the understanding of the enhancement of the body's defense mechanisms against respiratory tract infections. PMID:2117183

  12. Bacterial Activity in the Rhizosphere Analyzed at the Single-Cell Level by Monitoring Ribosome Contents and Synthesis Rates

    PubMed Central

    Ramos, Cayo; Mølbak, Lars; Molin, Søren

    2000-01-01

    The growth activity of Pseudomonas putida cells colonizing the rhizosphere of barley seedlings was estimated at the single-cell level by monitoring ribosomal contents and synthesis rates. Ribosomal synthesis was monitored by using a system comprising a fusion of the ribosomal Escherichia coli rrnBP1 promoter to a gene encoding an unstable variant of the green fluorescent protein (Gfp). Gfp expression in a P. putida strain carrying this system inserted into the chromosome was strongly dependent on the growth phase and growth rate of the strain, and cells growing exponentially at rates of ≥0.17 h−1 emitted growth rate-dependent green fluorescence detectable at the single-cell level. The single-cell ribosomal contents were very heterogeneous, as determined by quantitative hybridization with fluorescently labeled rRNA probes in P. putida cells extracted from the rhizosphere of 1-day-old barley seedlings grown under sterile conditions. After this, cells extracted from the root system had ribosomal contents similar to those found in starved cells. There was a significant decrease in the ribosomal content of P. putida cells when bacteria were introduced into nonsterile bulk or rhizosphere soil, and the Gfp monitoring system was not induced in cells extracted from either of the two soil systems. The monitoring system used permitted nondestructive in situ detection of fast-growing bacterial microcolonies on the sloughing root sheath cells of 1- and 2-day-old barley seedlings grown under sterile conditions, which demonstrated that it may be possible to use the unstable Gfp marker for studies of transient gene expression in plant-microbe systems. PMID:10653754

  13. Gamma-irradiated bacterial preparation having anti-tumor activity

    SciTech Connect

    Vass, A.A.; Tyndall, R.L.; Terzaghi-Howe, P.

    1999-11-16

    This application describes a bacterial preparation from Pseudomonas species isolated {number{underscore}sign}15 ATCC 55638 that has been exposed to gamma radiation exhibits cytotoxicity that is specific for neoplastic carcinoma cells. A method for obtaining a bacterial preparation having antitumor activity consists of suspending a bacterial isolate in media and exposing the suspension to gamma radiation. A bacterial preparation of an aged culture of an amoeba-associated bacteria exhibits anti-reverse transcriptase activity. A method for obtaining a bacterial preparation having anti-reverse transcriptase activity from an amoeba-associated bacterial isolate grown to stationary phase is disclosed.

  14. Gamma-irradiated bacterial preparation having anti-tumor activity

    DOEpatents

    Vass, Arpad A.; Tyndall, Richard L.; Terzaghi-Howe, Peggy

    1999-01-01

    A bacterial preparation from Pseudomonas species isolated #15 ATCC 55638 that has been exposed to gamma radiation exhibits cytotoxicity that is specific for neoplastic carcinoma cells. A method for obtaining a bacterial preparation having antitumor activity consists of suspending a bacterial isolate in media and exposing the suspension to gamma radiation. A bacterial preparation of an aged culture of an amoeba-associated bacteria exhibits anti-reverse transcriptase activity. A method for obtaining a bacterial preparation having anti-reverse transcriptase activity from an amoeba-associated bacterial isolate grown to stationary phase is disclosed.

  15. Antibacterial active compounds from Hypericum ascyron L. induce bacterial cell death through apoptosis pathway.

    PubMed

    Li, Xiu-Mei; Luo, Xue-Gang; Si, Chuan-Ling; Wang, Nan; Zhou, Hao; He, Jun-Fang; Zhang, Tong-Cun

    2015-01-01

    Hypericum ascyron L. has been used as a traditional medicine for the treatment of wounds, swelling, headache, nausea and abscesses in China for thousands of years. However, modern pharmacological studies are still necessary to provide a scientific basis to substantiate their traditional use. In this study, the mechanism underlying the antimicrobial effect of the antibacterial activity compounds from H. ascyron L. was investigated. Bioguided fractionation of the extract from H. ascyron L. afforded antibacterial activity fraction 8. The results of cup plate analysis and MTT assay showed that the MIC and MBC of fraction 8 is 5 mg/mL. Furthermore, using Annexin V-FITC/PI, TUNEL labeling and DNA gel electrophoresis, we found that cell death with apoptosis features similar to those in eucaryon could be induced in bacteria strains after exposure to the antibacterial activity compounds from H. ascyron L. at moderate concentration. In addition, we further found fraction 8 could disrupt the cell membrane potential indicate that fraction 8 exerts pro-apoptotic effects through a membrane-mediated apoptosis pathway. Finally, quercetin and kaempferol 3-O-β-(2″-acetyl)-galactopyranoside, were identified from fraction 8 by means of Mass spectrometry and Nuclear magnetic resonance. To our best knowledge, this study is the first to show that Kaempferol 3-O-β-(2″-acetyl)-galactopyranoside coupled with quercetin had significant antibacterial activity via apoptosis pathway, and it is also the first report that Kaempferol 3-O-β-(2″-acetyl)-galactopyranoside was found in clusiacea. Our data might provide a rational base for the use of H. ascyron L. in clinical, and throw light on the development of novel antibacterial drugs. PMID:25916905

  16. Fragmented Lactic Acid Bacterial Cells Activate Peroxisome Proliferator-Activated Receptors and Ameliorate Dyslipidemia in Obese Mice.

    PubMed

    Nakamura, Futoshi; Ishida, Yu; Sawada, Daisuke; Ashida, Nobuhisa; Sugawara, Tomonori; Sakai, Manami; Goto, Tsuyoshi; Kawada, Teruo; Fujiwara, Shigeru

    2016-03-30

    Recent studies suggest that peroxisome proliferator-activated receptor (PPAR) activation ameliorates metabolic disorders, including dyslipidemia. To identify an effective PPAR agonist, we screened the in vitro PPARα/γ activation ability of organic solvent extracts from food-oriented bacterial strains belonging to 5 genera and 32 species, including lactic acid bacteria, and of these, Lactobacillus amylovorus CP1563 demonstrated the highest PPARα/γ agonist activity. We also found that physical fragmentation of the strain could substitute organic solvent extraction for the expression of CP1563 activity in vitro. For functional food manufacturing, we selected the fragmented CP1563 and conducted subsequent animal experiments. In an obese mouse model, we found that treatment with fragmented CP1563 for 12 weeks decreased the levels of low-density lipoprotein (LDL)-cholesterol and triglyceride in plasma, significantly decreased the atherosclerosis index, and increased the plasma high-density lipoprotein (HDL)-cholesterol level. Thus, we conclude that fragmented CP1563 may be a candidate for the prevention and treatment of dyslipidemia. PMID:26927959

  17. Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts.

    PubMed

    Nakimbugwe, Dorothy; Masschalck, Barbara; Deckers, Daphne; Callewaert, Lien; Aertsen, Abram; Michiels, Chris W

    2006-06-01

    We have investigated the specificity of six different lysozymes for peptidoglycan substrates obtained by extraction of a number of gram-negative bacteria and Micrococcus lysodeikticus with chloroform/Tris-HCl buffer (chloroform/buffer). The lysozymes included two that are commercially available (hen egg white lysozyme or HEWL, and mutanolysin from Streptomyces globisporus or M1L), and four that were chromatographically purified (bacteriophage lambda lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). HEWL was much more effective on M. lysodeikticus than on any of the gram-negative cell walls, while the opposite was found for LaL. Also the gram-negative cell walls showed remarkable differences in susceptibility to the different lysozymes, even for closely related species like Escherichia coli and Salmonella Typhimurium. These differences could not be due to the presence of lysozyme inhibitors such as Ivy from E. coli in the cell wall substrates because we showed that chloroform extraction effectively removed this inhibitor. Interestingly, we found strong inhibitory activity to HEWL in the chloroform/buffer extracts of Salmonella Typhimurium, and to LaL in the extracts of Pseudomonas aeruginosa, suggesting that other lysozyme inhibitors than Ivy exist and are probably widespread in gram-negative bacteria. PMID:16684100

  18. Phylogenetic organization of bacterial activity.

    PubMed

    Morrissey, Ember M; Mau, Rebecca L; Schwartz, Egbert; Caporaso, J Gregory; Dijkstra, Paul; van Gestel, Natasja; Koch, Benjamin J; Liu, Cindy M; Hayer, Michaela; McHugh, Theresa A; Marks, Jane C; Price, Lance B; Hungate, Bruce A

    2016-09-01

    Phylogeny is an ecologically meaningful way to classify plants and animals, as closely related taxa frequently have similar ecological characteristics, functional traits and effects on ecosystem processes. For bacteria, however, phylogeny has been argued to be an unreliable indicator of an organism's ecology owing to evolutionary processes more common to microbes such as gene loss and lateral gene transfer, as well as convergent evolution. Here we use advanced stable isotope probing with (13)C and (18)O to show that evolutionary history has ecological significance for in situ bacterial activity. Phylogenetic organization in the activity of bacteria sets the stage for characterizing the functional attributes of bacterial taxonomic groups. Connecting identity with function in this way will allow scientists to begin building a mechanistic understanding of how bacterial community composition regulates critical ecosystem functions. PMID:26943624

  19. Phylogenetic organization of bacterial activity

    PubMed Central

    Morrissey, Ember M; Mau, Rebecca L; Schwartz, Egbert; Caporaso, J Gregory; Dijkstra, Paul; van Gestel, Natasja; Koch, Benjamin J; Liu, Cindy M; Hayer, Michaela; McHugh, Theresa A; Marks, Jane C; Price, Lance B; Hungate, Bruce A

    2016-01-01

    Phylogeny is an ecologically meaningful way to classify plants and animals, as closely related taxa frequently have similar ecological characteristics, functional traits and effects on ecosystem processes. For bacteria, however, phylogeny has been argued to be an unreliable indicator of an organism's ecology owing to evolutionary processes more common to microbes such as gene loss and lateral gene transfer, as well as convergent evolution. Here we use advanced stable isotope probing with 13C and 18O to show that evolutionary history has ecological significance for in situ bacterial activity. Phylogenetic organization in the activity of bacteria sets the stage for characterizing the functional attributes of bacterial taxonomic groups. Connecting identity with function in this way will allow scientists to begin building a mechanistic understanding of how bacterial community composition regulates critical ecosystem functions. PMID:26943624

  20. Bacterial Infection of endometrial stromal cells influences bovine herpersvirus 4 immediate early gene activation: a new insight into bacterial and viral interaction for uterine disease

    PubMed Central

    Donofrio, Gaetano; Ravanetti, Lara; Cavirani, Sandro; Herath, Shan; Capocefalo, Antonio; Sheldon, Iain Martin

    2009-01-01

    Experimental infection with the gammaherpesvirus Bovine herpesvirus 4 (BoHV-4) rarely establishes disease, yet BoHV-4 is commonly associated with uterine disease in cattle. Uterine disease involves co-infection with bacteria such as Escherichia coli, which stimulate the production of prostaglandin E2 (PGE2) by endometrial cells. BoHV-4 replication depends on Immediate Early 2 (IE2) gene transactivation, and in the present study, PGE2, E. coli or its lipopolysaccharide (LPS), up-regulated the IE2 gene promoter in uterine cells. Bacterial co-infection is important for BoHV-4 uterine disease. PMID:18577555

  1. SGLT1 activity in lung alveolar cells of diabetic rats modulates airway surface liquid glucose concentration and bacterial proliferation.

    PubMed

    Oliveira, Tales Lyra; Candeia-Medeiros, Návylla; Cavalcante-Araújo, Polliane M; Melo, Igor Santana; Fávaro-Pípi, Elaine; Fátima, Luciana Alves; Rocha, Antônio Augusto; Goulart, Luiz Ricardo; Machado, Ubiratan Fabres; Campos, Ruy R; Sabino-Silva, Robinson

    2016-01-01

    High glucose concentration in the airway surface liquid (ASL) is an important feature of diabetes that predisposes to respiratory infections. We investigated the role of alveolar epithelial SGLT1 activity on ASL glucose concentration and bacterial proliferation. Non-diabetic and diabetic rats were intranasally treated with saline, isoproterenol (to increase SGLT1 activity) or phlorizin (to decrease SGLT1 activity); 2 hours later, glucose concentration and bacterial proliferation (methicillin-resistant Sthaphylococcus aureus, MRSA and Pseudomonas aeruginosa, P. aeruginosa) were analyzed in bronchoalveolar lavage (BAL); and alveolar SGLT1 was analyzed by immunohistochemistry. BAL glucose concentration and bacterial proliferation increased in diabetic animals: isoproterenol stimulated SGLT1 migration to luminal membrane, and reduced (50%) the BAL glucose concentration; whereas phlorizin increased the BAL glucose concentration (100%). These regulations were accompanied by parallel changes of in vitro MRSA and P. aeruginosa proliferation in BAL (r = 0.9651 and r = 0.9613, respectively, Pearson correlation). The same regulations were observed in in vivo P. aeruginosa proliferation. In summary, the results indicate a relationship among SGLT1 activity, ASL glucose concentration and pulmonary bacterial proliferation. Besides, the study highlights that, in situations of pulmonary infection risk, such as in diabetic subjects, increased SGLT1 activity may prevent bacterial proliferation whereas decreased SGLT1 activity can exacerbate it. PMID:26902517

  2. SGLT1 activity in lung alveolar cells of diabetic rats modulates airway surface liquid glucose concentration and bacterial proliferation

    PubMed Central

    Oliveira, Tales Lyra; Candeia-Medeiros, Návylla; Cavalcante-Araújo, Polliane M.; Melo, Igor Santana; Fávaro-Pípi, Elaine; Fátima, Luciana Alves; Rocha, Antônio Augusto; Goulart, Luiz Ricardo; Machado, Ubiratan Fabres; Campos, Ruy R.; Sabino-Silva, Robinson

    2016-01-01

    High glucose concentration in the airway surface liquid (ASL) is an important feature of diabetes that predisposes to respiratory infections. We investigated the role of alveolar epithelial SGLT1 activity on ASL glucose concentration and bacterial proliferation. Non-diabetic and diabetic rats were intranasally treated with saline, isoproterenol (to increase SGLT1 activity) or phlorizin (to decrease SGLT1 activity); 2 hours later, glucose concentration and bacterial proliferation (methicillin-resistant Sthaphylococcus aureus, MRSA and Pseudomonas aeruginosa, P. aeruginosa) were analyzed in bronchoalveolar lavage (BAL); and alveolar SGLT1 was analyzed by immunohistochemistry. BAL glucose concentration and bacterial proliferation increased in diabetic animals: isoproterenol stimulated SGLT1 migration to luminal membrane, and reduced (50%) the BAL glucose concentration; whereas phlorizin increased the BAL glucose concentration (100%). These regulations were accompanied by parallel changes of in vitro MRSA and P. aeruginosa proliferation in BAL (r = 0.9651 and r = 0.9613, respectively, Pearson correlation). The same regulations were observed in in vivo P. aeruginosa proliferation. In summary, the results indicate a relationship among SGLT1 activity, ASL glucose concentration and pulmonary bacterial proliferation. Besides, the study highlights that, in situations of pulmonary infection risk, such as in diabetic subjects, increased SGLT1 activity may prevent bacterial proliferation whereas decreased SGLT1 activity can exacerbate it. PMID:26902517

  3. Bacterial RNAs activate innate immunity in Arabidopsis.

    PubMed

    Lee, Boyoung; Park, Yong-Soon; Lee, Soohyun; Song, Geun Cheol; Ryu, Choong-Min

    2016-01-01

    The common molecular patterns of microbes play a critical role in the regulation of plant innate immunity. However, little is known about the role of nucleic acids in this process in plants. We pre-infiltrated Arabidopsis leaves with total RNAs from Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) and subsequently inoculated these plants with the same bacterial cells. Total Pto DC3000 RNAs pre-infiltrated into Arabidopsis leaves elicited plant immune responses against Pto DC3000. However, sheared RNAs and RNase A application failed to induce immunity, suggesting that intact bacterial RNAs function in plant innate immunity. This notion was supported by the positive regulation of superoxide anion levels, callose deposition, two mitogen-activated protein kinases and defense-related genes observed in bacterial RNA-pre-treated leaves. Intriguingly, the Pto DC3000 population was not compromised in known pattern recognition receptor mutants for chitin, flagellin and elongation factor-Tu (EF-Tu). Plant defense-related mutant analyses further revealed that bacterial RNA-elicited innate immunity was normally required for salicylic and jasmonic acid signaling. Notably, among total RNAs, the abundant bacterial RNA species 16S and 23S ribosomal RNAs were the major determinants of this response. Our findings provide evidence that bacterial RNA serves as a microbe-associated molecular pattern in plants. PMID:26499893

  4. Visualization and quantification of archaeal and bacterial metabolically active cells in soil using fluorescence in situ hybridization method

    NASA Astrophysics Data System (ADS)

    Semenov, Mikhail; Manucharova, Natalia; Stepanov, Alexey

    2015-04-01

    The method of in situ hybridization using fluorescent labeled 16S rRNA-targeted oligonucleotide probes (FISH - fluorescence in situ hybridization) combines identification and quantification of groups of microorganisms at different phylogenetic levels, from domain to species. The FISH method enables to study the soil microbial community in situ, avoiding plating on nutrient media, and allows to identify and quantify living, metabolically active cells of Bacteria and Archaea. The full procedure consists of the following steps: desorption of the cells from the soil particles, fixation of cells, coating a fixed sample on the glass slide, hybridization with the specific probes and, finally, microscopic observation and cell counting. For the FISH analysis of Bacteria and Archaea, the paraformaldehyde-fixed samples were hybridized with Cy3-labeled Archaea-specific probe(Arc915) and 6-carboxyfluorescein (FAM)-labeled Bacteria-specific probe(EUB338). When a molecular probe is incorporated into a cell, it can hybridize solely with a complementary rRNA sequence. The hybridization can be visualized under the fluorescent microscope and counted. The application of FISH will be demonstrated by the abundance of metabolically active cells of Archaea and Bacteria depending on soil properties, depth and land use. The research was carried out at field and natural ecosystems of European part of Russia. Samples were collected within the soil profiles (3-6 horizons) of Chernozem and Kastanozem with distinct land use. Quantification of metabolically active cells in virgin and arable Chernozem revealed that the abundance of Archaea in topsoil of virgin Chernozem was doubled as compared with arable soil, but it leveled off in the deeper horizons. Plowing of Chernozem decreased an amount of archaeal and bacterial active cells simultaneously, however, Bacteria were more resistant to agrogenic impact than Archaea. In Kastanozem, a significant change in the abundance of metabolically active

  5. Human Interleukin-2 and Hen Egg White Lysozyme: Screening for Bacteriolytic Activity against Various Bacterial Cells

    PubMed Central

    Levashov, P. A.; Ovchinnikova, E. D.; Morozova, O. A.; Matolygina, D. A.; Osipova, H. E.; Cherdyntseva, T. A.; Savin, S. S.; Zakharova, G. S.; Alekseeva, A. A.; Belogurova, N. G.; Smirnov, S. A.; Tishkov, V. I.; Levashov, A. V.

    2016-01-01

    The bacteriolytic activity of interleukin-2 and hen egg white lysozyme against 34 different species of microorganisms has been studied. It was found that 6 species of microorganisms are lysed in the presence of interleukin-2. All interleukin-2-sensitive microorganisms belong either to the Enterobacteriaceae, Bacillaceae, or the Lactobacillaceae family. It was also found that 12 species of microorganisms are lysed in the presence of lysozyme, and 16 species of microorganisms are lysed in the presence of sodium dodecyl sulfate (SDS). The bacteriolytic activity of interleukin-2 and lysozyme was studied at various pH values. PMID:27099789

  6. Specific sorting of single bacterial cells with microfabricated fluorescence-activated cell sorting and tyramide signal amplification fluorescence in situ hybridization.

    PubMed

    Chen, Chun H; Cho, Sung H; Chiang, Hsin-I; Tsai, Frank; Zhang, Kun; Lo, Yu-Hwa

    2011-10-01

    When attempting to probe the genetic makeup of diverse bacterial communities that elude cell culturing, researchers face two primary challenges: isolation of rare bacteria from microbial samples and removal of contaminating cell-free DNA. We report a compact, low-cost, and high-performance microfabricated fluorescence-activated cell sorting (μFACS) technology in combination with a tyramide signal amplification fluorescence in situ hybridization (TSA-FISH) to address these two challenges. The TSA-FISH protocol that was adapted for flow cytometry yields a 10-30-fold enhancement in fluorescence intensity over standard FISH methods. The μFACS technology, capable of enhancing its sensitivity by ~18 dB through signal processing, was able to enrich TSA-FISH-labeled E. coli cells by 223-fold. The μFACS technology was also used to remove contaminating cell-free DNA. After two rounds of sorting on E. coli mixed with λ-phage DNA (10 ng/μL), we demonstrated over 100,000-fold reduction in λ-DNA concentration. The integrated μFACS and TSA-FISH technologies provide a highly effective and low-cost solution for research on the genomic complexity of bacteria as well as single-cell genomic analysis of other sample types. PMID:21809842

  7. Resonant activation: a strategy against bacterial persistence

    NASA Astrophysics Data System (ADS)

    Fu, Yan; Zhu, Meng; Xing, Jianhua

    2010-03-01

    A bacterial colony may develop a small number of cells genetically identical to, but phenotypically different from, other normally growing bacteria. These so-called persister cells keep themselves in a dormant state and thus are insensitive to antibiotic treatment, resulting in serious problems of drug resistance. In this paper, we proposed a novel strategy to 'kill' persister cells by triggering them to switch, in a fast and synchronized way, into normally growing cells that are susceptible to antibiotics. The strategy is based on resonant activation (RA), a well-studied phenomenon in physics where the internal noise of a system can constructively facilitate fast and synchronized barrier crossings. Through stochastic Gilliespie simulation with a generic toggle switch model, we demonstrated that RA exists in the phenotypic switching of a single bacterium. Further, by coupling single cell level and population level simulations, we showed that with RA, one can greatly reduce the time and total amount of antibiotics needed to sterilize a bacterial population. We suggest that resonant activation is a general phenomenon in phenotypic transition, and can find other applications such as cancer therapy.

  8. Electron Cryotomography of Bacterial Cells

    PubMed Central

    Chen, Songye; McDowall, Alasdair; Dobro, Megan J.; Briegel, Ariane; Ladinsky, Mark; Shi, Jian; Tocheva, Elitza I.; Beeby, Morgan; Pilhofer, Martin; Ding, H. Jane; Li, Zhuo; Gan, Lu; Morris, Dylan M.; Jensen, Grant J.

    2010-01-01

    While much is already known about the basic metabolism of bacterial cells, many fundamental questions are still surprisingly unanswered, including for instance how they generate and maintain specific cell shapes, establish polarity, segregate their genomes, and divide. In order to understand these phenomena, imaging technologies are needed that bridge the resolution gap between fluorescence light microscopy and higher-resolution methods such as X-ray crystallography and NMR spectroscopy. Electron cryotomography (ECT) is an emerging technology that does just this, allowing the ultrastructure of cells to be visualized in a near-native state, in three dimensions (3D), with "macromolecular" resolution (~4nm).1, 2 In ECT, cells are imaged in a vitreous, "frozen-hydrated" state in a cryo transmission electron microscope (cryoTEM) at low temperature (< -180°C). For slender cells (up to ~500 nm in thickness3), intact cells are plunge-frozen within media across EM grids in cryogens such as ethane or ethane/propane mixtures. Thicker cells and biofilms can also be imaged in a vitreous state by first "high-pressure freezing" and then, "cryo-sectioning" them. A series of two-dimensional projection images are then collected through the sample as it is incrementally tilted along one or two axes. A three-dimensional reconstruction, or "tomogram" can then be calculated from the images. While ECT requires expensive instrumentation, in recent years, it has been used in a few labs to reveal the structures of various external appendages, the structures of different cell envelopes, the positions and structures of cytoskeletal filaments, and the locations and architectures of large macromolecular assemblies such as flagellar motors, internal compartments and chemoreceptor arrays.1, 2 In this video article we illustrate how to image cells with ECT, including the processes of sample preparation, data collection, tomogram reconstruction, and interpretation of the results through

  9. Alveolar Epithelial Cells Are Critical in Protection of the Respiratory Tract by Secretion of Factors Able To Modulate the Activity of Pulmonary Macrophages and Directly Control Bacterial Growth

    PubMed Central

    Petursdottir, Dagbjort H.; Periolo, Natalia; Fernández, Carmen

    2013-01-01

    The respiratory epithelium is a physical and functional barrier actively involved in the clearance of environmental agents. The alveolar compartment is lined with membranous pneumocytes, known as type I alveolar epithelial cells (AEC I), and granular pneumocytes, type II alveolar epithelial cells (AEC II). AEC II are responsible for epithelial reparation upon injury and ion transport and are very active immunologically, contributing to lung defense by secreting antimicrobial factors. AEC II also secrete a broad variety of factors, such as cytokines and chemokines, involved in activation and differentiation of immune cells and are able to present antigen to specific T cells. Another cell type important in lung defense is the pulmonary macrophage (PuM). Considering the architecture of the alveoli, a good communication between the external and the internal compartments is crucial to mount effective responses. Our hypothesis is that being in the interface, AEC may play an important role in transmitting signals from the external to the internal compartment and in modulating the activity of PuM. For this, we collected supernatants from AEC unstimulated or stimulated in vitro with lipopolysaccharide (LPS). These AEC-conditioned media were used in various setups to test for the effects on a number of macrophage functions: (i) migration, (ii) phagocytosis and intracellular control of bacterial growth, and (iii) phenotypic changes and morphology. Finally, we tested the direct effect of AEC-conditioned media on bacterial growth. We found that AEC-secreted factors had a dual effect, on one hand controlling bacterial growth and on the other hand increasing macrophage activity. PMID:23147039

  10. In vivo activation of the intracrine vitamin D pathway in innate immune cells and mammary tissue during a bacterial infection.

    PubMed

    Nelson, Corwin D; Reinhardt, Timothy A; Beitz, Donald C; Lippolis, John D

    2010-01-01

    Numerous in vitro studies have shown that toll-like receptor signaling induces 25-hydroxyvitamin D(3) 1α-hydroxylase (1α-OHase; CYP27B1) expression in macrophages from various species. 1α-OHase is the primary enzyme that converts 25-hydroxyvitamin D(3) to 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). Subsequently, synthesis of 1,25(OH)(2)D(3) by 1α-OHase in macrophages has been shown to modulate innate immune responses of macrophages. Despite the numerous in vitro studies that have shown 1α-OHase expression is induced in macrophages, however, evidence that 1α-OHase expression is induced by pathogens in vivo is limited. The objective of this study was to evaluate 1α-OHase gene expression in macrophages and mammary tissue during an in vivo bacterial infection with Streptococcus uberis. In tissue and secreted cells from the infected mammary glands, 1α-OHase gene expression was significantly increased compared to expression in tissue and cells from the healthy mammary tissue. Separation of the cells by FACS9 revealed that 1α-OHase was predominantly expressed in the CD14(+) cells isolated from the infected mammary tissue. The 24-hydroxylase gene, a gene that is highly upregulated by 1,25(OH)(2)D(3), was significantly more expressed in tissue and cells from the infected mammary tissue than from the healthy uninfected mammary tissue thus indicating significant local 1,25(OH)(2)D(3) production at the infection site. In conclusion, this study provides the first in vivo evidence that 1α-OHase expression is upregulated in macrophages in response to bacterial infection and that 1α-OHase at the site of infection provides 1,25(OH)(2)D(3) for local regulation of vitamin D responsive genes. PMID:21124742

  11. Addition of Selenium Nanoparticles to Electrospun Silk Scaffold Improves the Mammalian Cell Activity While Reducing Bacterial Growth

    PubMed Central

    Chung, Stanley; Ercan, Batur; Roy, Amit K.; Webster, Thomas J.

    2016-01-01

    Silk possesses many beneficial wound healing properties, and electrospun scaffolds are especially applicable for skin applications, due to their smaller interstices and higher surface areas. However, purified silk promotes microbial growth. Selenium nanoparticles have shown excellent antibacterial properties and are a novel antimicrobial chemistry. Here, electrospun silk scaffolds were doped with selenium nanoparticles to impart antibacterial properties to the silk scaffolds. Results showed significantly improved bacterial inhibition and mild improvement in human dermal fibroblast metabolic activity. These results suggest that the addition of selenium nanoparticles to electrospun silk is a promising approach to improve wound healing with reduced infection, without relying on antibiotics. PMID:27471473

  12. 2'-O-Methylation within Bacterial RNA Acts as Suppressor of TLR7/TLR8 Activation in Human Innate Immune Cells.

    PubMed

    Rimbach, Katharina; Kaiser, Steffen; Helm, Mark; Dalpke, Alexander H; Eigenbrod, Tatjana

    2015-01-01

    Microbial RNA is an important stimulator of innate immune responses. Differences in posttranscriptional RNA modification profiles enable the immune system to discriminate between self and non-self nucleic acids. This principle may be exploited by certain bacteria to circumvent immune cell activation. In this regard, 2'-O-methylation of Escherichia coli tRNATyr at position 18 (Gm18) has recently been described to inhibit TLR7-mediated IFN-α production in human plasmacytoid dendritic cells (pDCs). Extending these findings, we now demonstrate that Gm18 also potently inhibits TLR7-independent human monocyte activation by RNA derived from a variety of bacterial strains. The half minimal inhibitory concentration values were similar to those found for IFN-α inhibition in pDCs. Mechanistically, 2'-O-methylated RNA impaired upstream signalling events, including MAP kinase and NFx03BA;B activation. Our results suggest that antagonizing effects of Gm18-modified RNA are due to competition with stimulatory RNA for receptor binding. The antagonistic effect was specific for RNA because the small molecule TLR7/8 agonist R848 was not inhibited. Despite the striking phenotype in human cells, 2'-O-methylated RNA did not interfere with TLR13 activation by bacterial 23S rRNA in murine DC and BMDM. Thus, we identify here Gm18 in E. coli tRNA(Tyr) as a universal suppressor of innate immune activation in the human but not the murine system. PMID:25823462

  13. Bacterial Heat Shock Protein Activity

    PubMed Central

    Maleki, Farajollah; Khosravi, Afra; Nasser, Ahmad; Taghinejad, Hamid

    2016-01-01

    Bacteria are exposed to different types of stress in their growth conditions. They have developed appropriate responses, modulated by the re-modeling of protein complexes and by phosphorylation dependent signal transduction systems, to adapt and to survive in a variety range of nature. Proteins are essential components for biologic activity in the eukaryotic and prokaryotic cell. Heat Shock Proteins (HSP) have been identified from various organisms and have critical role in cell hemostasis. Chaperone can sense environment and have different potential role in the organism evolution. PMID:27134861

  14. Antifungal activity of plant and bacterial ureases.

    PubMed

    Becker-Ritt, A B; Martinelli, A H S; Mitidieri, S; Feder, V; Wassermann, G E; Santi, L; Vainstein, M H; Oliveira, J T A; Fiuza, L M; Pasquali, G; Carlini, C R

    2007-12-01

    Ureases (EC 3.5.1.5) are nickel-dependent metalloenzymes that catalyze the hydrolysis of urea to ammonia and carbon dioxide. Produced by plants, fungi and bacteria, but not by animals, ureases share significant homology and similar mechanisms of catalysis, although differing in quaternary structures. While fungal and plant ureases are homo-oligomeric proteins of 90 kDa subunits, bacterial ureases are multimers of two (e.g. Helicobacter pylori) or three subunit complexes. It has been proposed that in plants these enzymes are involved in nitrogen bioavailability and in protection against pathogens. Previous studies by our group have shown that plant ureases, but not a bacterial (Bacillus pasteurii) urease, display insecticidal activity. Herein we demonstrate that (Glycine max) embryo-specific soybean urease, jackbean (Canavalia ensiformis) major urease and a recombinant H. pylori urease impair growth of selected phytopathogenic fungi at sub-micromolar concentrations. This antifungal property of ureases is not affected by treatment of the proteins with an irreversible inhibitor of the ureolytic activity. Scanning electron microscopy of urease-treated fungi suggests plasmolysis and cell wall injuries. Altogether, our data indicate that ureases probably contribute to the plant arsenal of defense compounds against predators and phytopathogens and that the urease defense mechanism is independent of ammonia release from urea. PMID:17825863

  15. Bacterial recognition pathways that lead to inflammasome activation

    PubMed Central

    Storek, Kelly M; Monack, Denise M

    2015-01-01

    Inflammasomes are multi-protein signaling platforms that upon activation trigger the maturation of the pro-inflammatory cytokines, interleukin-1β (IL-1β) and IL-18, and cell death. Inflammasome sensors detect microbial and host-derived molecules. Here, we review the mechanisms of inflammasome activation triggered by bacterial infection, primarily focusing on two model intracellular bacterial pathogens, Francisella novicida and Salmonella typhimurium. We discuss the complex relationship between bacterial recognition through direct and indirect detection by inflammasome sensors. We highlight regulation mechanisms that potentiate or limit inflammasome activation. We discuss the importance of caspase-1 and caspase-11 in host defense, and we examine the downstream consequences of inflammasome activation within the context of bacterial infections. PMID:25879288

  16. Electroporation of Functional Bacterial Effectors into Mammalian Cells

    DOE PAGESBeta

    Sontag, Ryan L.; Mihai, Cosmin; Orr, Galya; Savchenko, Alexei; Skarina, Tatiana; Cui, Hong; Cort, John R.; Adkins, Joshua N.; Brown, Roslyn N.

    2015-01-19

    Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

  17. Electroporation of Functional Bacterial Effectors into Mammalian Cells

    SciTech Connect

    Sontag, Ryan L.; Mihai, Cosmin; Orr, Galya; Savchenko, Alexei; Skarina, Tatiana; Cui, Hong; Cort, John R.; Adkins, Joshua N.; Brown, Roslyn N.

    2015-01-01

    Electroporation was used to insert purified bacterial virulence effector proteins directly into living eukaryotic cells. Protein localization was monitored by confocal immunofluorescence microscopy. This method allows for studies on trafficking, function, and protein-protein interactions using active exogenous proteins, avoiding the need for heterologous expression in eukaryotic cells.

  18. Bacterial vectors for active immunotherapy reach clinical and industrial stages

    PubMed Central

    Le Gouëllec, Audrey; Chauchet, Xavier; Polack, Benoit; Buffat, Laurent; Toussaint, Bertrand

    2012-01-01

    Active immunotherapy based on live attenuated bacterial vectors has matured in terms of industrial development and develops through a combination of three phenomena. First, active immunotherapy that stimulates an antigen-specific cytotoxic T-cell immune response has become a reality after several years of work. Second, there is still a need to identify vectors that can deliver antigens to the cytosol of antigen-presenting cells in vivo. Third, the recent progress in the understanding of bacterial lifestyle and in developing genetic engineering tools has enabled the design of bioengineered bugs that are capable of delivering antigens. Here, we review the mechanisms by which clinical bacterial vectors deliver antigens into the cytosol of antigen-presenting cells and summarize the development strategy of the three identified firms in this field. PMID:22894945

  19. Activation of phagocytic cells by Staphylococcus epidermidis biofilms: effects of extracellular matrix proteins and the bacterial stress protein GroEL on netosis and MRP-14 release.

    PubMed

    Dapunt, Ulrike; Gaida, Matthias M; Meyle, Eva; Prior, Birgit; Hänsch, Gertrud M

    2016-07-01

    The recognition and phagocytosis of free-swimming (planktonic) bacteria by polymorphonuclear neutrophils have been investigated in depth. However, less is known about the neutrophil response towards bacterial biofilms. Our previous work demonstrated that neutrophils recognize activating entities within the extracellular polymeric substance (EPS) of biofilms (the bacterial heat shock protein GroEL) and that this process does not require opsonization. Aim of this study was to evaluate the release of DNA by neutrophils in response to biofilms, as well as the release of the inflammatory cytokine MRP-14. Neutrophils were stimulated with Staphylococcus epidermidis biofilms, planktonic bacteria, extracted EPS and GroEL. Release of DNA and of MRP-14 was evaluated. Furthermore, tissue samples from patients suffering from biofilm infections were collected and evaluated by histology. MRP-14 concentration in blood samples was measured. We were able to show that biofilms, the EPS and GroEL induce DNA release. MRP-14 was only released after stimulation with EPS, not GroEL. Histology of tissue samples revealed MRP-14 positive cells in association with neutrophil infiltration and MRP-14 concentration was elevated in blood samples of patients suffering from biofilm infections. Our data demonstrate that neutrophil-activating entities are present in the EPS and that GroEL induces DNA release by neutrophils. PMID:27109773

  20. Campylobacter jejuni outer membrane vesicle-associated proteolytic activity promotes bacterial invasion by mediating cleavage of intestinal epithelial cell E-cadherin and occludin.

    PubMed

    Elmi, Abdi; Nasher, Fauzy; Jagatia, Heena; Gundogdu, Ozan; Bajaj-Elliott, Mona; Wren, Brendan; Dorrell, Nick

    2016-04-01

    Outer membrane vesicles (OMVs) play an important role in the pathogenicity of Gram-negative bacteria. Campylobacter jejuni produces OMVs that trigger IL-8, IL-6, hBD-3 and TNF-α responses from T84 intestinal epithelial cells and are cytotoxic to Caco-2 IECs and Galleria mellonella larvae. Proteomic analysis of 11168H OMVs identified the presence of three proteases, HtrA, Cj0511 and Cj1365c. In this study, 11168H OMVs were shown to possess proteolytic activity that was reduced by pretreatment with specific serine protease inhibitors. OMVs isolated from 11168H htrA, Cj0511 or Cj1365c mutants possess significantly reduced proteolytic activity. 11168H OMVs are able to cleave both E-cadherin and occludin, but this cleavage is reduced with OMVs pretreated with serine protease inhibitors and also with OMVs isolated from htrA or Cj1365c mutants. Co-incubation of T84 monolayers with 11168H OMVs results in a visible reduction in both E-cadherin and occludin. The addition of 11168H OMVs to the co-culture of live 11168H bacteria with T84 cells results in enhanced levels of bacterial adhesion and invasion in a time-dependent and dose-dependent manner. Further investigation of the cleavage of host cell structural proteins by C. jejuni OMVs should enhance our understanding of the interactions of this important pathogen with intestinal epithelial cells. PMID:26451973

  1. Effects of minimal exposures to atmospheric pressure plasma on the activity of Salmonella Typhimurium: Deactivation of bacterial motility and suppression of host-cell invasion.

    PubMed

    Park, Jin-Sung; Kim, Kijung; Han, Je-Hyun; Gweon, Bomi; Ko, Ung Hyun; Yoo, Suk Jae; Choe, Wonho; Shin, Jennifer H

    2016-09-01

    Atmospheric pressure plasma (APP) has been shown effective in sterilization by reducing the number of viable microbes during surface cleaning, food processing, or human tissue treatment. For safe conduct, the majority of previous research focused on complete abolition of microbes, which may require severe treatments. Our aim is to investigate the minimal treatment conditions necessary for effective inactivation of bacteria in such a manner that the APP treated bacteria would not be able to harm the host cells. For this, we ought to identify the objective criteria to make the bacteria dysfunctional. We choose the motile properties and the host-cell invasion capability as two measures to quantify the pathogenic state of bacteria. In this paper, we investigated how the APP treatment in a minimal dosage affects the activity of Salmonella Typhimurium. At 100 W and 15 kHz for 20 s, the APP treatment effectively suppressed active "run and tumble" type motility and induced formation of abnormally long structures. With 20 s exposure, the bacterial cells failed to cause pyroptosis in the host cells with >90% survival after 12 h of co-incubation. Our results suggest novel measures to evaluate the functional pathogenic state for identifying safe APP treatment conditions. PMID:27345896

  2. Influence of bacterial endotoxin on radiation-induced activation of human endothelial cells in vitro and in vivo: protective role of IL-10.

    PubMed

    Eissner, G; Lindner, H; Behrends, U; Kölch, W; Hieke, A; Klauke, I; Bornkamm, G W; Holler, E

    1996-09-27

    Previous work from our group has contributed to demonstrate the role of conditioning related release of proinflammatory cytokines in induction of acute graft-versus-host disease (GVHD) following allogeneic bone marrow transplantation (BMT). In the present report we show that ionizing radiation (IR) in a clinical relevant dose upregulates intercellular adhesion molecule 1 (ICAM-1) on cultured human microvascular endothelial cells (HMEC). Bacterial endotoxin (lipopolysaccharide, LPS) in a concentration corresponding to serum levels seen during clinical endotoxemia, is capable of further enhancing ICAM-1 expression on irradiated cells. Adhesion assays with freshly isolated peripheral blood mononuclear cells (PBMC) revealed that increased ICAM-1 on IR-treated endothelial cells led to an increased adhesion of PBMC. Again, this effect could be superinduced by LPS. Recombinant human interleukin 10 (IL-10), an antagonistic cytokine known to function as an LPS antagonist, was able to counteract the LPS-mediated enhancement of IR-triggered ICAM-1 induction and PBMC adhesion. In contrast, IL-10 could not inhibit irradiation caused effects. IL-10 seemed to interfere with the translocation of preformed intracellular ICAM-1 to the cell membrane. To investigate whether this superinductive function of IR and LPS on endothelial cells is of clinical relevance, mice were treated with total body irradiation (TBI) and inoculated with a single dose of LPS. Immunohistochemical analyses of murine tissues demonstrated that LPS superinduces IR-triggered ICAM-1 also in vivo. These findings may be of clinical importance as they suggest that the endothelium is activated after radiotherapy or TBI used for conditioning in bone marrow transplantation. The activated endothelium in turn may facilitate the accumulation of effector cells at sites of inflammation. PMID:8824483

  3. Identification of bacterial cells by chromosomal painting.

    PubMed Central

    Lanoil, B D; Giovannoni, S J

    1997-01-01

    Chromosomal painting is a technique for the microscopic localization of genetic material. It has been applied at the subcellular level to identify regions of eukaryotic chromosomes. Here we describe the development of bacterial chromosomal painting (BCP), a related technology for the identification of bacterial cells. Purified genomic DNAs from six bacterial strains were labeled by nick translation with the fluorochrome Fluor-X, Cy3, or Cy5. The average size of the labeled fragments was ca. 50 to 200 bp. The probes were hybridized to formaldehyde-fixed microbial cells attached to slides and visualized by fluorescence microscopy. In reciprocal comparisons, distantly related members of the class Proteobacteria (Escherichia coli and Oceanospirillum linum), different species of the genus Bacillus (B. subtilis and B. megaterium), and different serotypes of the subspecies Salmonella choleraesuis subsp. choleraesuis (serotype typhimurium LT2 and serotype typhi Ty2) could easily be distinguished. A combination of two probes, each labeled with a different fluorochrome, was used successfully to simultaneously identify two cell types in a mixture. Lysozyme treatment was required for the identification of Bacillus spp., and RNase digestion and pepsin digestion were found to enhance signal strength and specificity for all cell types tested. Chromosome in situ suppression, a technique that removes cross-hybridizing fragments from the probe, was necessary for the differentiation of the Salmonella serotypes but was not required to distinguish the more distantly related taxa. BCP may have applications in diverse branches of microbiology where the objective is the identification of bacterial cells. PMID:9055426

  4. Photo-Induced Effect on Bacterial Cells

    NASA Astrophysics Data System (ADS)

    El Batanouny, M. H.; Amin, Rehab M.; Naga, M. I.; Ibrahim, M. K.

    2010-04-01

    Bacterial resistance against antibiotics is an increasing problem in medicine. This stimulates study of other bactericidal regimens, one of which is photodynamic therapy (PDT), which involves the killing of bacterial species by low power laser light (LLL) in the presence of photosensitizing agent. It has already been shown that, various gram- negative and gram-positive bacteria can be killed by photodynamic therapy in vitro, using exogenous sensitizers. The mechanisms of laser action on bacteria are not adequately understood. Here, PDT on H. pylori, as an example of gram negative bacteria was studied. The ultra structure changes of the organism after PDT were examined under electron microscope. Neither Irradiation with laser without sensitizer nor sensitizing without laser has any lethal effect on bacterial cells. However, the successful lethal photosensitization was achieved by applying certain laser dose with the corresponding concentration of the photosensitizer. On the other hand, PDT has no significant effect on the genomic DNA of the cells.

  5. Bacterial Networks in Cells and Communities.

    PubMed

    Sourjik, Victor; Vorholt, Julia A

    2015-11-20

    Research on the bacterial regulatory networks is currently experiencing a true revival, driven by advances in methodology and by emergence of novel concepts. The biannual conference Bacterial Networks (BacNet15) held in May 2015, in Sant Feliu de Guíxols, Spain, covered progress in the studies of regulatory networks that control bacterial physiology, cell biology, stress responses, metabolism, collective behavior and evolution. It demonstrated how interdisciplinary approaches that combine molecular biology and biochemistry with the latest microscopy developments, whole cell (-omics) approaches and mathematical modeling can help understand design principles relevant in microbiology. It further showed how current biotechnology and medical microbiology could profit from our knowledge of and ability to engineer regulatory networks of bacteria. PMID:26506266

  6. Chicken NK-lysin is an alpha-helical cationic peptide that exerts its antibacterial activity through damage of bacterial cell membranes.

    PubMed

    Lee, Mi Ok; Jang, Hyun-Jun; Han, Jae Yong; Womack, James E

    2014-04-01

    The antimicrobial peptides (AMP) are important elements of the first line of defense against pathogens in animals, and an important constituent of innate immunity. Antimicrobial peptides act on a broad spectrum of microbial organisms. NK-Lysin is a cationic antibacterial peptide that was originally isolated from porcine intestinal tissue based on its antibacterial activity. We synthesized peptides corresponding to each helical region of chicken NK-lysin and analyzed their secondary structures in addition to their antimicrobial activity. Circular dichroism spectroscopy of the synthetic chicken NK-lysin (cNK-78) and 4 small peptides in negatively charged liposomes demonstrated transition in the conformation of α-helical peptides relative to the charged environment. Chicken NK-lysin inhibits the growth of a representative gram-negative bacterium, Escherichia coli. The antimicrobial activity of 2 peptides designated H23 and H34 was similar to that of mature NK-lysin, cNK-78. Microscopic analyses revealed the death of bacterium with disrupted membranes after peptide treatment, suggesting that chicken NK-lysin, an alpha-helical cationic peptide, exerts its antimicrobial activity by damaging the bacterial cell membrane. PMID:24706963

  7. Macrophages inhibit human osteosarcoma cell growth after activation with the bacterial cell wall derivative liposomal muramyl tripeptide in combination with interferon-γ

    PubMed Central

    2014-01-01

    Background In osteosarcoma, the presence of tumor-infiltrating macrophages positively correlates with patient survival in contrast to the negative effect of tumor-associated macrophages in patients with other tumors. Liposome-encapsulated muramyl tripeptide (L-MTP-PE) has been introduced in the treatment of osteosarcoma patients, which may enhance the potential anti-tumor activity of macrophages. Direct anti-tumor activity of human macrophages against human osteosarcoma cells has not been described so far. Hence, we assessed osteosarcoma cell growth after co-culture with human macrophages. Methods Monocyte-derived M1-like and M2-like macrophages were polarized with LPS + IFN-γ, L-MTP-PE +/− IFN-γ or IL-10 and incubated with osteosarcoma cells. Two days later, viable tumor cell numbers were analyzed. Antibody-dependent effects were investigated using the therapeutic anti-EGFR antibody cetuximab. Results M1-like macrophages inhibited osteosarcoma cell growth when activated with LPS + IFN-γ. Likewise, stimulation of M1-like macrophages with liposomal muramyl tripeptide (L-MTP-PE) inhibited tumor growth, but only when combined with IFN-γ. Addition of the tumor-reactive anti-EGFR antibody cetuximab did not further improve the anti-tumor activity of activated M1-like macrophages. The inhibition was mediated by supernatants of activated M1-like macrophages, containing TNF-α and IL-1β. However, specific blockage of these cytokines, nitric oxide or reactive oxygen species did not inhibit the anti-tumor effect, suggesting the involvement of other soluble factors released upon macrophage activation. While LPS + IFN-γ–activated M2-like macrophages had low anti-tumor activity, IL-10–polarized M2-like macrophages were able to reduce osteosarcoma cell growth in the presence of the anti-EGFR cetuximab involving antibody-dependent tumor cell phagocytosis. Conclusion This study demonstrates that human macrophages can be induced to exert direct anti

  8. Macrophage cell death upon intracellular bacterial infection

    PubMed Central

    Lai, Xin-He; Xu, Yunsheng; Chen, Xiao-Ming; Ren, Yi

    2015-01-01

    Macrophage-pathogen interaction is a complex process and the outcome of this tag-of-war for both sides is to live or die. Without attempting to be comprehensive, this review will discuss the complexity and significance of the interaction outcomes between macrophages and some facultative intracellular bacterial pathogens as exemplified by Francisella, Salmonella, Shigella and Yersinia. Upon bacterial infection, macrophages can die by a variety of ways, such as apoptosis, autophagic cell death, necrosis, necroptosis, oncosis, pyronecrosis, pyroptosis etc, which is the focus of this review. PMID:26690967

  9. Colon-targeted delivery of live bacterial cell biotherapeutics including microencapsulated live bacterial cells

    PubMed Central

    Prakash, Satya; Malgorzata Urbanska, Aleksandra

    2008-01-01

    There has been an ample interest in delivery of therapeutic molecules using live cells. Oral delivery has been stipulated as best way to deliver live cells to humans for therapy. Colon, in particular, is a part of gastrointestinal (GI) tract that has been proposed to be an oral targeted site. The main objective of these oral therapy procedures is to deliver live cells not only to treat diseases like colorectal cancer, inflammatory bowel disease, and other GI tract diseases like intestinal obstruction and gastritis, but also to deliver therapeutic molecules for overall therapy in various diseases such as renal failure, coronary heart disease, hypertension, and others. This review provides a comprehensive summary of recent advancement in colon targeted live bacterial cell biotherapeutics. Current status of bacterial cell therapy, principles of artificial cells and its potentials in oral delivery of live bacterial cell biotherapeutics for clinical applications as well as biotherapeutic future perspectives are also discussed in our review. PMID:19707368

  10. Single Cell Analysis of a Bacterial Sender-Receiver System.

    PubMed

    Ramalho, Tiago; Meyer, Andrea; Mückl, Andrea; Kapsner, Korbinian; Gerland, Ulrich; Simmel, Friedrich C

    2016-01-01

    Monitoring gene expression dynamics on the single cell level provides important information on cellular heterogeneity and stochasticity, and potentially allows for more accurate quantitation of gene expression processes. We here study bacterial senders and receivers genetically engineered with components of the quorum sensing system derived from Aliivibrio fischeri on the single cell level using microfluidics-based bacterial chemostats and fluorescence video microscopy. We track large numbers of bacteria over extended periods of time, which allows us to determine bacterial lineages and filter out subpopulations within a heterogeneous population. We quantitatively determine the dynamic gene expression response of receiver bacteria to varying amounts of the quorum sensing inducer N-3-oxo-C6-homoserine lactone (AHL). From this we construct AHL response curves and characterize gene expression dynamics of whole bacterial populations by investigating the statistical distribution of gene expression activity over time. The bacteria are found to display heterogeneous induction behavior within the population. We therefore also characterize gene expression in a homogeneous bacterial subpopulation by focusing on single cell trajectories derived only from bacteria with similar induction behavior. The response at the single cell level is found to be more cooperative than that obtained for the heterogeneous total population. For the analysis of systems containing both AHL senders and receiver cells, we utilize the receiver cells as 'bacterial sensors' for AHL. Based on a simple gene expression model and the response curves obtained in receiver-only experiments, the effective AHL concentration established by the senders and their 'sending power' is determined. PMID:26808777

  11. Single Cell Analysis of a Bacterial Sender-Receiver System

    PubMed Central

    Mückl, Andrea; Kapsner, Korbinian; Gerland, Ulrich; Simmel, Friedrich C.

    2016-01-01

    Monitoring gene expression dynamics on the single cell level provides important information on cellular heterogeneity and stochasticity, and potentially allows for more accurate quantitation of gene expression processes. We here study bacterial senders and receivers genetically engineered with components of the quorum sensing system derived from Aliivibrio fischeri on the single cell level using microfluidics-based bacterial chemostats and fluorescence video microscopy. We track large numbers of bacteria over extended periods of time, which allows us to determine bacterial lineages and filter out subpopulations within a heterogeneous population. We quantitatively determine the dynamic gene expression response of receiver bacteria to varying amounts of the quorum sensing inducer N-3-oxo-C6-homoserine lactone (AHL). From this we construct AHL response curves and characterize gene expression dynamics of whole bacterial populations by investigating the statistical distribution of gene expression activity over time. The bacteria are found to display heterogeneous induction behavior within the population. We therefore also characterize gene expression in a homogeneous bacterial subpopulation by focusing on single cell trajectories derived only from bacteria with similar induction behavior. The response at the single cell level is found to be more cooperative than that obtained for the heterogeneous total population. For the analysis of systems containing both AHL senders and receiver cells, we utilize the receiver cells as ‘bacterial sensors’ for AHL. Based on a simple gene expression model and the response curves obtained in receiver-only experiments, the effective AHL concentration established by the senders and their ‘sending power’ is determined. PMID:26808777

  12. Biosensors for Whole-Cell Bacterial Detection

    PubMed Central

    Rushworth, Jo V.; Hirst, Natalie A.; Millner, Paul A.

    2014-01-01

    SUMMARY Bacterial pathogens are important targets for detection and identification in medicine, food safety, public health, and security. Bacterial infection is a common cause of morbidity and mortality worldwide. In spite of the availability of antibiotics, these infections are often misdiagnosed or there is an unacceptable delay in diagnosis. Current methods of bacterial detection rely upon laboratory-based techniques such as cell culture, microscopic analysis, and biochemical assays. These procedures are time-consuming and costly and require specialist equipment and trained users. Portable stand-alone biosensors can facilitate rapid detection and diagnosis at the point of care. Biosensors will be particularly useful where a clear diagnosis informs treatment, in critical illness (e.g., meningitis) or to prevent further disease spread (e.g., in case of food-borne pathogens or sexually transmitted diseases). Detection of bacteria is also becoming increasingly important in antibioterrorism measures (e.g., anthrax detection). In this review, we discuss recent progress in the use of biosensors for the detection of whole bacterial cells for sensitive and earlier identification of bacteria without the need for sample processing. There is a particular focus on electrochemical biosensors, especially impedance-based systems, as these present key advantages in terms of ease of miniaturization, lack of reagents, sensitivity, and low cost. PMID:24982325

  13. Twenty Years of Active Bacterial Core Surveillance

    PubMed Central

    Schaffner, William; Farley, Monica M.; Lynfield, Ruth; Bennett, Nancy M.; Reingold, Arthur; Thomas, Ann; Harrison, Lee H.; Nichols, Megin; Petit, Susan; Miller, Lisa; Moore, Matthew R.; Schrag, Stephanie J.; Lessa, Fernanda C.; Skoff, Tami H.; MacNeil, Jessica R.; Briere, Elizabeth C.; Weston, Emily J.; Van Beneden, Chris

    2015-01-01

    Active Bacterial Core surveillance (ABCs) was established in 1995 as part of the Centers for Disease Control and Prevention Emerging Infections Program (EIP) network to assess the extent of invasive bacterial infections of public health importance. ABCs is distinctive among surveillance systems because of its large, population-based, geographically diverse catchment area; active laboratory-based identification of cases to ensure complete case capture; detailed collection of epidemiologic information paired with laboratory isolates; infrastructure that allows for more in-depth investigations; and sustained commitment of public health, academic, and clinical partners to maintain the system. ABCs has directly affected public health policies and practices through the development and evaluation of vaccines and other prevention strategies, the monitoring of antimicrobial drug resistance, and the response to public health emergencies and other emerging infections. PMID:26292067

  14. Adenylate Cyclase Toxin promotes bacterial internalisation into non phagocytic cells

    PubMed Central

    Martín, César; Etxaniz, Asier; Uribe, Kepa B.; Etxebarria, Aitor; González-Bullón, David; Arlucea, Jon; Goñi, Félix M.; Aréchaga, Juan; Ostolaza, Helena

    2015-01-01

    Bordetella pertussis causes whooping cough, a respiratory infectious disease that is the fifth largest cause of vaccine-preventable death in infants. Though historically considered an extracellular pathogen, this bacterium has been detected both in vitro and in vivo inside phagocytic and non-phagocytic cells. However the precise mechanism used by B. pertussis for cell entry, or the putative bacterial factors involved, are not fully elucidated. Here we find that adenylate cyclase toxin (ACT), one of the important toxins of B. pertussis, is sufficient to promote bacterial internalisation into non-phagocytic cells. After characterization of the entry route we show that uptake of “toxin-coated bacteria” proceeds via a clathrin-independent, caveolae-dependent entry pathway, allowing the internalised bacteria to survive within the cells. Intracellular bacteria were found inside non-acidic endosomes with high sphingomyelin and cholesterol content, or “free” in the cytosol of the invaded cells, suggesting that the ACT-induced bacterial uptake may not proceed through formation of late endolysosomes. Activation of Tyr kinases and toxin-induced Ca2+-influx are essential for the entry process. We hypothesize that B. pertussis might use ACT to activate the endocytic machinery of non-phagocytic cells and gain entry into these cells, in this way evading the host immune system. PMID:26346097

  15. Bacterial invasion of vascular cell types: vascular infectology and atherogenesis.

    PubMed

    Kozarov, Emil

    2012-01-01

    To portray the chronic inflammation in atherosclerosis, leukocytic cell types involved in the immune response to invading pathogens are often the focus. However, atherogenesis is a complex pathological deterioration of the arterial walls, where vascular cell types are participants with regards to deterioration and disease. Since other recent reviews have detailed the role of both the innate and adaptive immune response in atherosclerosis, herein we will summarize the latest developments regarding the association of bacteria with vascular cell types: infections as a risk factor for atherosclerosis; bacterial invasion of vascular cell types; the atherogenic sequelae of bacterial presence such as endothelial activation and blood clotting; and the identification of the species that are able to colonize this niche. The evidence of a polybacterial infectious component of the atheromatous lesions opens the doors for exploration of the new field of vascular infectology and for the study of atherosclerosis microbiome. PMID:22185451

  16. Fluorescence Activated Cell Sorting of Rickettsia prowazekii-Infected Host Cells Based on Bacterial Burden and Early Detection of Fluorescent Rickettsial Transformants

    PubMed Central

    Driskell, Lonnie O.; Tucker, Aimee M.; Woodard, Andrew; Wood, Raphael R.; Wood, David O.

    2016-01-01

    Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular bacterium that replicates only within the cytosol of a eukaryotic host cell. Despite the barriers to genetic manipulation that such a life style creates, rickettsial mutants have been generated by transposon insertion as well as by homologous recombination mechanisms. However, progress is hampered by the length of time required to identify and isolate R. prowazekii transformants. To reduce the time required and variability associated with propagation and harvesting of rickettsiae for each transformation experiment, characterized frozen stocks were used to generate electrocompetent rickettsiae. Transformation experiments employing these rickettsiae established that fluorescent rickettsial populations could be identified using a fluorescence activated cell sorter within one week following electroporation. Early detection was improved with increasing amounts of transforming DNA. In addition, we demonstrate that heterogeneous populations of rickettsiae-infected cells can be sorted into distinct sub-populations based on the number of rickettsiae per cell. Together our data suggest the combination of fluorescent reporters and cell sorting represent an important technical advance that will facilitate isolation of distinct R. prowazekii mutants and allow for closer examination of the effects of infection on host cells at various infectious burdens. PMID:27010457

  17. Expression of bacterial genes in plant cells.

    PubMed Central

    Fraley, R T; Rogers, S G; Horsch, R B; Sanders, P R; Flick, J S; Adams, S P; Bittner, M L; Brand, L A; Fink, C L; Fry, J S; Galluppi, G R; Goldberg, S B; Hoffmann, N L; Woo, S C

    1983-01-01

    Chimeric bacterial genes conferring resistance to aminoglycoside antibiotics have been inserted into the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid and introduced into plant cells by in vitro transformation techniques. The chimeric genes contain the nopaline synthase 5' and 3' regulatory regions joined to the genes for neomycin phosphotransferase type I or type II. The chimeric genes were cloned into an intermediate vector, pMON120, and inserted into pTiB6S3 by recombination and then introduced into petunia and tobacco cells by cocultivating A. tumefaciens cells with protoplast-derived cells. Southern hybridization was used to confirm the presence of the chimeric genes in the transformed plant tissues. Expression of the chimeric genes was determined by the ability of the transformed cells to proliferate on medium containing normally inhibitory levels of kanamycin (50 micrograms/ml) or other aminoglycoside antibiotics. Plant cells transformed by wild-type pTiB6S3 or derivatives carrying the bacterial neomycin phosphotransferase genes with their own promoters failed to grow under these conditions. The significance of these results for plant genetic engineering is discussed. Images PMID:6308651

  18. In vivo activation of the intracrine vitamin D pathway in innate immune cells and mammary tissue during a bacterial infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The active vitamin D metabolite, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is an important regulator of immune function. The enzyme that synthesizes 1,25(OH)2D3 from 25-hydroxyvitamin D3 is 1alpha-hydroxylase (1alpha-OHase; CYP27B1). Several in vitro studies have shown that TLR signaling induces expre...

  19. Role of Innate T Cells in Anti-Bacterial Immunity

    PubMed Central

    Gao, Yifang; Williams, Anthony P.

    2015-01-01

    Innate T cells are a heterogeneous group of αβ and γδ T cells that respond rapidly (<2 h) upon activation. These innate T cells also share a non MHC class I or II restriction requirement for antigen recognition. Three major populations within the innate T cell group are recognized, namely, invariant NKT cells, mucosal associated invariant T cells, and gamma delta T cells. These cells recognize foreign/self-lipid presented by non-classical MHC molecules, such as CD1d, MR1, and CD1a. They are activated during the early stages of bacterial infection and act as a bridge between the innate and adaptive immune systems. In this review, we focus on the functional properties of these three innate T cell populations and how they are purposed for antimicrobial defense. Furthermore, we address the mechanisms through which their effector functions are targeted for bacterial control and compare this in human and murine systems. Lastly, we speculate on future roles of these cell types in therapeutic settings such as vaccination. PMID:26124758

  20. Structure of a bacterial toxin-activating acyltransferase

    PubMed Central

    Greene, Nicholas P.; Hughes, Colin; Koronakis, Vassilis

    2015-01-01

    Secreted pore-forming toxins of pathogenic Gram-negative bacteria such as Escherichia coli hemolysin (HlyA) insert into host–cell membranes to subvert signal transduction and induce apoptosis and cell lysis. Unusually, these toxins are synthesized in an inactive form that requires posttranslational activation in the bacterial cytosol. We have previously shown that the activation mechanism is an acylation event directed by a specialized acyl-transferase that uses acyl carrier protein (ACP) to covalently link fatty acids, via an amide bond, to specific internal lysine residues of the protoxin. We now reveal the 2.15-Å resolution X-ray structure of the 172-aa ApxC, a toxin-activating acyl-transferase (TAAT) from pathogenic Actinobacillus pleuropneumoniae. This determination shows that bacterial TAATs are a structurally homologous family that, despite indiscernible sequence similarity, form a distinct branch of the Gcn5-like N-acetyl transferase (GNAT) superfamily of enzymes that typically use acyl-CoA to modify diverse bacterial, archaeal, and eukaryotic substrates. A combination of structural analysis, small angle X-ray scattering, mutagenesis, and cross-linking defined the solution state of TAATs, with intermonomer interactions mediated by an N-terminal α-helix. Superposition of ApxC with substrate-bound GNATs, and assay of toxin activation and binding of acyl-ACP and protoxin peptide substrates by mutated ApxC variants, indicates the enzyme active site to be a deep surface groove. PMID:26016525

  1. Expression and stabilization of bacterial luciferase in mammalian cells

    NASA Astrophysics Data System (ADS)

    Patterson, Stacey S.; Dionisi, Hebe M.; Gupta, Rakesh K.; Sayler, Gary S.

    2004-06-01

    Current mammalian bioreporters using either firefly luciferase (luc) or GFP constructs require lysis and/or exogenous excitation to evoke a measurable response. Consequently, these cells cannot serve as continuous, on-line monitoring devices for in vivo imaging. Bacterial luciferase, lux, produces a photonic reaction that is cyclic, resulting in autonomous signal generation without the requirement for exogenous substrates or external activation. Therefore, lux-based bioluminescent bioreporters are the only truly autonomous light-generating sensors in existence. Unfortunately, the bacterial lux system has not yet been efficiently expressed in mammalian cells. In this research, three approaches for optimal expression of the a and b subunits of the bacterial luciferase protein were compared and reporter signal stability was evaluated from stably transfected human embryonic kidney cells. Maximum light levels were obtained from cells expressing the luciferase subunits linked with an internal ribosomal entry site (IRES). Cells harboring this construct produced bioluminescence equaling 2.6 X 106 photons/sec compared to 7.2 X 104 photons/sec obtained from cells expressing the luciferase from a dual promoter vector and 3.5 X 104 photons/sec from a Lux fusion protein. Furthermore, the bioluminescence levels remained stable for more than forty cell passages (5 months) in the absence of antibiotic selection. After this time, bioluminescence signals dropped at a rate of approximately 5% per cell passage. These data indicate that mammalian cell lines can be engineered to efficiently express the bacterial lux system, thus lending themselves to possible long-term continuous monitoring or imaging applications in vivo.

  2. Induction of Human Regulatory T Cells with Bacterial Superantigens.

    PubMed

    Caserta, Stefano; Taylor, Amanda L; Terrazzini, Nadia; Llewelyn, Martin J

    2016-01-01

    Regulatory T cells (Tregs) that suppress the activation of immune effector cells limit immunopathology and are fast emerging as therapeutic targets for autoimmune and cancer disease. Tools enabling Treg in vitro-induction, expansion, and characterization and manipulation will help future clinical developments. In this chapter, we describe in detail how to use bacterial superantigens to induce human Tregs efficiently from peripheral blood mononuclear cells. How to assess human Treg phenotype and suppressive capacity are also described. Technical details, variations, and alternative experimental conditions are provided. PMID:26676048

  3. Production of Bacteriolytic Enzymes by Streptomyces globisporus Regulated by Exogenous Bacterial Cell Walls.

    PubMed

    Brönneke, V; Fiedler, F

    1994-03-01

    Mutanolysin biosynthesis and pigment production in Streptomyces globisporus ATCC 21553 were stimulated by adding bacterial cell walls to the medium. The increased bacteriolytic activity in the supernatant correlated with an increased de novo synthesis of mutanolysin and was between 4- and 20-fold higher than in cultures grown without bacterial cell walls. The increase in mutanolysin synthesis was brought about by enhanced transcription of the mutanolysin gene. The stimulation was only observed in medium which contained dextrin or starch as the carbon source. Glucose abolished the stimulation and also inhibited the low constitutive synthesis of mutanolysin. The induction of lytic activity was observed to require minimally 0.4 mg of bacterial cell walls per ml, whereas 0.6 mg of bacterial cell walls per ml yielded maximal lytic activity. Further supplements of bacterial cell walls did not result in enhanced lytic activity. The stimulation could be achieved independently of the phase of growth of the Streptomyces strain. Cultures grown in the presence of bacterial cell walls exhibited a higher growth yield. However, the accelerated growth was not the reason for the increased amount of mutanolysin produced. The growth of cultures with peptidoglycan monomers added to the medium instead of cell walls was similarly increased, but an effect on the biosynthesis of mutanolysin was not observed. All bacterial cell walls tested were capable of eliciting the stimulation of lytic activity, including cell walls of archaea, which contained pseudomurein. PMID:16349213

  4. BACTERIAL FOODBORNE INFECTIONS AFTER HEMATOPOIETIC CELL TRANSPLANTATION

    PubMed Central

    Boyle, Nicole; Podczervinski, Sara; Jordan, Kim; Stednick, Zach; Butler-Wu, Susan; McMillen, Kerry; Pergam, Steven A.

    2014-01-01

    Background Diarrhea, abdominal pain and fever are common among patients undergoing hematopoietic cell transplant (HCT), but such symptoms are also typical with foodborne infections. The burden of disease caused by foodborne infections in patients undergoing HCT is unknown. We sought to describe bacterial foodborne infection incidence post-transplant within a single-center population of HCT recipients. Methods All HCT recipients transplanted from 2001 through 2011 at the Fred Hutchinson Cancer Research Center in Seattle, WA were followed for one year post-transplant. Data were collected retrospectively using center databases, which include information from transplant, on-site examinations, outside records, and collected laboratory data. Patients were considered to have a bacterial foodborne infection if Campylobacter jejuni/coli, Listeria monocytogenes, E. coli 0157:H7, Salmonella species, Shigella species, Vibrio species or Yersinia species were isolated in culture within one-year post-transplant. Non-foodborne infections with these agents and patients with preexisting bacterial foodborne infection (within 30 days of transplant) were excluded from analyses. Results A total of 12/4069 (0.3%) patients developed a bacterial foodborne infection within one year post-transplant. Patients with infections had a median age at transplant of 50.5 years (interquartile range [IQR]: 35–57), and the majority were adults ≥18 years of age (9/12 [75%]), male gender (8/12 [67%]) and post-allogeneic transplant (8/12 [67%]). Infectious episodes occurred at an incidence rate of 1.0 per 100,000 patient-days (95% CI: 0.5–1.7) and at a median of 50.5 days after transplant (IQR: 26–58.5). The most frequent pathogen detected was Campylobacter jejuni/coli (5/12 [42%]) followed by Yersinia (3/12 [25%]), while Salmonella (2/12 [17%]) and Listeria (2/12 [17%]) showed equal frequencies; no cases of Shigella, Vibrio, or E. coli 0157:H7 were detected. Most patients were diagnosed via stool

  5. Studying bacterial quorum-sensing at the single cell level

    NASA Astrophysics Data System (ADS)

    Delfino Perez, Pablo; Pelakh, Leslie; Young, Jonathan; Johnson, Elaine; Hagen, Stephen

    2010-03-01

    Like many bacterial species, Vibrio fischeri can detect its own population density through a quorum sensing (QS) mechanism. The bacterium releases a signal molecule (AI, autoinducer), which accumulates at high population density and triggers a genetic switch. In V.fischeri this leads to bioluminescence. Little is known about how stochastic gene expression affects QS at the level of single cells. We are imaging the luminescence of individual V.fischeri cells in a flow chamber and directly measuring the intercell variability in AI activation of the QS circuit. Our single-cell luminescence experiments allow us to track cells over time and characterize variations in their response to AI levels. We find heterogeneous response to the external signal: at a given AI concentration some cells may be strongly luminescent while others are virtually dark. The analysis of noise in the individual cell response can eventually lead to a better understanding of how cells use QS to gather information about their environment.

  6. One Bacterial Cell, One Complete Genome

    SciTech Connect

    Woyke, Tanja; Tighe, Damon; Mavrommatis, Konstantinos; Clum, Alicia; Copeland, Alex; Schackwitz, Wendy; Lapidus, Alla; Wu, Dongying; McCutcheon, John P.; McDonald, Bradon R.; Moran, Nancy A.; Bristow, James; Cheng, Jan-Fang

    2010-04-26

    While the bulk of the finished microbial genomes sequenced to date are derived from cultured bacterial and archaeal representatives, the vast majority of microorganisms elude current culturing attempts, severely limiting the ability to recover complete or even partial genomes from these environmental species. Single cell genomics is a novel culture-independent approach, which enables access to the genetic material of an individual cell. No single cell genome has to our knowledge been closed and finished to date. Here we report the completed genome from an uncultured single cell of Candidatus Sulcia muelleri DMIN. Digital PCR on single symbiont cells isolated from the bacteriome of the green sharpshooter Draeculacephala minerva bacteriome allowed us to assess that this bacteria is polyploid with genome copies ranging from approximately 200?900 per cell, making it a most suitable target for single cell finishing efforts. For single cell shotgun sequencing, an individual Sulcia cell was isolated and whole genome amplified by multiple displacement amplification (MDA). Sanger-based finishing methods allowed us to close the genome. To verify the correctness of our single cell genome and exclude MDA-derived artifacts, we independently shotgun sequenced and assembled the Sulcia genome from pooled bacteriomes using a metagenomic approach, yielding a nearly identical genome. Four variations we detected appear to be genuine biological differences between the two samples. Comparison of the single cell genome with bacteriome metagenomic sequence data detected two single nucleotide polymorphisms (SNPs), indicating extremely low genetic diversity within a Sulcia population. This study demonstrates the power of single cell genomics to generate a complete, high quality, non-composite reference genome within an environmental sample, which can be used for population genetic analyzes.

  7. Role of Microglial Activation in the Pathophysiology of Bacterial Meningitis.

    PubMed

    Barichello, Tatiana; Generoso, Jaqueline S; Simões, Lutiana R; Goularte, Jessica A; Petronilho, Fabricia; Saigal, Priyanka; Badawy, Marwa; Quevedo, João

    2016-04-01

    Bacterial meningitis is a life-threatening infection associated with cognitive impairment in many survivors. The pathogen invades the central nervous system (CNS) by penetrating through the luminal side of the cerebral endothelium, which is an integral part of the blood-brain barrier. The replication of bacteria within the subarachnoid space occurs concomitantly with the release of their compounds that are highly immunogenic. These compounds known as pathogen-associated molecular patterns (PAMPs) may lead to both an increase in the inflammatory response in the host and also microglial activation. Microglia are the resident macrophages of the CNS which, when activated, can trigger a host of immunological pathways. Classical activation increases the production of pro-inflammatory cytokines, chemokines, and reactive oxygen species, while alternative activation is implicated in the inhibition of inflammation and restoration of homeostasis. The inflammatory response from classical microglial activation can facilitate the elimination of invasive microorganisms; however, excessive or extended microglial activation can result in neuronal damage and eventually cell death. This review aims to discuss the role of microglia in the pathophysiology of bacterial meningitis as well as the process of microglial activation by PAMPs and by endogenous constituents that are normally released from damaged cells known as danger-associated molecular patterns (DAMPs). PMID:25744564

  8. Mining bacterial genomes for novel arylesterase activity

    PubMed Central

    Wang, Lijun; Mavisakalyan, Valentina; Tillier, Elisabeth R. M.; Clark, Greg W.; Savchenko, Alexei V.; Yakunin, Alexander F.; Master, Emma R.

    2010-01-01

    Summary One hundred and seventy‐one genes encoding potential esterases from 11 bacterial genomes were cloned and overexpressed in Escherichia coli; 74 of the clones produced soluble proteins. All 74 soluble proteins were purified and screened for esterase activity; 36 proteins showed carboxyl esterase activity on short‐chain esters, 17 demonstrated arylesterase activity, while 38 proteins did not exhibit any activity towards the test substrates. Esterases from Rhodopseudomonas palustris (RpEST‐1, RpEST‐2 and RpEST‐3), Pseudomonas putida (PpEST‐1, PpEST‐2 and PpEST‐3), Pseudomonas aeruginosa (PaEST‐1) and Streptomyces avermitilis (SavEST‐1) were selected for detailed biochemical characterization. All of the enzymes showed optimal activity at neutral or alkaline pH, and the half‐life of each enzyme at 50°C ranged from < 5 min to over 5 h. PpEST‐3, RpEST‐1 and RpEST‐2 demonstrated the highest specific activity with pNP‐esters; these enzymes were also among the most stable at 50°C and in the presence of detergents, polar and non‐polar organic solvents, and imidazolium ionic liquids. Accordingly, these enzymes are particularly interesting targets for subsequent application trials. Finally, biochemical and bioinformatic analyses were compared to reveal sequence features that could be correlated to enzymes with arylesterase activity, facilitating subsequent searches for new esterases in microbial genome sequences. PMID:21255363

  9. Inhibition of /sup 125/I-labeled ristocetin binding to Micrococcus luteus cells by the peptides related to bacterial cell wall mucopeptide precursors: quantitative structure-activity relationships

    SciTech Connect

    Kim, K.H.; Martin, Y.; Otis, E.; Mao, J.

    1989-01-01

    Quantitative structure-activity relationships (QSAR) of N-Ac amino acids, N-Ac dipeptides, and N-Ac tripeptides in inhibition of /sup 125/I-labeled ristocetin binding to Micrococcus luteus cell wall have been developed to probe the details of the binding between ristocetin and N-acetylated peptides. The correlation equations indicate that (1) the binding is stronger for peptides in which the side chain of the C-terminal amino acid has a large molar refractivity (MR) value, (2) the binding is weaker for peptides with polar than for those with nonpolar C-terminal side chains, (3) the N-terminal amino acid in N-Ac dipeptides contributes 12 times that of the C-terminal amino acid to binding affinity, and (4) the interactions between ristocetin and the N-terminal amino acid of N-acetyl tripeptides appear to be much weaker than those with the first two amino acids.

  10. Metabolic Responses of Bacterial Cells to Immobilization.

    PubMed

    Żur, Joanna; Wojcieszyńska, Danuta; Guzik, Urszula

    2016-01-01

    In recent years immobilized cells have commonly been used for various biotechnological applications, e.g., antibiotic production, soil bioremediation, biodegradation and biotransformation of xenobiotics in wastewater treatment plants. Although the literature data on the physiological changes and behaviour of cells in the immobilized state remain fragmentary, it is well documented that in natural settings microorganisms are mainly found in association with surfaces, which results in biofilm formation. Biofilms are characterized by genetic and physiological heterogeneity and the occurrence of altered microenvironments within the matrix. Microbial cells in communities display a variety of metabolic differences as compared to their free-living counterparts. Immobilization of bacteria can occur either as a natural phenomenon or as an artificial process. The majority of changes observed in immobilized cells result from protection provided by the supports. Knowledge about the main physiological responses occurring in immobilized cells may contribute to improving the efficiency of immobilization techniques. This paper reviews the main metabolic changes exhibited by immobilized bacterial cells, including growth rate, biodegradation capabilities, biocatalytic efficiency and plasmid stability. PMID:27455220

  11. Production of Bacterial Cells from Methane

    PubMed Central

    Sheehan, Brian T.; Johnson, Marvin J.

    1971-01-01

    A mixed methane-oxidizing bacterial culture capable of stable and predictable growth in continuous culture was isolated. The culture consisted of two types of gram-negative nonsporulating rods resembling pseudomonads. The culture grew well at 45 C on an inorganic medium without asepsis. Specific metal requirements for Ca2+, Cu2+, MoO42−, Zn2+, Mn2+, Mg2+, and Fe3+ (or Fe2+) were shown. The cells grown in continuous culture contained 11.7 to 12.1% total nitrogen. From an animal nutrition standpoint, the distribution of amino acids was satisfactory. The continuous fermentation was operated over a range of steady-state dilution rates from 0.085 to 0.301 hr−1. The maximum specific growth rate for the culture, μmax, was 0.303 hr−1 (doubling time 2.29 hr). The average yield for all fermentations analyzed was 0.616 g (dry weight of cells per g of methane used and 0.215 g (dry weight) of cells per g of oxygen used. The yields on both methane and oxygen were higher for the oxygen-limited than for the methane-limited fermentations. The maximum productivity attained in the fermentor was 2.39 g (dry weight) of cells per hr per liter at a dilution rate of 0.187 hr−1 and a cell concentration of 12.8 g (dry weight) of cells per liter. The limit on maximum cell productivity was determined only by the mass transfer rate of oxygen in the fermentor. The simultaneous volumetric mass-transfer coefficients (kLa in hr−1) for oxygen and methane were determined. The results appear to indicate an oxygen to methane mass-transfer coefficient ratio of approximately 1.4. PMID:4928605

  12. Bacterial cell curvature through mechanical control of cell growth

    PubMed Central

    Cabeen, Matthew T; Charbon, Godefroid; Vollmer, Waldemar; Born, Petra; Ausmees, Nora; Weibel, Douglas B; Jacobs-Wagner, Christine

    2009-01-01

    The cytoskeleton is a key regulator of cell morphogenesis. Crescentin, a bacterial intermediate filament-like protein, is required for the curved shape of Caulobacter crescentus and localizes to the inner cell curvature. Here, we show that crescentin forms a single filamentous structure that collapses into a helix when detached from the cell membrane, suggesting that it is normally maintained in a stretched configuration. Crescentin causes an elongation rate gradient around the circumference of the sidewall, creating a longitudinal cell length differential and hence curvature. Such curvature can be produced by physical force alone when cells are grown in circular microchambers. Production of crescentin in Escherichia coli is sufficient to generate cell curvature. Our data argue for a model in which physical strain borne by the crescentin structure anisotropically alters the kinetics of cell wall insertion to produce curved growth. Our study suggests that bacteria may use the cytoskeleton for mechanical control of growth to alter morphology. PMID:19279668

  13. Microarray Analysis to Monitor Bacterial Cell Wall Homeostasis.

    PubMed

    Hong, Hee-Jeon; Hesketh, Andy

    2016-01-01

    Transcriptomics, the genome-wide analysis of gene transcription, has become an important tool for characterizing and understanding the signal transduction networks operating in bacteria. Here we describe a protocol for quantifying and interpreting changes in the transcriptome of Streptomyces coelicolor that take place in response to treatment with three antibiotics active against different stages of peptidoglycan biosynthesis. The results defined the transcriptional responses associated with cell envelope homeostasis including a generalized response to all three antibiotics involving activation of transcription of the cell envelope stress sigma factor σ(E), together with elements of the stringent response, and of the heat, osmotic, and oxidative stress regulons. Many antibiotic-specific transcriptional changes were identified, representing cellular processes potentially important for tolerance to each antibiotic. The principles behind the protocol are transferable to the study of cell envelope homeostatic mechanisms probed using alternative chemical/environmental insults or in other bacterial strains. PMID:27311662

  14. An Overview of Genetic Mechanisms in the Bacterial Cell.

    ERIC Educational Resources Information Center

    Metcalfe, Judith; Baumberg, Simon

    1988-01-01

    Outlines the genetic elements found in the bacterial cell which play a role in recombining DNA sequences. Provides a core structure to which the mechanisms occurring in and between bacterial cells can be related. Discusses the practicalities of recombinant DNA techniques. (Author/CW)

  15. Aerotactic Cell Density Variations in Bacterial Turbulence

    NASA Astrophysics Data System (ADS)

    Fernandez, Vicente; Smriga, Steven; Menolascina, Filippo; Rusconi, Roberto; Stocker, Roman

    2015-11-01

    Concentrated suspensions of motile bacteria such as Bacillus subtilis exhibit group dynamics much larger than the scale of an individual bacterium, visual similar to high Reynolds number turbulence. These suspensions represent a microscale realization of active matter. Individually, B. subtilis are also aerotactic, and will accumulate near oxygen sources. Using a microfluidic device for generating oxygen gradients, we investigate the relationship between individuals' attraction to oxygen and the collective motion resultant from hydrodynamic interactions. We focus on changes in density revealed by a fluorescently labeled sub-population of B. subtilis in the dense suspension. This approach allows us to examine changes in density during the onset of collective motion as well as fully developed bacterial turbulence.

  16. Bacterial-mediated DNA delivery to tumour associated phagocytic cells.

    PubMed

    Byrne, W L; Murphy, C T; Cronin, M; Wirth, T; Tangney, M

    2014-12-28

    Phagocytic cells including macrophages, dendritic cells and neutrophils are now recognised as playing a negative role in many disease settings including cancer. In particular, macrophages are known to play a pathophysiological role in multiple diseases and present a valid and ubiquitous therapeutic target. The technology to target these phagocytic cells in situ, both selectively and efficiently, is required in order to translate novel therapeutic modalities into clinical reality. We present a novel delivery strategy using non-pathogenic bacteria to effect gene delivery specifically to tumour-associated phagocytic cells. Non-invasive bacteria lack the ability to actively enter host cells, except for phagocytic cells. We exploit this natural property to effect 'passive transfection' of tumour-associated phagocytic cells following direct administration of transgene-loaded bacteria to tumour regions. Using an in vitro-differentiated human monocyte cell line and two in vivo mouse models (an ovarian cancer ascites and a solid colon tumour model) proof of delivery is demonstrated with bacteria carrying reporter constructs. The results confirm that the delivery strategy is specific for phagocytic cells and that the bacterial vector itself recruits more phagocytic cells to the tumour. While proof of delivery to phagocytic cells is demonstrated in vivo for solid and ascites tumour models, this strategy may be applied to other settings, including non-cancer related disease. PMID:25466954

  17. Messenger Functions of the Bacterial Cell Wall-derived Muropeptides

    PubMed Central

    Boudreau, Marc A.; Fisher, Jed. F.; Mobashery, Shahriar

    2012-01-01

    Bacterial muropeptides are soluble peptidoglycan structures central to recycling of the bacterial cell wall, and messengers in diverse cell-signaling events. Bacteria sense muropeptides as signals that antibiotics targeting cell-wall biosynthesis are present, and eukaryotes detect muropeptides during the innate immune response to bacterial infection. This review summarizes the roles of bacterial muropeptides as messengers, with a special emphasis on bacterial muropeptide structures and the relationship of structure to the biochemical events that the muropeptides elicit. Muropeptide sensing and recycling in both Gram-positive and Gram-negative bacteria is discussed, followed by muropeptide sensing by eukaryotes as a crucial event to the innate immune response of insects (via peptidoglycan-recognition proteins) and mammals (through Nod-like receptors) to bacterial invasion. PMID:22409164

  18. Trogocytosis-associated cell to cell spread of intracellular bacterial pathogens

    PubMed Central

    Steele, Shaun; Radlinski, Lauren; Taft-Benz, Sharon; Brunton, Jason; Kawula, Thomas H

    2016-01-01

    Macrophages are myeloid-derived phagocytic cells and one of the first immune cell types to respond to microbial infections. However, a number of bacterial pathogens are resistant to the antimicrobial activities of macrophages and can grow within these cells. Macrophages have other immune surveillance roles including the acquisition of cytosolic components from multiple types of cells. We hypothesized that intracellular pathogens that can replicate within macrophages could also exploit cytosolic transfer to facilitate bacterial spread. We found that viable Francisella tularensis, as well as Salmonella enterica bacteria transferred from infected cells to uninfected macrophages along with other cytosolic material through a transient, contact dependent mechanism. Bacterial transfer occurred when the host cells exchanged plasma membrane proteins and cytosol via a trogocytosis related process leaving both donor and recipient cells intact and viable. Trogocytosis was strongly associated with infection in mice, suggesting that direct bacterial transfer occurs by this process in vivo. DOI: http://dx.doi.org/10.7554/eLife.10625.001 PMID:26802627

  19. A Noninvasive Technique for Monitoring Peroxidative and H2O2-Scavenging Activities during Interactions between Bacterial Plant Pathogens and Suspension Cells.

    PubMed Central

    Baker, C. J.; Harmon, G. L.; Glazener, J. A.; Orlandi, E. W.

    1995-01-01

    Stimulation of active oxygen metabolism occurs during the early stages of interactions involving bacteria and plant cell suspensions. Although many cellular processes are known to affect active oxygen metabolism in plants, it is not known which of these factors affect active oxygen levels during plant-bacteria interactions. Extracellular peroxidases have been shown to participate in both the production and utilization of active oxygen species such as H2O2 and superoxide. Catalase and other scavenging mechanisms also affect the overall level of active oxygen. In this study the luminol-dependent chemiluminescent reaction previously used to measure H2O2 levels in suspension cells was modified to allow the assay of both peroxidase and H2O2-scavenging activity. The early stages of the interactions between tobacco (Nicotiana tabacum) and Pseudomonas syringae pv syringae, as well as between soybean (Glycine max) and P. syringae pv glycinea, were investigated. This method of monitoring peroxidase and H2O2-scavenging activity proved to be rapid, sensitive, and nonintrusive, allowing the processing of multiple samples using intact cells or cell-free preparations. The results from the study demonstrate that the scavenging activities can be significant and must be considered when studying active oxygen production in biological interactions. PMID:12228480

  20. Modification of Bacterial Effector Proteins Inside Eukaryotic Host Cells

    PubMed Central

    Popa, Crina M.; Tabuchi, Mitsuaki; Valls, Marc

    2016-01-01

    Pathogenic bacteria manipulate their hosts by delivering a number of virulence proteins -called effectors- directly into the plant or animal cells. Recent findings have shown that such effectors can suffer covalent modifications inside the eukaryotic cells. Here, we summarize the recent reports where effector modifications by the eukaryotic machinery have been described. We restrict our focus on proteins secreted by the type III or type IV systems, excluding other bacterial toxins. We describe the known examples of effectors whose enzymatic activity is triggered by interaction with plant and animal cell factors, including GTPases, E2-Ubiquitin conjugates, cyclophilin and thioredoxins. We focus on the structural interactions with these factors and their influence on effector function. We also review the described examples of host-mediated post-translational effector modifications which are required for proper subcellular location and function. These host-specific covalent modifications include phosphorylation, ubiquitination, SUMOylation, and lipidations such as prenylation, fatty acylation and phospholipid binding. PMID:27489796

  1. Development and validation of a high-throughput cell-based screen to identify activators of a bacterial two-component signal transduction system.

    PubMed

    van Rensburg, Julia J; Fortney, Kate R; Chen, Lan; Krieger, Andrew J; Lima, Bruno P; Wolfe, Alan J; Katz, Barry P; Zhang, Zhong-Yin; Spinola, Stanley M

    2015-07-01

    CpxRA is a two-component signal transduction system (2CSTS) found in many drug-resistant Gram-negative bacteria. In response to periplasmic stress, CpxA autophosphorylates and donates a phosphoryl group to its cognate response regulator, CpxR. Phosphorylated CpxR (CpxR-P) upregulates genes involved in membrane repair and downregulates multiple genes that encode virulence factors, which are trafficked across the cell membrane. Mutants that constitutively activate CpxRA in Salmonella enterica serovar Typhimurium and Haemophilus ducreyi are avirulent in mice and humans, respectively. Thus, the activation of CpxRA has high potential as a novel antimicrobial/antivirulence strategy. Using a series of Escherichia coli strains containing a CpxR-P-responsive lacZ reporter and deletions in genes encoding CpxRA system components, we developed and validated a novel cell-based high-throughput screen (HTS) for CpxRA activators. A screen of 36,000 compounds yielded one hit compound that increased reporter activity in wild-type cells. This is the first report of a compound that activates, rather than inhibits, a 2CSTS. The activity profile of the compound against CpxRA pathway mutants in the presence of glucose suggested that the compound inhibits CpxA phosphatase activity. We confirmed that the compound induced the accumulation of CpxR-P in treated cells. Although the hit compound contained a nitro group, a derivative lacking this group retained activity in serum and had lower cytotoxicity than that of the initial hit. This HTS is amenable for the screening of larger libraries to find compounds that activate CpxRA by other mechanisms, and it could be adapted to find activators of other two-component systems. PMID:25870061

  2. Bacterial biomass and activity in the marginal ice zone of the northern Barents Sea

    NASA Astrophysics Data System (ADS)

    Tammert, Helen; Olli, Kalle; Sturluson, Maria; Hodal, Helene

    2008-10-01

    Bacteria in the Arctic Waters are well adapted to low temperatures and play a key role in the transformation of organic matter. However, the activity of planktonic bacteria at cellular level remains poorly understood. In this study, we use fluorescent markers (4',6'-diamidino-2-phenylindole (DAPI), 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), Live/Dead BacLight viability kit) to discriminate between bacterial cells with a variety of physiological activities in the 0-200 m water column and sinking particles. During two field studies (July 2003 and 2004), we covered nine stations in the northern Barents Sea. The median bacterial abundance (DAPI staining) in the upper 50 m layer was 0.9×10 6 cells ml -1 (range 0.2-3.2×10 6 cells ml -1) in 2003 and 0.5×10 6 cells ml -1 (range 0.2-1.0×10 6 cells ml -1) in 2004. Bacteria with sufficient electron transport activity to be stained with CTC were on average 10% of the total count and ca. 20% of the total cells had intact cell membranes. In the water column, proxies of substrate availability (POC, PON, chlorophyll a, primary production) and bacterial production (thymidine and leucine uptake) correlated strongly with total bacterial count, CTC-stained cells and cells with 'leaky' membrane (stained with propidium iodine), but not with the concentration of cells with intact cell membrane. Contrary to expectations, the proportion of CTC-stained bacteria was not higher in the sinking particles (captured with sediment traps) compared to the ambient water. However, out of the bacteria with intact cell membranes, a higher proportion scored as CTC positive in the aggregates compared to the ambient water. Bacterial cells with 'leaky' cell membranes formed the largest part of total cell count in all samples, and accumulated in sites with high microbial activity (sinking aggregates, chlorophyll maxima, layers of high primary and bacterial production). We hypothesize that the source of the bacterial cells with 'leaky' cell membranes

  3. Structure of a bacterial cell surface decaheme electron conduit

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves decaheme cytochromes that are located on the bacterial cell surface at the termini of trans-outer-membrane electron transfer conduits...

  4. Mechanics of Bacterial Cells and Initial Surface Colonisation.

    PubMed

    Aguayo, Sebastian; Bozec, Laurent

    2016-01-01

    The mechanical properties of bacterial cells play an important role in crucial bacterial processes such as cell growth, colonisation and biofilm formation. Recent developments in the field of nanotechnology and atomic force microscopy (AFM) have made it possible to observe, characterise and understand the nanomechanic behaviour of live bacterial cells as never before. Unlike traditional techniques, AFM makes it possible to employ living bacteria in their physiological environment with minimal or no sample preparation. The technique of AFM nanoindentation opens new possibilities to study bacterial cell wall stiffness under different mechanical and buffer conditions. Also, by attaching bacterial cells to functionalised AFM cantilevers, single-cell force spectroscopy (SCFS) can be used to measure the adhesion of bacteria to biological and non-biological substrates at the nano-newton and pico-newton scale, and provide specific information on receptor-ligand interactions. By studying the biophysics of the bacterial-surface interaction with the abovementioned techniques, it has been possible to gain new insight on the early stages of bacterial colonisation and biofilm formation. PMID:27193547

  5. Shared catalysis in virus entry and bacterial cell wall depolymerization.

    PubMed

    Cohen, Daniel N; Sham, Yuk Y; Haugstad, Greg D; Xiang, Ye; Rossmann, Michael G; Anderson, Dwight L; Popham, David L

    2009-04-01

    Bacterial virus entry and cell wall depolymerization require the breakdown of peptidoglycan (PG), the peptide-cross-linked polysaccharide matrix that surrounds bacterial cells. Structural studies of lysostaphin, a PG lytic enzyme (autolysin), have suggested that residues in the active site facilitate hydrolysis, but a clear mechanism for this reaction has remained unsolved. The active-site residues and a structural pattern of beta-sheets are conserved among lysostaphin homologs (such as LytM of Staphylococcus aureus) and the C-terminal domain of gene product 13 (gp13), a protein at the tail tip of the Bacillus subtilis bacteriophage varphi29. gp13 activity on PG and muropeptides was assayed using high-performance liquid chromatography, and gp13 was found to be a d,d-endopeptidase that cleaved the peptide cross-link. Computational modeling of the B. subtilis cross-linked peptide into the gp13 active site suggested that Asp195 may facilitate scissile-bond activation and that His247 is oriented to mediate nucleophile generation. To our knowledge, this is the first model of a Zn(2)(+) metallopeptidase and its substrate. Residue Asp195 of gp13 was found to be critical for Zn(2)(+) binding and catalysis by substitution mutagenesis with Ala or Cys. Circular dichroism and particle-induced X-ray emission spectroscopy showed that the general protein folding and Zn(2)(+) binding were maintained in the Cys mutant but reduced in the Ala mutant. These findings together support a model in which the Asp195 and His247 in gp13 and homologous residues in the LytM and lysostaphin active sites facilitate hydrolysis of the peptide substrate that cross-links PG. Thus, these autolysins and phage-entry enzymes have a shared chemical mechanism of action. PMID:19361422

  6. Polyclonal Mucosa-Associated Invariant T Cells Have Unique Innate Functions in Bacterial Infection

    PubMed Central

    Chua, Wei-Jen; Truscott, Steven M.; Eickhoff, Christopher S.; Blazevic, Azra

    2012-01-01

    Mucosa-associated invariant T (MAIT) cells are a unique population of αβ T cells in mammals that reside preferentially in mucosal tissues and express an invariant Vα paired with limited Vβ T-cell receptor (TCR) chains. Furthermore, MAIT cell development is dependent upon the expression of the evolutionarily conserved major histocompatibility complex (MHC) class Ib molecule MR1. Using in vitro assays, recent studies have shown that mouse and human MAIT cells are activated by antigen-presenting cells (APCs) infected with diverse microbes, including numerous bacterial strains and yeasts, but not viral pathogens. However, whether MAIT cells play an important, and perhaps unique, role in controlling microbial infection has remained unclear. To probe MAIT cell function, we show here that purified polyclonal MAIT cells potently inhibit intracellular bacterial growth of Mycobacterium bovis BCG in macrophages (MΦ) in coculture assays, and this inhibitory activity was dependent upon MAIT cell selection by MR1, secretion of gamma interferon (IFN-γ), and an innate interleukin 12 (IL-12) signal from infected MΦ. Surprisingly, however, the cognate recognition of MR1 by MAIT cells on the infected MΦ was found to play only a minor role in MAIT cell effector function. We also report that MAIT cell-deficient mice had higher bacterial loads at early times after infection compared to wild-type (WT) mice, demonstrating that MAIT cells play a unique role among innate lymphocytes in protective immunity against bacterial infection. PMID:22778103

  7. Mucosal SIV Vaccines Comprising Inactivated Virus Particles and Bacterial Adjuvants Induce CD8+ T-Regulatory Cells that Suppress SIV-Positive CD4+ T-Cell Activation and Prevent SIV Infection in the Macaque Model

    PubMed Central

    Andrieu, Jean-Marie; Chen, Song; Lai, Chunhui; Guo, Weizhong; Lu, Wei

    2014-01-01

    A new paradigm of mucosal vaccination against human immunodeficiency virus (HIV) infection has been investigated in the macaque model. A vaccine consisting of inactivated simian immunodeficiency virus (SIV)mac239 particles together with a living bacterial adjuvant (either the Calmette and Guerin bacillus, Lactobacillus plantarum or Lactobacillus rhamnosus) was administered to macaques via the vaginal or oral/intragastric route. In contrast to all established human and veterinary vaccines, these three vaccine regimens did not elicit SIV-specific antibodies nor cytotoxic T-lymphocytes but induced a previously unrecognized population of non-cytolytic MHCIb/E-restricted CD8+ T-regulatory cells that suppressed the activation of SIV-positive CD4+ T-lymphocytes. SIV reverse transcription was thereby blocked in inactivated CD4+ T-cells; the initial burst of virus replication was prevented and the vaccinated macaques were protected from a challenge infection. For 3–14 months after intragastric immunization, 24 macaques were challenged intrarectally with a high dose of SIVmac239 or with the heterologous strain SIV B670 (both strains grown on macaques PBMC). Twenty-three of these animals were found to be protected for up to 48 months while all 24 control macaques became infected. This protective effect against SIV challenge together with the concomitant identification of a robust ex vivo correlate of protection suggests a new approach for developing an HIV vaccine in humans. The induction of this new class of CD8+ T-regulatory cells could also possibly be used therapeutically for suppressing HIV replication in infected patients and this novel tolerogenic vaccine paradigm may have potential applications for treating a wide range of immune disorders and is likely to may have profound implications across immunology generally. PMID:25071760

  8. Host-induced bacterial cell wall decomposition mediates pattern-triggered immunity in Arabidopsis

    PubMed Central

    Liu, Xiaokun; Grabherr, Heini M; Willmann, Roland; Kolb, Dagmar; Brunner, Frédéric; Bertsche, Ute; Kühner, Daniel; Franz-Wachtel, Mirita; Amin, Bushra; Felix, Georg; Ongena, Marc; Nürnberger, Thorsten; Gust, Andrea A

    2014-01-01

    Peptidoglycans (PGNs) are immunogenic bacterial surface patterns that trigger immune activation in metazoans and plants. It is generally unknown how complex bacterial structures such as PGNs are perceived by plant pattern recognition receptors (PRRs) and whether host hydrolytic activities facilitate decomposition of bacterial matrices and generation of soluble PRR ligands. Here we show that Arabidopsis thaliana, upon bacterial infection or exposure to microbial patterns, produces a metazoan lysozyme-like hydrolase (lysozyme 1, LYS1). LYS1 activity releases soluble PGN fragments from insoluble bacterial cell walls and cleavage products are able to trigger responses typically associated with plant immunity. Importantly, LYS1 mutant genotypes exhibit super-susceptibility to bacterial infections similar to that observed on PGN receptor mutants. We propose that plants employ hydrolytic activities for the decomposition of complex bacterial structures, and that soluble pattern generation might aid PRR-mediated immune activation in cell layers adjacent to infection sites. DOI: http://dx.doi.org/10.7554/eLife.01990.001 PMID:24957336

  9. Osmotic Pressure, Bacterial Cell Walls, and Penicillin: A Demonstration.

    ERIC Educational Resources Information Center

    Lennox, John E.

    1984-01-01

    An easily constructed apparatus that models the effect of penicillin on the structure of bacterial cells is described. Background information and procedures for using the apparatus during a classroom demonstration are included. (JN)

  10. Lung dendritic cells facilitate extrapulmonary bacterial dissemination during pneumococcal pneumonia

    PubMed Central

    Rosendahl, Alva; Bergmann, Simone; Hammerschmidt, Sven; Goldmann, Oliver; Medina, Eva

    2013-01-01

    Streptococcus pneumoniae is a leading cause of bacterial pneumonia worldwide. Given the critical role of dendritic cells (DCs) in regulating and modulating the immune response to pathogens, we investigated here the role of DCs in S. pneumoniae lung infections. Using a well-established transgenic mouse line which allows the conditional transient depletion of DCs, we showed that ablation of DCs resulted in enhanced resistance to intranasal challenge with S. pneumoniae. DCs-depleted mice exhibited delayed bacterial systemic dissemination, significantly reduced bacterial loads in the infected organs and lower levels of serum inflammatory mediators than non-depleted animals. The increased resistance of DCs-depleted mice to S. pneumoniae was associated with a better capacity to restrict pneumococci extrapulmonary dissemination. Furthermore, we demonstrated that S. pneumoniae disseminated from the lungs into the regional lymph nodes in a cell-independent manner and that this direct way of dissemination was much more efficient in the presence of DCs. We also provide evidence that S. pneumoniae induces expression and activation of matrix metalloproteinase-9 (MMP-9) in cultured bone marrow-derived DCs. MMP-9 is a protease involved in the breakdown of extracellular matrix proteins and is critical for DC trafficking across extracellular matrix and basement membranes during the migration from the periphery to the lymph nodes. MMP-9 was also significantly up-regulated in the lungs of mice after intranasal infection with S. pneumoniae. Notably, the expression levels of MMP-9 in the infected lungs were significantly decreased after depletion of DCs suggesting the involvement of DCs in MMP-9 production during pneumococcal pneumonia. Thus, we propose that S. pneumoniae can exploit the DC-derived proteolysis to open tissue barriers thereby facilitating its own dissemination from the local site of infection. PMID:23802100

  11. Bacterial adhesion to uroepithelial cells: a morphologic study.

    PubMed

    Marrie, T J; Lam, J; Costerton, J W

    1980-08-01

    Urethral and midstream urine samples from healthy women and from patients with urinary tract infections (UTI) were examined by electron microscopy. Urethral urine samples from healthy subjects contained sparsely and densely colonized uroepithelial cells. The latter had morphologically heterogeneous bacteria adherent to each other and to the epithelial cell by a ruthenium red-positive fibrous matrix, which was present on the surface of all bacteria examined. Urethral urine samples from patients with UTI often had two distinct microcolonies of morphologically similar bacteria adherent to the same uroepithelial cell. Midstream urine samples from these patients contained large microcolonies of morphologically identical bacteria. Urine from patients with catheter-associated infections contained few uroepithelial cells and two distinct varieties of bacterial microcolonies--one of intact homogeneous cells and another of a mixture of damaged and intact bacteria. These in vivo observations indicate that the bacterial surface matrix participates in bacterial adhesion to uroepithelial cells and in bacteria-bacteria adhesion. PMID:6774033

  12. Role for erbin in bacterial activation of Nod2.

    PubMed

    Kufer, T A; Kremmer, E; Banks, D J; Philpott, D J

    2006-06-01

    Intracellular peptidoglycan (PG) recognition in human cells is mediated by the NACHT-LRR proteins Nod1 and Nod2. Elicitation of these proteins by PG motifs released from invasive bacteria triggers signaling events, resulting in the activation of the NF-kappaB pathway. In order to decipher the molecular components involved in Nod2 signal transduction, we set out to identify new interaction partners of Nod2 by using a yeast two-hybrid screen. Besides the known interaction partner RIP2, the screen identified the leucine-rich repeat (LRR)- and PDZ domain-containing family member Erbin as a binding partner of Nod2. Erbin showed a specific interaction with Nod2 in coimmunoprecipitation experiments with human HEK 293T cells. Immunofluorescence microscopy with a newly generated anti-Nod2 monoclonal antibody showed that Erbin and Nod2 partially colocalize in human cells. Subsequent analysis of the Erbin/Nod2 interaction revealed that the LRR of Erbin and the caspase activating and recruiting domains of Nod2 were necessary for this interaction. No significant interaction was observed with a Walker B box mutant of Nod2 or a Crohn's disease-associated frameshift mutant of Nod2, indicating that complex formation is dependent on the activity of the molecule. In addition, a change in the dynamics of the Erbin/Nod2 complex was observed during Shigella flexneri infection. Furthermore, ectopic expression of increasing amounts of Erbin or short hairpin RNA-mediated knockdown of Erbin showed a negative influence of Erbin on Nod2/muramyl-dipeptide-mediated NF-kappaB activation. These results implicate Erbin as a potential negative regulator of Nod2 and show that bacterial infection has an impact on Nod2/Erbin complex formation within cells. PMID:16714539

  13. Effect of flow and active mixing on bacterial growth in a colon-like geometry

    NASA Astrophysics Data System (ADS)

    Cremer, Jonas; Segota, Igor; Arnoldini, Markus; Groisman, Alex; Hwa, Terence

    The large intestine harbors bacteria from hundreds of species, with bacterial densities reaching up to 1012 cells per gram. Many different factors influence bacterial growth dynamics and thus bacterial density and microbiota composition. One dominant force is flow which can in principle lead to a washout of bacteria from the proximal colon. Active mixing by Contractions of the colonic wall together with bacterial growth might counteract such flow-forces and allow high bacterial densities to occur. As a step towards understanding bacterial growth in the presence of mixing and flow, we constructed an in-vitro setup where controlled wall-deformations of a channel emulate Contractions. We investigate growth along the channel under a steady nutrient inflow. In the limits of no or very frequent Contractions, the device behaves like a plug-flow reactor and a chemostat respectively. Depending on mixing and flow, we observe varying spatial gradients in bacterial density along the channel. Active mixing by deformations of the channel wall is shown to be crucial in maintaining a steady-state bacterial population in the presence of flow. The growth-dynamics is quantitatively captured by a simple mathematical model, with the effect of mixing described by an effective diffusion term.

  14. Impedance spectroscopy of micro-Droplets reveals activation of Bacterial Mechanosensitive Channels in Hypotonic Solutions

    NASA Astrophysics Data System (ADS)

    Ebrahimi, Aida; Alam, Muhammad A.

    Rapid detection of bacterial pathogens is of great importance in healthcare, food safety, environmental monitoring, and homeland security. Most bacterial detection platforms rely on binary fission (i.e. cell growth) to reach a threshold cell population that can be resolved by the sensing method. Since cell division depends on the bacteria type, the detection time of such methods can vary from hours to days. In contrast, in this work, we show that bacteria cells can be detected within minutes by relying on activation of specific protein channels, i.e. mechanosensitive channels (MS channels). When cells are exposed to hypotonic solutions, MS channels allow efflux of solutes to the external solution which leads to release the excessive membrane tension. Release of the cytoplasmic solutes, in turn, results in increase of the electrical conductance measured by droplet-based impedance sensing. The approach can be an effective technique for fast, pre-screening of bacterial contamination at ultra-low concentration.

  15. Ubiquitin Activates Patatin-Like Phospholipases from Multiple Bacterial Species

    PubMed Central

    Anderson, David M.; Sato, Hiromi; Dirck, Aaron T.; Feix, Jimmy B.

    2014-01-01

    Phospholipase A2 enzymes are ubiquitously distributed throughout the prokaryotic and eukaryotic kingdoms and are utilized in a wide array of cellular processes and physiological and immunological responses. Several patatin-like phospholipase homologs of ExoU from Pseudomonas aeruginosa were selected on the premise that ubiquitin activation of this class of bacterial enzymes was a conserved process. We found that ubiquitin activated all phospholipases tested in both in vitro and in vivo assays via a conserved serine-aspartate catalytic dyad. Ubiquitin chains versus monomeric ubiquitin were superior in inducing catalysis, and ubiquitin-like proteins failed to activate phospholipase activity. Toxicity studies in a prokaryotic dual-expression system grouped the enzymes into high- and low-toxicity classes. Toxicity measured in eukaryotic cells also suggested a two-tiered classification but was not predictive of the severity of cellular damage, suggesting that each enzyme may correspond to unique properties perhaps based on its specific biological function. Additional studies on lipid binding preference suggest that some enzymes in this family may be differentially sensitive to phosphatidyl-4,5-bisphosphate in terms of catalytic activation enhancement and binding affinity. Further analysis of the function and amino acid sequences of this enzyme family may lead to a useful approach to formulating a unifying model of how these phospholipases behave after delivery into the cytoplasmic compartment. PMID:25404699

  16. Bacterial cell biology outside the streetlight.

    PubMed

    Bulgheresi, Silvia

    2016-09-01

    As much as vertical transmission of microbial symbionts requires their deep integration into the host reproductive and developmental biology, symbiotic lifestyle might profoundly affect bacterial growth and proliferation. This review describes the reproductive oddities displayed by bacteria associated - more or less intimately - with multicellular eukaryotes. PMID:27306428

  17. Bacterial cell biology outside the streetlight

    PubMed Central

    2016-01-01

    Summary As much as vertical transmission of microbial symbionts requires their deep integration into the host reproductive and developmental biology, symbiotic lifestyle might profoundly affect bacterial growth and proliferation. This review describes the reproductive oddities displayed by bacteria associated – more or less intimately – with multicellular eukaryotes. PMID:27306428

  18. Size of Suspended Bacterial Cells and Association of Heterotrophic Activity with Size Fractions of Particles in Estuarine and Coastal Waters †

    PubMed Central

    Palumbo, Anthony V.; Ferguson, Randolph L.; Rublee, Parke A.

    1984-01-01

    The size of bacteria and the size distribution of heterotrophic activity were examined in estuarine, neritic, and coastal waters. The data indicated the small size of suspended marine bacteria and the predominance of free-living cells in numerical abundance and in the incorporation of dissolved amino acids. The average per-cell volume of suspended marine bacteria in all environments was less than 0.1 μm3. Cell volume ranged from 0.072 to 0.096 μm3 at salinities of 0 to 34.3‰ in the Newport River estuary, N.C., and from 0.078 to 0.096 μm3 in diverse areas of the Gulf of Mexico. Thus, the free-living bacteria were too small to be susceptible to predation by copepods. In the Newport River estuary, ca. 93 to 99% of the total number of cells and 75 to 97% of incorporated tritium (from 3H-labeled mixed amino acids) retained by a 0.2-μm-pore-size filter passed through a 3.0-μm-pore-size filter. Although the amino acid turnover rate per cell was higher for the bacteria in the >3.0-μm size fraction than in the <3.0-μm size fraction, the small number of bacteria associated with the >3.0-μm size particles resulted in the low relative contribution of attached bacteria to total heterotrophic activity in the estuary. For coastal and neritic samples, collected off the coast of Georgia and northeast Florida and in the plume of the Mississippi River, 56 to 98% of incorporated label passed through a 3.0-μm-pore-size filter. The greatest activity in the >3.0-μm fraction in the Georgia Bight was at nearshore stations and in the bottom samples. Our data were consistent with the hypothesis that resuspension of bottom material is an important factor in influencing the proportion of heterotrophic activity attributable to particle-associated bacteria. PMID:16346582

  19. Bacterial effectors target the plant cell nucleus to subvert host transcription

    PubMed Central

    Canonne, Joanne; Rivas, Susana

    2012-01-01

    In order to promote virulence, Gram-negative bacteria have evolved the ability to inject so-called type III effector proteins into host cells. The plant cell nucleus appears to be a subcellular compartment repeatedly targeted by bacterial effectors. In agreement with this observation, mounting evidence suggests that manipulation of host transcription is a major strategy developed by bacteria to counteract plant defense responses. It has been suggested that bacterial effectors may adopt at least three alternative, although not mutually exclusive, strategies to subvert host transcription. T3Es may (1) act as transcription factors that directly activate transcription in host cells, (2) affect histone packing and chromatin configuration, and/or (3) directly target host transcription factor activity. Here, we provide an overview on how all these strategies may lead to host transcriptional re-programming and, as a result, to improved bacterial multiplication inside plant cells. PMID:22353865

  20. Mechanics of swimming of multi-body bacterial swarmers using non-labeled cell tracking algorithm

    NASA Astrophysics Data System (ADS)

    Phuyal, Kiran; Kim, Min Jun

    2013-01-01

    To better understand the survival strategy of bacterial swarmers and the mechanical advantages offered by the linear chain (head-tail) attachment of the multiple bacterial bodies in an individual swarmer cell at low Reynolds number, a non-labeled cell tracking algorithm was used to quantify the mechanics of multi-body flagellated bacteria, Serratia marcescens, swimming in a motility buffer that originally exhibited the swarming motility. Swarming is a type of bacterial motility that is characterized by the collective coordinated motion of differentiated swarmer cells on a two-dimensional surface such as agar. In this study, the bacterial swarmers with multiple cell bodies (2, 3, and 4) were extracted from the swarm plate, and then tracked individually after resuspending in the motility medium. Their motion was investigated and compared with individual undifferentiated swimming bacterial cells. The swarmers when released into the motility buffer swam actively without tumbles. Their speeds, orientations, and the diffusive properties were studied by tracking the individual cell trajectories over a short distance in two-dimensional field when the cells are swimming at a constant depth in a bulk aqueous environment. At short time scales, the ballistic trajectory was dominant for both multi-body swarmers and undifferentiated cells.

  1. RNA-seq based transcriptomic analysis of single bacterial cells.

    PubMed

    Wang, Jiangxin; Chen, Lei; Chen, Zixi; Zhang, Weiwen

    2015-11-01

    Gene-expression heterogeneity among individual cells determines the fate of a bacterial population. Here we report the first bacterial single-cell RNA sequencing (RNA-seq), BaSiC RNA-seq, a method integrating RNA isolation, cDNA synthesis and amplification, and RNA-seq analysis of the whole transcriptome of single cyanobacterium Synechocystis sp. PCC 6803 cells which typically contain approximately 5-7 femtogram total RNA per cell. We applied the method to 3 Synechocystis single cells at 24 h and 3 single cells at 72 h after nitrogen-starvation stress treatment, as well as their bulk-cell controls under the same conditions, to determine the heterogeneity upon environmental stress. With 82-98% and 31-48% of all putative Synechocystis genes identified in single cells of 24 and 72 h, respectively, the results demonstrated that the method could achieve good identification of the transcripts in single bacterial cells. In addition, the preliminary results from nitrogen-starved cells also showed a possible increasing gene-expression heterogeneity from 24 h to 72 h after nitrogen starvation stress. Moreover, preliminary analysis of single-cell transcriptomic datasets revealed that genes from the "Mobile elements" functional category have the most significant increase of gene-expression heterogeneity upon stress, which was further confirmed by single-cell RT-qPCR analysis of gene expression in 24 randomly selected cells. PMID:26331465

  2. Bacterial Cellulose as a Substrate for Microbial Cell Culture

    PubMed Central

    Yin, Na; Santos, Thiago M. A.; Auer, George K.; Crooks, John A.; Oliver, Piercen M.

    2014-01-01

    Bacterial cellulose (BC) has a range of structural and physicochemical properties that make it a particularly useful material for the culture of bacteria. We studied the growth of 14 genera of bacteria on BC substrates produced by Acetobacter xylinum and compared the results to growth on the commercially available biopolymers agar, gellan, and xanthan. We demonstrate that BC produces rates of bacterial cell growth that typically exceed those on the commercial biopolymers and yields cultures with higher titers of cells at stationary phase. The morphology of the cells did not change during growth on BC. The rates of nutrient diffusion in BC being higher than those in other biopolymers is likely a primary factor that leads to higher growth rates. Collectively, our results suggest that the use of BC may open new avenues in microbiology by facilitating bacterial cell culture and isolation. PMID:24441155

  3. Defining heterogeneity within bacterial populations via single cell approaches.

    PubMed

    Davis, Kimberly M; Isberg, Ralph R

    2016-08-01

    Bacterial populations are heterogeneous, which in many cases can provide a selective advantage during changes in environmental conditions. In some instances, heterogeneity exists at the genetic level, in which significant allelic variation occurs within a population seeded by a single cell. In other cases, heterogeneity exists due to phenotypic differences within a clonal, genetically identical population. A variety of mechanisms can drive this latter strategy. Stochastic fluctuations can drive differential gene expression, but heterogeneity in gene expression can also be driven by environmental changes sensed by individual cells residing in distinct locales. Utilizing multiple single cell approaches, workers have started to uncover the extent of heterogeneity within bacterial populations. This review will first describe several examples of phenotypic and genetic heterogeneity, and then discuss many single cell approaches that have recently been applied to define heterogeneity within bacterial populations. PMID:27273675

  4. Efficient Immobilization and Patterning of Live Bacterial Cells

    PubMed Central

    Suo, Zhiyong; Avci, Recep; Yang, Xinghong; Pascual, David W.

    2008-01-01

    A monolayer of live bacterial cells has been patterned onto substrates through the interaction between CFA/I fimbriae and the corresponding antibody. Patterns of live bacteria have been prepared with cellular resolution on silicon and gold substrates for Salmonella enterica serovar Typhimurium as a model with high specificity and efficiency. The immobilized cells are capable of dividing in growth medium to form a self-sustaining bacterial monolayer on the patterned areas. Interestingly, the immobilized cells can alter their orientation on the substrate, from lying-down to standing-up, as a response to the cell density increase during incubation. This method was successfully used to sort a targeted bacterial species from a mixed culture within 2 h. PMID:18321142

  5. Conductivity and Dielectric Dispersion of Gram-Positive Bacterial Cells

    PubMed

    van der Wal A; Minor; Norde; Zehnder; Lyklema

    1997-02-01

    The conductivity of bacterial cell suspensions has been studied over a wide range of ionic strengths and is interpreted in terms of their cell wall properties. The experimental data have been analyzed after improving the high kappaa double-layer theory of Fixman, by accounting for ionic mobility in the hydrodynamically stagnant layer, i.e., in the bacterial wall. Static conductivity and dielectric dispersion measurements both show that the counterions in the porous gel-like cell wall give rise to a considerable surface conductance. From a comparison of the mobile charge with the total cell wall charge it is inferred that the mobilities of the ions in the bacterial wall are of the same order but somewhat lower than those in the bulk electrolyte solution. The occurrence of surface conductance reduces the electrophoretic mobility in electrophoresis studies. If this effect is not taken into account, the zeta-potential will be underestimated, especially at low electrolyte concentrations. PMID:9056304

  6. (p)ppGpp and the bacterial cell cycle.

    PubMed

    Nazir, Aanisa; Harinarayanan, Rajendran

    2016-06-01

    Genes of the Rel/Spo homolog (RSH) superfamily synthesize and/or hydrolyse the modified nucleotides pppGpp/ ppGpp (collectively referred to as (p)ppGpp) and are prevalent across diverse bacteria and in plant chloroplasts. Bacteria accumulate (p)ppGpp in response to nutrient deprivation (generically called the stringent response) and elicit appropriate adaptive responses mainly through the regulation of transcription. Although at different concentrations (p)ppGpp affect the expression of distinct set of genes, the two well-characterized responses are reduction in expression of the protein synthesis machinery and increase in the expression of genes coding for amino acid biosynthesis. In Escherichia coli, the cellular (p)ppGpp level inversely correlates with the growth rate and increasing its concentration decreases the steady state growth rate in a defined growth medium. Since change in growth rate must be accompanied by changes in cell cycle parameters set through the activities of the DNA replication and cell division apparatus, (p)ppGpp could coordinate protein synthesis (cell mass increase) with these processes. Here we review the role of (p)ppGpp in bacterial cell cycle regulation. PMID:27240988

  7. [Cashmere goat bacterial artificial chromosome recombination and cell transfection system].

    PubMed

    Huang, Tian; Cao, Zhongyang; Yang, Yaohui; Cao, Gengsheng

    2016-03-01

    The Cashmere goat is mainly used to produce cashmere, which is very popular for its delicate fiber, luscious softness and natural excellent warm property. Keratin associated protein (KAP) and bone morphogenetic protein (BMP) of the Cashmere goat play an important role in the proliferation and development of cashmere fiber follicle cells. Bacterial artificial chromosome containing kap6.3, kap8.1 and bmp4 genes were used to increase the production and quality of Cashmere. First, we constructed bacterial artificial chromosomes by homology recombination. Then Tol2 transposon was inserted into bacterial artificial chromosomes that were then transfected into Cashmere goat fibroblasts by Amaxa Nucleofector technology according to the manufacture's instructions. We successfully constructed the BAC-Tol2 vectors containing target genes. Each vector contained egfp report gene with UBC promoter, Neomycin resistant gene for cell screening and two loxp elements for resistance removing after transfected into cells. The bacterial artificial chromosome-Tol2 vectors showed a high efficiency of transfection that can reach 1% to 6% with a highest efficiency of 10%. We also obtained Cashmere goat fibroblasts integrated exogenous genes (kap6.3, kap8.1 and bmp4) preparing for the clone of Cashmere goat in the future. Our research demonstrates that the insertion of Tol2 transposons into bacterial artificial chromosomes improves the transfection efficiency and accuracy of bacterial artificial chromosome error-free recombination. PMID:27349114

  8. Attachment of motile bacterial cells to prealigned holed microarrays.

    PubMed

    Rozhok, Sergey; Fan, Zhifang; Nyamjav, Dorjderem; Liu, Chang; Mirkin, Chad A; Holz, Richard C

    2006-12-19

    Construction of biomotors is an exciting area of scientific research that holds great promise for the development of new technologies with broad potential applications in areas such as the energy industry and medicine. Herein, we demonstrate the fabrication of prealigned microarrays of motile Escherichia coli bacterial cells on SiOx substrates. To prepare these arrays, holed surfaces with a gold layer on the bottom of the holes were utilized. The attachment of bacteria to the holes was achieved via nonspecific interactions using poly-l-lysine hydrobromide (PLL). Our data suggest that a single motile bacterial cell can be selectively attached to an individual hole on a surface and bacterial cell binding can be controlled by altering the pH, with the greatest occupancy occurring at pH 7.8. Cells attached to hole arrays remained motile for at least 4 h. These data indicate that holed surface structures provide a promising footprint for the attachment of motile bacterial cells to form high-density site-specific functional bacterial microarrays. PMID:17154612

  9. Characterization of CCN and IN activity of bacterial isolates collected in Atlanta, GA

    NASA Astrophysics Data System (ADS)

    Purdue, Sara; Waters, Samantha; Karthikeyan, Smruthi; Konstantinidis, Kostas; Nenes, Athanasios

    2016-04-01

    Characterization of CCN activity of bacteria, other than a few select types such as Pseudomonas syringae, is limited, especially when looked at in conjunction with corresponding IN activity. The link between these two points is especially important for bacteria as those that have high CCN activity are likely to form an aqueous phase required for immersion freezing. Given the high ice nucleation temperature of bacterial cells, especially in immersion mode, it is important to characterize the CCN and IN activity of many different bacterial strains. To this effect, we developed a droplet freezing assay (DFA) which consists of an aluminum cold plate, cooled by a continuous flow of an ethylene glycol-water mixture, in order to observe immersion freezing of the collected bacteria. Here, we present the initial results on the CCN and IN activities of bacterial samples we have collected in Atlanta, GA. Bacterial strains were collected and isolated from rainwater samples taken from different storms throughout the year. We then characterized the CCN activity of each strain using a DMT Continuous Flow Streamwise Thermal Gradient CCN Counter by exposing the aerosolized bacteria to supersaturations ranging from 0.05% to 0.6%. Additionally, using our new DFA, we characterized the IN activity of each bacterial strain at temperatures ranging from -20oC to 0oC. The combined CCN and IN activity gives us valuable information on how some uncharacterized bacteria contribute to warm and mixed-phase cloud formation in the atmosphere.

  10. Peptidotriazoles with antimicrobial activity against bacterial and fungal plant pathogens.

    PubMed

    Güell, Imma; Micaló, Lluís; Cano, Laura; Badosa, Esther; Ferre, Rafael; Montesinos, Emilio; Bardají, Eduard; Feliu, Lidia; Planas, Marta

    2012-01-01

    We designed and prepared peptidotriazoles based on the antimicrobial peptide BP100 (LysLysLeuPheLysLysIleLeuLysTyrLeu-NH(2)) by introducing a triazole ring in the peptide backbone or onto the side chain of a selected residue. These compounds were screened for their in vitro growth inhibition of bacterial and fungal phytopathogens, and for their cytotoxic effects on eukaryotic cells and tobacco leaves. Their proteolytic susceptibility was also analyzed. The antibacterial activity and the hemolysis were influenced by the amino acid that was modified with the triazole as well as by the absence of presence of a substituent in this heterocyclic ring. We identified sequences active against the bacteria Xanthomonas axonopodis pv. vesicatoria, Erwinia amylovora, Pseudomonas syringae pv. syringae (MIC of 1.6-12.5 μM), and against the fungi Fusarium oxysporum (MIC<6.2-12.5 μM) with low hemolytic activity (0-23% at 50 μM), high stability to protease digestion and no phytotoxicity. These peptidotriazoles constitute good candidates to design new antimicrobial agents. PMID:22198367

  11. Extracellular enzyme activity in anaerobic bacterial cultures: evidence of pullulanase activity among mesophilic marine bacteria.

    PubMed

    Arnosti, C; Repeta, D J

    1994-03-01

    The extracellular enzymatic activity of a mixed culture of anaerobic marine bacteria enriched on pullulan [alpha(1,6)-linked maltotriose units] was directly assessed with a combination of gel permeation chromatography (GPC) and nuclear magnetic resonance spectroscopy (NMR). Hydrolysis products of pullulan were separated by GPC into three fractions with molecular weights of > or = 10,000, approximately 5,000, and < or = 1,200. NMR spectra of these fractions demonstrated that pullulan was rapidly and specifically hydrolyzed at alpha(1,6) linkages by pullulanase enzymes, most likely type II pullulanase. Although isolated pullulanase enzymes have been shown to hydrolyze pullulan completely to maltotriose (S. H. Brown, H. R. Costantino, and R. M. Kelly, Appl. Environ. Microbiol. 56:1985-1991, 1990; M. Klingeberg, H. Hippe, and G. Antranikian, FEMS Microbiol. Lett. 69:145-152, 1990; R. Koch, P. Zablowski, A. Spreinat, and G. Antranikian, FEMS Microbiol. Lett. 71:21-26, 1990), the smallest carbohydrate detected in the bacterial cultures consisted of two maltotriose units linked through one alpha(1,6) linkage. Either the final hydrolysis step was closely linked to substrate uptake, or specialized porins similar to maltoporin might permit direct transport of large oligosaccharides into the bacterial cell. This is the first report of pullulanase activity among mesophilic marine bacteria. The combination of GPC and NMR could easily be used to assess other types of extracellular enzyme activity in bacterial cultures. PMID:8161177

  12. Nanomechanical Response of Bacterial Cells to Cationic Antimicrobial Peptides

    NASA Astrophysics Data System (ADS)

    Lu, Shun; Walters, Grant; Parg, Richard; Dutcher, John

    2014-03-01

    The effectiveness of antimicrobial compounds can be easily screened, however their mechanism of action is much more difficult to determine. Many compounds act by compromising the mechanical integrity of the bacterial cell envelope, and our study introduces an atomic force microscopy (AFM)-based creep deformation technique to evaluate changes in the time-dependent mechanical properties of Pseudomonas aeruginosa PAO1 bacterial cells upon exposure to two different but structurally related antimicrobial peptides: polymyxin B and polymyxin B nonapeptide. We observed a distinctive signature for the loss of integrity of the bacterial cell envelope following exposure to the peptides. Measurements performed before and after exposure, as well as time-resolved measurements and those performed at different concentrations, revealed large changes to the viscoelastic parameters that are consistent with differences in the membrane permeabilizing effects of the peptides. The AFM creep deformation measurement provides new, unique insight into the kinetics and mechanism of action of antimicrobial peptides on bacteria.

  13. Bacterial cell identification in differential interference contrast microscopy images

    PubMed Central

    2013-01-01

    Background Microscopy image segmentation lays the foundation for shape analysis, motion tracking, and classification of biological objects. Despite its importance, automated segmentation remains challenging for several widely used non-fluorescence, interference-based microscopy imaging modalities. For example in differential interference contrast microscopy which plays an important role in modern bacterial cell biology. Therefore, new revolutions in the field require the development of tools, technologies and work-flows to extract and exploit information from interference-based imaging data so as to achieve new fundamental biological insights and understanding. Results We have developed and evaluated a high-throughput image analysis and processing approach to detect and characterize bacterial cells and chemotaxis proteins. Its performance was evaluated using differential interference contrast and fluorescence microscopy images of Rhodobacter sphaeroides. Conclusions Results demonstrate that the proposed approach provides a fast and robust method for detection and analysis of spatial relationship between bacterial cells and their chemotaxis proteins. PMID:23617824

  14. Metal Toxicity Affects Fungal and Bacterial Activities in Soil Differently

    PubMed Central

    Rajapaksha, R. M. C. P.; Tobor-Kapłon, M. A; Bååth, E.

    2004-01-01

    Although the toxic effect of heavy metals on soil microorganism activity is well known, little is known about the effects on different organism groups. The influence of heavy metal addition on total, bacterial, and fungal activities was therefore studied for up to 60 days in a laboratory experiment using forest soil contaminated with different concentrations of Zn or Cu. The effects of the metals differed between the different activity measurements. During the first week after metal addition, the total activity (respiration rate) decreased by 30% at the highest level of contamination and then remained stable during the 60 days of incubation. The bacterial activity (thymidine incorporation rate) decreased during the first days with the level of metal contamination, resulting in a 90% decrease at the highest level of contamination. Bacterial activity then slowly recovered to values similar to those of the control soil. The recovery was faster when soil pH, which had decreased due to metal addition, was restored to control values by liming. Fungal activity (acetate-in-ergosterol incorporation rate) initially increased with the level of metal contamination, being up to 3 and 7 times higher than that in the control samples during the first week at the highest levels of Zn and Cu addition, respectively. The positive effect of metal addition on fungal activity then decreased, but fungal activity was still higher in contaminated than in control soil after 35 days. This is the first direct evidence that fungal and bacterial activities in soil are differently affected by heavy metals. The different responses of bacteria and fungi to heavy metals were reflected in an increase in the relative fungal/bacterial ratio (estimated using phospholipid fatty acid analysis) with increased metal load. PMID:15128558

  15. Metal toxicity affects fungal and bacterial activities in soil differently.

    PubMed

    Rajapaksha, R M C P; Tobor-Kapłon, M A; Bååth, E

    2004-05-01

    Although the toxic effect of heavy metals on soil microorganism activity is well known, little is known about the effects on different organism groups. The influence of heavy metal addition on total, bacterial, and fungal activities was therefore studied for up to 60 days in a laboratory experiment using forest soil contaminated with different concentrations of Zn or Cu. The effects of the metals differed between the different activity measurements. During the first week after metal addition, the total activity (respiration rate) decreased by 30% at the highest level of contamination and then remained stable during the 60 days of incubation. The bacterial activity (thymidine incorporation rate) decreased during the first days with the level of metal contamination, resulting in a 90% decrease at the highest level of contamination. Bacterial activity then slowly recovered to values similar to those of the control soil. The recovery was faster when soil pH, which had decreased due to metal addition, was restored to control values by liming. Fungal activity (acetate-in-ergosterol incorporation rate) initially increased with the level of metal contamination, being up to 3 and 7 times higher than that in the control samples during the first week at the highest levels of Zn and Cu addition, respectively. The positive effect of metal addition on fungal activity then decreased, but fungal activity was still higher in contaminated than in control soil after 35 days. This is the first direct evidence that fungal and bacterial activities in soil are differently affected by heavy metals. The different responses of bacteria and fungi to heavy metals were reflected in an increase in the relative fungal/bacterial ratio (estimated using phospholipid fatty acid analysis) with increased metal load. PMID:15128558

  16. Myeloid-Derived Suppressor Cells in Bacterial Infections.

    PubMed

    Ost, Michael; Singh, Anurag; Peschel, Andreas; Mehling, Roman; Rieber, Nikolaus; Hartl, Dominik

    2016-01-01

    Myeloid-derived suppressor cells (MDSCs) comprise monocytic and granulocytic innate immune cells with the capability of suppressing T- and NK-cell responses. While the role of MDSCs has been studied in depth in malignant diseases, the understanding of their regulation and function in infectious disease conditions has just begun to evolve. Here we summarize and discuss the current view how MDSCs participate in bacterial infections and how this knowledge could be exploited for potential future therapeutics. PMID:27066459

  17. Myeloid-Derived Suppressor Cells in Bacterial Infections

    PubMed Central

    Ost, Michael; Singh, Anurag; Peschel, Andreas; Mehling, Roman; Rieber, Nikolaus; Hartl, Dominik

    2016-01-01

    Myeloid-derived suppressor cells (MDSCs) comprise monocytic and granulocytic innate immune cells with the capability of suppressing T- and NK-cell responses. While the role of MDSCs has been studied in depth in malignant diseases, the understanding of their regulation and function in infectious disease conditions has just begun to evolve. Here we summarize and discuss the current view how MDSCs participate in bacterial infections and how this knowledge could be exploited for potential future therapeutics. PMID:27066459

  18. Nanomechanical Response of Bacterial Cells to Antimicrobial Peptides

    NASA Astrophysics Data System (ADS)

    Parg, Richard; Dutcher, John

    2015-03-01

    The effectiveness of antimicrobial compounds can be easily screened, however their mechanism of action is much more difficult to determine. Many compounds act by compromising the mechanical integrity of the bacterial cell envelope, and we have developed an atomic force microscopy (AFM)-based creep deformation technique to evaluate changes in the time-dependent mechanical properties of bacterial cells upon exposure to antimicrobial peptides. Measurements performed before and after exposure, as well as time-resolved measurements and those performed at different antimicrobial concentrations, revealed large changes to the viscoelastic parameters including a distinctive signature for the loss of integrity of the bacterial cell envelope. Our previous experiments have focused on Pseudomonas aeruginosaPAO1 bacterial cells in Milli-Q water, for which the cells can withstand the large osmotic pressure. In the present study we have focused on performing the measurements in buffer to obtain more biologically relevant results. The AFM creep deformation measurement provides new, unique insight into the kinetics and mechanism of action of antimicrobial peptides on bacteria.

  19. Regulation of bacterial metabolic activity by dissolved organic carbon and viruses

    NASA Astrophysics Data System (ADS)

    Xu, Jie; Jing, Hongmei; Sun, Mingming; Harrison, Paul J.; Liu, Hongbin

    2013-12-01

    regulation of bacterial metabolic activity by viruses and dissolved organic carbon (DOC) was examined using natural microbial communities in three treatments (active viruses, inactive viruses, and virus free) at two contrasting coastal sites (pristine vs. eutrophic) with substantial differences in environmental conditions during the wet and dry seasons. Our results showed that net growth rates and production of bacterioplankton were reduced primarily by viruses via repressing metabolically active bacteria with high nucleic acid (HNA) content which had a high capacity for incorporating carbon, while bacterial respiration was primarily regulated by DOC lability. The quality of organic matter played a more important role in regulating bacterial growth efficiency (BGE) than the supply of organic matter in eutrophic coastal waters. The lack of HMW-DOC and high carbon demand in the virus-free treatment resulted in a significant increase in cell-specific bacterial respiration, which was responsible for the lowest bacterial growth efficiency among the three treatments. The presence of viruses did not necessarily lower bacterial growth efficiency since virus-induced mortality alleviated bacterial carbon demand and enhanced carbon cycling. Virus-induced mortality was greater in relatively pristine waters than eutrophic waters, likely since the high supply of substrates alleviated the pressure of viral infection, through extracellular proteases produced by bacteria, which might result in the hydrolytic destruction or modification of viral capsids. An important implication of our results was that the input of riverine DOC and nutrients improved bacterial metabolic activity by alleviating virus-induced mortality of bacteria in estuarine and coastal waters.

  20. A simple and novel modification of comet assay for determination of bacteriophage mediated bacterial cell lysis.

    PubMed

    Khairnar, Krishna; Sanmukh, Swapnil; Chandekar, Rajshree; Paunikar, Waman

    2014-07-01

    The comet assay is the widely used method for in vitro toxicity testing which is also an alternative to the use of animal models for in vivo testing. Since, its inception in 1984 by Ostling and Johansson, it is being modified frequently for a wide range of application. In spite of its wide applicability, unfortunately there is no report of its application in bacteriophages research. In this study, a novel application of comet assay for the detection of bacteriophage mediated bacterial cell lysis was described. The conventional methods in bacteriophage research for studying bacterial lysis by bacteriophages are plaque assay method. It is time consuming, laborious and costly. The lytic activity of bacteriophage devours the bacterial cell which results in the release of bacterial genomic material that gets detected by ethidium bromide staining method by the comet assay protocol. The objective of this study was to compare efficacy of comet assay with different assay used to study phage mediated bacterial lysis. The assay was performed on culture isolates (N=80 studies), modified comet assay appear to have relatively higher sensitivity and specificity than other assay. The results of the study showed that the application of comet assay can be an economical, time saving and less laborious alternative to conventional plaque assay for the detection of bacteriophage mediated bacterial cell lysis. PMID:24681053

  1. Collective chemotaxis and segregation of active bacterial colonies.

    PubMed

    Ben Amar, M

    2016-01-01

    Still recently, bacterial fluid suspensions have motivated a lot of works, both experimental and theoretical, with the objective to understand their collective dynamics from universal and simple rules. Since some species are active, most of these works concern the strong interactions that these bacteria exert on a forced flow leading to instabilities, chaos and turbulence. Here, we investigate the self-organization of expanding bacterial colonies under chemotaxis, proliferation and eventually active-reaction. We propose a simple model to understand and quantify the physical properties of these living organisms which either give cohesion or on the contrary dispersion to the colony. Taking into account the diffusion and capture of morphogens complicates the model since it induces a bacterial density gradient coupled to bacterial density fluctuations and dynamics. Nevertheless under some specific conditions, it is possible to investigate the pattern formation as a usual viscous fingering instability. This explains the similarity and differences of patterns according to the physical bacterial suspension properties and explain the factors which favor compactness or branching. PMID:26888040

  2. The active bacterial community in a pristine confined aquifer

    NASA Astrophysics Data System (ADS)

    Flynn, Theodore M.; Sanford, Robert A.; Santo Domingo, Jorge W.; Ashbolt, Nicholas J.; Levine, Audrey D.; Bethke, Craig M.

    2012-09-01

    This study of the active bacteria residing in a pristine confined aquifer provides unexpected insights into the ecology of iron-reducing and sulfate-reducing bacteria in the subsurface. At 18 wells, we trapped the microbes that attached to aquifer sediment and used molecular techniques to examine the bacterial populations. We used multivariate statistics to compare the composition of bacterial communities among the wells with respect to the chemistry of the groundwater. We found groundwater at each well was considerably richer in ferrous iron than sulfide, indicating iron-reducing bacteria should, by established criteria, dominate the sulfate reducers. Our results show, however, that areas where groundwater contains more than a negligible amount of sulfate (>0.03 mM), populations related to sulfate reducers of the generaDesulfobacter and Desulfobulbus were of nearly equal abundance with putative iron reducers related to Geobacter, Geothrix, and Desulfuromonas. Whereas sulfate is a key discriminant of bacterial community structure, we observed no statistical relationship between the distribution of bacterial populations in this aquifer and the concentration of either ferrous iron or dissolved sulfide. These results call into question the validity of using the relative concentration of these two ions to predict the nature of bacterial activity in an aquifer. Sulfate reducers and iron reducers do not appear to be segregated into discrete zones in the aquifer, as would be predicted by the theory of competitive exclusion. Instead, we find the two groups coexist in the subsurface in what we suggest is a mutualistic relationship.

  3. Collective chemotaxis and segregation of active bacterial colonies

    PubMed Central

    Amar, M. Ben

    2016-01-01

    Still recently, bacterial fluid suspensions have motivated a lot of works, both experimental and theoretical, with the objective to understand their collective dynamics from universal and simple rules. Since some species are active, most of these works concern the strong interactions that these bacteria exert on a forced flow leading to instabilities, chaos and turbulence. Here, we investigate the self-organization of expanding bacterial colonies under chemotaxis, proliferation and eventually active-reaction. We propose a simple model to understand and quantify the physical properties of these living organisms which either give cohesion or on the contrary dispersion to the colony. Taking into account the diffusion and capture of morphogens complicates the model since it induces a bacterial density gradient coupled to bacterial density fluctuations and dynamics. Nevertheless under some specific conditions, it is possible to investigate the pattern formation as a usual viscous fingering instability. This explains the similarity and differences of patterns according to the physical bacterial suspension properties and explain the factors which favor compactness or branching. PMID:26888040

  4. Mechanistic Analysis of Activation of the Innate Immune Sensor PKR by Bacterial RNA.

    PubMed

    Hull, Chelsea M; Bevilacqua, Philip C

    2015-11-01

    The protein kinase PKR (protein kinase R) is a sensor in innate immunity. PKR autophosphorylates in the presence of double-stranded RNA enabling it to phosphorylate its substrate, eIF2α (eukaryotic initiation factor 2α), halting cellular translation. Classical activators of PKR are long viral double-stranded RNAs, but recently, PKR has been found to be activated by bacterial RNA. However, the features of bacterial RNA that activate PKR are unknown. We studied the Bacillus subtilis trp 5'-UTR (untranslated region), which is an indirect riboswitch with secondary and tertiary RNA structures that regulate gene function. Additionally, the trp 5'-UTR binds a protein, TRAP (tryptophan RNA-binding attenuation protein), which recognizes l-tryptophan. We present the first evidence that multiple structural features in this RNA, which are typical of bacterial RNAs, activate PKR in TRAP-free and TRAP/l-Trp-bound forms. Segments from the 5'-UTR, including the terminator 5'-stem-loop and Shine-Dalgarno blocking hairpins, demonstrated 5'-triphosphate and flanking RNA tail dependence on PKR activation. Disruption of long-distance tertiary interactions in the 5'-UTR led to partial loss in activation, consistent with highly base-paired regions in bacterial RNA activating PKR. One physiological change a bacterial RNA would face in a human cell is a decrease in the concentration of free magnesium. Upon lowering the magnesium concentration to human physiological conditions of 0.5mM, the trp 5'-UTR continued to activate PKR potently. Moreover, total RNA from Escherichia coli, depleted of rRNA, also activated PKR under these ionic conditions. This study demonstrates that PKR can signal the presence of bacterial RNAs under physiological ionic conditions and offers a potential explanation for the apparent absence of riboswitches in the human genome. PMID:26026708

  5. Micro-magnet arrays for specific single bacterial cell positioning

    NASA Astrophysics Data System (ADS)

    Pivetal, Jérémy; Royet, David; Ciuta, Georgeta; Frenea-Robin, Marie; Haddour, Naoufel; Dempsey, Nora M.; Dumas-Bouchiat, Frédéric; Simonet, Pascal

    2015-04-01

    In various contexts such as pathogen detection or analysis of microbial diversity where cellular heterogeneity must be taken into account, there is a growing need for tools and methods that enable microbiologists to analyze bacterial cells individually. One of the main challenges in the development of new platforms for single cell studies is to perform precise cell positioning, but the ability to specifically target cells is also important in many applications. In this work, we report the development of new strategies to selectively trap single bacterial cells upon large arrays, based on the use of micro-magnets. Escherichia coli bacteria were used to demonstrate magnetically driven bacterial cell organization. In order to provide a flexible approach adaptable to several applications in the field of microbiology, cells were magnetically and specifically labeled using two different strategies, namely immunomagnetic labeling and magnetic in situ hybridization. Results show that centimeter-sized arrays of targeted, isolated bacteria can be successfully created upon the surface of a flat magnetically patterned hard magnetic film. Efforts are now being directed towards the integration of a detection tool to provide a complete micro-system device for a variety of microbiological applications.

  6. Bacterial expression of human kynurenine 3-monooxygenase: solubility, activity, purification.

    PubMed

    Wilson, K; Mole, D J; Binnie, M; Homer, N Z M; Zheng, X; Yard, B A; Iredale, J P; Auer, M; Webster, S P

    2014-03-01

    Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington's disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification. PMID:24316190

  7. MAIT cells are licensed through granzyme exchange to kill bacterially sensitized targets

    PubMed Central

    Kurioka, A; Ussher, J E; Cosgrove, C; Clough, C; Fergusson, J R; Smith, K; Kang, Y-H; Walker, L J; Hansen, T H; Willberg, C B; Klenerman, P

    2015-01-01

    Mucosal-associated invariant T (MAIT) cells are an innate-like T-cell population restricted by the non-polymorphic, major histocompatibility complex class I-related protein 1, MR1. MAIT cells are activated by a broad range of bacteria through detection of riboflavin metabolites bound by MR1, but their direct cytolytic capacity upon recognition of cognate target cells remains unclear. We show that resting human MAIT cells are uniquely characterized by a lack of granzyme (Gr) B and low perforin expression, key granule proteins required for efficient cytotoxic activity, but high levels of expression of GrA and GrK. Bacterial activation of MAIT cells rapidly induced GrB and perforin, licensing these cells to kill their cognate target cells. Using a novel flow cytometry-based killing assay, we show that licensed MAIT cells, but not ex vivo MAIT cells from the same donors, can efficiently kill Escherichia coli-exposed B-cell lines in an MR1- and degranulation-dependent manner. Finally, we show that MAIT cells are highly proliferative in response to antigenic and cytokine stimulation, maintaining high expression of GrB, perforin, and GrA, but reduced expression of GrK following antigenic proliferation. The tightly regulated cytolytic capacity of MAIT cells may have an important role in the control of intracellular bacterial infections, such as Mycobacterium tuberculosis. PMID:25269706

  8. Bacterial Cell Wall Polymer-Induced Granulomatous Inflammation

    PubMed

    Sartor; Herfarth; Van Tol EAF

    1996-04-01

    Local or systemic injection of peptidoglycan-polysaccharide polymers, which are the primary structural components of cell walls of nearly all bacteria, leads to acute inflammation, which can develop into chronic, spontaneously relapsing, granulomatous inflammation in a number of organs. Evolution into chronic granulomatous inflammation is dependent upon persistence of poorly biodegradable cell wall polymers within tissues, genetically determined host susceptibility, and generation of a T-lymphocyte-mediated immune response. Intraperitoneal injection of peptidoglycan-polysaccharide fragments from group A streptococci or selected intestinal bacteria into susceptible Lewis rats leads to chronic, spontaneously reactivating erosive arthritis and hepatic granulomas. Subserosal (intramural) injection of poorly biodegradable cell wall fragments into the distal intestine of Lewis rats induces chronic, spontaneously relapsing granulomatous enterocolitis with associated arthritis, hepatic granulomas, anemia, and leukocytosis. Chronic inflammation does not occur in T-lymphocyte-deficient rats and is prevented by cyclosporin-A therapy and degradation of peptidoglycan by the muralytic enzyme, mutanolysin. Moreover, resistant Buffalo and Fischer F344 rats, the latter sharing identical MHC antigens with Lewis rats, develop only acute inflammation with no chronic granulomatous response. Peptidoglycan-polysaccharide polymers activate almost every limb of the inflammatory response. Blockade of specific pathways suggests that interleukin-1, transforming growth factor-beta, plasma kallikrein, and T lymphocytes are dominant mediators of peptidoglycan-polysaccharide-induced arthritis, hepatic granulomas, and enterocolitis. Because of the similarity of immune mechanisms of these rat models to human disease, bacterial cell wall-induced inflammation provides unique opportunities to study pathogenic mechanisms of granuloma formation in response to ubiquitous microbial agents and to test

  9. Staphylococcal Enterotoxin B Primes Cytokine Secretion and Lytic Activity in Response to Native Bacterial Antigens

    PubMed Central

    Mason, Kevin M.; Dryden, Tricia D.; Bigley, Nancy J.; Fink, Pamela S.

    1998-01-01

    Superantigens stimulate T-lymphocyte proliferation and cytokine production, but the effects of superantigen exposure on cell function within a complex, highly regulated immune response remain to be determined. In this study, we demonstrate that superantigen exposure significantly alters the murine host response to bacterial antigens in an in vitro coculture system. Two days after exposure to the superantigen staphylococcal enterotoxin B, splenocytes cultured with Streptococcus mutans produced significantly greater amounts of gamma interferon (IFN-γ) and interleukin-12 than did sham-injected controls. The majority of IFN-γ production appeared to be CD8+ T-cell derived since depletion of this cell type dramatically reduced the levels of IFN-γ. To study host cell damage that may occur following superantigen exposure, we analyzed cytotoxicity to “bystander” fibroblast cells cultured with splenocytes in the presence of bacterial antigens. Prior host exposure to staphylococcal enterotoxin B significantly enhanced fibroblast cytotoxicity in the presence of bacteria. Neutralization of IFN-γ decreased the amount of cytotoxicity observed. However, a greater reduction was evident when splenocyte-bacterium cocultures were separated from the bystander cell monolayer via a permeable membrane support. Increased cytotoxicity appears to be primarily dependent upon cell-cell contact. Collectively, these data indicate that overproduction of inflammatory cytokines may alter the activity of cytotoxic immune cells. Superantigen exposure exacerbates cytokine production and lytic cell activity when immune cells encounter bacteria in vitro and comparable activities could possibly occur in vivo. PMID:9784507

  10. Comparison of Hemagglutination and Hemolytic Activity of Various Bacterial Clinical Isolates Against Different Human Blood Groups

    PubMed Central

    HRV, Rajkumar; Devaki, Ramakrishna

    2016-01-01

    Among the various pathogenic determinants shown by microorganisms hemagglutination and hemolysin production assume greater significance in terms of laboratory identification. This study evaluated the hemagglutination and hemolytic activity of various bacterial isolates against different blood groups. One hundred and fifty bacterial strains, isolated from clinical specimens like urine, pus, blood, and other body fluids were tested for their hemagglutinating and hemolytic activity against human A, B, AB, and O group red blood cells. Among the 150 isolates 81 were Escherichia coli, 18 were Klebsiella pneumoniae, 19 were Pseudomonas aeruginosa, 10 were Pseudomonas spp, six were Proteus mirabilis, and the rest 16 were Staphylococcus aureus. Nearly 85% of the isolates agglutinated A group cells followed by B and AB group (59.3% and 60.6% respectively). Least number of isolates agglutinated O group cells (38.0%). When the hemolytic activity was tested, out of these 150 isolates 79 (52.6%) hemolyzed A group cells, 61 (40.6%) hemolyzed AB group cells, 46 (30.6%) hemolyzed B group cells, and 57 (38.6%) isolates hemolyzed O group cells. Forty-six percent of the isolates exhibited both hemagglutinating and hemolytic property against A group cells, followed by B and AB group cells (28.6% and 21.3% respectively). Least number of isolates i.e., 32 (21.3%) showed both the properties against O group cells. The isolates showed wide variation in their hemagglutination and hemolytic properties against different combinations of human blood group cells. The study highlights the importance of selection of the type of cells especially when human RBCs are used for studying the hemagglutination and hemolytic activity of bacterial isolates because these two properties are considered as characteristic of pathogenic strains. PMID:27014523

  11. Comparison of Hemagglutination and Hemolytic Activity of Various Bacterial Clinical Isolates Against Different Human Blood Groups.

    PubMed

    Hrv, Rajkumar; Devaki, Ramakrishna; Kandi, Venkataramana

    2016-01-01

    Among the various pathogenic determinants shown by microorganisms hemagglutination and hemolysin production assume greater significance in terms of laboratory identification. This study evaluated the hemagglutination and hemolytic activity of various bacterial isolates against different blood groups. One hundred and fifty bacterial strains, isolated from clinical specimens like urine, pus, blood, and other body fluids were tested for their hemagglutinating and hemolytic activity against human A, B, AB, and O group red blood cells. Among the 150 isolates 81 were Escherichia coli, 18 were Klebsiella pneumoniae, 19 were Pseudomonas aeruginosa, 10 were Pseudomonas spp, six were Proteus mirabilis, and the rest 16 were Staphylococcus aureus. Nearly 85% of the isolates agglutinated A group cells followed by B and AB group (59.3% and 60.6% respectively). Least number of isolates agglutinated O group cells (38.0%). When the hemolytic activity was tested, out of these 150 isolates 79 (52.6%) hemolyzed A group cells, 61 (40.6%) hemolyzed AB group cells, 46 (30.6%) hemolyzed B group cells, and 57 (38.6%) isolates hemolyzed O group cells. Forty-six percent of the isolates exhibited both hemagglutinating and hemolytic property against A group cells, followed by B and AB group cells (28.6% and 21.3% respectively). Least number of isolates i.e., 32 (21.3%) showed both the properties against O group cells. The isolates showed wide variation in their hemagglutination and hemolytic properties against different combinations of human blood group cells. The study highlights the importance of selection of the type of cells especially when human RBCs are used for studying the hemagglutination and hemolytic activity of bacterial isolates because these two properties are considered as characteristic of pathogenic strains. PMID:27014523

  12. Sorption of heavy metals by prepared bacterial cell surfaces

    SciTech Connect

    Churchill, S.A.; Walters, J.V.; Churchill, P.F.

    1995-10-01

    Prepared biomass from two Gram-negative and one Gram-positive bacterial strains was examined for single, binary, and quaternary mixtures of polyvalent metal cation binding to cell surfaces. The biosorption of {sub 24}Cr{sup 3+}, {sub 27}Co{sup 2+}, {sub 28}Ni{sup 2+}, and {sub 29}Cu{sup 2+} for each bacterial cell type was evaluated using a batch equilibrium method. The binding of each metal by all three bacterial cells could be described by the Freundlich sorption model. The isotherm binding constants suggest that E. coli cells are the most efficient at binding copper, chromium, and nickel; and M. luteus adsorbs cobalt most efficiently. The K-values for copper bound to P. aeruginosa and E. coli are > 2-fold and > 8-fold greater, respectively, than previous reported for intact cells. The general metal-affinity series observed was Cr{sup 3+} > Cu{sup 2+} > Ni{sup 2+} > Co{sup 2+}. There was a marked lower affinity of all biosorbents for Co{sup 2+} and Ni{sup 2+}. M. luteus and E. coli had a strong preference for Co{sup 2+} over Ni{sup 2+}. Metal-binding enhancement could be ascribed to increased cell barrier surface porosity to metal-bearing solutions.

  13. Metatranscriptomics reveals overall active bacterial composition in caries lesions

    PubMed Central

    Simón-Soro, Aurea; Guillen-Navarro, Miriam; Mira, Alex

    2014-01-01

    Background Identifying the microbial species in caries lesions is instrumental to determine the etiology of dental caries. However, a significant proportion of bacteria in carious lesions have not been cultured, and the use of molecular methods has been limited to DNA-based approaches, which detect both active and inactive or dead microorganisms. Objective To identify the RNA-based, metabolically active bacterial composition of caries lesions at different stages of disease progression in order to provide a list of potential etiological agents of tooth decay. Design Non-cavitated enamel caries lesions (n=15) and dentin caries lesions samples (n=12) were collected from 13 individuals. RNA was extracted and cDNA was constructed, which was used to amplify the 16S rRNA gene. The resulting 780 bp polymerase chain reaction products were pyrosequenced using Titanium-plus chemistry, and the sequences obtained were used to determine the bacterial composition. Results A mean of 4,900 sequences of the 16S rRNA gene with an average read length of 661 bp was obtained per sample, giving a comprehensive view of the active bacterial communities in caries lesions. Estimates of bacterial diversity indicate that the microbiota of cavities is highly complex, each sample containing between 70 and 400 metabolically active species. The composition of these bacterial consortia varied among individuals and between caries lesions of the same individuals. In addition, enamel and dentin lesions had a different bacterial makeup. Lactobacilli were found almost exclusively in dentin cavities. Streptococci accounted for 40% of the total active community in enamel caries, and 20% in dentin caries. However, Streptococcus mutans represented only 0.02–0.73% of the total bacterial community. Conclusions The data indicate that the etiology of dental caries is tissue dependent and that the disease has a clear polymicrobial origin. The low proportion of mutans streptococci detected confirms that they

  14. Studies on bacterial cell wall inhibitors. VI. Screening method for the specific inhibitors of peptidoglycan synthesis.

    PubMed

    Omura, S; Tanaka, H; Oiwa, R; Nagai, T; Koyama, Y; Takahashi, Y

    1979-10-01

    A screening method was established for selecting new specific inhibitors of bacterial cell wall peptidoglycan synthesis. In the primary test, culture broths of soil isolates were selected based on relative microbial activity. A culture, to be retained, must be active against Bacillus subtilis and lack activities against Acholeplasma laidawii. In the secondary test, inhibitors of bacterial cell wall synthesis were identified by their ability to prevent the incorporation of meso-[3H]diaminopimelic acid but not to prevent the incorporation of L-[4C]leucine into the acid-insoluble macromolecular fraction of growing cells of Bacillus sp. ATCC 21206 (Dpm-). As the tertiary test, inhibitors with molecular weights under 1,000 were selected by passage through a Diaflo UM-2 membrane. By this screening procedure, six known antibiotics and one new one were picked out from ten thousand soil isolates. PMID:528376

  15. Resistance to antibiotics targeted to the bacterial cell wall

    PubMed Central

    Nikolaidis, I; Favini-Stabile, S; Dessen, A

    2014-01-01

    Peptidoglycan is the main component of the bacterial cell wall. It is a complex, three-dimensional mesh that surrounds the entire cell and is composed of strands of alternating glycan units crosslinked by short peptides. Its biosynthetic machinery has been, for the past five decades, a preferred target for the discovery of antibacterials. Synthesis of the peptidoglycan occurs sequentially within three cellular compartments (cytoplasm, membrane, and periplasm), and inhibitors of proteins that catalyze each stage have been identified, although not all are applicable for clinical use. A number of these antimicrobials, however, have been rendered inactive by resistance mechanisms. The employment of structural biology techniques has been instrumental in the understanding of such processes, as well as the development of strategies to overcome them. This review provides an overview of resistance mechanisms developed toward antibiotics that target bacterial cell wall precursors and its biosynthetic machinery. Strategies toward the development of novel inhibitors that could overcome resistance are also discussed. PMID:24375653

  16. Subdiffraction localization of a nanostructured photosensitizer in bacterial cells

    PubMed Central

    Delcanale, Pietro; Pennacchietti, Francesca; Maestrini, Giulio; Rodríguez-Amigo, Beatriz; Bianchini, Paolo; Diaspro, Alberto; Iagatti, Alessandro; Patrizi, Barbara; Foggi, Paolo; Agut, Monserrat; Nonell, Santi; Abbruzzetti, Stefania; Viappiani, Cristiano

    2015-01-01

    Antibacterial treatments based on photosensitized production of reactive oxygen species is a promising approach to address local microbial infections. Given the small size of bacterial cells, identification of the sites of binding of the photosensitizing molecules is a difficult issue to address with conventional microscopy. We show that the excited state properties of the naturally occurring photosensitizer hypericin can be exploited to perform STED microscopy on bacteria incubated with the complex between hypericin and apomyoglobin, a self-assembled nanostructure that confers very good bioavailability to the photosensitizer. Hypericin fluorescence is mostly localized at the bacterial wall, and accumulates at the polar regions of the cell and at sites of cell wall growth. While these features are shared by Gram-negative and Gram-positive bacteria, only the latter are effectively photoinactivated by light exposure. PMID:26494535

  17. Thiolactomycin-Based Inhibitors of Bacterial β-Ketoacyl-ACP Synthases with in Vivo Activity.

    PubMed

    Bommineni, Gopal R; Kapilashrami, Kanishk; Cummings, Jason E; Lu, Yang; Knudson, Susan E; Gu, Chendi; Walker, Stephen G; Slayden, Richard A; Tonge, Peter J

    2016-06-01

    β-Ketoacyl-ACP synthases (KAS) are key enzymes involved in the type II bacterial fatty acid biosynthesis (FASII) pathway and are putative targets for antibacterial discovery. Several natural product KAS inhibitors have previously been reported, including thiolactomycin (TLM), which is produced by Nocardia spp. Here we describe the synthesis and characterization of optically pure 5R-thiolactomycin (TLM) analogues that show improved whole cell activity against bacterial strains including methicillin-resistant Staphylococcus aureus (MRSA) and priority pathogens such as Francisella tularensis and Burkholderia pseudomallei. In addition, we identify TLM analogues with in vivo efficacy against MRSA and Klebsiella pneumoniae in animal models of infection. PMID:27187871

  18. Antibiofilm activity of Dendrophthoe falcata against different bacterial pathogens.

    PubMed

    Karthikeyan, Alagarsamy; Rameshkumar, Ramakrishnan; Sivakumar, Nallusamy; Al Amri, Issa S; Karutha Pandian, Shunmugiah; Ramesh, Manikandan

    2012-12-01

    Dendrophthoe falcata is a hemiparasitic plant commonly used for ailments such as ulcers, asthma, impotence, paralysis, skin diseases, menstrual troubles, pulmonary tuberculosis, and wounds. In this context, the validations of the traditional claim that the leaf extract of D. falcata possesses antibiofilm and anti-quorum sensing activity against different bacterial pathogens were assessed. The bacterial biofilms were quantified by crystal violet staining. Among the 17 bacterial pathogens screened, the methanolic fraction of the leaf extract clearly demonstrated antibiofilm activity for Proteus mirabilis, Vibrio vulnificus, Aeromonas hydrophila, Shigella sonnei, Chromobacterium violaceum ATCC 12472, Vibrio parahaemolyticus, Vibrio harveyi, Vibrio alginolyticus, Vibrio cholerae, and Proteus vulgaris. At biofilm inhibitory concentrations, biofilm formation was reduced by up to 70-90 %. Furthermore, the potential quorum-sensing activity of the leaf extract was tested by agar well diffusion using Chromobacterium violaceum (ATCC 12472 & CV O26) reporter strains. The inhibition of violacein production may be due to direct or indirect interference on QS by active constituents or the interactive effect of different phytocompounds present in the extracts. This is the first report on antibiofilm and QS activity of D. falcata leaf extracts, signifying the scope for development of complementary medicine for biofilm-associated infections. PMID:23115018

  19. Exploring bacterial cell biology with single-molecule tracking and super-resolution imaging

    PubMed Central

    Gahlmann, Andreas; Moerner, W. E.

    2014-01-01

    The ability to detect single molecules in live bacterial cells enables us to probe biological events one molecule at a time and thereby gain knowledge of the activities of intracellular molecules that remain obscure in conventional ensemble-averaged measurements. Single-molecule fluorescence tracking and super-resolution imaging are thus providing a new window into bacterial cells and facilitating the elucidation of cellular processes at an unprecedented level of sensitivity, specificity and spatial resolution. In this Review, we consider what these technologies have taught us about the bacterial cytoskeleton, nucleoid organization and the dynamic processes of transcription and translation, and we also highlight the methodological improvements that are needed to address a number of experimental challenges in the field. PMID:24336182

  20. Reducing systems protecting the bacterial cell envelope from oxidative damage.

    PubMed

    Arts, Isabelle S; Gennaris, Alexandra; Collet, Jean-François

    2015-06-22

    Exposure of cells to elevated levels of reactive oxygen species (ROS) damages DNA, membrane lipids and proteins, which can potentially lead to cell death. In proteins, the sulfur-containing residues cysteine and methionine are particularly sensitive to oxidation, forming sulfenic acids and methionine sulfoxides, respectively. The presence of protection mechanisms to scavenge ROS and repair damaged cellular components is therefore essential for cell survival. The bacterial cell envelope, which constitutes the first protection barrier from the extracellular environment, is particularly exposed to the oxidizing molecules generated by the host cells to kill invading microorganisms. Therefore, the presence of oxidative stress defense mechanisms in that compartment is crucial for cell survival. Here, we review recent findings that led to the identification of several reducing pathways protecting the cell envelope from oxidative damage. We focus in particular on the mechanisms that repair envelope proteins with oxidized cysteine and methionine residues and we discuss the major questions that remain to be solved. PMID:25957772

  1. Bacterial cells enhance laser driven ion acceleration

    PubMed Central

    Dalui, Malay; Kundu, M.; Trivikram, T. Madhu; Rajeev, R.; Ray, Krishanu; Krishnamurthy, M.

    2014-01-01

    Intense laser produced plasmas generate hot electrons which in turn leads to ion acceleration. Ability to generate faster ions or hotter electrons using the same laser parameters is one of the main outstanding paradigms in the intense laser-plasma physics. Here, we present a simple, albeit, unconventional target that succeeds in generating 700 keV carbon ions where conventional targets for the same laser parameters generate at most 40 keV. A few layers of micron sized bacteria coating on a polished surface increases the laser energy coupling and generates a hotter plasma which is more effective for the ion acceleration compared to the conventional polished targets. Particle-in-cell simulations show that micro-particle coated target are much more effective in ion acceleration as seen in the experiment. We envisage that the accelerated, high-energy carbon ions can be used as a source for multiple applications. PMID:25102948

  2. Antiviral activity and specific modes of action of bacterial prodigiosin against Bombyx mori nucleopolyhedrovirus in vitro.

    PubMed

    Zhou, Wei; Zeng, Cheng; Liu, RenHua; Chen, Jie; Li, Ru; Wang, XinYan; Bai, WenWen; Liu, XiaoYuan; Xiang, TingTing; Zhang, Lin; Wan, YongJi

    2016-05-01

    Prodigiosin, the tripyrrole red pigment, is a bacterial secondary metabolite with multiple bioactivities; however, the antiviral activity has not been reported yet. In the present study, we found the antiviral activity of bacterial prodigiosin on Bombyx mori nucleopolyhedrovirus (BmNPV)-infected cells in vitro, with specific modes of action. Prodigiosin at nontoxic concentrations selectively killed virus-infected cells, inhibited viral gene transcription, especially viral early gene ie-1, and prevented virus-mediated membrane fusion. Under prodigiosin treatment, both progeny virus production and viral DNA replication were significantly inhibited. Fluorescent assays showed that prodigiosin predominantly located in cytoplasm which suggested it might interact with cytoplasm factors to inhibit virus replication. In conclusion, the present study clearly indicates that prodigiosin possesses significant antiviral activity against BmNPV. PMID:26685856

  3. Cooperation between Monocyte-Derived Cells and Lymphoid Cells in the Acute Response to a Bacterial Lung Pathogen

    PubMed Central

    Brown, Andrew S.; Yang, Chao; Fung, Ka Yee; Bachem, Annabell; Bourges, Dorothée; Bedoui, Sammy; Hartland, Elizabeth L.; van Driel, Ian R.

    2016-01-01

    Legionella pneumophila is the causative agent of Legionnaires’ disease, a potentially fatal lung infection. Alveolar macrophages support intracellular replication of L. pneumophila, however the contributions of other immune cell types to bacterial killing during infection are unclear. Here, we used recently described methods to characterise the major inflammatory cells in lung after acute respiratory infection of mice with L. pneumophila. We observed that the numbers of alveolar macrophages rapidly decreased after infection coincident with a rapid infiltration of the lung by monocyte-derived cells (MC), which, together with neutrophils, became the dominant inflammatory cells associated with the bacteria. Using mice in which the ability of MC to infiltrate tissues is impaired it was found that MC were required for bacterial clearance and were the major source of IL12. IL12 was needed to induce IFNγ production by lymphoid cells including NK cells, memory T cells, NKT cells and γδ T cells. Memory T cells that produced IFNγ appeared to be circulating effector/memory T cells that infiltrated the lung after infection. IFNγ production by memory T cells was stimulated in an antigen-independent fashion and could effectively clear bacteria from the lung indicating that memory T cells are an important contributor to innate bacterial defence. We also determined that a major function of IFNγ was to stimulate bactericidal activity of MC. On the other hand, neutrophils did not require IFNγ to kill bacteria and alveolar macrophages remained poorly bactericidal even in the presence of IFNγ. This work has revealed a cooperative innate immune circuit between lymphoid cells and MC that combats acute L. pneumophila infection and defines a specific role for IFNγ in anti-bacterial immunity. PMID:27300652

  4. Wingless homolog Wnt11 suppresses bacterial invasion and inflammation in intestinal epithelial cells.

    PubMed

    Liu, Xingyin; Wu, Shaoping; Xia, Yinglin; Li, Xi Emma; Xia, Yuxuan; Zhou, Zhongren David; Sun, Jun

    2011-12-01

    Wnt11 plays an essential role in gastrointestinal epithelial proliferation, and previous investigations have focused on development and immune responses. However, the roles of how enteric bacteria regulate Wnt11 and how Wnt11 modulates the host response to pathogenic bacteria remain unexplored. This study investigated the effects of Salmonella infection on Wnt activation in intestinal epithelial cells. We found that Wnt11 mRNA and protein expression were elevated after Salmonella colonization. Wnt11 protein secretion in epithelial cells was also elevated after bacterial infection. Furthermore, we demonstrated that pathogenic Salmonella regulated Wnt11 expression and localization in vivo. We found a decrease in Salmonella invasion in cells with Wnt11 overexpression compared with cells with normal Wnt11 level. IL-8 mRNA in Wnt11-transfected cells was low; however, it was enhanced in cells with a low level of Wnt11 expression. Functionally, Wnt11 overexpression inhibited Salmonella-induced apoptosis. AvrA is a known bacterial effector protein that stabilizes β-catenin, the downstream regulator of Wnt signaling, and inhibits bacterially induced intestinal inflammation. We observed that Wnt11 expression, secretion, and transcriptional activity were regulated by Salmonella AvrA. Overall, Wnt11 is involved in the protection of the host intestinal cells by blocking the invasion of pathogenic bacteria, suppressing inflammation, and inhibiting apoptosis. Wnt11 is a novel and important contributor to intestinal homeostasis and host defense. PMID:21903761

  5. Toxicity of a polymer-graphene oxide composite against bacterial planktonic cells, biofilms, and mammalian cells

    NASA Astrophysics Data System (ADS)

    Mejías Carpio, Isis E.; Santos, Catherine M.; Wei, Xin; Rodrigues, Debora F.

    2012-07-01

    It is critical to develop highly effective antimicrobial agents that are not harmful to humans and do not present adverse effects on the environment. Although antimicrobial studies of graphene-based nanomaterials are still quite limited, some researchers have paid particular attention to such nanocomposites as promising candidates for the next generation of antimicrobial agents. The polyvinyl-N-carbazole (PVK)-graphene oxide (GO) nanocomposite (PVK-GO), which contains only 3 wt% of GO well-dispersed in a 97 wt% PVK matrix, presents excellent antibacterial properties without significant cytotoxicity to mammalian cells. The high polymer content in this nanocomposite makes future large-scale material manufacturing possible in a high-yield process of adiabatic bulk polymerization. In this study, the toxicity of PVK-GO was assessed with planktonic microbial cells, biofilms, and NIH 3T3 fibroblast cells. The antibacterial effects were evaluated against two Gram-negative bacteria: Escherichia coli and Cupriavidus metallidurans; and two Gram-positive bacteria: Bacillus subtilis and Rhodococcus opacus. The results show that the PVK-GO nanocomposite presents higher antimicrobial effects than the pristine GO. The effectiveness of the PVK-GO in solution was demonstrated as the nanocomposite ``encapsulated'' the bacterial cells, which led to reduced microbial metabolic activity and cell death. The fact that the PVK-GO did not present significant cytotoxicity to fibroblast cells offers a great opportunity for potential applications in important biomedical and industrial fields.It is critical to develop highly effective antimicrobial agents that are not harmful to humans and do not present adverse effects on the environment. Although antimicrobial studies of graphene-based nanomaterials are still quite limited, some researchers have paid particular attention to such nanocomposites as promising candidates for the next generation of antimicrobial agents. The polyvinyl

  6. Bacterial-induced cell reprogramming to stem cell-like cells: new premise in host-pathogen interactions

    PubMed Central

    Hess, Samuel; Rambukkana, Anura

    2015-01-01

    Bacterial pathogens employ a myriad of strategies to alter host tissue cell functions for bacterial advantage during infection. Recent advances revealed a fusion of infection biology with stem cell biology by demonstrating developmental reprogramming of lineage committed host glial cells to progenitor/stem cell-like cells by an intracellular bacterial pathogen Mycobacterium leprae. Acquisition of migratory and immunomodulatory properties of such reprogrammed cells provides an added advantage for promoting bacterial spread. This presents a previously unseen sophistication of cell manipulation by hijacking the genomic plasticity of host cells by a human bacterial pathogen. The rationale for such extreme fate conversion of host cells may be directly linked to the exceedingly passive obligate life style of M. leprae with a degraded genome and host cell dependence for both bacterial survival and dissemination, particularly the use of host-derived stem cell-like cells as a vehicle for spreading infection without being detected by immune cells. Thus, this unexpected link between cell reprogramming and infection opens up a new premise in host-pathogen interactions. Furthermore, such bacterial ingenuity could also be harnessed for developing natural ways of reprogramming host cells for repairing damaged tissues from infection, injury and diseases. PMID:25541240

  7. Water Diffusion from a Bacterial Cell in Low-Moisture Foods.

    PubMed

    Syamaladevi, Roopesh M; Tang, Juming; Zhong, QingPing

    2016-09-01

    We used a Fick's unsteady state diffusion equation to estimate the time required for a single spherical shaped bacterium (assuming Enterococcus faecium as the target microorganism) in low-moisture foods to equilibrate with the environment. We generated water sorption isotherms of freeze-dried E. faecium. The water activity of bacterial cells at given water content increased considerably as temperature increased from 20 to 80 °C, as observed in the sorption isotherms of bacterial cells. When the water vapor diffusion coefficient was assumed as between 10(-12) and 10(-10) m(2) /s for bacterial cells, the predicted equilibration times (teq ) ranged from 8.24×10(-4) to 8.24×10(-2) s. Considering a cell membrane barrier with a lower water diffusion coefficient (10(-15) m(2) /s) around the bacterial cell with a water diffusion coefficient of 10(-12) m(2) /s, the teq predicted using COMSOL Multiphysics program was 3.8×10(-1) s. This result suggests that a single bacterium equilibrates rapidly (within seconds) with change in environmental humidity and temperature. PMID:27505687

  8. Antibacterial activity and mechanism of action of auranofin against multi-drug resistant bacterial pathogens.

    PubMed

    Thangamani, Shankar; Mohammad, Haroon; Abushahba, Mostafa F N; Sobreira, Tiago J P; Hedrick, Victoria E; Paul, Lake N; Seleem, Mohamed N

    2016-01-01

    Traditional methods employed to discover new antibiotics are both a time-consuming and financially-taxing venture. This has led researchers to mine existing libraries of clinical molecules in order to repurpose old drugs for new applications (as antimicrobials). Such an effort led to the discovery of auranofin, a drug initially approved as an anti-rheumatic agent, which also possesses potent antibacterial activity in a clinically achievable range. The present study demonstrates auranofin's antibacterial activity is a complex process that involves inhibition of multiple biosynthetic pathways including cell wall, DNA, and bacterial protein synthesis. We also confirmed that the lack of activity of auranofin observed against Gram-negative bacteria is due to the permeability barrier conferred by the outer membrane. Auranofin's ability to suppress bacterial protein synthesis leads to significant reduction in the production of key methicillin-resistant Staphylococcus aureus (MRSA) toxins. Additionally, auranofin is capable of eradicating intracellular MRSA present inside infected macrophage cells. Furthermore, auranofin is efficacious in a mouse model of MRSA systemic infection and significantly reduces the bacterial load in murine organs including the spleen and liver. Collectively, this study provides valuable evidence that auranofin has significant promise to be repurposed as a novel antibacterial for treatment of invasive bacterial infections. PMID:26936660

  9. Antibacterial activity and mechanism of action of auranofin against multi-drug resistant bacterial pathogens

    PubMed Central

    Thangamani, Shankar; Mohammad, Haroon; Abushahba, Mostafa F. N.; Sobreira, Tiago J. P.; Hedrick, Victoria E.; Paul, Lake N.; Seleem, Mohamed N.

    2016-01-01

    Traditional methods employed to discover new antibiotics are both a time-consuming and financially-taxing venture. This has led researchers to mine existing libraries of clinical molecules in order to repurpose old drugs for new applications (as antimicrobials). Such an effort led to the discovery of auranofin, a drug initially approved as an anti-rheumatic agent, which also possesses potent antibacterial activity in a clinically achievable range. The present study demonstrates auranofin’s antibacterial activity is a complex process that involves inhibition of multiple biosynthetic pathways including cell wall, DNA, and bacterial protein synthesis. We also confirmed that the lack of activity of auranofin observed against Gram-negative bacteria is due to the permeability barrier conferred by the outer membrane. Auranofin’s ability to suppress bacterial protein synthesis leads to significant reduction in the production of key methicillin-resistant Staphylococcus aureus (MRSA) toxins. Additionally, auranofin is capable of eradicating intracellular MRSA present inside infected macrophage cells. Furthermore, auranofin is efficacious in a mouse model of MRSA systemic infection and significantly reduces the bacterial load in murine organs including the spleen and liver. Collectively, this study provides valuable evidence that auranofin has significant promise to be repurposed as a novel antibacterial for treatment of invasive bacterial infections. PMID:26936660

  10. Extracellular Heme Uptake and the Challenges of Bacterial Cell Membranes

    PubMed Central

    Smith, Aaron D.; Wilks, Angela

    2013-01-01

    In bacteria, the fine balance of maintaining adequate iron levels while preventing the deleterious effects of excess iron has led to the evolution of sophisticated cellular mechanisms to obtain, store, and regulate iron. Iron uptake provides a significant challenge given its limited bioavailability and need to be transported across the bacterial cell wall and membranes. Pathogenic bacteria have circumvented the iron-availability issue by utilizing the hosts' heme-containing proteins as a source of iron. Once internalized, iron is liberated from the porphyrin enzymatically for cellular processes within the bacterial cell. Heme, a lipophilic and toxic molecule, poses a significant challenge in terms of transport given its chemical reactivity. As such, pathogenic bacteria have evolved sophisticated membrane transporters to coordinate, sequester, and transport heme. Recent advances in the biochemical and structural characterization of the membrane-bound heme transport proteins are discussed in the context of ligand coordination, protein–protein interaction, and heme transfer. PMID:23046657

  11. ULTRASOUND INCREASES THE RATE OF BACTERIAL CELL GROWTH

    PubMed Central

    Pitt, William G.; Ross, S. Aaron

    2006-01-01

    Ultrasound was employed to increase the growth rate of bacterial cells attached to surfaces. Staphylococcus epidermidis, Pseudomonas aeruginosa and Escherichia coli cells adhered to and grew on a polyethylene surface in the presence of ultrasound. It was found that low frequency ultrasound (70 kHz) of low acoustic intensity (<2 W/cm2) increased the growth rate of the cells compared to growth without ultrasound. However, at high intensity levels, cells were partially removed from the surface. Ultrasound also enhanced planktonic growth of S. epidermidis and other planktonic bacteria. It is hypothesized that ultrasound increases the rate of transport of oxygen and nutrients to the cells and increases the rate of transport of waste products away from the cells, thus enhancing their growth. PMID:12790676

  12. Influence of bacterial endotoxin on radiation-induced activation of human endothelial cells in vitro and in vivo: interleukin-10 protects against transendothelial migration.

    PubMed

    Lindner, H; Holler, E; Gerbitz, A; Johnson, J P; Bornkamm, G W; Eissner, G

    1997-11-15

    To extend previous studies on the anti-inflammatory role of interleukin (IL)-10 in vivo, mice pretreated with IL-10 were subjected to ionizing radiation (IR), lipopolysaccharide (LPS), or both and assessed for the expression of the intercellular adhesion molecule 1 (ICAM-1) in immunohistochemical analyses. IL-10 was able to almost fully protect LPS+IR-treated animals against ICAM-1 up-regulation. Because LPS and IR also increased adhesion of peripheral blood mononuclear cells, transendothelial migration assays were performed to investigate the functional significance of these findings. IR was found to induce transendothelial migration, and this effect could be enhanced by cotreatment with LPS, in the same fashion as peripheral blood mononuclear cell adhesion. Also in this system, IL-10 proved to act as a potent LPS antagonist. Finally, in vivo immunohistochemical analyses revealed an infiltration of CD3+ T lymphocytes into organs that were the target of transplant-related complications after LPS+IR treatment. This infiltration could also be completely reversed by IL-10 pretreatment. PMID:9371683

  13. Human NAIP and mouse NAIP1 recognize bacterial type III secretion needle protein for inflammasome activation.

    PubMed

    Yang, Jieling; Zhao, Yue; Shi, Jianjin; Shao, Feng

    2013-08-27

    Inflammasome mediated by central nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) protein is critical for defense against bacterial infection. Here we show that type III secretion system (T3SS) needle proteins from several bacterial pathogens, including Salmonella typhimurium, enterohemorrhagic Escherichia coli, Shigella flexneri, and Burkholderia spp., can induce robust inflammasome activation in both human monocyte-derived and mouse bone marrow macrophages. Needle protein activation of human NRL family CARD domain containing 4 (NLRC4) inflammasome requires the sole human neuronal apoptosis inhibitory protein (hNAIP). Among the seven mouse NAIPs, NAIP1 functions as the mouse counterpart of hNAIP. We found that NAIP1 recognition of T3SS needle proteins was more robust in mouse dendritic cells than in bone marrow macrophages. Needle proteins, as well as flagellin and rod proteins from five different bacteria, exhibited differential and cell type-dependent inflammasome-stimulating activity. Comprehensive profiling of the three types of NAIP ligands revealed that NAIP1 sensing of the needle protein dominated S. flexneri-induced inflammasome activation, particularly in dendritic cells. hNAIP/NAIP1 and NAIP2/5 formed a large oligomeric complex with NLRC4 in the presence of corresponding bacterial ligands, and could support reconstitution of the NLRC4 inflammasome in a ligand-specific manner. PMID:23940371

  14. The Role of Lipid Domains in Bacterial Cell Processes

    PubMed Central

    Barák, Imrich; Muchová, Katarína

    2013-01-01

    Membranes are vital structures for cellular life forms. As thin, hydrophobic films, they provide a physical barrier separating the aqueous cytoplasm from the outside world or from the interiors of other cellular compartments. They maintain a selective permeability for the import and export of water-soluble compounds, enabling the living cell to maintain a stable chemical environment for biological processes. Cell membranes are primarily composed of two crucial substances, lipids and proteins. Bacterial membranes can sense environmental changes or communication signals from other cells and they support different cell processes, including cell division, differentiation, protein secretion and supplementary protein functions. The original fluid mosaic model of membrane structure has been recently revised because it has become apparent that domains of different lipid composition are present in both eukaryotic and prokaryotic cell membranes. In this review, we summarize different aspects of phospholipid domain formation in bacterial membranes, mainly in Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. We describe the role of these lipid domains in membrane dynamics and the localization of specific proteins and protein complexes in relation to the regulation of cellular function. PMID:23429192

  15. Bacterial communities and enzyme activities of PAHs polluted soils.

    PubMed

    Andreoni, V; Cavalca, L; Rao, M A; Nocerino, G; Bernasconi, S; Dell'Amico, E; Colombo, M; Gianfreda, L

    2004-11-01

    Three soils (i.e. a Belgian soil, B-BT, a German soil, G, and an Italian agricultural soil, I-BT) with different properties and hydrocarbon-pollution history with regard to their potential to degrade phenanthrene were investigated. A chemical and microbiological evaluation of soils was done using measurements of routine chemical properties, bacterial counts and several enzyme activities. The three soils showed different levels of polycyclic aromatic hydrocarbons (PAHs), being their contamination strictly associated to their pollution history. High values of enzyme activities and culturable heterotrophic bacteria were detected in the soil with no or negligible presence of organic pollutants. Genetic diversity of soil samples and enrichment cultures was measured as bands on denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA sequences from the soil and enrichment community DNAs. When analysed by Shannon index (H'), the highest genetic biodiversity (H'=2.87) was found in the Belgian soil B-BT with a medium-term exposition to PAHs and the poorest biodiversity (H'=0.85) in the German soil with a long-term exposition to alkanes and PAHs and where absence, or lower levels of enzyme activities were measured. For the Italian agricultural soil I-BT, containing negligible amounts of organic pollutants but the highest Cu content, a Shannon index=2.13 was found. The enrichment of four mixed cultures capable of degrading solid phenanthrene in batch liquid systems was also studied. Phenanthrene degradation rates in batch systems were culture-dependent, and simple (one-slope) and complex (two-slope) kinetic behaviours were observed. The presence of common bands of microbial species in the cultures and in the native soil DNA indicated that those strains could be potential in situ phenanthrene degraders. Consistent with this assumption are the decrease of PAH and phenanthrene contents of Belgian soil B-BT and the isolation of phenanthrene-degrading bacteria. From

  16. The impact of metabolic state on Cd adsorption onto bacterial cells

    USGS Publications Warehouse

    Johnson, K.J.; Ams, D.A.; Wedel, A.N.; Szymanowski, J.E.S.; Weber, D.L.; Schneegurt, M.A.; Fein, J.B.

    2007-01-01

    This study examines the effect of bacterial metabolism on the adsorption of Cd onto Gram-positive and Gram-negative bacterial cells. Metabolically active Gram-positive cells adsorbed significantly less Cd than non-metabolizing cells. Gram-negative cells, however, showed no systematic difference in Cd adsorption between metabolizing and non-metabolizing cells. The effect of metabolism on Cd adsorption to Gram-positive cells was likely due to an influx of protons in and around the cell wall from the metabolic proton motive force, promoting competition between Cd and protons for adsorption sites on the cell wall. The relative lack of a metabolic effect on Cd adsorption onto Gram-negative compared to Gram-positive cells suggests that Cd binding in Gram-negative cells is focused in a region of the cell wall that is not reached, or is unaffected by this proton flux. Thermodynamic modeling was used to estimate that proton pumping causes the pH in the cell wall of metabolizing Gram-positive bacteria to decrease from the bulk solution value of 7.0 to approximately 5.7. ?? 2007 The Authors.

  17. Electroporation of Functional Bacterial Effectors into Mammalian Cells

    PubMed Central

    Sontag, Ryan L.; Mihai, Cosmin; Orr, Galya; Savchenko, Alexei; Skarina, Tatiana; Cui, Hong; Cort, John R.; Adkins, Joshua N.; Brown, Roslyn N.

    2015-01-01

    The study of protein interactions in the context of living cells can generate critical information about localization, dynamics, and interacting partners. This information is particularly valuable in the context of host-pathogen interactions. Many pathogen proteins function within host cells in a variety of way such as, enabling evasion of the host immune system and survival within the intracellular environment. To study these pathogen-protein host-cell interactions, several approaches are commonly used, including: in vivo infection with a strain expressing a tagged or mutant protein, or introduction of pathogen genes via transfection or transduction. Each of these approaches has advantages and disadvantages. We sought a means to directly introduce exogenous proteins into cells. Electroporation is commonly used to introduce nucleic acids into cells, but has been more rarely applied to proteins although the biophysical basis is exactly the same. A standard electroporator was used to introduce affinity-tagged bacterial effectors into mammalian cells. Human epithelial and mouse macrophage cells were cultured by traditional methods, detached, and placed in 0.4 cm gap electroporation cuvettes with an exogenous bacterial pathogen protein of interest (e.g. Salmonella Typhimurium GtgE). After electroporation (0.3 kV) and a short (4 hr) recovery period, intracellular protein was verified by fluorescently labeling the protein via its affinity tag and examining spatial and temporal distribution by confocal microscopy. The electroporated protein was also shown to be functional inside the cell and capable of correct subcellular trafficking and protein-protein interaction. While the exogenous proteins tended to accumulate on the surface of the cells, the electroporated samples had large increases in intracellular effector concentration relative to incubation alone. The protocol is simple and fast enough to be done in a parallel fashion, allowing for high

  18. Bending forces plastically deform growing bacterial cell walls.

    PubMed

    Amir, Ariel; Babaeipour, Farinaz; McIntosh, Dustin B; Nelson, David R; Jun, Suckjoon

    2014-04-22

    Cell walls define a cell's shape in bacteria. The walls are rigid to resist large internal pressures, but remarkably plastic to adapt to a wide range of external forces and geometric constraints. Currently, it is unknown how bacteria maintain their shape. In this paper, we develop experimental and theoretical approaches and show that mechanical stresses regulate bacterial cell wall growth. By applying a precisely controllable hydrodynamic force to growing rod-shaped Escherichia coli and Bacillus subtilis cells, we demonstrate that the cells can exhibit two fundamentally different modes of deformation. The cells behave like elastic rods when subjected to transient forces, but deform plastically when significant cell wall synthesis occurs while the force is applied. The deformed cells always recover their shape. The experimental results are in quantitative agreement with the predictions of the theory of dislocation-mediated growth. In particular, we find that a single dimensionless parameter, which depends on a combination of independently measured physical properties of the cell, can describe the cell's responses under various experimental conditions. These findings provide insight into how living cells robustly maintain their shape under varying physical environments. PMID:24711421

  19. Uniform dose atmospheric pressure microplasma exposure of individual bacterial cells

    NASA Astrophysics Data System (ADS)

    Rutherford, David; Mahony, Charles; Spence, Sarah; Perez-Martin, Fatima; Kelsey, Colin; Hamilton, Neil; Diver, Declan; Bennet, Euan; Potts, Hugh; Mariotti, Davide; McDowell, David; Maguire, Paul

    2015-09-01

    Plasma - bacteria interactions have been studied for some time with a view to using plasma exposure for wound healing, sterilization and decontamination. While high efficacy has been demonstrated, important fundamental mechanisms are not understood and may be critical for ultimate acceptance. The dose variation across the exposed population and the impact of non-lethal exposure on subsequent bacterial growth are important issues. We demonstrate that individual bacterial cells can remain viable after exposure to a uniform plasma dose. Each bacteria cell (E coli) is delivered to the atmospheric pressure plasma in an aerosolised droplet (d ~ 10 micron). The estimated plasma density is 1E13 - 1E14 cm-3, gas temperature <400 K, and exposure times vary between 0.04 and 0.1ms. Droplet evaporation in flight is ~2 micron and plasma - cell interactions are mediated by the surrounding liquid (Ringers solution) where plasma-induced droplet surface chemistry and charging is known to occur. We report the cell viability and recovery dynamics of individual exposed cells as well as impact on DNA and membrane components with reference to measured plasma parameters. This research was funded by EPSRC (Grants: EP/K006088/1 & EP/K006142/1).

  20. Detecting Bacterial Surface Organelles on Single Cells Using Optical Tweezers.

    PubMed

    Zakrisson, Johan; Singh, Bhupender; Svenmarker, Pontus; Wiklund, Krister; Zhang, Hanqing; Hakobyan, Shoghik; Ramstedt, Madeleine; Andersson, Magnus

    2016-05-10

    Bacterial cells display a diverse array of surface organelles that are important for a range of processes such as intercellular communication, motility and adhesion leading to biofilm formation, infections, and bacterial spread. More specifically, attachment to host cells by Gram-negative bacteria are mediated by adhesion pili, which are nanometers wide and micrometers long fibrous organelles. Since these pili are significantly thinner than the wavelength of visible light, they cannot be detected using standard light microscopy techniques. At present, there is no fast and simple method available to investigate if a single cell expresses pili while keeping the cell alive for further studies. In this study, we present a method to determine the presence of pili on a single bacterium. The protocol involves imaging the bacterium to measure its size, followed by predicting the fluid drag based on its size using an analytical model, and thereafter oscillating the sample while a single bacterium is trapped by an optical tweezer to measure its effective fluid drag. Comparison between the predicted and the measured fluid drag thereby indicate the presence of pili. Herein, we verify the method using polymer coated silica microspheres and Escherichia coli bacteria expressing adhesion pili. Our protocol can in real time and within seconds assist single cell studies by distinguishing between piliated and nonpiliated bacteria. PMID:27088225

  1. Virus and Bacterial Cell Chemical Analysis by NanoSIMS

    SciTech Connect

    Weber, P; Holt, J

    2008-07-28

    In past work for the Department of Homeland Security, the LLNL NanoSIMS team has succeeded in extracting quantitative elemental composition at sub-micron resolution from bacterial spores using nanometer-scale secondary ion mass spectrometry (NanoSIMS). The purpose of this task is to test our NanoSIMS capabilities on viruses and bacterial cells. This initial work has proven successful. We imaged Tobacco Mosaic Virus (TMV) and Bacillus anthracis Sterne cells using scanning electron microscopy (SEM) and then analyzed those samples by NanoSIMS. We were able resolve individual viral particles ({approx}18 nm by 300 nm) in the SEM and extract correlated elemental composition in the NanoSIMS. The phosphorous/carbon ratio observed in TMV is comparable to that seen in bacterial spores (0.033), as was the chlorine/carbon ratio (0.11). TMV elemental composition is consistent from spot to spot, and TMV is readily distinguished from debris by NanoSIMS analysis. Bacterial cells were readily identified in the SEM and relocated in the NanoSIMS for elemental analysis. The Ba Sterne cells were observed to have a measurably lower phosphorous/carbon ratio (0.005), as compared to the spores produced in the same run (0.02). The chlorine/carbon ratio was approximately 2.5X larger in the cells (0.2) versus the spores (0.08), while the fluorine/carbon ratio was approximately 10X lower in the cells (0.008) than the spores (0.08). Silicon/carbon ratios for both cells and spores encompassed a comparable range. The initial data in this study suggest that high resolution analysis is useful because it allows the target agent to be analyzed separate from particulates and other debris. High resolution analysis would also be useful for trace sample analysis. The next step in this work is to determine the potential utility of elemental signatures in these kinds of samples. We recommend bulk analyses of media and agent samples to determine the range of media compositions in use, and to determine how

  2. Toxicity of a polymer-graphene oxide composite against bacterial planktonic cells, biofilms, and mammalian cells.

    PubMed

    Mejías Carpio, Isis E; Santos, Catherine M; Wei, Xin; Rodrigues, Debora F

    2012-08-01

    It is critical to develop highly effective antimicrobial agents that are not harmful to humans and do not present adverse effects on the environment. Although antimicrobial studies of graphene-based nanomaterials are still quite limited, some researchers have paid particular attention to such nanocomposites as promising candidates for the next generation of antimicrobial agents. The polyvinyl-N-carbazole (PVK)-graphene oxide (GO) nanocomposite (PVK-GO), which contains only 3 wt% of GO well-dispersed in a 97 wt% PVK matrix, presents excellent antibacterial properties without significant cytotoxicity to mammalian cells. The high polymer content in this nanocomposite makes future large-scale material manufacturing possible in a high-yield process of adiabatic bulk polymerization. In this study, the toxicity of PVK-GO was assessed with planktonic microbial cells, biofilms, and NIH 3T3 fibroblast cells. The antibacterial effects were evaluated against two Gram-negative bacteria: Escherichia coli and Cupriavidus metallidurans; and two Gram-positive bacteria: Bacillus subtilis and Rhodococcus opacus. The results show that the PVK-GO nanocomposite presents higher antimicrobial effects than the pristine GO. The effectiveness of the PVK-GO in solution was demonstrated as the nanocomposite "encapsulated" the bacterial cells, which led to reduced microbial metabolic activity and cell death. The fact that the PVK-GO did not present significant cytotoxicity to fibroblast cells offers a great opportunity for potential applications in important biomedical and industrial fields. PMID:22751735

  3. Effects of Bacterial Microflora of the Lower Digestive Tract of Free-Range Waterfowl on Influenza Virus Activation

    PubMed Central

    King, Marcus D.; Guentzel, M. Neal; Arulanandam, Bernard P.; Bodour, Adria A.; Brahmakshatriya, Vinayak; Lupiani, Blanca; Chambers, James P.

    2011-01-01

    Proteolytic cleavage activation of influenza virus hemagglutinin (HA0) is required for cell entry via receptor-mediated endocytosis. Despite numerous studies describing bacterial protease-mediated influenza A viral activation in mammals, very little is known about the role of intestinal bacterial flora of birds in hemagglutinin cleavage/activation. Therefore, the cloaca of wild waterfowl was examined for (i) representative bacterial types and (ii) their ability to cleave in a “trypsin-like” manner the precursor viral hemagglutinin molecule (HA0). Using radiolabeled HA0, bacterial secretion-mediated trypsin-like conversion of HA0 to HA1 and HA2 peptide products was observed to various degrees in 42 of 44 bacterial isolates suggestive of influenza virus activation in the cloaca of wild waterfowl. However, treatment of uncleaved virus with all bacterial isolates gave rise to substantially reduced emergent virus progeny compared with what was expected. Examination of two isolates exhibiting pronounced trypsin-like conversion of HA0 to HA1 and HA2 peptide products and low infectivity revealed lipase activity to be present. Because influenza virus possesses a complex lipid envelope, the presence of lipid hydrolase activity could in part account for the observed less-than-expected level of viable progeny. A thorough characterization of respective isolate protease HA0 hydrolysis products as well as other resident activities (i.e., lipase) is ongoing such that the role of these respective contributors in virus activation/inactivation can be firmly established. PMID:21531837

  4. Bacterial actin and tubulin homologs in cell growth and division.

    PubMed

    Busiek, Kimberly K; Margolin, William

    2015-03-16

    In contrast to the elaborate cytoskeletal machines harbored by eukaryotic cells, such as mitotic spindles, cytoskeletal structures detectable by typical negative stain electron microscopy are generally absent from bacterial cells. As a result, for decades it was thought that bacteria lacked cytoskeletal machines. Revolutions in genomics and fluorescence microscopy have confirmed the existence not only of smaller-scale cytoskeletal structures in bacteria, but also of widespread functional homologs of eukaryotic cytoskeletal proteins. The presence of actin, tubulin, and intermediate filament homologs in these relatively simple cells suggests that primitive cytoskeletons first arose in bacteria. In bacteria such as Escherichia coli, homologs of tubulin and actin directly interact with each other and are crucial for coordinating cell growth and division. The function and direct interactions between these proteins will be the focus of this review. PMID:25784047

  5. Cell surface energy, contact angles and phase partition. II. Bacterial cells in biphasic aqueous mixtures.

    PubMed

    Gerson, D F; Akit, J

    1980-11-01

    Partition coefficients in biphasic mixtures of poly(ethylene glycol) and Dextran are compared to cell surface energies obtained from contact angles of each liquid phase on cell layers. Linear relationships are observed between these two independent measurements for a variety of bacterial cells. The results demonstrate the importance of interfacial phenomena and contact angles in the phase-partition process. PMID:6159003

  6. Disturbance opens recruitment sites for bacterial colonization in activated sludge.

    PubMed

    Vuono, David C; Munakata-Marr, Junko; Spear, John R; Drewes, Jörg E

    2016-01-01

    Little is known about the role of immigration in shaping bacterial communities or the factors that may dictate success or failure of colonization by bacteria from regional species pools. To address these knowledge gaps, the influence of bacterial colonization into an ecosystem (activated sludge bioreactor) was measured through a disturbance gradient (successive decreases in the parameter solids retention time) relative to stable operational conditions. Through a DNA sequencing approach, we show that the most abundant bacteria within the immigrant community have a greater probability of colonizing the receiving ecosystem, but mostly as low abundance community members. Only during the disturbance do some of these bacterial populations significantly increase in abundance beyond background levels and in few cases become dominant community members post-disturbance. Two mechanisms facilitate the enhanced enrichment of immigrant populations during disturbance: (i) the availability of resources left unconsumed by established species and (ii) the increased availability of niche space for colonizers to establish and displace resident populations. Thus, as a disturbance decreases local diversity, recruitment sites become available to promote colonization. This work advances our understanding of microbial resource management and diversity maintenance in complex ecosystems. PMID:25727891

  7. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications.

    PubMed

    Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

    2016-02-01

    Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies. PMID:26907343

  8. Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications

    PubMed Central

    Yeo, Chew Chieng; Abu Bakar, Fauziah; Chan, Wai Ting; Espinosa, Manuel; Harikrishna, Jennifer Ann

    2016-01-01

    Toxin-antitoxin (TA) systems are found in nearly all prokaryotic genomes and usually consist of a pair of co-transcribed genes, one of which encodes a stable toxin and the other, its cognate labile antitoxin. Certain environmental and physiological cues trigger the degradation of the antitoxin, causing activation of the toxin, leading either to the death or stasis of the host cell. TA systems have a variety of functions in the bacterial cell, including acting as mediators of programmed cell death, the induction of a dormant state known as persistence and the stable maintenance of plasmids and other mobile genetic elements. Some bacterial TA systems are functional when expressed in eukaryotic cells and this has led to several innovative applications, which are the subject of this review. Here, we look at how bacterial TA systems have been utilized for the genetic manipulation of yeasts and other eukaryotes, for the containment of genetically modified organisms, and for the engineering of high expression eukaryotic cell lines. We also examine how TA systems have been adopted as an important tool in developmental biology research for the ablation of specific cells and the potential for utility of TA systems in antiviral and anticancer gene therapies. PMID:26907343

  9. Activation of Protein Tyrosine Kinases by Coxiella burnetii: Role in Actin Cytoskeleton Reorganization and Bacterial Phagocytosis

    PubMed Central

    Meconi, Sonia; Capo, Christian; Remacle-Bonnet, Maryse; Pommier, Gilbert; Raoult, Didier; Mege, Jean-Louis

    2001-01-01

    Coxiella burnetii, the agent of Q fever, is an obligate intracellular microorganism that grows in monocytes/macrophages. The internalization of virulent organisms by monocytes is lower than that of avirulent variants and is associated with actin cytoskeleton reorganization. We studied the activation of protein tyrosine kinases (PTKs) by C. burnetii in THP-1 monocytes. Virulent organisms induced early PTK activation and the tyrosine phosphorylation of several endogenous substrates, including Hck and Lyn, two Src-related kinases. PTK activation reflects C. burnetii virulence since avirulent variants were unable to stimulate PTK. We also investigated the role of PTK activation in C. burnetii-stimulated F-actin reorganization. Tyrosine-phosphorylated proteins were colocalized with F-actin inside cell protrusions induced by C. burnetii, and PTK activity was increased in Triton X-100-insoluble fractions. In addition, lavendustin A, a PTK inhibitor, and PP1, a Src kinase inhibitor, prevented C. burnetii-induced cell protrusions and F-actin reorganization. We finally assessed the role of PTK activation in bacterial phagocytosis. Pretreatment of THP-1 cells with lavendustin A and PP1 upregulated the uptake of virulent C. burnetii but had no effect on the phagocytosis of avirulent organisms. Thus, it is likely that PTK activation by C. burnetii negatively regulates bacterial uptake by interfering with cytoskeleton organization. PMID:11254615

  10. Human mesenchymal stem cells: New sojourn of bacterial pathogens.

    PubMed

    Kohli, Sakshi; Singh, Yadvir; Sowpati, Divya Tej; Ehtesham, Nasreen Z; Dobrindt, Ulrich; Hacker, Jörg; Hasnain, Seyed E

    2015-05-01

    Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tuberculosis), is the leading infectious disease which claims one human life every 15-20s globally. The persistence of this deadly disease in human population can be attributed to the ability of the bacterium to stay in latent form. M. tuberculosis possesses a plethora of mechanisms not only to survive latently under harsh conditions inside the host but also modulate the host immune cells in its favour. Various M. tuberculosis gene families have also been described to play a role in this process. Recently, human bone marrow derived mesenchymal stem cells (MSCs) have been reported as a niche for dormant M. tuberculosis. MSCs possess abilities to alter the host immune response. The bacterium finds this self-renewal and immune privileged nature of MSCs very favourable not only to modulate the host immune system, with some help from its own genes, but also to avoid the external drug pressure. We suggest that the MSCs not only provide a resting place for M. tuberculosis but could also, by virtue of their intrinsic ability to disseminate in the body, explain the genesis of extra-pulmonary TB. A similar exploitation of stem cells by other bacterial pathogens is a distinct possibility. It may be likely that other intracellular bacterial pathogens adopt this strategy to 'piggy-back' on to ovarian stem cells to ensure vertical transmission and successful propagation to the next generation. PMID:25648374

  11. Tiny cells meet big questions: a closer look at bacterial cell biology

    PubMed Central

    Goley, Erin D.

    2013-01-01

    While studying actin assembly as a graduate student with Matt Welch at the University of California at Berkeley, my interest was piqued by reports of surprising observations in bacteria: the identification of numerous cytoskeletal proteins, actin homologues fulfilling spindle-like functions, and even the presence of membrane-bound organelles. Curiosity about these phenomena drew me to Lucy Shapiro's lab at Stanford University for my postdoctoral research. In the Shapiro lab, and now in my lab at Johns Hopkins, I have focused on investigating the mechanisms of bacterial cytokinesis. Spending time as both a eukaryotic cell biologist and a bacterial cell biologist has convinced me that bacterial cells present the same questions as eukaryotic cells: How are chromosomes organized and accurately segregated? How is force generated for cytokinesis? How is polarity established? How are signals transduced within and between cells? These problems are conceptually similar between eukaryotes and bacteria, although their solutions can differ significantly in specifics. In this Perspective, I provide a broad view of cell biological phenomena in bacteria, the technical challenges facing those of us who peer into bacterial cells, and areas of common ground as research in eukaryotic and bacterial cell biology moves forward. PMID:23580191

  12. Bacterial Riboswitches and Ribozymes Potently Activate the Human Innate Immune Sensor PKR.

    PubMed

    Hull, Chelsea M; Anmangandla, Ananya; Bevilacqua, Philip C

    2016-04-15

    The innate immune system provides the first line of defense against pathogens through the recognition of nonspecific patterns in RNA to protect the cell in a generalized way. The human RNA-activated protein kinase, PKR, is a dsRNA binding protein and an essential sensor in the innate immune response, which recognizes viral and bacterial pathogens through their RNAs. Upon activation via RNA-dependent autophosphorylation, PKR phosphorylates the eukaryotic initiation factor eIF2α, leading to termination of translation. PKR has a well-characterized role in recognizing viral RNA, where it binds long stretches of double-stranded RNA nonsequence specifically to promote activation; however, the mechanism by which bacterial RNA activates PKR and the mode by which self RNA avoids activating PKR are unknown. We characterized activation of PKR by three functional bacterial RNAs with pseudoknots and extensive tertiary structure: the cyclic di-GMP riboswitch, the glmS riboswitch-ribozyme, and the twister ribozyme, two of which are ligand-activated. These RNAs were found to activate PKR with comparable potency to long dsRNA. Enzymatic structure mapping in the absence and presence of PKR reveals a clear PKR footprint and provides a structural basis for how these bacterial RNAs activate PKR. In the case of the cyclic di-GMP riboswitch and the glmS riboswitch-ribozyme, PKR appears to dimerize on the peripheral double-stranded regions of the native RNA tertiary structure. Overall, these results provide new insights into how PKR acts as an innate immune signaling protein for the presence of bacteria and suggest a reason for the apparent absence of protein-free riboswitches and ribozymes in the human genome. PMID:27011290

  13. Microfabricated ratchet structures for concentrating and patterning motile bacterial cells

    NASA Astrophysics Data System (ADS)

    Yub Kim, Sang; Lee, Eun Se; Lee, Ho Jae; Lee, Se Yeon; Kuk Lee, Sung; Kim, Taesung

    2010-09-01

    We present a novel microfabricated concentrator for Escherichia coli that can be a stand-alone and self-contained microfluidic device because it utilizes the motility of cells. First of all, we characterize the motility of E. coli cells and various ratcheting structures that can guide cells to move in a desired direction in straight and circular channels. Then, we combine these ratcheting microstructures with the intrinsic tendency of cells to swim on the right side in microchannels to enhance the concentration rates up to 180 fold until the concentrators are fully filled with cells. Furthermore, we demonstrate that cells can be positioned and concentrated with a constant spacing distance on a surface, allowing spatial patterning of motile cells. These results can be applied to biosorption or biosensor devices that are powered by motile cells because they can be highly concentrated without any external mechanical and electrical energy sources. Hence, we believe that the concentrator design holds considerable potential to be applied for concentrating and patterning other motile microbes and providing a versatile structure for motility study of bacterial cells.

  14. Bending forces plastically deform growing bacterial cell walls

    PubMed Central

    Amir, Ariel; Babaeipour, Farinaz; McIntosh, Dustin B.; Nelson, David R.; Jun, Suckjoon

    2014-01-01

    Cell walls define a cell’s shape in bacteria. The walls are rigid to resist large internal pressures, but remarkably plastic to adapt to a wide range of external forces and geometric constraints. Currently, it is unknown how bacteria maintain their shape. In this paper, we develop experimental and theoretical approaches and show that mechanical stresses regulate bacterial cell wall growth. By applying a precisely controllable hydrodynamic force to growing rod-shaped Escherichia coli and Bacillus subtilis cells, we demonstrate that the cells can exhibit two fundamentally different modes of deformation. The cells behave like elastic rods when subjected to transient forces, but deform plastically when significant cell wall synthesis occurs while the force is applied. The deformed cells always recover their shape. The experimental results are in quantitative agreement with the predictions of the theory of dislocation-mediated growth. In particular, we find that a single dimensionless parameter, which depends on a combination of independently measured physical properties of the cell, can describe the cell’s responses under various experimental conditions. These findings provide insight into how living cells robustly maintain their shape under varying physical environments. PMID:24711421

  15. Following the Fate of Bacterial Cells Experiencing Sudden Chromosome Loss

    PubMed Central

    Elbaz, Maya

    2015-01-01

    ABSTRACT Chromosomal DNA is a constant source of information, essential for any given cell to respond and adapt to changing conditions. Here, we investigated the fate of exponentially growing bacterial cells experiencing a sudden and rapid loss of their entire chromosome. Utilizing Bacillus subtilis cells harboring an inducible copy of the endogenous toxin yqcG, which encodes an endonuclease, we induced the formation of a population of cells that lost their genetic information simultaneously. Surprisingly, these DNA-less cells, termed DLCs, did not lyse immediately and exhibited normal cellular morphology for a period of at least 5 h after DNA loss. This cellular integrity was manifested by their capacity to maintain an intact membrane and membrane potential and cell wall architecture similar to those of wild-type cells. Unlike growing cells that exhibit a dynamic profile of macromolecules, DLCs displayed steady protein and RNA reservoirs. Remarkably, following DLCs by time lapse microscopy revealed that they succeeded in synthesizing proteins, elongating, and dividing, apparently forming de novo Z rings at the midcell position. Taken together, the persistence of key cellular events in DLCs indicates that the information to carry out lengthy processes is harbored within the remaining molecular components. PMID:25922388

  16. Vaginal epithelial cells regulate membrane adhesiveness to co-ordinate bacterial adhesion.

    PubMed

    Younes, Jessica A; Klappe, Karin; Kok, Jan Willem; Busscher, Henk J; Reid, Gregor; van der Mei, Henny C

    2016-04-01

    Vaginal epithelium is colonized by different bacterial strains and species. The bacterial composition of vaginal biofilms controls the balance between health and disease. Little is known about the relative contribution of the epithelial and bacterial cell surfaces to bacterial adhesion and whether and how adhesion is regulated over cell membrane regions. Here, we show that bacterial adhesion forces with cell membrane regions not located above the nucleus are stronger than with regions above the nucleus both for vaginal pathogens and different commensal and probiotic lactobacillus strains involved in health. Importantly, adhesion force ratios over membrane regions away from and above the nucleus coincided with the ratios between numbers of adhering bacteria over both regions. Bacterial adhesion forces were dramatically decreased by depleting the epithelial cell membrane of cholesterol or sub-membrane cortical actin. Thus, epithelial cells can regulate membrane regions to which bacterial adhesion is discouraged, possibly to protect the nucleus. PMID:26477544

  17. Active and Secretory IgA-Coated Bacterial Fractions Elucidate Dysbiosis in Clostridium difficile Infection

    PubMed Central

    Moya, Andrés; Vázquez-Castellanos, Jorge F.; Artacho, Alejandro; Chen, Xinhua; Kelly, Ciaran

    2016-01-01

    ABSTRACT The onset of Clostridium difficile infection (CDI) has been associated with treatment with wide-spectrum antibiotics. Antibiotic treatment alters the activity of gut commensals and may result in modified patterns of immune responses to pathogens. To study these mechanisms during CDI, we separated bacteria with high cellular RNA content (the active bacteria) and their inactive counterparts by fluorescence-activated cell sorting (FACS) of the fecal bacterial suspension. The gut dysbiosis due to the antibiotic treatment may result in modification of immune recognition of intestinal bacteria. The immune recognition patterns were assessed by FACS of bacterial fractions either coated or not with intestinal secretory immunoglobulin A (SIgA). We described the taxonomic distributions of these four bacterial fractions (active versus inactive and SIgA coated versus non-SIgA coated) by massive 16S rRNA gene amplicon sequencing and quantified the proportion of C. difficile toxin genes in the samples. The overall gut microbiome composition was more robustly influenced by antibiotics than by the C. difficile toxins. Bayesian networks revealed that the C. difficile cluster was preferentially SIgA coated during CDI. In contrast, in the CDI-negative group Fusobacterium was the characteristic genus of the SIgA-opsonized fraction. Lactobacillales and Clostridium cluster IV were mostly inactive in CDI-positive patients. In conclusion, although the proportion of C. difficile in the gut is very low, it is able to initiate infection during the gut dysbiosis caused by environmental stress (antibiotic treatment) as a consequence of decreased activity of the protective bacteria. IMPORTANCE C. difficile is a major enteric pathogen with worldwide distribution. Its expansion is associated with broad-spectrum antibiotics which disturb the normal gut microbiome. In this study, the DNA sequencing of highly active bacteria and bacteria opsonized by intestinal secretory immunoglobulin

  18. Active and Secretory IgA-Coated Bacterial Fractions Elucidate Dysbiosis in Clostridium difficile Infection.

    PubMed

    Džunková, Mária; Moya, Andrés; Vázquez-Castellanos, Jorge F; Artacho, Alejandro; Chen, Xinhua; Kelly, Ciaran; D'Auria, Giuseppe

    2016-01-01

    The onset of Clostridium difficile infection (CDI) has been associated with treatment with wide-spectrum antibiotics. Antibiotic treatment alters the activity of gut commensals and may result in modified patterns of immune responses to pathogens. To study these mechanisms during CDI, we separated bacteria with high cellular RNA content (the active bacteria) and their inactive counterparts by fluorescence-activated cell sorting (FACS) of the fecal bacterial suspension. The gut dysbiosis due to the antibiotic treatment may result in modification of immune recognition of intestinal bacteria. The immune recognition patterns were assessed by FACS of bacterial fractions either coated or not with intestinal secretory immunoglobulin A (SIgA). We described the taxonomic distributions of these four bacterial fractions (active versus inactive and SIgA coated versus non-SIgA coated) by massive 16S rRNA gene amplicon sequencing and quantified the proportion of C. difficile toxin genes in the samples. The overall gut microbiome composition was more robustly influenced by antibiotics than by the C. difficile toxins. Bayesian networks revealed that the C. difficile cluster was preferentially SIgA coated during CDI. In contrast, in the CDI-negative group Fusobacterium was the characteristic genus of the SIgA-opsonized fraction. Lactobacillales and Clostridium cluster IV were mostly inactive in CDI-positive patients. In conclusion, although the proportion of C. difficile in the gut is very low, it is able to initiate infection during the gut dysbiosis caused by environmental stress (antibiotic treatment) as a consequence of decreased activity of the protective bacteria. IMPORTANCE C. difficile is a major enteric pathogen with worldwide distribution. Its expansion is associated with broad-spectrum antibiotics which disturb the normal gut microbiome. In this study, the DNA sequencing of highly active bacteria and bacteria opsonized by intestinal secretory immunoglobulin A (SIg

  19. Multitarget magnetic activated cell sorter

    PubMed Central

    Adams, Jonathan D.; Kim, Unyoung; Soh, H. Tom

    2008-01-01

    Magnetic selection allows high-throughput sorting of target cells based on surface markers, and it is extensively used in biotechnology for a wide range of applications from in vitro diagnostics to cell-based therapies. However, existing methods can only perform separation based on a single parameter (i.e., the presence or absence of magnetization), and therefore, the simultaneous sorting of multiple targets at high levels of purity, recovery, and throughput remains a challenge. In this work, we present an alternative system, the multitarget magnetic activated cell sorter (MT-MACS), which makes use of microfluidics technology to achieve simultaneous spatially-addressable sorting of multiple target cell types in a continuous-flow manner. We used the MT-MACS device to purify 2 types of target cells, which had been labeled via target-specific affinity reagents with 2 different magnetic tags with distinct saturation magnetization and size. The device was engineered so that the combined effects of the hydrodynamic force produced from the laminar flow and the magnetophoretic force produced from patterned ferromagnetic structures within the microchannel result in the selective purification of the differentially labeled target cells into multiple independent outlets. We demonstrate here the capability to simultaneously sort multiple magnetic tags with >90% purity and >5,000-fold enrichment and multiple bacterial cell types with >90% purity and >500-fold enrichment at a throughput of 109 cells per hour. PMID:19015523

  20. Structure of a Bacterial Cell Surface Decaheme Electron Conduit

    SciTech Connect

    Clarke, Thomas A.; Edwards, Marcus; Gates, Andrew J.; Hall, Andrea; White, Gaye; Bradley, Justin; Reardon, Catherine L.; Shi, Liang; Beliaev, Alex S.; Marshall, Matthew J.; Wang, Zheming; Watmough, Nicholas; Fredrickson, Jim K.; Zachara, John M.; Butt, Julea N.; Richardson, David J.

    2011-05-23

    Some bacterial species are able to utilize extracellular mineral forms of iron and manganese as respiratory electron acceptors. In Shewanella oneidensis this involves deca-heme cytochromes that are located on the bacterial cell surface at the termini of trans-outermembrane (OM) electron transfer conduits. The cell surface cytochromes can potentially play multiple roles in mediating electron transfer directly to insoluble electron sinks, catalyzing electron exchange with flavin electron shuttles or participating in extracellular inter-cytochrome electron exchange along ‘nanowire’ appendages. We present a 3.2 Å crystal structure of one of these deca-heme cytochromes, MtrF, that allows the spatial organization of the ten hemes to be visualized for the first time. The hemes are organized across four domains in a unique crossed conformation, in which a staggered 65 Å octa-heme chain transects the length of the protein and is bisected by a planar 45 Å tetra-heme chain that connects two extended Greek key split β-barrel domains. The structure provides molecular insight into how reduction of insoluble substrate (e.g. minerals), soluble substrates (e.g. flavins) and cytochrome redox partners might be possible in tandem at different termini of a trifurcated electron transport chain on the cell surface.

  1. Bacterial activities driving arsenic speciation and solubility in marine sediments

    NASA Astrophysics Data System (ADS)

    Battaglia-Brunet, F.; Seby, F.; Crouzet, C.; Joulian, C.; Mamindy-Pajany, Y.; Guezennec, A. G.; Hurel, C.; Marmier, N.; Bataillard, P.

    2012-04-01

    Harbour and marina sediments represent particular environments, with high concentrations in organic carbon and pollutants. Over 50 million m3 of marine sediments are dredged every year in French maritime and commercial ports, to maintain the water depth suitable for navigation, and the most part of them is discharged in deeper sea zones. The present study aimed to elucidate, using a range of complementary approaches, the influence of bacterial activity on arsenic speciation and mobility in marina sediments. Two sites were considered: L'Estaque, impacted by metallurgical activities and by the commercial port of Marseille, and St-Mandrier, less polluted, affected by classical chemical pollutants associated to professional and recreational boating. Arsenic concentration was noticeably higher in l'Estaque sediment (200-350 mg/kg) than in St-Mandrier sediment (15-50 mg/kg). In the solid phases, As(III) was the dominant species in L'Estaque sediment, whereas As(V) was the main form in St Mandrier sediment. At both sites, arsenic was the major trace element detected in interstitial water. Free sulfide and thio-arsenic complexes were detected in the interstitial water of l'Estaque sediment, suggesting a role of sulfate-reduction bacterial activity on arsenic solubility. Anaerobic microcosm experiments confirmed this hypothesis, as stimulation of sulfate-reduction induced a dramatic increase of arsenic concentration in the liquid phase, linked to the formation of soluble thio-arsenic complexes. Nevertheless, microcosms performed in aerobic conditions showed that bacterial activity globally decreased the transfer of arsenic from the sediment toward the overlying water. A red-brown fine layer developed at the sediment-water interface. Altogether, these results suggest that the sediment-water interface zone and the close transition area between aerobic and anaerobic conditions host intense biogeochemical reactions involving As, Fe and S species. These reactions most probably

  2. Protein, cell and bacterial response to atmospheric pressure plasma grafted hyaluronic acid on poly(methylmethacrylate).

    PubMed

    D'Sa, Raechelle A; Raj, Jog; Dickinson, Peter J; McMahon, M Ann S; McDowell, David A; Meenan, Brian J

    2015-11-01

    Hyaluronic acid (HA) has been immobilised on poly(methyl methacrylate) (PMMA) surfaces using a novel dielectric barrier discharge (DBD) plasma process for the purposes of repelling protein, cellular and bacterial adhesion in the context of improving the performance of ophthalmic devices. Grafting was achieved by the following steps: (1) treatment of the PMMA with a DBD plasma operating at atmospheric pressure, (2) amine functionalisation of the activated polymer surface by exposure to a 3-aminopropyltrimethoxysilane (APTMS) linker molecule and (3) reaction of HA with the surface bound amine. The mechanism and effectiveness of the grafting process was verified by surface analysis. XPS data indicates that the APTMS linker molecule binds to PMMA via the Si-O chemistry and has the required pendant amine moiety. The carboxylic acid moiety on HA then binds with this -NH2 group via standard carbodiimide chemistry. ToF-SIMS confirms the presence of a coherent HA layer the microstructure of which is verified by AFM. The plasma grafted HA coating surfaces showed a pronounced decrease in protein and cellular adhesion when tested with bovine serum albumin and human corneal epithelial cells, respectively. The ability of these coatings to resist bacterial adhesion was established using Staphylococcus aureus NTC8325. Interestingly, the coatings did not repel bacterial adhesion, indicating that the mechanism of adhesion of bacterial cells is different to that for the surface interactions of mammalian cells. It is proposed that this difference is a consequence of the specific HA conformation that occurs under the conditions employed here. Hence, it is apparent that the microstructure/architecture of the HA coatings is an important factor in fabricating surfaces intended to repel proteins, mammalian and bacterial cells. PMID:26449450

  3. Depth variation of bacterial extracellular enzyme activity and population diversity in the northeastern North Atlantic Ocean

    NASA Astrophysics Data System (ADS)

    Davey, Katherine E.; Kirby, Richard R.; Turley, Carol M.; Weightman, Andrew J.; Fry, John C.

    Distinct profiles of extracellular proteolytic enzyme activity were observed in the water column of the North Atlantic, with maximum potential proteolytic activity occurring in the top 35 m. The proteolytic enzyme Vmax values varied significantly and decreased from 1.46 nM min -1 in surface waters to 0.365 nM min -1 at 100 m. In contrast, Km values increased with depth from about 70 to 360 μM. Cell-associated enzymes accounted for the majority of the observed proteolytic activity. Dissolved enzymes comprised only 30-40% of the total extracellular enzyme activity and exhibited a low substrate affinity ( Km=˜1000 μM). These observations indicate clear stratification of bacterial associated extracellular enzyme activity, with the maximum activity in surface waters. This is consistent with some environmental changes in the water column, especially algal biomass and nitrate concentration. Bacterial mediated nitrogen remineralization in surface waters was approximately three times the total nitrogen demand of phytoplankton and bacteria. We determined bacterial population diversity using 16S rRNA sequence analysis and found evidence for stratification, with a higher representation of the Cytophaga/Flexibacter/Bacteriodes group at 5 m compared to 100 m. No similar stratification was observed among the α-proteobacterial SAR11 cluster, which were especially prevalent in the PRIME eddy. However, sequences phylogenetically related to another marine cluster, SAR122, were only observed at 100 m. We suggest that stratification of proteolytic activity within the water column may be explained at least in part, by differences in the composition of the bacterial community.

  4. Neuronal cells' behavior on polypyrrole coated bacterial nanocellulose three-dimensional (3D) scaffolds.

    PubMed

    Muller, D; Silva, J P; Rambo, C R; Barra, G M O; Dourado, F; Gama, F M

    2013-01-01

    In this work, polypyrrole (PPy) was in situ polymerized onto the surface of bacterial nanocellulose (BNC) produced by Gluconacetobacter xylinus, by chemical oxidation in aqueous medium using ammonium persulfate. Composites (BNC/PPy) were produced with varying concentrations of pyrrole (Py). The produced BNC/PPy membranes were used as a template for the seeding of PC12 rat neuronal cells. Cell suspensions were directly seeded onto the surfaces of the BNC/PPy membranes. The Py concentration affected the behavior of neuronal cells that adhered and grew significantly more on BNC/PPy comparatively to BNC. Scanning electron microscopy (SEM) micrographs revealed that PC12 cells adhered on the surface of the BNC and BNC/PPy membranes. Conductive PPy coatings on nanofibers acting as an active interface for tissue engineering may be used to regulate cell activity through electrical stimulations. PMID:23796037

  5. In vitro interactions of biomedical polyurethanes with macrophages and bacterial cells.

    PubMed

    Visai, Livia; Rindi, Simonetta; Speziale, Pietro; Petrini, Paola; Farè, Silvia; Tanzi, M Cristina

    2002-01-01

    Three commercial medical-grade polyurethanes (PUs), a poly-ether-urethane (Pellethane), and two poly-carbonate-urethanes, the one aromatic (Bionate) and the other aliphatic (Chronoflex), were tested for macrophages and bacterial cells adhesion, in the presence or absence of adhesive plasma proteins. All the experiments were carried out on PUs films obtained by solvent casting. The wettability of these films was analysed by measuring static contact angles against water. The ability of the selected PUs to adsorb human fibronectin (Fn) and fibrinogen (Fbg) was checked by ELISA with biotin-labelled proteins. All PUs were able to adsorb Fn and Fbg (Fn > Fbg). Fn adsorption was in the order: Pellethane > Chronoflex > Bionate, the highest Fbg adsorption being detected onto Bionate (Bionate > Chronoflex > Pellethane). The human macrophagic line J111, and the two main bacterial strains responsible for infection in humans (Staphylococcus aureus Newman and Staphylococcus epidermidis 14852) were incubated in turn with the three PUs, uncoated or coated with plasma proteins. No macrophage or bacterial adhesion was observed onto uncoated PUs. PUs coated with plasma, Fn or Fbg promoted bacterial adhesion (S. aureus > S. epidermidis), whereas macrophage adhered more onto PUs coated with Fn or plasma. The coating with Fbg did not promote cell adhesion. Pellethane showed the highest macrophage activation (i.e. spreading), followed, in the order, by Bionate and Chronoflex. PMID:11939455

  6. Solar cell activation system

    SciTech Connect

    Apelian, L.

    1983-07-05

    A system for activating solar cells involves the use of phosphorescent paint, the light from which is amplified by a thin magnifying lens and used to activate solar cells. In a typical system, a member painted with phosphorescent paint is mounted adjacent a thin magnifying lens which focuses the light on a predetermined array of sensitive cells such as selenium, cadmium or silicon, mounted on a plastic board. A one-sided mirror is mounted adjacent the cells to reflect the light back onto said cells for purposes of further intensification. The cells may be coupled to rechargeable batteries or used to directly power a small radio or watch.

  7. Functionalization of whole‐cell bacterial reporters with magnetic nanoparticles

    PubMed Central

    Zhang, Dayi; Fakhrullin, Rawil F.; Özmen, Mustafa; Wang, Hui; Wang, Jian; Paunov, Vesselin N.; Li, Guanghe; Huang, Wei E.

    2011-01-01

    Summary We developed a biocompatible and highly efficient approach for functionalization of bacterial cell wall with magnetic nanoparticles (MNPs). Three Acinetobacter baylyi ADP1 chromosomally based bioreporters, which were genetically engineered to express bioluminescence in response to salicylate, toluene/xylene and alkanes, were functionalized with 18 ± 3 nm iron oxide MNPs to acquire magnetic function. The efficiency of MNPs functionalization of Acinetobacter bioreporters was 99.96 ± 0.01%. The MNPs‐functionalized bioreporters (MFBs) can be remotely controlled and collected by an external magnetic field. The MFBs were all viable and functional as good as the native cells in terms of sensitivity, specificity and quantitative response. More importantly, we demonstrated that salicylate sensing MFBs can be applied to sediments and garden soils, and semi‐quantitatively detect salicylate in those samples by discriminably recovering MFBs with a permanent magnet. The magnetically functionalized cells are especially useful to complex environments in which the indigenous cells, particles and impurities may interfere with direct measurement of bioreporter cells and conventional filtration is not applicable to distinguish and harvest bioreporters. The approach described here provides a powerful tool to remotely control and selectively manipulate MNPs‐functionalized cells in water and soils. It would have a potential in the application of environmental microbiology, such as bioremediation enhancement and environment monitoring and assessment. PMID:21255376

  8. Imaging the action of antimicrobial peptides on living bacterial cells

    PubMed Central

    Gee, Michelle L.; Burton, Matthew; Grevis-James, Alistair; Hossain, Mohammed Akhter; McArthur, Sally; Palombo, Enzo A.; Wade, John D.; Clayton, Andrew H. A.

    2013-01-01

    Antimicrobial peptides hold promise as broad-spectrum alternatives to conventional antibiotics. The mechanism of action of this class of peptide is a topical area of research focused predominantly on their interaction with artificial membranes. Here we compare the interaction mechanism of a model antimicrobial peptide with single artificial membranes and live bacterial cells. The interaction kinetics was imaged using time-lapse fluorescence lifetime imaging of a fluorescently-tagged melittin derivative. Interaction with the synthetic membranes resulted in membrane pore formation. In contrast, the interaction with bacteria led to transient membrane disruption and corresponding leakage of the cytoplasm, but surprisingly with a much reduced level of pore formation. The discovery that pore formation is a less significant part of lipid-peptide interaction in live bacteria highlights the mechanistic complexity of these interactions in living cells compared to simple artificial systems. PMID:23532056

  9. High-efficiency transformation of bacterial cells by electroporation.

    PubMed Central

    Calvin, N M; Hanawalt, P C

    1988-01-01

    We have developed a method for efficiently generating transient pores in the outer membranes of Escherichia coli K-12 derivatives by using a new type of electroporation apparatus. The pores are large enough and persist long enough to facilitate the equilibration of plasmid molecules between the intracellular and extracellular spaces. The method has been used to transform bacterial cells with an efficiency greater than 10(9) transformants per microgram of plasmid. It has also been used to extract intact plasmid from transformed cells with efficiencies comparable to those of the traditional alkaline lysis or CsCl equilibrium density gradient techniques. The technique is simple and rapid, allowing a transformation or the preparation of microgram quantities of plasmid to be accomplished in minutes. PMID:3286620

  10. Secretion of a bacterial protein by mammalian cells.

    PubMed

    Clément, J M; Jehanno, M

    1995-12-15

    The MalE protein is a periplasmic maltooligosaccharide binding protein from Escherichia coli. This protein is widely used as a model for protein export in bacteria and as a vector for the export and one-step affinity purification of foreign polypeptides. Expression of MalE was studied in various animal cell lines. The protein was exported into the culture medium, following the classical pathway of eukaryotic protein secretion. This was shown by a combination of approaches including the use of inhibitors of the Golgi complex and immunocytological methods. The signal sequence of MalE is required for secretion and a specific signal can be added to MalE that targets it to the endoplasmic reticulum. This work opens the way to the study of the secretion of a bacterial protein and to its use as a vector for protein secretion and purification from mammalian cells. PMID:8590643

  11. Circular Dichroism studies on the interactions of antimicrobial peptides with bacterial cells

    NASA Astrophysics Data System (ADS)

    Avitabile, Concetta; D'Andrea, Luca Domenico; Romanelli, Alessandra

    2014-03-01

    Studying how antimicrobial peptides interact with bacterial cells is pivotal to understand their mechanism of action. In this paper we explored the use of Circular Dichroism to detect the secondary structure of two antimicrobial peptides, magainin 2 and cecropin A, with E. coli bacterial cells. The results of our studies allow us to gain two important information in the context of antimicrobial peptides- bacterial cells interactions: peptides fold mainly due to interaction with LPS, which is the main component of the Gram negative bacteria outer membrane and the time required for the folding on the bacterial cells depends on the peptide analyzed.

  12. Bacterial Folates Provide an Exogenous Signal for C. elegans Germline Stem Cell Proliferation.

    PubMed

    Chaudhari, Snehal N; Mukherjee, Madhumati; Vagasi, Alexandra S; Bi, Gaofeng; Rahman, Mohammad M; Nguyen, Christine Q; Paul, Ligi; Selhub, Jacob; Kipreos, Edward T

    2016-07-11

    Here we describe an in vitro primary culture system for Caenorhabditis elegans germline stem cells. This culture system was used to identify a bacterial folate as a positive regulator of germ cell proliferation. Folates are a family of B-complex vitamins that function in one-carbon metabolism to allow the de novo synthesis of amino acids and nucleosides. We show that germ cell proliferation is stimulated by the folate 10-formyl-tetrahydrofolate-Glun both in vitro and in animals. Other folates that can act as vitamins to rescue folate deficiency lack this germ cell stimulatory activity. The bacterial folate precursor dihydropteroate also promotes germ cell proliferation in vitro and in vivo, despite its inability to promote one-carbon metabolism. The folate receptor homolog FOLR-1 is required for the stimulation of germ cells by 10-formyl-tetrahydrofolate-Glun and dihydropteroate. This work defines a folate and folate-related compound as exogenous signals to modulate germ cell proliferation. PMID:27404357

  13. CAP-D3 Promotes Bacterial Clearance in Human Intestinal Epithelial Cells by Repressing Expression of Amino Acid Transporters

    PubMed Central

    Kemp, Jacqueline R.; Nickerson, Kourtney P.; Deutschman, Emily; Kim, Yeojung; West, Gail; Sadler, Tammy; Stylianou, Eleni; Krokowski, Dawid; Hatzoglou, Maria; de la Motte, Carol; Rubin, Brian P.; Fiocchi, Claudio

    2015-01-01

    BACKGROUND & AIMS Defects in colonic epithelial barrier defenses are associated with ulcerative colitis (UC). The proteins that regulate bacterial clearance in the colonic epithelium have not been completely identified. The chromosome-associated protein D3 (dCAP-D3), regulates responses to bacterial infection. We examined whether CAP-D3 promotes bacterial clearance in human colonic epithelium. METHODS Clearance of Salmonella or adherent-invasive Escherichia coli LF82 was assessed by gentamycin protection assays in HT-29 and Caco-2 cells expressing small hairpin RNAs against CAP-D3. We used immunoblot assays to measure levels of CAP-D3 in colonic epithelial cells from patients with UC and healthy individuals (controls). RNA sequencing identified genes activated by CAP-D3. We analyzed the roles of CAP-D3 target genes in bacterial clearance using gentamycin protection and immunofluorescence assays and studies with pharmacologic inhibitors. RESULTS CAP-D3 expression was reduced in colonic epithelial cells from patients with active UC. Reduced CAP-D3 expression decreased autophagy and impaired intracellular bacterial clearance by HT-29 and Caco-2 colonic epithelial cells. Lower levels of CAP-D3 increased transcription of genes encoding SLC7A5 and SLC3A2, whose products heterodimerize to form an amino acid transporter in HT-29 cells following bacterial infection; levels of SLC7A5–SLC3A2 were increased in tissues from patients with UC, compared with controls. Reduced CAP-D3 in HT-29 cells resulted in earlier recruitment of SLC7A5 to Salmonella-containing vacuoles, increased activity of mTORC1, and increased survival of bacteria. Inhibition of SLC7A5–SLC3A2 or mTORC1 activity rescued the bacterial clearance defects of CAP-D3– deficient cells. CONCLUSIONS CAP-D3 downregulates transcription of genes that encode amino acid transporters (SLC7A5 and SLC3A2) to promote bacterial autophagy by colon epithelial cells. Levels of CAP-D3 protein are reduced in patients with

  14. A common clathrin-mediated machinery coordinates cell-cell adhesion and bacterial internalization

    PubMed Central

    Bonazzi, Matteo; Kühbacher, Andreas; Toledo-Arana, Alejandro; Mallet, Adeline; Vasudevan, Lavanya; Pizarro-Cerdá, Javier; Brodsky, Frances M.; Cossart, Pascale

    2013-01-01

    Invasive bacterial pathogens often target cellular proteins involved in adhesion as a first event during infection. For example, Listeria monocytogenes uses the bacterial protein InlA to interact with E-cadherin, hijack the host adherens junction machinery, and invade non-phagocytic cells by a clathrin-dependent mechanism. Here we investigate a potential role for clathrin in cell-cell adhesion. We observed that the initial steps of adherens junction formation trigger the phosphorylation of clathrin, and its transient localization at forming cell-cell contacts. Furthermore, we show that clathrin serves as a hub for the recruitment of proteins that are necessary for the actin rearrangements that accompany the maturation of adherens junctions. Using an InlA/E-cadherin chimera, we show that adherent cells expressing the chimera form adherens junctions with cells expressing E-cadherin. To model bacterial invasion, we demonstrate that non-adherent cells expressing the InlA chimera can be internalized by E-cadherin-expressing adherent cells. Together these results reveal that a common clathrin-mediated machinery may regulate internalization and cell adhesion and that the relative mobility of one of the interacting partners plays an important role in the commitment to either one of these processes. PMID:22984946

  15. Inhibitory Activities of Gatifloxacin (AM-1155), a Newly Developed Fluoroquinolone, against Bacterial and Mammalian Type II Topoisomerases

    PubMed Central

    Takei, Masaya; Fukuda, Hideyuki; Yasue, Tokutaro; Hosaka, Masaki; Oomori, Yasuo

    1998-01-01

    We determined the inhibitory activities of gatifloxacin against Staphylococcus aureus topoisomerase IV, Escherichia coli DNA gyrase, and HeLa cell topoisomerase II and compared them with those of several quinolones. The inhibitory activities of quinolones against these type II topoisomerases significantly correlated with their antibacterial activities or cytotoxicities (correlation coefficient [r] = 0.926 for S. aureus, r = 0.972 for E. coli, and r = 0.648 for HeLa cells). Gatifloxacin possessed potent inhibitory activities against bacterial type II topoisomerases (50% inhibitory concentration [IC50] = 13.8 μg/ml for S. aureus topoisomerase IV; IC50 = 0.109 μg/ml for E. coli DNA gyrase) but the lowest activity against HeLa cell topoisomerase II (IC50 = 265 μg/ml) among the quinolones tested. There was also a significant correlation between the inhibitory activities of quinolones against S. aureus topoisomerase IV and those against E. coli DNA gyrase (r = 0.969). However, the inhibitory activity against HeLa cell topoisomerase II did not correlate with that against either bacterial enzyme. The IC50 of gatifloxacin for HeLa cell topoisomerase II was 19 and was more than 2,400 times higher than that for S. aureus topoisomerase IV and that for E. coli DNA gyrase. These ratios were higher than those for other quinolones, indicating that gatifloxacin possesses a higher selectivity for bacterial type II topoisomerases. PMID:9756776

  16. Lipid biomarkers and bacterial lipase activities as indicators of organic matter and bacterial dynamics in contrasted regimes at the DYFAMED site, NW Mediterranean

    NASA Astrophysics Data System (ADS)

    Bourguet, Nicolas; Goutx, Madeleine; Ghiglione, Jean-François; Pujo-Pay, Mireille; Mével, Geneviève; Momzikoff, André; Mousseau, Laure; Guigue, Catherine; Garcia, Nicole; Raimbault, Patrick; Pete, Romain; Oriol, Louise; Lefèvre, Dominique

    2009-08-01

    This study investigated the relationships between dissolved organic matter (DOM) composition and bacterial dynamics on short time scale during spring mesotrophic (March 2003) and summer oligotrophic (June 2003) regimes, in a 0-500 m depth water column with almost no advection, at the DYFAMED site, NW Mediterranean. DOM was characterized by analyzing dissolved organic carbon (DOC), colored dissolved organic matter (CDOM) and lipid class biotracers. Bacterial dynamic was assessed through the measurement of in situ bacterial lipase activity, abundance, production and bacterial community structure. We made the assumption that by coupling the ambient concentration of hydrolysable acyl-lipids with the measurement of their in situ bacterial hydrolysis rates (i.e. the free fatty acids release rate) would provide new insights about bacterial response to change in environmental conditions. The seasonal transition from spring to summer was accompanied by a significant accumulation of excess DOC (+5 μM) (ANOVA, p<0.05, n=8) in the upper layer (0-50 m). In this layer, the free fatty acids release rate to the bacterial carbon demand (BCD) ratio increased from 0.6±0.3 in March to 1.3±1.0 in June (ANOVA, p<0.05, n=8) showing that more uncoupling between the hydrolysis of the acyl-lipids and the BCD occurred during the evolution of the season, and that free fatty acids contributed to the excess DOC. The increase of lipolysis index and CDOM absorbance (from 0.24±0.17 to 0.39±0.13 and from 0.076±0.039 to 0.144±0.068; ANOVA, p<0.05, n=8, respectively), and the higher contribution of triglycerides, wax esters and phospholipids (from <5% to 12-31%) to the lipid pool reflected the change in the DOM quality. In addition to a strong increase of bacterial lipase activity per cell (51.4±29.4-418.3±290.6 Ag C cell -1 h -1), a significant percentage of ribotypes (39%) was different between spring and summer in the deep chlorophyll maximum (DCM) layer in particular, suggesting a shift

  17. Activity and bacterial diversity of snow around Russian Antarctic stations.

    PubMed

    Lopatina, Anna; Krylenkov, Vjacheslav; Severinov, Konstantin

    2013-11-01

    The diversity and temporal dynamics of bacterial communities in pristine snow around two Russian Antarctic stations was investigated. Taxonomic analysis of rDNA libraries revealed that snow communities were dominated by bacteria from a small number of operational taxonomic units (OTUs) that underwent dramatic swings in abundance between the 54th (2008-2009) and 55th (2009-2010) Russian Antarctic expeditions. Moreover, analysis of the 55th expedition samples indicated that there was very little, if any, correspondence in abundance of clones belonging to the same OTU present in rDNA and rRNA libraries. The latter result suggests that most rDNA clones originate from bacteria that are not alive and/or active and may have been deposited on the snow surface from the atmosphere. In contrast, clones most abundant in rRNA libraries (mostly belonging to Variovorax, Janthinobacterium, Pseudomonas, and Sphingomonas genera) may be considered as endogenous Antarctic snow inhabitants. PMID:24012540

  18. Substrate Trafficking And Dioxygen Activation in Bacterial Multicomponent Monooxygenases

    SciTech Connect

    Murray, L.J.; Lippard, S.J.

    2009-06-03

    Non-heme carboxylate-bridged diiron centers in the hydroxylase components of the bacterial multicomponent monooxygenases process four substrates during catalysis: electrons, protons, dioxygen, and hydrocarbons. Understanding how protein-protein interactions mediate the transport of these substrates to the diiron center to achieve the selective oxidation of the hydrocarbon is a significant challenge. In this Account, we summarize our current knowledge of these processes with a focus on the methane monooxygenase system. We also describe recent results for the toluene/o-xylene monooxygenase and phenol hydroxylase systems from Pseudomonas sporium OX1. The observation in these latter systems of a diiron(III) oxygenated intermediate having different Moessbauer parameters from analogous species in other carboxylate-bridged diiron proteins is discussed. The results indicate that the ability of the protein framework to tune the reactivity of the diiron center at structurally similar active sites is substantially more complex than previously recognized.

  19. Assessment of denitrifying bacterial composition in activated sludge.

    PubMed

    Srinandan, C S; Shah, Mrinal; Patel, Bhavita; Nerurkar, Anuradha S

    2011-10-01

    The abundance and structure of denitrifying bacterial community in different activated sludge samples were assessed, where the abundance of denitrifying functional genes showed nirS in the range of 10(4)-10(5), nosZ with 10(4)-10(6) and 16S rRNA gene in the range 10(9)-10(10) copy number per ml of sludge. The culturable approach revealed Pseudomonas sp. and Alcaligenes sp. to be numerically high, whereas culture independent method showed betaproteobacteria to dominate the sludge samples. Comamonas sp. and Pseudomonas fluorescens isolates showed efficient denitrification, while Pseudomonas mendocina, Pseudomonas stutzeri and Brevundimonas diminuta accumulated nitrite during denitrification. Numerically dominant RFLP OTUs of the nosZ gene from the fertilizer factory sludge samples clustered with the known isolates of betaproteobacteria. The data also suggests the presence of different truncated denitrifiers with high numbers in sludge habitat. PMID:21868215

  20. Bacterial lifestyle shapes the regulation of stringent response activation

    PubMed Central

    Boutte, Cara C.; Crosson, Sean

    2014-01-01

    Bacteria inhabit enormously diverse niches and have a correspondingly large array of regulatory mechanisms to adapt to often inhospitable and variable environments. The stringent response allows bacteria to quickly reprogram transcription in response to changes in nutrient availability. Although the proteins controlling this response are conserved in almost all bacterial species, recent work has illuminated considerable diversity in the starvation cues and regulatory mechanisms that activate stringent signaling proteins in bacteria from different environments. In this review we describe the signals and genetic circuitries that control the stringent signaling systems of a copiotroph, a bacteriovore, an oligotroph and a mammalian pathogen – Escherichia coli, Myxococcus xanthus, Caulobacter crescentus and Mycobacterium tuberculosis, respectively – and discuss how control of the stringent response in these species is adapted to their particular lifestyles. PMID:23419217

  1. Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms.

    PubMed

    Turnbull, Lynne; Toyofuku, Masanori; Hynen, Amelia L; Kurosawa, Masaharu; Pessi, Gabriella; Petty, Nicola K; Osvath, Sarah R; Cárcamo-Oyarce, Gerardo; Gloag, Erin S; Shimoni, Raz; Omasits, Ulrich; Ito, Satoshi; Yap, Xinhui; Monahan, Leigh G; Cavaliere, Rosalia; Ahrens, Christian H; Charles, Ian G; Nomura, Nobuhiko; Eberl, Leo; Whitchurch, Cynthia B

    2016-01-01

    Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs. PMID:27075392

  2. Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms

    PubMed Central

    Turnbull, Lynne; Toyofuku, Masanori; Hynen, Amelia L.; Kurosawa, Masaharu; Pessi, Gabriella; Petty, Nicola K.; Osvath, Sarah R.; Cárcamo-Oyarce, Gerardo; Gloag, Erin S.; Shimoni, Raz; Omasits, Ulrich; Ito, Satoshi; Yap, Xinhui; Monahan, Leigh G.; Cavaliere, Rosalia; Ahrens, Christian H.; Charles, Ian G.; Nomura, Nobuhiko; Eberl, Leo; Whitchurch, Cynthia B.

    2016-01-01

    Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs. PMID:27075392

  3. Bacterial Virulence Proteins as Tools to Rewire Kinase Pathways in Yeast and Immune Cells

    PubMed Central

    Wei, Ping; Wong, Wilson W.; Park, Jason S.; Corcoran, Ethan E.; Peisajovich, Sergio G.; Onuffer, James J.; Weiss, Arthur; Lim, Wendell A.

    2012-01-01

    Bacterial pathogens have evolved specific effector proteins that, by interfacing with host kinase signaling pathways, provide a mechanism to evade immune responses during infection1,2. Although these effectors are responsible for pathogen virulence, we realized that they might also serve as valuable synthetic biology reagents for engineering cellular behavior. Here, we have exploited two effector proteins, the Shigella flexneri OspF protein3 and Yersinia pestis YopH protein4, to systematically rewire kinase-mediated responses in both yeast and mammalian immune cells. Bacterial effector proteins can be directed to selectively inhibit specific mitogen activated protein kinase (MAPK) pathways in yeast by artificially targeting them to pathway specific complexes. Moreover, we show that unique properties of the effectors generate novel pathway behaviors: OspF, which irreversibly inactivates MAPKs4, was used to construct a synthetic feedback circuit that displays novel frequency-dependent input filtering. Finally, we show that effectors can be used in T cells, either as feedback modulators to precisely tune the T cell response amplitude, or as an inducible pause switch that can temporarily disable T cell activation. These studies demonstrate how pathogens could provide a rich toolkit of parts to engineer cells for therapeutic or biotechnological applications. PMID:22820255

  4. Identification of Active Bacterial Communities in Drinking Water Using 16S rRNA-Based Sequence Analyses

    EPA Science Inventory

    DNA-based methods have considerably increased our understanding of the bacterial diversity of water distribution systems (WDS). However, as DNA may persist after cell death, the use of DNA-based methods cannot be used to describe metabolically-active microbes. In contrast, intra...

  5. Bacterial glycosidases for the production of universal red blood cells.

    PubMed

    Liu, Qiyong P; Sulzenbacher, Gerlind; Yuan, Huaiping; Bennett, Eric P; Pietz, Greg; Saunders, Kristen; Spence, Jean; Nudelman, Edward; Levery, Steven B; White, Thayer; Neveu, John M; Lane, William S; Bourne, Yves; Olsson, Martin L; Henrissat, Bernard; Clausen, Henrik

    2007-04-01

    Enzymatic removal of blood group ABO antigens to develop universal red blood cells (RBCs) was a pioneering vision originally proposed more than 25 years ago. Although the feasibility of this approach was demonstrated in clinical trials for group B RBCs, a major obstacle in translating this technology to clinical practice has been the lack of efficient glycosidase enzymes. Here we report two bacterial glycosidase gene families that provide enzymes capable of efficient removal of A and B antigens at neutral pH with low consumption of recombinant enzymes. The crystal structure of a member of the alpha-N-acetylgalactosaminidase family reveals an unusual catalytic mechanism involving NAD+. The enzymatic conversion processes we describe hold promise for achieving the goal of producing universal RBCs, which would improve the blood supply while enhancing the safety of clinical transfusions. PMID:17401360

  6. Synthetic genomics and the construction of a synthetic bacterial cell.

    PubMed

    Glass, John I

    2012-01-01

    The first synthetic cellular organism was created in 2010 and based on a very small, very simple bacterium called Mycoplasma mycoides. The bacterium was called synthetic because its DNA genome was chemically synthesized rather than replicated from an existing template DNA, as occurs in all other known cellular life on Earth. The experiment was undertaken in order to develop a system that would allow creation of a minimal bacterial cell that could lead to a better understand of the first principles of cellular life. The effort resulted in new synthetic genomics techniques called genome assembly and genome transplantation. The ability of scientists to design and build bacteria opens new possibilities for creating microbes to solve human problems. PMID:23502559

  7. Type III secretion: a bacterial device for close combat with cells of their eukaryotic host.

    PubMed

    Cornelis, G R

    2000-05-29

    Salmonella, Shigella, Yersinia, Pseudomonas aeruginosa, enteropathogenic Escherichia coli and several plant-pathogenic Gram-negative bacteria use a new type of systems called 'type III secretion' to attack their host. These systems are activated by contact with a eukaryotic cell membrane and they allow bacteria to inject bacterial proteins across the two bacterial membranes and the eukaryotic cell membrane to reach a given compartment and destroy or subvert the target cell. These systems consist of a secretion apparatus made up of about 25 individual proteins and a set of proteins released by this apparatus. Some of these released proteins are 'effectors' that are delivered by extracellular bacteria into the cytosol of the target cell while the others are 'translocators' that help the 'effectors' to cross the membrane of the eukaryotic cell. Most of the 'effectors' act on the cytoskeleton or on intracellular signalling cascades. One of the proteins injected by the enteropathogenic E. coli serves as a membrane receptor for the docking of the bacterium itself at the surface of the cell. PMID:10874740

  8. Involvement of the cell-specific pigment genes pks and sult in bacterial defense response of sea urchins Strongylocentrotus intermedius.

    PubMed

    Kiselev, Konstantin V; Ageenko, Natalya V; Kurilenko, Valeria V

    2013-03-26

    Bacterial infections are one of the most important problems in mass aquaculture, causing the loss of millions of juvenile organisms. We isolated 22 bacterial strains from the cavity fluid of the sea urchin Strongylocentrotus pallidus and used phylogenetic analysis based on 16S rRNA gene sequences to separate the bacterial strains into 9 genera (Aliivibrio, Bizionia, Colwellia, Olleya, Paenibacillus, Photobacterium, Pseudoalteromonas, Shewanella, and Vibrio). Incubating Strongylocentrotus intermedius larvae with a strain from each of the 9 bacterial genera, we investigated the viability of the larvae, the amount of pigment cells, and the level of polyketide synthase (pks) and sulfotransferase (sult) gene expression. Results of the assay on sea urchin development showed that all bacterial strains, except Pseudoalteromonas and Bizionia, suppressed sea urchin development (resulting in retardation of the embryos' development with cellular disorders) and reduced cell viability. We found that pks expression in the sea urchin larvae after incubation with the bacteria of 9 tested genera was significantly increased, while the sult expression was increased only after the treatment with Pseudoalteromonas and Shewanella. Shikimic acid, which is known to activate the biosynthesis of naphthoquinone pigments, increased the tolerance of the sea urchin embryos to the bacteria. In conclusion, we show that the cell-specific pigment genes pks and sult are involved in the bacterial defense response of sea urchins. PMID:23548362

  9. Microelectrode Measurements of the Activity Distribution in Nitrifying Bacterial Aggregates

    PubMed Central

    de Beer, D.; van den Heuvel, J. C.; Ottengraf, S. P. P.

    1993-01-01

    Microelectrodes for ammonium, oxygen, nitrate, and pH were used to study nitrifying aggregates grown in a fluidized-bed reactor. Local reactant fluxes and distribution of microbial activity could be determined from the microprofiles. The interfacial fluxes of the reactants closely reflected the stoichiometry of bacterial nitrification. Both ammonium consumption and nitrate production were localized in the outer shells, with a thickness of approximately 100 to 120 μm, of the aggregates. Under conditions in which ammonium and oxygen penetrated the whole aggregate, nitrification was restricted to this zone; oxygen was consumed in the central parts of the aggregates as well, probably because of oxidation of dead biomass. A sudden increase of the oxygen concentration to saturation (pure oxygen) was inhibitory to nitrification. The pH profiles showed acidification in the aggregates, but not to an inhibitory level. The distribution of activity was determined by the penetration depth of oxygen during aggregate development in the reactor. Mass transfer was significantly limited by the boundary layer surrounding the aggregates. Microelectrode measurements showed that the thickness of this layer was correlated with the diffusion coefficient of the species. Determination of the distribution of nitrifying activity required the use of ammonium or nitrate microelectrodes, whereas the use of oxygen microelectrodes alone would lead to erroneous results. Images PMID:16348875

  10. Mechanisms of Oxidative Protein Folding in the Bacterial Cell Envelope

    PubMed Central

    2010-01-01

    Abstract Disulfide-bond formation is important for the correct folding of a great number of proteins that are exported to the cell envelope of bacteria. Bacterial cells have evolved elaborate systems to promote the joining of two cysteines to form a disulfide bond and to repair misoxidized proteins. In the past two decades, significant advances have occurred in our understanding of the enzyme systems (DsbA, DsbB, DsbC, DsbG, and DsbD) used by the gram-negative bacterium Escherichia coli to ensure that correct pairs of cysteines are joined during the process of protein folding. However, a number of fundamental questions about these processes remain, especially about how they occur inside the cell. In addition, recent recognition of the increasing diversity among bacteria in the disulfide bond–forming capacity and in the systems for introducing disulfide bonds into proteins is raising new questions. We review here the marked progress in this field and discuss important questions that remain for future studies. Antioxid. Redox Signal. 13, 1231–1246. PMID:20367276

  11. Control of Bacterial Persister Cells by Trp/Arg-Containing Antimicrobial Peptides▿

    PubMed Central

    Chen, Xi; Zhang, Mi; Zhou, Chunhui; Kallenbach, Neville R.; Ren, Dacheng

    2011-01-01

    Persister cells are dormant phenotypic variants inherent in a bacterial population. They play important roles in chronic infections and present great challenges to therapy due to extremely enhanced tolerance to antibiotics compared to that of normal cells of the same genotype. In this study, we report that cationic membrane-penetrating peptides containing various numbers of arginine and tryptophan repeats are effective in killing persister cells of Escherichia coli HM22, a hyper-persister producer. The activities of three linear peptides [(RW)n-NH2, where n is 2, 3, or 4] and a dendrimeric peptide, (RW)4D, in killing bacterial persisters were compared. Although the dendrimeric peptide (RW)4D requires a lower threshold to kill planktonic persisters, octameric peptide (RW)4-NH2 is the most effective against planktonic persister cells at high concentrations. For example, treatment with 80 μM (RW)4-NH2 for 60 min led to a 99.7% reduction in the number of viable persister cells. The viability of persister cells residing in surface-attached biofilms was also significantly reduced by (RW)4-NH2 and (RW)4D. These two peptides were also found to significantly enhance the susceptibility of biofilm cells to ofloxacin. The potency of (RW)4-NH2 was further marked by its ability to disperse and kill preformed biofilms harboring high percentages of persister cells. Interestingly, approximately 70% of the dispersed cells were found to have lost their intrinsic tolerance and become susceptible to ampicillin if not killed directly by this peptide. These results are helpful for better understanding the activities of these peptides and may aid in future development of more effective therapies of chronic infections. PMID:21622798

  12. Crystal structure of MraY, an essential membrane enzyme for bacterial cell wall synthesis.

    PubMed

    Chung, Ben C; Zhao, Jinshi; Gillespie, Robert A; Kwon, Do-Yeon; Guan, Ziqiang; Hong, Jiyong; Zhou, Pei; Lee, Seok-Yong

    2013-08-30

    MraY (phospho-MurNAc-pentapeptide translocase) is an integral membrane enzyme that catalyzes an essential step of bacterial cell wall biosynthesis: the transfer of the peptidoglycan precursor phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl phosphate. MraY has long been considered a promising target for the development of antibiotics, but the lack of a structure has hindered mechanistic understanding of this critical enzyme and the enzyme superfamily in general. The superfamily includes enzymes involved in bacterial lipopolysaccharide/teichoic acid formation and eukaryotic N-linked glycosylation, modifications that are central in many biological processes. We present the crystal structure of MraY from Aquifex aeolicus (MraYAA) at 3.3 Å resolution, which allows us to visualize the overall architecture, locate Mg(2+) within the active site, and provide a structural basis of catalysis for this class of enzyme. PMID:23990562

  13. Enhancement of bacterial denitrification for nitrate removal in groundwater with electrical stimulation from microbial fuel cells

    NASA Astrophysics Data System (ADS)

    Zhang, Baogang; Liu, Ye; Tong, Shuang; Zheng, Maosheng; Zhao, Yinxin; Tian, Caixing; Liu, Hengyuan; Feng, Chuanping

    2014-12-01

    Electricity generated from the microbial fuel cell (MFC) is applied to the bioelectrical reactor (BER) directly as electrical stimulation means for enhancement of bacterial denitrification to remove nitrate effectively from groundwater. With maximum power density of 502.5 mW m-2 and voltage outputs ranging from 500 mV to 700 mV, the nitrate removal is accelerated, with less intermediates accumulation, compared with control sets without electrical stimulation. Denitrification bacteria proliferations and activities are promoted as its number and Adenosine-5'-triphosphate (ATP) concentration increased one order of magnitude (3.5 × 107 in per milliliter biofilm solution) and about 1.5 folds, respectively. Effects of electricity from MFCs on enhancement of bacterial behaviors are demonstrated for the first time. These results indicate that MFCs can be applied in the in-situ bioremediation of nitrate polluted groundwater for efficiency improvement.

  14. Live cell imaging of SOS and prophage dynamics in isogenic bacterial populations.

    PubMed

    Helfrich, Stefan; Pfeifer, Eugen; Krämer, Christina; Sachs, Christian Carsten; Wiechert, Wolfgang; Kohlheyer, Dietrich; Nöh, Katharina; Frunzke, Julia

    2015-11-01

    Almost all bacterial genomes contain DNA of viral origin, including functional prophages or degenerated phage elements. A frequent but often unnoted phenomenon is the spontaneous induction of prophage elements (SPI) even in the absence of an external stimulus. In this study, we have analyzed SPI of the large, degenerated prophage CGP3 (187 kbp), which is integrated into the genome of the Gram-positive Corynebacterium glutamicum ATCC 13032. Time-lapse fluorescence microscopy of fluorescent reporter strains grown in microfluidic chips revealed the sporadic induction of the SOS response as a prominent trigger of CGP3 SPI but also displayed a considerable fraction (∼30%) of RecA-independent SPI. Whereas approx. 20% of SOS-induced cells recovered from this stress and resumed growth, the spontaneous induction of CGP3 always led to a stop of growth and likely cell death. A carbon source starvation experiment clearly emphasized that SPI only occurs in actively proliferating cells, whereas sporadic SOS induction was still observed in resting cells. These data highlight the impact of sporadic DNA damage on the activity of prophage elements and provide a time-resolved, quantitative description of SPI as general phenomenon of bacterial populations. PMID:26235130

  15. Bacterial Peptide Recognition and Immune Activation Facilitated by Human Peptide Transporter PEPT2

    PubMed Central

    Swaan, Peter W.; Bensman, Timothy; Bahadduri, Praveen M.; Hall, Mark W.; Sarkar, Anasuya; Bao, Shengying; Khantwal, Chandra M.; Ekins, Sean; Knoell, Daren L.

    2008-01-01

    Microbial detection requires the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) that are distributed on the cell surface and within the cytosol. The nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family functions as an intracellular PRR that triggers the innate immune response. The mechanism by which PAMPs enter the cytosol to interact with NLRs, particularly muropeptides derived from the bacterial proteoglycan cell wall, is poorly understood. PEPT2 is a proton-dependent transporter that mediates the active translocation of di- and tripeptides across epithelial tissues, including the lung. Using computational tools, we initially established that bacterial dipeptides, particularly γ-D-glutamyl-meso-diaminopimelic acid (γ-iE-DAP), are suitable substrates for PEPT2. We then determined in primary cultures of human upper airway epithelia and transiently transfected CHO-PEPT2 cell lines that γ-iE-DAP uptake was mediated by PEPT2 with an affinity constant of approximately 193 μM, whereas muramyl dipeptide was not transported. Exposure to γ-iE-DAP at the apical surface of differentiated, polarized cultures resulted in activation of the innate immune response in an NOD1- and RIP2-dependent manner, resulting in release of IL-6 and IL-8. Based on these findings we report that PEPT2 plays a vital role in microbial recognition by NLR proteins, particularly with regard to airborne pathogens, thereby participating in host defense in the lung. PMID:18474668

  16. Super-Resolution Microscopy and Tracking of DNA-Binding Proteins in Bacterial Cells

    PubMed Central

    Uphoff, Stephan

    2016-01-01

    Summary The ability to detect individual fluorescent molecules inside living cells has enabled a range of powerful microscopy techniques that resolve biological processes on the molecular scale. These methods have also transformed the study of bacterial cell biology, which was previously obstructed by the limited spatial resolution of conventional microscopy. In the case of DNA-binding proteins, super-resolution microscopy can visualize the detailed spatial organization of DNA replication, transcription, and repair processes by reconstructing a map of single-molecule localizations. Furthermore, DNA binding activities can be observed directly by tracking protein movement in real time. This allows identifying subpopulations of DNA-bound and diffusing proteins, and can be used to measure DNA-binding times in vivo. This chapter provides a detailed protocol for super-resolution microscopy and tracking of DNA-binding proteins in Escherichia coli cells. The protocol covers the construction of cell strains and describes data acquisition and analysis procedures, such as super-resolution image reconstruction, mapping single-molecule tracks, computing diffusion coefficients to identify molecular subpopulations with different mobility, and analysis of DNA-binding kinetics. While the focus is on the study of bacterial chromosome biology, these approaches are generally applicable to other molecular processes and cell types. PMID:27283312

  17. Peptidomimetic Small Molecules Disrupt Type IV Secretion System Activity in Diverse Bacterial Pathogens

    PubMed Central

    Shaffer, Carrie L.; Good, James A. D.; Kumar, Santosh; Krishnan, K. Syam; Gaddy, Jennifer A.; Loh, John T.; Chappell, Joseph; Almqvist, Fredrik

    2016-01-01

    ABSTRACT Bacteria utilize complex type IV secretion systems (T4SSs) to translocate diverse effector proteins or DNA into target cells. Despite the importance of T4SSs in bacterial pathogenesis, the mechanism by which these translocation machineries deliver cargo across the bacterial envelope remains poorly understood, and very few studies have investigated the use of synthetic molecules to disrupt T4SS-mediated transport. Here, we describe two synthetic small molecules (C10 and KSK85) that disrupt T4SS-dependent processes in multiple bacterial pathogens. Helicobacter pylori exploits a pilus appendage associated with the cag T4SS to inject an oncogenic effector protein (CagA) and peptidoglycan into gastric epithelial cells. In H. pylori, KSK85 impedes biogenesis of the pilus appendage associated with the cag T4SS, while C10 disrupts cag T4SS activity without perturbing pilus assembly. In addition to the effects in H. pylori, we demonstrate that these compounds disrupt interbacterial DNA transfer by conjugative T4SSs in Escherichia coli and impede vir T4SS-mediated DNA delivery by Agrobacterium tumefaciens in a plant model of infection. Of note, C10 effectively disarmed dissemination of a derepressed IncF plasmid into a recipient bacterial population, thus demonstrating the potential of these compounds in mitigating the spread of antibiotic resistance determinants driven by conjugation. To our knowledge, this study is the first report of synthetic small molecules that impair delivery of both effector protein and DNA cargos by diverse T4SSs. PMID:27118587

  18. Fluid dynamics and noise in bacterial cell–cell and cell–surface scattering

    PubMed Central

    Drescher, Knut; Dunkel, Jörn; Cisneros, Luis H.; Ganguly, Sujoy; Goldstein, Raymond E.

    2011-01-01

    Bacterial processes ranging from gene expression to motility and biofilm formation are constantly challenged by internal and external noise. While the importance of stochastic fluctuations has been appreciated for chemotaxis, it is currently believed that deterministic long-range fluid dynamical effects govern cell–cell and cell–surface scattering—the elementary events that lead to swarming and collective swimming in active suspensions and to the formation of biofilms. Here, we report direct measurements of the bacterial flow field generated by individual swimming Escherichia coli both far from and near to a solid surface. These experiments allowed us to examine the relative importance of fluid dynamics and rotational diffusion for bacteria. For cell–cell interactions it is shown that thermal and intrinsic stochasticity drown the effects of long-range fluid dynamics, implying that physical interactions between bacteria are determined by steric collisions and near-field lubrication forces. This dominance of short-range forces closely links collective motion in bacterial suspensions to self-organization in driven granular systems, assemblages of biofilaments, and animal flocks. For the scattering of bacteria with surfaces, long-range fluid dynamical interactions are also shown to be negligible before collisions; however, once the bacterium swims along the surface within a few microns after an aligning collision, hydrodynamic effects can contribute to the experimentally observed, long residence times. Because these results are based on purely mechanical properties, they apply to a wide range of microorganisms. PMID:21690349

  19. Rates of dissolved organic carbon (DOC) production and bacterial activity in the eastern North Atlantic Subtropical Gyre during summer

    NASA Astrophysics Data System (ADS)

    Teira, E.; Pazó, M. J.; Quevedo, M.; Fuentes, M. V.; Niell, F. X.; Fernández, E.

    2003-04-01

    Rates of particulate organic carbon production, dissolved organic carbon production (DOC) and bacterial production were measured at 8 stations located in the eastern North Atlantic Subtropical Gyre during August 1998. Euphotic-depth-integrated particulate organic carbon (POC) production rate was on average 27 mg C m-2 h-1. The corresponding averaged integrated DOC production rate was 5 mg C m-2 h-1, i.e., about 20 % of total primary production. No statistically significant relationship was found between the rates of DOC and POC production, suggesting that other processes besides phytoplankton exudation, such as cell lysis or protist grazing, could substantially contribute to the release of DOC. Euphotic-depth-integrated bacterial biomass and production were, on average, 214 mg C m-2 and 1.4 mg C m-2 h-1, respectively. The lack of correlation between the rates of DOC release and bacterial activity, and a bacterial carbon demand (BCD, calculated by using an estimated bacterial growth efficiency ranging from 11 to 18%) in excess of DOC production suggest the existence of additional organic carbon sources (both allochthonous and/or autochthonous reservoirs), apart from in situ phytoplankton-derived DOC production, for the maintenance of bacterial activity in this region during summer.

  20. Bacterial Infections in Hematopoietic Stem Cell Transplant Recipients

    PubMed Central

    Balletto, Elisa; Mikulska, Małgorzata

    2015-01-01

    Bacterial infections are major complications after Hematopoietic Stem Cell Transplant (HSCT). They consist mainly of bloodstream infections (BSI), followed by pneumonia and gastrointestinal infections, including typhlitis and Clostridium difficile infection. Microbiological data come mostly from BSI. Coagulase negative staphylococci and Enterobacteriaceae are the most frequent pathogens causing approximately 25% of BSI each, followed by enterococci, P. aeruginosa and viridans streptococci. Bacterial pneumonia is frequent after HSCT, and Gram-negatives are predominant. Clostridium difficile infection affects approximately 15% of HSCT recipients, being more frequent in case of allogeneic than autologous HSCT. The epidemiology and the prevalence of resistant strains vary significantly between transplant centres. In some regions, multi-drug resistant (MDR) Gram-negative rods are increasingly frequent. In others, vancomycin-resistant enterococci are predominant. In the era of increasing resistance to antibiotics, the efficacy of fluoroquinolone prophylaxis and standard treatment of febrile neutropenia have been questioned. Therefore, a thorough evaluation of local epidemiology is mandatory to decide the need for prophylaxis and the choice of the best regimen for empirical treatment of febrile neutropenia. For the latter, individualised approach has been proposed, consisting of either escalation or de-escalation strategy. De-escalation strategy is recommended since resistant bacteria should be covered upfront, mainly in patients with severe clinical presentation and previous infection or colonisation with a resistant pathogen. Non-pharmacological interventions, such as screening for resistant bacteria, applying isolation and contact precautions should be put in place to limit the spread of MDR bacteria. Antimicrobial stewardship program should be implemented in transplant centres. PMID:26185610

  1. Cell Wall Nonlinear Elasticity and Growth Dynamics: How Do Bacterial Cells Regulate Pressure and Growth?

    NASA Astrophysics Data System (ADS)

    Deng, Yi

    In my thesis, I study intact and bulging Escherichia coli cells using atomic force microscopy to separate the contributions of the cell wall and turgor pressure to the overall cell stiffness. I find strong evidence of power--law stress--stiffening in the E. coli cell wall, with an exponent of 1.22±0.12, such that the wall is significantly stiffer in intact cells (E = 23±8 MPa and 49±20 MPa in the axial and circumferential directions) than in unpressurized sacculi. These measurements also indicate that the turgor pressure in living cells E. coli is 29±3 kPa. The nonlinearity in cell elasticity serves as a plausible mechanism to balance the mechanical protection and tension measurement sensitivity of the cell envelope. I also study the growth dynamics of the Bacillus subtilis cell wall to help understand the mechanism of the spatiotemporal order of inserting new cell wall material. High density fluorescent markers are used to label the entire cell surface to capture the morphological changes of the cell surface at sub-cellular to diffraction-limited spatial resolution and sub-minute temporal resolution. This approach reveals that rod-shaped chaining B. subtilis cells grow and twist in a highly heterogeneous fashion both spatially and temporally. Regions of high growth and twisting activity have a typical length scale of 5 μm, and last for 10-40 minutes. Motivated by the quantification of the cell wall growth dynamics, two microscopy and image analysis techniques are developed and applied to broader applications beyond resolving bacterial growth. To resolve densely distributed quantum dots, we present a fast and efficient image analysis algorithm, namely Spatial Covariance Reconstruction (SCORE) microscopy that takes into account the blinking statistics of the fluorescence emitters. We achieve sub-diffraction lateral resolution of 100 nm from 5 to 7 seconds of imaging, which is at least an order of magnitude faster than single-particle localization based methods

  2. Endogenous signals released from necrotic cells augment inflammatory responses to bacterial endotoxin

    PubMed Central

    El Mezayen, Rabab; El Gazzar, Mohamed; Seeds, Michael C.; McCall, Charles E.; Dreskin, Stephen C.; Nicolls, Mark R.

    2011-01-01

    Stressed cells undergoing necrosis release molecules that acts as endogenous danger signals to alert and activate innate immune cells. Both HMGB1 and HSP70 are induced in activated monocytes/macrophages and also are released from stressed or injured cells. We investigated whether HMGB1 and HSP70 released from necrotic monocytes/macrophages, can act as danger signals to mediate proinflammatory cytokine responses to bacterial endotoxin or lipopolysaccharide (LPS). We show that cell lysate, obtained from necrotic cells directly stimulates the proinflammatory cytokine and chemokine responses in human monocyte/macrophage cell line, THP-1, as revealed by the induction of TNF-α, IL-6 and IL-8 mRNA expression and protein production. In the presence of LPS, necrotic cell lysate induced a more robust increase in all three proteins. We found that HMGB1 and HSP70 were indeed present in the necrotic cell lysate and were responsible for the significant induction of the proinflammatory cytokine expression, as neutralization with antibodies against both proteins blocked the increase in the cytokine production seen after incubating LPS-stimulated cells with the necrotic cell lysate. We also found that the newly identified triggering receptor expressed on myeloid cells-1 (TREM-1) was involved in mediating the HMGB1- and HSP70-induced cytokine production. Blocking TREM-1 on THP-1 cells with a recombinant chimera prevented the increase in cytokine production, while simultaneous blocking of TLR4 and TREM-1 completely abolished the proinflammatory response, suggesting that TREM-1 synergizes with TLR4 to mediate the effects of such signals from necrotic cells. In addition, blocking HMGB1 or HSP70 simultaneously with TREM-1 did not decrease the cytokine level further, confirming the involvement of TREM-1 in mediating the effect of HMGB1 and HSP70. Although the interaction of HMGB1 and HSP70 with TREM-1 induced IκBα and p38 expression, both of which are required for the inflammatory

  3. The General Phosphotransferase System Proteins Localize to Sites of Strong Negative Curvature in Bacterial Cells

    PubMed Central

    Govindarajan, Sutharsan; Elisha, Yair; Nevo-Dinur, Keren; Amster-Choder, Orna

    2013-01-01

    ABSTRACT The bacterial cell poles are emerging as subdomains where many cellular activities take place, but the mechanisms for polar localization are just beginning to unravel. The general phosphotransferase system (PTS) proteins, enzyme I (EI) and HPr, which control preferential use of carbon sources in bacteria, were recently shown to localize near the Escherichia coli cell poles. Here, we show that EI localization does not depend on known polar constituents, such as anionic lipids or the chemotaxis receptors, and on the cell division machinery, nor can it be explained by nucleoid occlusion or localized translation. Detection of the general PTS proteins at the budding sites of endocytotic-like membrane invaginations in spherical cells and their colocalization with the negative curvature sensor protein DivIVA suggest that geometric cues underlie localization of the PTS system. Notably, the kinetics of glucose uptake by spherical and rod-shaped E. coli cells are comparable, implying that negatively curved “pole-like” sites support not only the localization but also the proper functioning of the PTS system in cells with different shapes. Consistent with the curvature-mediated localization model, we observed the EI protein from Bacillus subtilis at strongly curved sites in both B. subtilis and E. coli. Taken together, we propose that changes in cell architecture correlate with dynamic survival strategies that localize central metabolic systems like the PTS to subcellular domains where they remain active, thus maintaining cell viability and metabolic alertness. PMID:24129255

  4. Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals

    PubMed Central

    Tizzano, Marco; Gulbransen, Brian D.; Vandenbeuch, Aurelie; Clapp, Tod R.; Herman, Jake P.; Sibhatu, Hiruy M.; Churchill, Mair E. A.; Silver, Wayne L.; Kinnamon, Sue C.; Finger, Thomas E.

    2010-01-01

    The upper respiratory tract is continually assaulted with harmful dusts and xenobiotics carried on the incoming airstream. Detection of such irritants by the trigeminal nerve evokes protective reflexes, including sneezing, apnea, and local neurogenic inflammation of the mucosa. Although free intra-epithelial nerve endings can detect certain lipophilic irritants (e.g., mints, ammonia), the epithelium also houses a population of trigeminally innervated solitary chemosensory cells (SCCs) that express T2R bitter taste receptors along with their downstream signaling components. These SCCs have been postulated to enhance the chemoresponsive capabilities of the trigeminal irritant-detection system. Here we show that transduction by the intranasal solitary chemosensory cells is necessary to evoke trigeminally mediated reflex reactions to some irritants including acyl–homoserine lactone bacterial quorum-sensing molecules, which activate the downstream signaling effectors associated with bitter taste transduction. Isolated nasal chemosensory cells respond to the classic bitter ligand denatonium as well as to the bacterial signals by increasing intracellular Ca2+. Furthermore, these same substances evoke changes in respiration indicative of trigeminal activation. Genetic ablation of either Gα-gustducin or TrpM5, essential elements of the T2R transduction cascade, eliminates the trigeminal response. Because acyl–homoserine lactones serve as quorum-sensing molecules for Gram-negative pathogenic bacteria, detection of these substances by airway chemoreceptors offers a means by which the airway epithelium may trigger an epithelial inflammatory response before the bacteria reach population densities capable of forming destructive biofilms. PMID:20133764

  5. Role of eukaryotic-like serine/threonine kinases in bacterial cell division and morphogenesis.

    PubMed

    Manuse, Sylvie; Fleurie, Aurore; Zucchini, Laure; Lesterlin, Christian; Grangeasse, Christophe

    2016-01-01

    Bacteria possess a repertoire of versatile protein kinases modulating diverse aspects of their physiology by phosphorylating proteins on various amino acids including histidine, cysteine, aspartic acid, arginine, serine, threonine and tyrosine. One class of membrane serine/threonine protein kinases possesses a catalytic domain sharing a common fold with eukaryotic protein kinases and an extracellular mosaic domain found in bacteria only, named PASTA for 'Penicillin binding proteins And Serine/Threonine kinase Associated'. Over the last decade, evidence has been accumulating that these protein kinases are involved in cell division, morphogenesis and developmental processes in Firmicutes and Actinobacteria. However, observations differ from one species to another suggesting that a general mechanism of activation of their kinase activity is unlikely and that species-specific regulation of cell division is at play. In this review, we survey the latest research on the structural aspects and the cellular functions of bacterial serine/threonine kinases with PASTA motifs to illustrate the diversity of the regulatory mechanisms controlling bacterial cell division and morphogenesis. PMID:26429880

  6. Upregulation of TMEM16A Protein in Bronchial Epithelial Cells by Bacterial Pyocyanin

    PubMed Central

    Caci, Emanuela; Scudieri, Paolo; Di Carlo, Emma; Morelli, Patrizia; Bruno, Silvia; De Fino, Ida; Bragonzi, Alessandra; Gianotti, Ambra; Sondo, Elvira; Ferrera, Loretta; Palleschi, Alessandro; Santambrogio, Luigi; Ravazzolo, Roberto; Galietta, Luis J. V.

    2015-01-01

    Induction of mucus hypersecretion in the airway epithelium by Th2 cytokines is associated with the expression of TMEM16A, a Ca2+-activated Cl- channel. We asked whether exposure of airway epithelial cells to bacterial components, a condition that mimics the highly infected environment occurring in cystic fibrosis (CF), also results in a similar response. In cultured human bronchial epithelial cells, treatment with pyocyanin or with a P. aeruginosa culture supernatant caused a significant increase in TMEM16A function. The Ca2+-dependent Cl- secretion, triggered by stimulation with UTP, was particularly enhanced by pyocyanin in cells from CF patients. Increased expression of TMEM16A protein and of MUC5AC mucin by bacterial components was demonstrated by immunofluorescence in CF and non-CF cells. We also investigated TMEM16A expression in human bronchi by immunocytochemistry. We found increased TMEM16A staining in the airways of CF patients. The strongest signal was observed in CF submucosal glands. Our results suggest that TMEM16A expression/function is upregulated in CF lung disease, possibly as a response towards the presence of bacteria in the airways. PMID:26121472

  7. Proteasomal Degradation of Nod2 Protein Mediates Tolerance to Bacterial Cell Wall Components*

    PubMed Central

    Lee, Kyoung-Hee; Biswas, Amlan; Liu, Yuen-Joyce; Kobayashi, Koichi S.

    2012-01-01

    The innate immune system serves as the first line of defense by detecting microbes and initiating inflammatory responses. Although both Toll-like receptor (TLR) and nucleotide binding domain and leucine-rich repeat (NLR) proteins are important for this process, their excessive activation is hazardous to hosts; thus, tight regulation is required. Endotoxin tolerance is refractory to repeated lipopolysaccharide (LPS) stimulation and serves as a host defense mechanism against septic shock caused by an excessive TLR4 response during Gram-negative bacterial infection. Gram-positive bacteria as well as their cell wall components also induce shock. However, the mechanism underlying tolerance is not understood. Here, we show that activation of Nod2 by its ligand, muramyl dipeptide (MDP) in the bacterial cell wall, induces rapid degradation of Nod2, which confers MDP tolerance in vitro and in vivo. Nod2 is constitutively associated with a chaperone protein, Hsp90, which is required for Nod2 stability and protects Nod2 from degradation. Upon MDP stimulation, Hsp90 rapidly dissociates from Nod2, which subsequently undergoes ubiquitination and proteasomal degradation. The SOCS-3 protein induced by Nod2 activation further facilitates this degradation process. Therefore, Nod2 protein stability is a key factor in determining responsiveness to MDP stimulation. This indicates that TLRs and NLRs induce a tolerant state through distinct molecular mechanisms that protect the host from septic shock. PMID:23019338

  8. Colorimetric detection of Shewanella oneidensis based on immunomagnetic capture and bacterial intrinsic peroxidase activity

    NASA Astrophysics Data System (ADS)

    Wen, Junlin; Zhou, Shungui; Chen, Junhua

    2014-06-01

    Rapid detection and enumeration of target microorganisms is considered as a powerful tool for monitoring bioremediation process that typically involves cleaning up polluted environments with functional microbes. A novel colorimetric assay is presented based on immunomagnetic capture and bacterial intrinsic peroxidase activity for rapidly detecting Shewanella oneidensis, an important model organism for environmental bioremediation because of its remarkably diverse respiratory abilities. Analyte bacteria captured on the immunomagnetic beads provided a bacterial out-membrane peroxidase-amplified colorimetric readout of the immunorecognition event by oxidizing 3, 3', 5, 5'-tetramethylbenzidine (TMB) in the present of hydrogen peroxide. The high-efficiency of immunomagnetic capture and signal amplification of peroxidase activity offers an excellent detection performance with a wide dynamic range between 5.0 × 103 and 5.0 × 106 CFU/mL toward target cells. Furthermore, this method was demonstrated to be feasible in detecting S. oneidensis cells spiked in environmental samples. The proposed colorimetric assay shows promising environmental applications for rapid detection of target microorganisms.

  9. Colorimetric detection of Shewanella oneidensis based on immunomagnetic capture and bacterial intrinsic peroxidase activity

    PubMed Central

    Wen, Junlin; Zhou, Shungui; Chen, Junhua

    2014-01-01

    Rapid detection and enumeration of target microorganisms is considered as a powerful tool for monitoring bioremediation process that typically involves cleaning up polluted environments with functional microbes. A novel colorimetric assay is presented based on immunomagnetic capture and bacterial intrinsic peroxidase activity for rapidly detecting Shewanella oneidensis, an important model organism for environmental bioremediation because of its remarkably diverse respiratory abilities. Analyte bacteria captured on the immunomagnetic beads provided a bacterial out-membrane peroxidase-amplified colorimetric readout of the immunorecognition event by oxidizing 3, 3′, 5, 5′-tetramethylbenzidine (TMB) in the present of hydrogen peroxide. The high-efficiency of immunomagnetic capture and signal amplification of peroxidase activity offers an excellent detection performance with a wide dynamic range between 5.0 × 103 and 5.0 × 106 CFU/mL toward target cells. Furthermore, this method was demonstrated to be feasible in detecting S. oneidensis cells spiked in environmental samples. The proposed colorimetric assay shows promising environmental applications for rapid detection of target microorganisms. PMID:24898751

  10. Gustatory-mediated avoidance of bacterial lipopolysaccharides via TRPA1 activation in Drosophila

    PubMed Central

    Soldano, Alessia; Alpizar, Yeranddy A; Boonen, Brett; Franco, Luis; López-Requena, Alejandro; Liu, Guangda; Mora, Natalia; Yaksi, Emre; Voets, Thomas; Vennekens, Rudi; Hassan, Bassem A; Talavera, Karel

    2016-01-01

    Detecting pathogens and mounting immune responses upon infection is crucial for animal health. However, these responses come at a high metabolic price (McKean and Lazzaro, 2011, Kominsky et al., 2010), and avoiding pathogens before infection may be advantageous. The bacterial endotoxins lipopolysaccharides (LPS) are important immune system infection cues (Abbas et al., 2014), but it remains unknown whether animals possess sensory mechanisms to detect them prior to infection. Here we show that Drosophila melanogaster display strong aversive responses to LPS and that gustatory neurons expressing Gr66a bitter receptors mediate avoidance of LPS in feeding and egg laying assays. We found the expression of the chemosensory cation channel dTRPA1 in these cells to be necessary and sufficient for LPS avoidance. Furthermore, LPS stimulates Drosophila neurons in a TRPA1-dependent manner and activates exogenous dTRPA1 channels in human cells. Our findings demonstrate that flies detect bacterial endotoxins via a gustatory pathway through TRPA1 activation as conserved molecular mechanism. DOI: http://dx.doi.org/10.7554/eLife.13133.001 PMID:27296646

  11. Increases in Calmodulin Abundance and Stabilization of Activated iNOS Mediate Bacterial Killing in RAW 264.7 Macrophages

    SciTech Connect

    Smallwood, Heather S.; Shi, Liang; Squier, Thomas C.

    2006-08-01

    The rapid activation of macrophages in response to bacterial antigens is central to the innate immune system that permits the recognition and killing of pathogens to limit infection. To understand regulatory mechanisms underlying macrophage activation, we have investigated changes in the abundance of calmodulin (CaM) and iNOS in response to the bacterial cell wall component lipopolysaccharide (LPS) using RAW 264.7 macrophages. Critical to these measurements was the ability to differentiate free iNOS from the CaM-bound (active) form of iNOS associated with nitric oxide generation. We observe a rapid two-fold increase in CaM abundance during the first 30 minutes that is blocked by inhibition of NF?B nuclear translocation or protein synthesis. A similar two-fold increase in the abundance of the complex between CaM and iNOS is observed with the same time dependence. In contrast, there are no detectable increases in the CaM-free (i.e., inactive) form of iNOS within the first hour; it remains at a very low abundance during the initial phase of macrophage activation. Increasing cellular CaM levels in stably transfected cells results in a corresponding increase in the abundance of the CaM/iNOS complex that promotes effective bacterial killing following challenge by Salmonella typhimurium. Thus, LPS-dependent increases in CaM abundance function in the stabilization and activation of iNOS on the rapid time-scale associated with macrophage activation and bacterial killing. These results explain how CaM and iNOS coordinately function to form a stable complex that is part of a rapid host-response that functions within the first 30 minutes following bacterial infection to up-regulate the innate immune system involving macrophage activation.

  12. Molecular Architecture of the Bacterial Flagellar Motor in Cells

    PubMed Central

    2015-01-01

    The flagellum is one of the most sophisticated self-assembling molecular machines in bacteria. Powered by the proton-motive force, the flagellum rapidly rotates in either a clockwise or counterclockwise direction, which ultimately controls bacterial motility and behavior. Escherichia coli and Salmonella enterica have served as important model systems for extensive genetic, biochemical, and structural analysis of the flagellum, providing unparalleled insights into its structure, function, and gene regulation. Despite these advances, our understanding of flagellar assembly and rotational mechanisms remains incomplete, in part because of the limited structural information available regarding the intact rotor–stator complex and secretion apparatus. Cryo-electron tomography (cryo-ET) has become a valuable imaging technique capable of visualizing the intact flagellar motor in cells at molecular resolution. Because the resolution that can be achieved by cryo-ET with large bacteria (such as E. coli and S. enterica) is limited, analysis of small-diameter bacteria (including Borrelia burgdorferi and Campylobacter jejuni) can provide additional insights into the in situ structure of the flagellar motor and other cellular components. This review is focused on the application of cryo-ET, in combination with genetic and biophysical approaches, to the study of flagellar structures and its potential for improving the understanding of rotor–stator interactions, the rotational switching mechanism, and the secretion and assembly of flagellar components. PMID:24697492

  13. Macroscopic and spectroscopic analysis of lanthanide adsorption to bacterial cells

    NASA Astrophysics Data System (ADS)

    Ngwenya, Bryne T.; Mosselmans, J. Fred W.; Magennis, Marisa; Atkinson, Kirk D.; Tourney, Janette; Olive, Valerie; Ellam, Robert M.

    2009-06-01

    This study was designed to combine surface complexation modelling of macroscopic adsorption data with X-ray Absorption Spectroscopic (XAS) measurements to identify lanthanide sorption sites on the bacterial surface. The adsorption of selected representatives for light (La and Nd), middle (Sm and Gd) and heavy (Er and Yb) lanthanides was measured as a function of pH, and biomass samples exposed to 4 mg/L lanthanide at pH 3.5 and 6 were analysed using XAS. Surface complexation modelling was consistent with the light lanthanides adsorbing to phosphate sites, whereas the adsorption of middle and heavy lanthanides could be modelled equally well by carboxyl and phosphate sites. The existence of such mixed mode coordination was confirmed by Extended X-ray Absorption Fine Structure (EXAFS) analysis, which was also consistent with adsorption to phosphate sites at low pH, with secondary involvement of carboxyl sites at high adsorption density (high pH). Thus, the two approaches yield broadly consistent information with regard to surface site identity and lanthanide coordination environment. Furthermore, spectroscopic analysis suggests that coordination to phosphate sites is monodentate at the metal/biomass ratios used. Based on the best-fitting p Ka site, we infer that the phosphate sites are located on N-acetylglucosamine phosphate, the most likely polymer on gram-negative cells with potential phosphate sites that deprotonate around neutral pH.

  14. Graphene oxide exhibits broad-spectrum antimicrobial activity against bacterial phytopathogens and fungal conidia by intertwining and membrane perturbation

    NASA Astrophysics Data System (ADS)

    Chen, Juanni; Peng, Hui; Wang, Xiuping; Shao, Feng; Yuan, Zhaodong; Han, Heyou

    2014-01-01

    To understand the interaction mechanism between graphene oxide (GO) and typical phytopathogens, a particular investigation was conducted about the antimicrobial activity of GO against two bacterial pathogens (P. syringae and X. campestris pv. undulosa) and two fungal pathogens (F. graminearum and F. oxysporum). The results showed that GO had a powerful effect on the reproduction of all four pathogens (killed nearly 90% of the bacteria and repressed 80% macroconidia germination along with partial cell swelling and lysis at 500 μg mL-1). A mutual mechanism is proposed in this work that GO intertwinds the bacteria and fungal spores with a wide range of aggregated graphene oxide sheets, resulting in the local perturbation of their cell membrane and inducing the decrease of the bacterial membrane potential and the leakage of electrolytes of fungal spores. It is likely that GO interacts with the pathogens by mechanically wrapping and locally damaging the cell membrane and finally causing cell lysis, which may be one of the major toxicity actions of GO against phytopathogens. The antibacterial mode proposed in this study suggests that the GO may possess antibacterial activity against more multi-resistant bacterial and fungal phytopathogens, and provides useful information about the application of GO in resisting crop diseases.To understand the interaction mechanism between graphene oxide (GO) and typical phytopathogens, a particular investigation was conducted about the antimicrobial activity of GO against two bacterial pathogens (P. syringae and X. campestris pv. undulosa) and two fungal pathogens (F. graminearum and F. oxysporum). The results showed that GO had a powerful effect on the reproduction of all four pathogens (killed nearly 90% of the bacteria and repressed 80% macroconidia germination along with partial cell swelling and lysis at 500 μg mL-1). A mutual mechanism is proposed in this work that GO intertwinds the bacteria and fungal spores with a wide range

  15. Role of liquid membrane phenomenon in the anti-bacterial activity of Cefuroxime Sodium

    PubMed Central

    Nagesh, C.; Shankaraiah, M. M.; Venkatesh, J. S.; Setty, S. Ramachandra

    2010-01-01

    The role of liquid membrane phenomenon has been studied in the anti bacterial activity of cephalosporins i.e. Cefuroxime sodium. In our earlier publication [1] it was reported that hydraulic permeability data obtained to demonstrate the existence of liquid membrane in series with supporting membrane generated by Cefuroxime sodium. Transport of selected permeants (glucose, PABA, glycine, and ions like Mg++, NH4+, PO4-, Ca++, Na+, K+ and Cl-) through liquid membrane generated by Cefuroxime sodium in series with supporting membrane has been studied. The results indicated that the liquid membrane generated by Cefuroxime sodium inhibit the transport of various essential bio-molecules and permeants in to the cell. This modification in permeability of different permeants in the presence of the liquid membranes is likely to play significant role in the biological actions of Cefuroxime sodium. The anti-bacterial activity of Cefuroxime sodium further confirmed that the generation of liquid membrane by Cefuroxime sodium is also contributing for the antibacterial activity of them. PMID:24825969

  16. Comparative study of HOCl-inflicted damage to bacterial DNA ex vivo and within cells.

    PubMed

    Suquet, Christine; Warren, Jeffrey J; Seth, Nimulrith; Hurst, James K

    2010-01-15

    The prospects for using bacterial DNA as an intrinsic probe for HOCl and secondary oxidants/chlorinating agents associated with it has been evaluated using both in vitro and in vivo studies. Single-strand and double-strand breaks occurred in bare plasmid DNA that had been exposed to high levels of HOCl, although these reactions were very inefficient compared to polynucleotide chain cleavage caused by the OH.-generating reagent, peroxynitrite. Plasmid nicking was not increased when intact Escherichia coli were exposed to HOCl; rather, the amount of recoverable plasmid diminished in a dose-dependent manner. At concentration levels of HOCl exceeding lethal doses, genomic bacterial DNA underwent extensive fragmentation and the amount of precipitable DNA-protein complexes increased several-fold. The 5-chlorocytosine content of plasmid and genomic DNA isolated from HOCl-exposed E. coli was also slightly elevated above controls, as measured by mass spectrometry of the deaminated product, 5-chlorouracil. However, the yields were not dose-dependent over the bactericidal concentration range. Genomic DNA recovered from E. coli that had been subjected to phagocytosis by human neutrophils occasionally showed small increases in 5-chlorocytosine content when compared to analogous cellular reactions where myeloperoxidase activity was inhibited by azide ion. Overall, the amount of isolable 5-chlorouracil from the HOCl-exposed bacterial cells was far less than the damage manifested in polynucleotide bond cleavage and cross-linking. PMID:19850004

  17. A dynamin-like protein involved in bacterial cell membrane surveillance under environmental stress.

    PubMed

    Sawant, Prachi; Eissenberger, Kristina; Karier, Laurence; Mascher, Thorsten; Bramkamp, Marc

    2016-09-01

    In ever-changing natural environments, bacteria are continuously challenged with numerous biotic and abiotic stresses. Accordingly, they have evolved both specific and more general mechanisms to counteract stress-induced damage and ensure survival. In the soil habitat of Bacillus subtilis, peptide antibiotics and bacteriophages are among the primary stressors that affect the integrity of the cytoplasmic membrane. Dynamin-like proteins (DLPs) play a major role in eukaryotic membrane re-modelling processes, including antiviral activities, but the function of the corresponding bacterial homologues was so far poorly understood. Here, we report on the protective function of a bacterial DLP, DynA from B. subtilis. We provide evidence that DynA plays an important role in a membrane surveillance system that counteracts membrane pore formation provoked by antibiotics and phages. In unstressed cells, DynA is a highly dynamic membrane-associated protein. Upon membrane damage, DynA localizes into large and static assemblies, where DynA acts locally to counteract stress-induced pores, presumably by inducing lipid bilayer fusion and sealing membrane gaps. Thus, lack of DynA increases the sensitivity to antibiotic exposure and phage infection. Taken together, our work suggests that DynA, and potentially other bacterial DLPs, contribute to the innate immunity of bacteria against membrane stress. PMID:26530236

  18. Streptomyces: A Screening Tool for Bacterial Cell Division Inhibitors

    PubMed Central

    Jani, Charul; Tocheva, Elitza I.; McAuley, Scott; Craney, Arryn; Jensen, Grant J.; Nodwell, Justin

    2016-01-01

    Cell division is essential for spore formation but not for viability in the filamentous streptomycetes bacteria. Failure to complete cell division instead blocks spore formation, a phenotype that can be visualized by the absence of gray (in Streptomyces coelicolor) and green (in Streptomyces venezuelae) spore-associated pigmentation. Despite the lack of essentiality, the streptomycetes divisome is similar to that of other prokaryotes. Therefore, the chemical inhibitors of sporulation in model streptomycetes may interfere with the cell division in rod-shaped bacteria as well. To test this, we investigated 196 compounds that inhibit sporulation in S. coelicolor. We show that 19 of these compounds cause filamentous growth in Bacillus subtilis, consistent with impaired cell division. One of the compounds is a DNA-damaging agent and inhibits cell division by activating the SOS response. The remaining 18 act independently of known stress responses and may therefore act on the divisome or on divisome positioning and stability. Three of the compounds (Fil-1, Fil-2, and Fil-3) confer distinct cell division defects on B. subtilis. They also block B. subtilis sporulation, which is mechanistically unrelated to the sporulation pathway of streptomycetes but is also dependent on the divisome. We discuss ways in which these differing phenotypes can be used in screens for cell division inhibitors. PMID:25256667

  19. K+ efflux is the Common Trigger of NLRP3 inflammasome Activation by Bacterial Toxins and Particulate Matter

    PubMed Central

    Muñoz-Planillo, Raúl; Kuffa, Peter; Martínez-Colón, Giovanny; Smith, Brenna L.; Rajendiran, Thekkelnaycke M.; Núñez, Gabriel

    2013-01-01

    Summary The NLRP3 inflammasome is an important component of the innate immune system. However, its mechanism of activation remains largely unknown. We show that NLRP3 activators including bacterial pore-forming toxins, nigericin, ATP and particulate matter caused mitochondrial perturbation or the opening of a large membrane pore; but this was not required for NLRP3 activation. Furthermore, reactive oxygen species generation or a change in cell volume was not necessary for NLRP3 activation. Instead, the only common activity induced by all NLRP3 agonists was the permeation of the cell membrane to K+ and Na+. Notably, reduction of the intracellular K+ concentration was sufficient to activate NLRP3 whereas an increase in intracellular Na+ modulated, but was not strictly required for inflammasome activation. These results provide a unifying model for the activation of the NLRP3 inflammasome in which a drop in cytosolic K+ is the common step that is necessary and sufficient for caspase-1 activation. PMID:23809161

  20. Metatranscriptomic Analysis of Groundwater Reveals an Active Anammox Bacterial Population

    NASA Astrophysics Data System (ADS)

    Jewell, T. N. M.; Karaoz, U.; Thomas, B. C.; Banfield, J. F.; Brodie, E.; Williams, K. H.; Beller, H. R.

    2014-12-01

    Groundwater is a major natural resource, yet little is known about the contribution of microbial anaerobic ammonium oxidation (anammox) activity to subsurface nitrogen cycling. During anammox, energy is generated as ammonium is oxidized under anaerobic conditions to dinitrogen gas, using nitrite as the final electron acceptor. This process is a global sink for fixed nitrogen. Only a narrow range of monophyletic bacteria within the Planctomycetes carries out anammox, and the full extent of their metabolism, and subsequent impact on nitrogen cycling and microbial community structure, is still unknown. Here, we employ a metatranscriptomic analysis on enriched mRNA to identify the abundance and activity of a population of anammox bacteria within an aquifer at Rifle, CO. Planktonic biomass was collected over a two-month period after injection of up to 1.5 mM nitrate. Illumina-generated sequences were mapped to a phylogenetically binned Rifle metagenome database. We identified transcripts for genes with high protein sequence identities (81-98%) to those of anammox strain KSU-1 and to two of the five anammox bacteria genera, Brocadia and Kuenenia, suggesting an active, if not diverse, anammox population. Many of the most abundant anammox transcripts mapped to a single scaffold, indicative of a single dominant anammox species. Transcripts of the genes necessary for the anammox pathway were present, including an ammonium transporter (amtB), nitrite/formate transporter, nitrite reductase (nirK), and hydrazine oxidoreductase (hzoB). The form of nitrite reductase encoded by anammox is species-dependent, and we only identified nirK, with no evidence of anammox nirS. In addition to the anammox pathway we saw evidence of the anammox bacterial dissimilatory nitrate reduction to ammonium pathway (narH, putative nrfA, and nrfB), which provides an alternate means of generating substrates for anammox from nitrate, rather than relying on an external pool. Transcripts for hydroxylamine

  1. Testing an agent-based model of bacterial cell motility: How nutrient concentration affects speed distribution

    NASA Astrophysics Data System (ADS)

    Garcia, V.; Birbaumer, M.; Schweitzer, F.

    2011-08-01

    We revisit a recently proposed agent-based model of active biological motion and compare its predictions with own experimental findings for the speed distribution of bacterial cells, Salmonella typhimurium. Agents move according to a stochastic dynamics and use energy stored in an internal depot for metabolism and active motion. We discuss different assumptions of how the conversion from internal to kinetic energy d( v) may depend on the actual speed, to conclude that d 2 v ξ with either ξ = 2 or 1 < ξ < 2 are promising hypotheses. To test these, we compare the model's prediction with the speed distribution of bacteria which were obtained in media of different nutrient concentration and at different times. We find that both hypotheses are in line with the experimental observations, with ξ between 1.67 and 2.0. Regarding the influence of a higher nutrient concentration, we conclude that the take-up of energy by bacterial cells is indeed increased. But this energy is not used to increase the speed, with 40 μm/s as the most probable value of the speed distribution, but is rather spend on metabolism and growth.

  2. The Bacterial Alarmone (p)ppGpp Activates the Type III Secretion System in Erwinia amylovora

    PubMed Central

    Ancona, Veronica; Lee, Jae Hoon; Chatnaparat, Tiyakhon; Oh, Jinrok; Hong, Jong-In

    2015-01-01

    ABSTRACT The hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS) is a key pathogenicity factor in Erwinia amylovora. Previous studies have demonstrated that the T3SS in E. amylovora is transcriptionally regulated by a sigma factor cascade. In this study, the role of the bacterial alarmone ppGpp in activating the T3SS and virulence of E. amylovora was investigated using ppGpp mutants generated by Red recombinase cloning. The virulence of a ppGpp-deficient mutant (ppGpp0) as well as a dksA mutant of E. amylovora was completely impaired, and bacterial growth was significantly reduced, suggesting that ppGpp is required for full virulence of E. amylovora. Expression of T3SS genes was greatly downregulated in the ppGpp0 and dksA mutants. Western blotting showed that accumulations of the HrpA protein in the ppGpp0 and dksA mutants were about 10 and 4%, respectively, of that in the wild-type strain. Furthermore, higher levels of ppGpp resulted in a reduced cell size of E. amylovora. Moreover, serine hydroxamate and α-methylglucoside, which induce amino acid and carbon starvation, respectively, activated hrpA and hrpL promoter activities in hrp-inducing minimal medium. These results demonstrated that ppGpp and DksA play central roles in E. amylovora virulence and indicated that E. amylovora utilizes ppGpp as an internal messenger to sense environmental/nutritional stimuli for regulation of the T3SS and virulence. IMPORTANCE The type III secretion system (T3SS) is a key pathogenicity factor in Gram-negative bacteria. Fully elucidating how the T3SS is activated is crucial for comprehensively understanding the function of the T3SS, bacterial pathogenesis, and survival under stress conditions. In this study, we present the first evidence that the bacterial alarmone ppGpp-mediated stringent response activates the T3SS through a sigma factor cascade, indicating that ppGpp acts as an internal messenger to sense environmental/nutritional stimuli for

  3. Bacterial genotoxins: The long journey to the nucleus of mammalian cells.

    PubMed

    Frisan, Teresa

    2016-03-01

    Bacterial protein genotoxins target the DNA of eukaryotic cells, causing DNA single and double strand breaks. The final outcome of the intoxication is induction of DNA damage responses and activation of DNA repair pathways. When the damage is beyond repair, the target cell either undergoes apoptosis or enters a permanent quiescent stage, known as cellular senescence. In certain instances, intoxicated cells can survive and proliferate. This event leads to accumulation of genomic instability and acquisition of malignant traits, underlining the carcinogenic potential of these toxins. The toxicity is dependent on the toxins' internalization and trafficking from the extracellular environment to the nucleus, and requires a complex interaction with several cellular membrane compartments: the plasma membrane, the endosomes, the trans Golgi network and the endoplasmic reticulum, and finally the nucleus. This review will discuss the current knowledge of the bacterial genotoxins internalization pathways and will highlight the issues that still remain unanswered. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale. PMID:26299818

  4. Molecular Mechanism of Holin Transmembrane Domain I in Pore Formation and Bacterial Cell Death.

    PubMed

    Lella, Muralikrishna; Kamilla, Soumya; Jain, Vikas; Mahalakshmi, Radhakrishnan

    2016-04-15

    Bacterial cell lysis during bacteriophage infection is timed by perfect orchestration between components of the holin-endolysin cassette. In bacteria, progressively accumulating holin in the inner membrane, retained in its inactive form by antiholin, is triggered into active hole formation, resulting in the canonical host cell lysis. However, the molecular mechanism of regulation and physical basis of pore formation in the mycobacterial cell membrane by D29 mycobacteriophage holin, particularly in the nonexistence of a known antiholin, is poorly understood. In this study, we report, for the first time, the use of fluorescence resonance transfer measurements to demonstrate that the first transmembrane domain (TM1) of D29 holin undergoes a helix ↔ β-hairpin conformational interconversion. We validate that this structural malleability is mediated by a centrally positioned proline and is responsible for controlled TM1 self-association in membrana, in the presence of a proton gradient across the lipid membrane. We demonstrate that TM1 is sufficient for bacterial growth inhibition. The biological effect of D29 holin structural alteration is presented as a holin self-regulatory mechanism, and its implications are discussed in the context of holin function. PMID:26701742

  5. Modification of N-glycosylation sites allows secretion of bacterial chondroitinase ABC from mammalian cells

    PubMed Central

    Muir, Elizabeth M.; Fyfe, Ian; Gardiner, Sonya; Li, Li; Warren, Philippa; Fawcett, James W.; Keynes, Roger J.; Rogers, John H.

    2010-01-01

    Although many eukaryotic proteins have been secreted by transfected bacterial cells, little is known about how a bacterial protein is treated as it passes through the secretory pathway when expressed in a eukaryotic cell. The eukaryotic N-glycosylation system could interfere with folding and secretion of prokaryotic proteins whose sequence has not been adapted for glycosylation in structurally appropriate locations. Here we show that such interference does indeed occur for chondroitinase ABC from the bacterium Proteus vulgaris, and can be overcome by eliminating potential N-glycosylation sites. Chondroitinase ABC was heavily glycosylated when expressed in mammalian cells or in a mammalian translation system, and this process prevented secretion of functional enzyme. Directed mutagenesis of selected N-glycosylation sites allowed efficient secretion of active chondroitinase. As these proteoglycans are known to inhibit regeneration of axons in the mammalian central nervous system, the modified chondroitinase gene is a potential tool for gene therapy to promote neural regeneration, ultimately in human spinal cord injury. PMID:19900493

  6. Nanomechanical Response of Pseudomonas aeruginosa PAO1 Bacterial Cells to Cationic Antimicrobial Peptides

    NASA Astrophysics Data System (ADS)

    Lu, Shun; Walters, Grant; Dutcher, John

    2013-03-01

    We have used an atomic force microscopy (AFM)-based creep deformation technique to study changes to the viscoelastic properties of individual Gram-negative Pseudomonas aeruginosa PAO1 cells as a function of time of exposure to two cationic peptides: polymyxin B (PMB), a cyclic antimicrobial peptide, and the structurally-related compound, polymyxin B nonapeptide (PMBN). The measurements provide a direct measure of the mechanical integrity of the bacterial cell envelope, and the results can be understood in terms of simple viscoelastic models of arrangements of springs and dashpots, which can be ascribed to different components within the bacterial cell. Time-resolved creep deformation experiments reveal abrupt changes to the viscoelastic properties of P. aeruginosa bacterial cells after exposure to both PMB and PMBN, with quantitatively different changes for the two cationic peptides. These measurements provide new insights into the kinetics and mechanism of action of antimicrobial peptides on bacterial cells.

  7. Grazing activity and ruminal bacterial population associated with frothy bloat in steers grazing winter wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two grazing experiments were designed to elucidate the shifts in rumen bacterial populations (Exp. 1) and grazing activities (Exp. 2) in wheat forage diets between bloated and non-bloated steers. In Exp. 1, the bacterial DNA density was greatest for Ruminococcus flavefaciens, Streptococcus bovis, a...

  8. Efficiency of fluorescence in situ hybridization for bacterial cell identification in temporary river sediments with contrasting water content.

    PubMed

    Fazi, Stefano; Amalfitano, Stefano; Pizzetti, Ilaria; Pernthaler, Jakob

    2007-09-01

    We studied the efficiency of two hybridization techniques for the analysis of benthic bacterial community composition under varying sediment water content. Microcosms were set up with sediments from four European temporary rivers. Wet sediments were dried, and dry sediments were artificially rewetted. The percentage of bacterial cells detected by fluorescence in situ hybridization with fluorescently monolabeled probes (FISH) significantly increased from dry to wet sediments, showing a positive correlation with the community activity measured via incorporation of (3)H leucine. FISH and signal amplification by catalyzed reporter deposition (CARD-FISH) could significantly better detect cells with low activity in dried sediments. Through the application of an optimized cell permeabilization protocol, the percentage of hybridized cells by CARD-FISH showed comparable values in dry and wet conditions. This approach was unrelated to (3)H leucine incorporation rates. Moreover, the optimized protocol allowed a significantly better visualization of Gram-positive Actinobacteria in the studied samples. CARD-FISH is, therefore, proposed as an effective technique to compare bacterial communities residing in sediments with contrasting water content, irrespective of differences in the activity state of target cells. Considering the increasing frequencies of flood and drought cycles in European temporary rivers, our approach may help to better understand the dynamics of microbial communities in such systems. PMID:17452089

  9. Bacterial IMPDH gene used for the selection of mammalian cell transfectants.

    SciTech Connect

    Baccam, M.; Huberman, E.; Energy Systems

    2003-06-01

    Stable cell transfection is used for the expression of exogenous genes or cDNAs in eukaryotic cells. Selection of these transfectants requires a dominant selectable marker. A variety of such markers has been identified and is currently in use. However, many of these are not suitable for all cell types or require unique conditions. Here we describe a simple and versatile dominant selectable marker that involves bacterial IMP dehydrogenase (IMPDH), an enzyme essential for the replication of mammalian and bacterial cells. Although IMPDH is evolutionarily conserved, the bacterial enzyme is orders of magnitude more resistant to the toxic effect of the drug mycophenolic acid, which is an IMPDH inhibitor. We have demonstrated that transfection of human, monkey or Chinese hamster cell lines with an expression vector containing bacterial IMPDH and mycophenolic acid treatment resulted in the selection of colonies with a strikingly increased resistance to mycophenolic acid toxicity. Analysis of cells derived from these colonies indicated that the acquisition of this resistance was associated with bacterial IMPDH protein expression. As a proof of principle, we showed that mammalian cell transfection with a hicistronic IMPDH/GFP expression vector and mycophenolic acid treatment can he used to successfully select transfectants that express the fluorescent protein. These results indicate that bacterial IMPDH is a practical dominant selectable marker that can be used for the selection of transfectants that express exogenous genes or cDNAs in mammalian cells.

  10. Nε−Lysine Acetylation of a Bacterial Transcription Factor Inhibits Its DNA-Binding Activity

    PubMed Central

    Thao, Sandy; Chen, Chien-Sheng; Zhu, Heng; Escalante-Semerena, Jorge C.

    2010-01-01

    Evidence suggesting that eukaryotes and archaea use reversible Nε-lysine (Nε-Lys) acetylation to modulate gene expression has been reported, but evidence for bacterial use of Nε-Lys acetylation for this purpose is lacking. Here, we report data in support of the notion that bacteria can control gene expression by modulating the acetylation state of transcription factors (TFs). We screened the E. coli proteome for substrates of the bacterial Gcn5-like protein acetyltransferase (Pat). Pat acetylated four TFs, including the RcsB global regulatory protein, which controls cell division, and capsule and flagellum biosynthesis in many bacteria. Pat acetylated residue Lys180 of RcsB, and the NAD+-dependent Sir2 (sirtuin)-like protein deacetylase (CobB) deacetylated acetylated RcsB (RcsBAc), demonstrating that Nε-Lys acetylation of RcsB is reversible. Analysis of RcsBAc and variant RcsB proteins carrying substitutions at Lys180 provided biochemical and physiological evidence implicating Lys180 as a critical residue for RcsB DNA-binding activity. These findings further the likelihood that reversible Nε-Lys acetylation of transcription factors is a mode of regulation of gene expression used by all cells. PMID:21217812

  11. Release of Toll-Like Receptor-2-Activating Bacterial Lipoproteins in Shigella flexneri Culture Supernatants

    PubMed Central

    Aliprantis, Antonios O.; Weiss, David S.; Radolf, Justin D.; Zychlinsky, Arturo

    2001-01-01

    Shigella spp. cause dysentery, a severe form of bloody diarrhea. Apoptosis, or programmed cell death, is induced during Shigella infections and has been proposed to be a key event in the pathogenesis of dysentery. Here, we describe a novel cytotoxic activity in the sterile-culture supernatants of Shigella flexneri. An identical activity was identified in purified S. flexneri endotoxin, defined here as a mixture of lipopolysaccharide (LPS) and endotoxin-associated proteins (EP). Separation of endotoxin into EP and LPS revealed the activity to partition exclusively to the EP fraction. Biochemical characterization of S. flexneri EP and culture supernatants, including enzymatic deactivation, reverse-phase high-pressure liquid chromatography analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a Toll-like receptor-2 (TLR2) activation assay, indicates that the cytotoxic component is a mixture of bacterial lipoproteins (BLP). We show that biologically active BLP are liberated into culture supernatants of actively growing S. flexneri. In addition, our data indicate that BLP, and not LPS, are the component of endotoxin of gram-negative organisms responsible for activating TLR2. The activation of apoptosis by BLP shed from S. flexneri is discussed as a novel aspect of the interaction of bacteria with the host. PMID:11553567

  12. Salt Reduction in a Model High-Salt Akawi Cheese: Effects on Bacterial Activity, pH, Moisture, Potential Bioactive Peptides, Amino Acids, and Growth of Human Colon Cells.

    PubMed

    Gandhi, Akanksha; Shah, Nagendra P

    2016-04-01

    This study evaluated the effects of sodium chloride reduction and its substitution with potassium chloride on Akawi cheese during storage for 30 d at 4 °C. Survival of probiotic bacteria (Lactobacillus acidophilus, Lactobacillus casei, and Bifidobacterium longum) and starter bacteria (Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus), angiotensin-converting enzyme-inhibitory and antioxidant activities, and concentrations of standard amino acids as affected by storage in different brine solutions (10% NaCl, 7.5% NaCl, 7.5% NaCl+KCl [1:1], 5% NaCl, and 5% NaCl+KCl [1:1]) were investigated. Furthermore, viability of human colon cells and human colon cancer cells as affected by the extract showing improved peptide profiles, highest release of amino acids and antioxidant activity (that is, from cheese brined in 7.5% NaCl+KCl) was evaluated. Significant increase was observed in survival of probiotic bacteria in cheeses with low salt after 30 d. Calcium content decreased slightly during storage in all cheeses brined in various solutions. Further, no significant changes were observed in ACE-inhibitory activity and antioxidant activity of cheeses during storage. Interestingly, concentrations of 4 essential amino acids (phenylalanine, tryptophan, valine, and leucine) increased significantly during storage in brine solutions containing 7.5% total salt. Low concentration of cheese extract (100 μg/mL) significantly improved the growth of normal human colon cells, and reduced the growth of human colon cancer cells. Overall, the study revealed that cheese extracts from reduced-NaCl brine improved the growth of human colon cells, and the release of essential amino acids, but did not affect the activities of potential bioactive peptides. PMID:26919457

  13. Adenoviral augmentation of elafin protects the lung against acute injury mediated by activated neutrophils and bacterial infection.

    PubMed

    Simpson, A J; Wallace, W A; Marsden, M E; Govan, J R; Porteous, D J; Haslett, C; Sallenave, J M

    2001-08-01

    During acute pulmonary infection, tissue injury may be secondary to the effects of bacterial products or to the effects of the host inflammatory response. An attractive strategy for tissue protection in this setting would combine antimicrobial activity with inhibition of human neutrophil elastase (HNE), a key effector of neutrophil-mediated tissue injury. We postulated that genetic augmentation of elafin (an endogenous inhibitor of HNE with intrinsic antimicrobial activity) could protect the lung against acute inflammatory injury without detriment to host defense. A replication-deficient adenovirus encoding elafin cDNA significantly protected A549 cells against the injurious effects of both HNE and whole activated human neutrophils in vitro. Intratracheal replication-deficient adenovirus encoding elafin cDNA significantly protected murine lungs against injury mediated by Pseudomonas aeruginosa in vivo. Genetic augmentation of elafin therefore has the capacity to protect the lung against the injurious effects of both bacterial pathogens resistant to conventional antibiotics and activated neutrophils. PMID:11466403

  14. Keratinocyte Production of Cathelicidin Provides Direct Activity against Bacterial Skin Pathogens

    PubMed Central

    Braff, Marissa H.; Zaiou, Mohamed; Fierer, Joshua; Nizet, Victor; Gallo, Richard L.

    2005-01-01

    Immune defense at an interface with the external environment reflects the functions of physical and chemical barriers provided by epithelial and immune cells. Resident epithelial cells, such as keratinocytes, produce numerous peptides with direct antimicrobial activity but also provide a physical barrier against invading pathogens and signal the recruitment of circulating immune cells, such as neutrophils. Antimicrobial peptides such as cathelicidin are produced constitutively by neutrophils and are inducible in keratinocytes in response to infection. The multiplicity of antimicrobial peptides and their cellular sources has resulted in an incomplete understanding of the role of cathelicidin production by epithelial cells in cutaneous immune defense. Therefore, this study sought to evaluate keratinocyte antimicrobial activity and the potential contribution of keratinocyte cathelicidin to host protection against two leading human skin pathogens. Wild-type mice and those with a targeted deletion of the cathelicidin gene, Cnlp, were rendered neutropenic prior to cutaneous infection. Interestingly, Cnlp-deficient mice remained more susceptible to group A streptococcus infection than mice with Cnlp intact, suggesting the involvement of epithelial cell-derived cathelicidin in host immune defense. Keratinocytes were then isolated in culture and found to inhibit the growth of Staphylococcus aureus, an effect that was partially dependent on their ability to synthesize and activate cathelicidin. Further, lentivirus-mediated delivery of activated human cathelicidin enhanced keratinocyte antimicrobial activity. Combined, these data illustrate the potential contribution of keratinocyte cathelicidin to the innate immune defense of skin against bacterial pathogens and highlight the need to consider epithelial antimicrobial function in the diagnosis and therapy of skin infection. PMID:16177355

  15. Bacterial biomass, metabolic state, and activity in stream sediments: relation to environmental variables and multiple assay comparisons.

    PubMed

    Bott, T L; Kaplan, L A

    1985-08-01

    Bacterial biomass, metabolic condition, and activity were measured over a 16-month period in the surface sediments of the following four field sites with differing dissolved organic matter regimes: a woodlot spring seep, a meadow spring seep, a second-order stream, and a third-order stream. Total bacterial biomass was measured by lipid phosphate and epifluorescence microscopic counts (EMC), and viable biomass was measured by C most probable number, EMC with 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride reduction, and ATP. Bacterial metabolic condition was determined from the percentage of respiring cells, poly-beta-hydroxybutyrate concentrations, and adenylate energy charge. Activity measures included C-lipid synthesis, P-phospholipid synthesis, the rate of uptake of algal lysate dissolved organic carbon, and respiration, from which biosynthesis was calculated (dissolved organic carbon uptake corrected for respiration). Total bacterial biomass (from EMC) ranged from 0.012 to 0.354 mug of C/mg of dry sediment and was usually lowest in the third-order stream. The percentage of cells respiring was less than 25% at all sites, indicating that most bacteria were dormant or dead. Adenylate energy charge was measured only in the third-order stream and was uniformly low. Poly-beta-hydroxybutyrate concentrations were greater in the woodlot spring seep than in the second- and third-order streams. Uptake of algal lysate dissolved organic carbon ranged from undetectable levels to 166 mg of C . m . h. Little community respiration could be attributed to algal lysate metabolism. Phospholipid synthesis ranged from 0.006 to 0.354 pmol . mg of dry sediment . h. Phospholipid synthesis rates were used to estimate bacterial turnover at the study sites. An estimated 375 bacterial generations per year were produced in the woodlot spring seep, and 67 per year were produced in the third-order stream. PMID:16346867

  16. Facile method to stain the bacterial cell surface for super-resolution fluorescence microscopy

    SciTech Connect

    Gunsolus, Ian L.; Hu, Dehong; Mihai, Cosmin; Lohse, Samuel E.; Lee, Chang-Soo; Torelli, Marco; Hamers, Robert J.; Murphy, Catherine; Orr, Galya; Haynes, Christy L.

    2014-01-01

    A method to fluorescently stain the surfaces of both Gram-negative and Gram-positive bacterial cells compatible with super-resolution fluorescence microscopy is presented. This method utilizes a commercially-available fluorescent probe to label primary amines at the surface of the cell. We demonstrate efficient staining of two bacterial strains, the Gram-negative Shewanella oneidensis MR-1 and the Gram-positive Bacillus subtilis 168. Using structured illumination microscopy and stochastic optical reconstruction microscopy, which require high quantum yield or specialized dyes, we show that this staining method may be used to resolve the bacterial cell surface with sub-diffraction-limited resolution. We further use this method to identify localization patterns of nanomaterials, specifically cadmium selenide quantum dots, following interaction with bacterial cells.

  17. Probing interaction of gram-positive and gram-negative bacterial cells with ZnO nanorods.

    PubMed

    Jain, Aanchal; Bhargava, Richa; Poddar, Pankaj

    2013-04-01

    In the present work, the physiological effects of the ZnO nanorods on the Gram positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Escherichia coli and Aerobacter aerogenes) bacterial cells have been studied. The analysis of bacterial growth curves for various concentrations of ZnO nanorods indicates that Gram positive and Gram negative bacterial cells show inhibition at concentrations of ~64 and ~256 μg/mL respectively. The marked difference in susceptibility towards nanorods was also validated by spread plate and disk diffusion methods. In addition, the scanning electron micrographs show a clear damage to the cells via changed morphology of the cells from rod to coccoid etc. The confocal optical microscopy images of these cells also demonstrate the reduction in live cell count in the presence of ZnO nanorods. These, results clearly indicate that the antibacterial activity of ZnO nanorods is higher towards Gram positive bacterium than Gram negative bacterium which indicates that the structure of the cell wall might play a major role in the interaction with nanostructured materials and shows high sensitivity to the particle concentration. PMID:23827568

  18. Controlled delivery of bioactive molecules into live cells using the bacterial mechanosensitive channel MscL

    PubMed Central

    Doerner, Julia F.; Febvay, Sebastien; Clapham, David E.

    2013-01-01

    Bacterial mechanosensitive channels are some of the largest pores in nature. In particular, MscL, with a pore diameter > 25 Å, allows passage of large organic ions and small proteins. Functional MscL reconstitution into lipids has been proposed for applications in vesicular-based drug release. Here we show that these channels can be functionally expressed in mammalian cells to afford rapid controlled uptake of membrane impermeable molecules. We first demonstrate that MscL gating in response to increased membrane tension is preserved in mammalian cell membranes. Molecular delivery is controlled by adopting an established method of MscL charge-induced activation. We then determine pore size limitations using fluorescently labeled model cargoes. Finally, we activate MscL to introduce the cell-impermeable bi-cyclic peptide phalloidin, a specific marker for actin filaments, into cells. We propose that MscL will be a useful tool for gated and controlled delivery of bioactive molecules into cells. PMID:22871809

  19. Dipeptide-Based Metabolic Labeling of Bacterial Cells for Endogenous Antibody Recruitment

    PubMed Central

    2016-01-01

    The number of antibiotic-resistant bacterial infections has increased dramatically over the past decade. To combat these pathogens, novel antimicrobial strategies must be explored and developed. We previously reported a strategy based on hapten-modified cell wall analogues to induce recruitment of endogenous antibodies to bacterial cell surfaces. Cell surface remodeling using unnatural single d-amino acid cell wall analogues led to modification at the C-terminus of the peptidoglycan stem peptide. During peptidoglycan processing, installed hapten-displaying amino acids can be subsequently removed by cell wall enzymes. Herein, we disclose a two-step dipeptide peptidoglycan remodeling strategy aimed at introducing haptens at an alternative site within the stem peptide to improve retention and diminish removal by cell wall enzymes. Through this redesigned strategy, we determined size constraints of peptidoglycan remodeling and applied these constraints to attain hapten–linker conjugates that produced high levels of antibody recruitment to bacterial cell surfaces. PMID:27294199

  20. Phase Diagram of Collective Motion of Bacterial Cells in a Shallow Circular Pool

    NASA Astrophysics Data System (ADS)

    Wakita, Jun-ichi; Tsukamoto, Shota; Yamamoto, Ken; Katori, Makoto; Yamada, Yasuyuki

    2015-12-01

    The collective motion of bacterial cells in a shallow circular pool is systematically studied using the bacterial species Bacillus subtilis. The ratio of cell length to pool diameter (i.e., the reduced cell length) ranges from 0.06 to 0.43 in our experiments. Bacterial cells in a circular pool show various types of collective motion depending on the cell density in the pool and the reduced cell length. The motion is classified into six types, which we call random motion, turbulent motion, one-way rotational motion, two-way rotational motion, random oscillatory motion, and ordered oscillatory motion. Two critical values of reduced cell lengths are evaluated, at which drastic changes in collective motion are induced. A phase diagram is proposed in which the six phases are arranged.

  1. Nano-engineered living bacterial motors for active microfluidic mixing.

    PubMed

    Al-Fandi, M; Jaradat, M A K; Fandi, K; Beech, J P; Tegenfeldt, J O; Yih, T C

    2010-09-01

    Active micromixers with rotating elements are attractive microfluidic actuators in many applications because of their mixing ability at a short distance. However, miniaturising the impeller design poses technical challenges including the fabrication and driving means. As a possible solution inspired by macro magnetic bar-stirrers, this study proposes the use of tethered, rotating bacteria as mixing elements. A tethered cell is a genetically engineered, harmless Escherichia coli (E. coli) attached to a surface by a single, shortened flagellum. The tethered flagellum acts as a pivot around which the entire cell body smoothly rotates. Videomicroscopy, image analysis and computational fluid dynamics (CFD) are utilised to demonstrate a proof-of-concept for the micro mixing process. Flow visualisation experiments show that a approximately 3 microm long tethered E. coli rotating at approximately 240 rpm can circulate a 1 microm polystyrene bead in the adjacent area at an average speed of nearly 4 microm/s. The Peclet (Peb) number for the stirred bead is evaluated to approximately 4. CFD simulations show that the rotary motion of a tethered E. coli rotating at 240 rpm can generate fluid velocities, up to 37 microm/s bordering the cell envelop. Based on these simulations, the Strouhal number (St) is calculated to about 2. This hybrid bio-inorganic micromxer could be used as a local, disposable mixer. PMID:20726672

  2. Sulfotransferase-independent genotoxicity of illudin S and its acylfulvene derivatives in bacterial and mammalian cells.

    PubMed

    Glatt, Hansruedi; Pietsch, Kathryn E; Sturla, Shana J; Meinl, Walter

    2014-01-01

    Acylfulvenes are a class of antitumor agents derived from illudin S, a sesquiterpenoid toxin isolated from mushrooms of the genus Omphalotus. Although DNA appears to be their major target, no data concerning mutagenicity of acylfulvenes are available in the literature, and limited data have been published on illudin S. Enzyme-mediated biotransformations have been demonstrated to influence the cytotoxicity of acylfulvenes. Illudin S and some acylfulvenes [e.g., (-)-6-hydroxymethylacylfulvene (HMAF)] are allylic alcohols with potential for enhanced cytotoxicity and genotoxicity by means of metabolic sulfation. Therefore, we studied the influence of various heterologously expressed human sulfotransferases (SULTs) on biological activities of illudin S and HMAF in bacterial and mammalian cells. (-)-Acylfulvene (AF) was tested as a congener lacking an allylic hydroxyl group. We found: (1) all three compounds were mutagenic in standard Salmonella typhimurium strains TA98, TA100 and TA104; (2) they induced gene mutations (at the hypoxanthine phosphoribosyl transferase locus) and sister chromatid exchange (SCE) in Chinese hamster V79 cells; (3) these effects were practically unaffected when human SULTs were expressed in the target bacteria or mammalian cells (using SCE as the endpoint); (4) illudin S demonstrated 40-600 times higher genotoxic activities than the semisynthetic acylfulvenes studied; it was positive in the SCE test even at a concentration of 0.3 nM; (5) genotoxicity in mammalian cells was observed at substantially lower concentrations of the compounds than required for a positive result in the bacterial test (400 nM with illudin S). We conclude that illudin S, HMAF and AF are potent genotoxicants and human SULTs do not play a significant role in their bioactivation. PMID:23881331

  3. Influence of dietary nucleotide restriction on bacterial sepsis and phagocytic cell function in mice.

    PubMed

    Kulkarni, A D; Fanslow, W C; Drath, D B; Rudolph, F B; Van Buren, C T

    1986-02-01

    Although enzyme defects in purine metabolism have revealed the importance of these substrates to maintenance of a normal immune response, the role of exogenous nucleotides on the cells that mediate the host defense system has remained largely unexplored. Recent investigations have revealed that dietary nucleotides are vital to the maintenance of cell-mediated responses to antigen stimulation. To test the influence of dietary nucleotide deprivation on resistance to infection, Balb/c mice were maintained on chow, a nucleotide-free (NF) diet, or an NF diet repleted with adenine, uracil, or RNA. Mice on the NF diet suffered 100% mortality following intravenous challenge with Staphylococcus aureus, while chow-fed and RNA- or uracil-repleted mice demonstrated significantly greater resistance to this bacterial challenge. Macrophages from mice on the NF diet had decreased phagocytic activity as measured by uptake of radiolabeled bacteria compared with mice maintained on the NF diet supplemented with adenine, uracil, or RNA. No change in S aureus antibody response was noted on the various diets. Although the mechanism of this suppression of nonspecific immunity remains unclear, provision of nucleotides to defined diets appears vital to maintain host resistance to bacterial challenge. PMID:3947217

  4. Cell fate regulation governed by a repurposed bacterial histidine kinase

    SciTech Connect

    Childers, W. Seth; Xu, Qingping; Mann, Thomas H.; Mathews, Irimpan I.; Blair, Jimmy A.; Deacon, Ashley M.; Shapiro, Lucy; Stock, Ann M.

    2014-10-28

    One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR) DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK~P over DivK, which is modulated by an allosteric intramolecular interaction between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK)-RR systems and coupling a complex network of signaling proteins for cell-fate regulation.

  5. Cell Fate Regulation Governed by a Repurposed Bacterial Histidine Kinase

    PubMed Central

    Mann, Thomas H.; Mathews, Irimpan I.; Blair, Jimmy A.; Deacon, Ashley M.; Shapiro, Lucy

    2014-01-01

    One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR) DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK∼P over DivK, which is modulated by an allosteric intramolecular interaction between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK)-RR systems and coupling a complex network of signaling proteins for cell-fate regulation. PMID:25349992

  6. Cell fate regulation governed by a repurposed bacterial histidine kinase

    DOE PAGESBeta

    Childers, W. Seth; Xu, Qingping; Mann, Thomas H.; Mathews, Irimpan I.; Blair, Jimmy A.; Deacon, Ashley M.; Shapiro, Lucy; Stock, Ann M.

    2014-10-28

    One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR) DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK~P over DivK, which is modulated by an allosteric intramolecular interactionmore » between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK)-RR systems and coupling a complex network of signaling proteins for cell-fate regulation.« less

  7. Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB

    PubMed Central

    Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D.; Garner, Ethan C.; Walker, Suzanne

    2014-01-01

    Summary The bacterial actin homolog MreB, which is critical for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids, but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis. PMID:25402772

  8. S9-DEPENDENT ACTIVATION OF 1-NITROPYRENE AND 3-NITROFLUORANTHENE IN BACTERIAL MUTAGENICITY ASSAYS

    EPA Science Inventory

    Nitro-substituted polycyclic aromatic hydrocarbons (N02PAH) such as 1-nitropyrene (NP) and 3-nitrofluoranthene (3-NFA), both widespread environmental mutagens, generally require activation by bacterial nitroreductases for maximal expression of their mutagenicity in the Ames Salmo...

  9. DNA-crosslinker cisplatin eradicates bacterial persister cells.

    PubMed

    Chowdhury, Nityananda; Wood, Thammajun L; Martínez-Vázquez, Mariano; García-Contreras, Rodolfo; Wood, Thomas K

    2016-09-01

    For all bacteria, nearly every antimicrobial fails since a subpopulation of the bacteria enter a dormant state known as persistence, in which the antimicrobials are rendered ineffective due to the lack of metabolism. This tolerance to antibiotics makes microbial infections the leading cause of death worldwide and makes treating chronic infections, including those of wounds problematic. Here, we show that the FDA-approved anti-cancer drug cisplatin [cis-diamminodichloroplatinum(II)], which mainly forms intra-strand DNA crosslinks, eradicates Escherichia coli K-12 persister cells through a growth-independent mechanism. Additionally, cisplatin is more effective at killing Pseudomonas aeruginosa persister cells than mitomycin C, which forms inter-strand DNA crosslinks, and cisplatin eradicates the persister cells of several pathogens including enterohemorrhagic E. coli, Staphylococcus aureus, and P. aeruginosa. Cisplatin was also highly effective against clinical isolates of S. aureus and P. aeruginosa. Therefore, cisplatin has broad spectrum activity against persister cells. Biotechnol. Bioeng. 2016;113: 1984-1992. © 2016 Wiley Periodicals, Inc. PMID:26914280

  10. Yeast cell wall extract induces disease resistance against bacterial and fungal pathogens in Arabidopsis thaliana and Brassica crop.

    PubMed

    Narusaka, Mari; Minami, Taichi; Iwabuchi, Chikako; Hamasaki, Takashi; Takasaki, Satoko; Kawamura, Kimito; Narusaka, Yoshihiro

    2015-01-01

    Housaku Monogatari (HM) is a plant activator prepared from a yeast cell wall extract. We examined the efficacy of HM application and observed that HM treatment increased the resistance of Arabidopsis thaliana and Brassica rapa leaves to bacterial and fungal infections. HM reduced the severity of bacterial leaf spot and anthracnose on A. thaliana and Brassica crop leaves with protective effects. In addition, gene expression analysis of A. thaliana plants after treatment with HM indicated increased expression of several plant defense-related genes. HM treatment appears to induce early activation of jasmonate/ethylene and late activation of salicylic acid (SA) pathways. Analysis using signaling mutants revealed that HM required SA accumulation and SA signaling to facilitate resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola and the fungal pathogen Colletotrichum higginsianum. In addition, HM-induced resistance conferred chitin-independent disease resistance to bacterial pathogens in A. thaliana. These results suggest that HM contains multiple microbe-associated molecular patterns that activate defense responses in plants. These findings suggest that the application of HM is a useful tool that may facilitate new disease control methods. PMID:25565273

  11. Yeast Cell Wall Extract Induces Disease Resistance against Bacterial and Fungal Pathogens in Arabidopsis thaliana and Brassica Crop

    PubMed Central

    Narusaka, Mari; Minami, Taichi; Iwabuchi, Chikako; Hamasaki, Takashi; Takasaki, Satoko; Kawamura, Kimito; Narusaka, Yoshihiro

    2015-01-01

    Housaku Monogatari (HM) is a plant activator prepared from a yeast cell wall extract. We examined the efficacy of HM application and observed that HM treatment increased the resistance of Arabidopsis thaliana and Brassica rapa leaves to bacterial and fungal infections. HM reduced the severity of bacterial leaf spot and anthracnose on A. thaliana and Brassica crop leaves with protective effects. In addition, gene expression analysis of A. thaliana plants after treatment with HM indicated increased expression of several plant defense-related genes. HM treatment appears to induce early activation of jasmonate/ethylene and late activation of salicylic acid (SA) pathways. Analysis using signaling mutants revealed that HM required SA accumulation and SA signaling to facilitate resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola and the fungal pathogen Colletotrichum higginsianum. In addition, HM-induced resistance conferred chitin-independent disease resistance to bacterial pathogens in A. thaliana. These results suggest that HM contains multiple microbe-associated molecular patterns that activate defense responses in plants. These findings suggest that the application of HM is a useful tool that may facilitate new disease control methods. PMID:25565273

  12. Mechanism of cell integration on biomaterial implant surfaces in the presence of bacterial contamination.

    PubMed

    Yue, Chongxia; van der Mei, Henny C; Kuijer, Roel; Busscher, Henk J; Rochford, Edward T J

    2015-11-01

    Bacterial contamination during biomaterial implantation is often unavoidable, yielding a combat between cells and bacteria. Here we aim to determine the modulatory function of bacterial components on stem-cell, fibroblast, and osteoblast adhesion to a titanium alloy, including the role of toll-like-receptors (TLRs). Presence of heat-sacrificed Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, or Pseudomonas aeruginosa induced dose and cell-type dependent responses. Stem-cells were most sensitive to bacterial presence, demonstrating decreased adhesion number yet increased adhesion effort with a relatively large focal adhesion contact area. Blocking TLRs had no effect on stem-cell adhesion in presence of S. aureus, but blocking both TLR2 and TLR4 induced an increased adhesion effort in presence of E. coli. Neither lipopolysaccharide, lipoteichoic acid, nor bacterial DNA provoked the same cell response as did whole bacteria. Herewith we suggest a new mechanism as to how biomaterials are integrated by cells despite the unavoidable presence of bacterial contamination. Stimulation of host cell integration of implant surfaces may open a new window to design new biomaterials with enhanced healing, thereby reducing the risk of biomaterial-associated infection of both "hardware-based" implants as well as of tissue-engineered constructs, known to suffer from similarly high infection risks as currently prevailing in "hardware-based" implants. PMID:25966819

  13. Bacterial Surface Appendages Strongly Impact Nanomechanical and Electrokinetic Properties of Escherichia coli Cells Subjected to Osmotic Stress

    PubMed Central

    Francius, Grégory; Polyakov, Pavel; Merlin, Jenny; Abe, Yumiko; Ghigo, Jean-Marc; Merlin, Christophe; Beloin, Christophe; Duval, Jérôme F. L.

    2011-01-01

    The physicochemical properties and dynamics of bacterial envelope, play a major role in bacterial activity. In this study, the morphological, nanomechanical and electrohydrodynamic properties of Escherichia coli K-12 mutant cells were thoroughly investigated as a function of bulk medium ionic strength using atomic force microscopy (AFM) and electrokinetics (electrophoresis). Bacteria were differing according to genetic alterations controlling the production of different surface appendages (short and rigid Ag43 adhesins, longer and more flexible type 1 fimbriae and F pilus). From the analysis of the spatially resolved force curves, it is shown that cells elasticity and turgor pressure are not only depending on bulk salt concentration but also on the presence/absence and nature of surface appendage. In 1 mM KNO3, cells without appendages or cells surrounded by Ag43 exhibit large Young moduli and turgor pressures (∼700–900 kPa and ∼100–300 kPa respectively). Under similar ionic strength condition, a dramatic ∼50% to ∼70% decrease of these nanomechanical parameters was evidenced for cells with appendages. Qualitatively, such dependence of nanomechanical behavior on surface organization remains when increasing medium salt content to 100 mM, even though, quantitatively, differences are marked to a much smaller extent. Additionally, for a given surface appendage, the magnitude of the nanomechanical parameters decreases significantly when increasing bulk salt concentration. This effect is ascribed to a bacterial exoosmotic water loss resulting in a combined contraction of bacterial cytoplasm together with an electrostatically-driven shrinkage of the surface appendages. The former process is demonstrated upon AFM analysis, while the latter, inaccessible upon AFM imaging, is inferred from electrophoretic data interpreted according to advanced soft particle electrokinetic theory. Altogether, AFM and electrokinetic results clearly demonstrate the intimate

  14. Improvement of antimicrobial activity of graphene oxide/bacterial cellulose nanocomposites through the electrostatic modification.

    PubMed

    Yang, Xiao-Ning; Xue, Dong-Dong; Li, Jia-Ying; Liu, Miao; Jia, Shi-Ru; Chu, Li-Qiang; Wahid, Fazli; Zhang, Yu-Ming; Zhong, Cheng

    2016-01-20

    Graphene oxide (GO) has an attracting and ever-growing interest in various research fields for its fascinating nanostructures. In this study, bacterial cellulose (BC) was used as a matrix to synthesize GO-based materials by a mechanical mixing method. The modification of GO with PEI significantly improved the bonding force between GO nanofillers and BC matrix. The morphology of the nanocomposites had a significant effect on the mechanical properties, hydrophilic properties as well as the antibacterial activity. After the modification, the GO-PEI/BC showed a strong antimicrobial effect on Saccharomyces cerevisiae due to the effective direct contacts between the nanofillers of the composites and the cell surfaces. This study demonstrates that the morphology of the nanocomposites has a great effect on physiochemical properties and the interactions between the microorganism and the nanocomposites. PMID:26572458

  15. NAIPs: building an innate immune barrier against bacterial pathogens. NAIPs function as sensors that initiate innate immunity by detection of bacterial proteins in the host cell cytosol.

    PubMed

    Kofoed, Eric M; Vance, Russell E

    2012-07-01

    The innate immune system of mammals encodes several families of immune detector proteins that monitor the cytosol for signs of pathogen invasion. One important but poorly understood family of cytosolic immunosurveillance proteins is the NLR (nucleotide-binding domain, leucine-rich repeat containing) proteins. Recent work has demonstrated that one subfamily of NLRs, the NAIPs (NLR family, apoptosis inhibitory proteins), are activated by specific interaction with bacterial ligands, such as flagellin. NAIP activation leads to assembly of a large multiprotein complex called the inflammasome, which initiates innate immune responses by activation of the Caspase-1 protease. NAIPs therefore appear to detect pathogen molecules via a simple and direct receptor-ligand mechanism. Interestingly, other NLR family members appear to detect pathogens indirectly, perhaps by responding to host cell "stress" caused by the pathogen. Thus, the NLR family may have evolved surprisingly diverse mechanisms for detecting pathogens. PMID:22513803

  16. [Algicidal activity against red-tide algaes by marine bacterial strain N3 isolated from a HABs area, southern China].

    PubMed

    Shi, Rong-jun; Huang, Hong-hui; Qi, Zhan-hui; Hu, Wei-an; Tian, Zi-yang; Dai, Ming

    2013-05-01

    A marine algicidal bacterium N3 was isolated from a HABs area in Mirs Bay, a subtropical bay, in southern China. Algicidal activity and algicidal mode against Phaeodactylum tricornutum, Scrippsiella trochoidea, Prorocentrum micans and Skeletonema costatum were observed by the liquid infection method. The results showed that there were no algicidal activities against P. tricornutum and S. costatum. However, when the bacterial volume fractions were 2% and 10% , S. trochoidea and P. micans could be killed, respectively. S. trochoidea cells which were exposed to strain N3 became irregular in shape and the cellular components lost their integrity and were decomposed. While, the P. micans cells became inflated and the cellular components aggregated, followed by cell lysis. Strain N3 killed S. trochoidea and P. micans directly, and the algicidal activities of the bacterial strain N3 was concentration-dependent. To S. trochoidea, 2% (V/V) of bacteria in algae showed the strongest algicidal activity, all of the S. trochoidea cells were killed within 120 h. But the growth rates of cells, in the 1% and 0. 1% treatment groups, were only slightly lower than that in the control group. In all treatment groups, the densities of strain N3 were in declining trends. While, to P. micans, 10% and 5% of bacteria in algae showed strong algicidal activities, 78% and 70% of the S. trochoidea were killed within 120 h, respectively. However, the number of S. trochoidea after exposure to 1% of bacterial cultures still increased up to 5 incubation days. And in the three treatment groups, the densities of strain N3 experienced a decrease process. The isolated strain N3 was identified as Bacillus sp. by morphological observation, physiological and biochemical characterization, and homology comparisons based on 16S rRNA sequences. PMID:23914549

  17. Bacterial diversity is strongly associated with historical penguin activity in an Antarctic lake sediment profile.

    PubMed

    Zhu, Renbin; Shi, Yu; Ma, Dawei; Wang, Can; Xu, Hua; Chu, Haiyan

    2015-01-01

    Current penguin activity in Antarctica affects the geochemistry of sediments and their microbial communities; the effects of historical penguin activity are less well understood. Here, bacterial diversity in ornithogenic sediment was investigated using high-throughput pyrosequencing. The relative abundances of dominant phyla were controlled by the amount of historical penguin guano deposition. Significant positive correlations were found between both the bacterial richness and diversity, and the relative penguin number (p < 0.01); this indicated that historical penguin activity drove the vertical distribution of the bacterial communities. The lowest relative abundances of individual phyla corresponded to lowest number of penguin population at 1,800-2,300 yr BP during a drier and colder period; the opposite was observed during a moister and warmer climate (1,400-1,800 yr BP). This study shows that changes in the climate over millennia affected penguin populations and the outcomes of these changes affect the sediment bacterial community today. PMID:26601753

  18. Determination of bacterial activity by use of an evanescent-wave fiber-optic sensor.

    PubMed

    John, M Shelly; Kishen, Anil; Sing, Lim Chu; Asundi, Anand

    2002-12-01

    A novel technique based on fiber-optic evanescent-wave spectroscopy is proposed for the detection of bacterial activity in human saliva. The sensor determines th e specific concentration of Streptococcus mutans in saliva, which is a major causative factor in dental caries. In this design, one prepares the fiber-optic bacterial sensor by replacing a portion of the cladding region of a multimode fiber with a dye-encapsulated xerogel, using the solgel technique. The exponential decay of the evanescent wave at the core-cladding interface of a multimode fiber is utilized for the determination of bacterial activity in saliva. The acidogenic profile of Streptococcus mutans is estimated by use of evanescent-waveabsorption spectra at various levels of bacterial activity. PMID:12477126

  19. Distributed Classifier Based on Genetically Engineered Bacterial Cell Cultures

    PubMed Central

    2015-01-01

    We describe a conceptual design of a distributed classifier formed by a population of genetically engineered microbial cells. The central idea is to create a complex classifier from a population of weak or simple classifiers. We create a master population of cells with randomized synthetic biosensor circuits that have a broad range of sensitivities toward chemical signals of interest that form the input vectors subject to classification. The randomized sensitivities are achieved by constructing a library of synthetic gene circuits with randomized control sequences (e.g., ribosome-binding sites) in the front element. The training procedure consists in reshaping of the master population in such a way that it collectively responds to the “positive” patterns of input signals by producing above-threshold output (e.g., fluorescent signal), and below-threshold output in case of the “negative” patterns. The population reshaping is achieved by presenting sequential examples and pruning the population using either graded selection/counterselection or by fluorescence-activated cell sorting (FACS). We demonstrate the feasibility of experimental implementation of such system computationally using a realistic model of the synthetic sensing gene circuits. PMID:25349924

  20. Peptidoglycan at its peaks: how chromatographic analyses can reveal bacterial cell-wall structure and assembly

    PubMed Central

    Desmarais, Samantha M.; De Pedro, Miguel A.; Cava, Felipe; Huang, Kerwyn Casey

    2013-01-01

    The peptidoglycan (PG) cell wall is a unique macromolecule responsible for both shape determination and cellular integrity under osmotic stress in virtually all bacteria. A quantitative understanding of the relationships between PG architecture, morphogenesis, immune system activation, and pathogenesis can provide molecular-scale insights into the function of proteins involved in cell-wall synthesis and cell growth. High Performance Liquid Chromatography (HPLC) has played an important role in our understanding of the structural and chemical complexity of the cell wall by providing an analytical method to quantify differences in chemical composition. Here, we present a primer on the basic chemical features of wall structure that can be revealed through HPLC, along with a description of the applications of HPLC PG analyses for interpreting the effects of genetic and chemical perturbations to a variety of bacterial species in different environments. We describe the physical consequences of different PG compositions on cell shape, and review complementary experimental and computational methodologies for PG analysis. Finally, we present a partial list of future targets of development for HPLC and related techniques. PMID:23679048

  1. Different binarization processes validated against manual counts of fluorescent bacterial cells.

    PubMed

    Tamminga, Gerrit G; Paulitsch-Fuchs, Astrid H; Jansen, Gijsbert J; Euverink, Gert-Jan W

    2016-09-01

    State of the art software methods (such as fixed value approaches or statistical approaches) to create a binary image of fluorescent bacterial cells are not as accurate and precise as they should be for counting bacteria and measuring their area. To overcome these bottlenecks, we introduce biological significance to obtain a binary image from a greyscale microscopic image. Using our biological significance approach we are able to automatically count about the same number of cells as an individual researcher would do by manual/visual counting. Using the fixed value or statistical approach to obtain a binary image leads to about 20% less cells in automatic counting. In our procedure we included the area measurements of the bacterial cells to determine the right parameters for background subtraction and threshold values. In an iterative process the threshold and background subtraction values were incremented until the number of particles smaller than a typical bacterial cell is less than the number of bacterial cells with a certain area. This research also shows that every image has a specific threshold with respect to the optical system, magnification and staining procedure as well as the exposure time. The biological significance approach shows that automatic counting can be performed with the same accuracy, precision and reproducibility as manual counting. The same approach can be used to count bacterial cells using different optical systems (Leica, Olympus and Navitar), magnification factors (200× and 400×), staining procedures (DNA (Propidium Iodide) and RNA (FISH)) and substrates (polycarbonate filter or glass). PMID:27380963

  2. MAIT cells are activated during human viral infections.

    PubMed

    van Wilgenburg, Bonnie; Scherwitzl, Iris; Hutchinson, Edward C; Leng, Tianqi; Kurioka, Ayako; Kulicke, Corinna; de Lara, Catherine; Cole, Suzanne; Vasanawathana, Sirijitt; Limpitikul, Wannee; Malasit, Prida; Young, Duncan; Denney, Laura; Moore, Michael D; Fabris, Paolo; Giordani, Maria Teresa; Oo, Ye Htun; Laidlaw, Stephen M; Dustin, Lynn B; Ho, Ling-Pei; Thompson, Fiona M; Ramamurthy, Narayan; Mongkolsapaya, Juthathip; Willberg, Christian B; Screaton, Gavin R; Klenerman, Paul

    2016-01-01

    Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize bacterial ligands. Here, we demonstrate that MAIT cells are also activated during human viral infections in vivo. MAIT cells activation was observed during infection with dengue virus, hepatitis C virus and influenza virus. This activation-driving cytokine release and Granzyme B upregulation-is TCR-independent but dependent on IL-18 in synergy with IL-12, IL-15 and/or interferon-α/β. IL-18 levels and MAIT cell activation correlate with disease severity in acute dengue infection. Furthermore, HCV treatment with interferon-α leads to specific MAIT cell activation in vivo in parallel with an enhanced therapeutic response. Moreover, TCR-independent activation of MAIT cells leads to a reduction of HCV replication in vitro mediated by IFN-γ. Together these data demonstrate MAIT cells are activated following viral infections, and suggest a potential role in both host defence and immunopathology. PMID:27337592

  3. Lysozyme-coated silver nanoparticles for differentiating bacterial strains on the basis of antibacterial activity

    NASA Astrophysics Data System (ADS)

    Ashraf, Sumaira; Chatha, Mariyam Asghar; Ejaz, Wardah; Janjua, Hussnain Ahmed; Hussain, Irshad

    2014-10-01

    Lysozyme, an antibacterial enzyme, was used as a stabilizing ligand for the synthesis of fairly uniform silver nanoparticles adopting various strategies. The synthesized particles were characterized using UV-visible spectroscopy, FTIR, dynamic light scattering (DLS), and TEM to observe their morphology and surface chemistry. The silver nanoparticles were evaluated for their antimicrobial activity against several bacterial species and various bacterial strains within the same species. The cationic silver nanoparticles were found to be more effective against Pseudomonas aeruginosa 3 compared to other bacterial species/strains investigated. Some of the bacterial strains of the same species showed variable antibacterial activity. The difference in antimicrobial activity of these particles has led to the conclusion that antimicrobial products formed from silver nanoparticles may not be equally effective against all the bacteria. This difference in the antibacterial activity of silver nanoparticles for different bacterial strains from the same species may be due to the genome islands that are acquired through horizontal gene transfer (HGT). These genome islands are expected to possess some genes that may encode enzymes to resist the antimicrobial activity of silver nanoparticles. These silver nanoparticles may thus also be used to differentiate some bacterial strains within the same species due to variable silver resistance of these variants, which may not possible by simple biochemical tests.

  4. Lysozyme-coated silver nanoparticles for differentiating bacterial strains on the basis of antibacterial activity

    PubMed Central

    2014-01-01

    Lysozyme, an antibacterial enzyme, was used as a stabilizing ligand for the synthesis of fairly uniform silver nanoparticles adopting various strategies. The synthesized particles were characterized using UV-visible spectroscopy, FTIR, dynamic light scattering (DLS), and TEM to observe their morphology and surface chemistry. The silver nanoparticles were evaluated for their antimicrobial activity against several bacterial species and various bacterial strains within the same species. The cationic silver nanoparticles were found to be more effective against Pseudomonas aeruginosa 3 compared to other bacterial species/strains investigated. Some of the bacterial strains of the same species showed variable antibacterial activity. The difference in antimicrobial activity of these particles has led to the conclusion that antimicrobial products formed from silver nanoparticles may not be equally effective against all the bacteria. This difference in the antibacterial activity of silver nanoparticles for different bacterial strains from the same species may be due to the genome islands that are acquired through horizontal gene transfer (HGT). These genome islands are expected to possess some genes that may encode enzymes to resist the antimicrobial activity of silver nanoparticles. These silver nanoparticles may thus also be used to differentiate some bacterial strains within the same species due to variable silver resistance of these variants, which may not possible by simple biochemical tests. PMID:25435831

  5. Attachment of bacterial pathogens to a bacterial cellulose-derived plant cell wall model: a proof of concept.

    PubMed

    Tan, Michelle S F; Wang, Yi; Dykes, Gary A

    2013-11-01

    This study aimed to establish, as a proof of concept, whether bacterial cellulose (BC)-derived plant cell wall models could be used to investigate foodborne bacterial pathogen attachment. Attachment of two strains each of Salmonella enterica and Listeria monocytogenes to four BC-derived plant cell wall models (namely, BC, BC-pectin [BCP], BC-xyloglucan [BCX], and BC-pectin-xyloglucan [BCPX]) was investigated. Chemical analysis indicated that the BCPX composite (31% cellulose, 45.6% pectin, 23.4% xyloglucan) had a composition typical of plant cell walls. The Salmonella strains attached in significantly (p<0.05) higher numbers (~6 log colony-forming units [CFU]/cm(2)) to the composites than the Listeria strains (~5 log CFU/cm(2)). Strain-specific differences were also apparent with one Salmonella strain, for example, attaching in significantly (p<0.05) higher numbers to the BCX composite than to the other composites. This study highlights the potential usefulness of these composites to understand attachment of foodborne bacteria to fresh produce. PMID:23941519

  6. Cell order in bacterial swarms arises from reversals of moving direction

    NASA Astrophysics Data System (ADS)

    Wu, Yilin; Jiang, Yi; Kaiser, Dale; Alber, Mark

    2010-03-01

    Bacterial swarms are a beautiful example of the emergent behavior of systems of self-propelled rods. In swarming rod-shaped bacteria cells move smoothly even though they are packed together in high density. Experimental evidence shows that long-distance signaling is not required for bacterial swarming. It naturally raises the question how a swarm develops its order. Using a biomechanical model, we show here that regular periodic reversals of gliding direction in general systems of self-propelled rod shaped bacteria can lead to the extensive ordering of cells. We also show that an optimal reversal period and an optimal cell shape exist for producing such order. Given the observations of reversing behavior in several bacterial species,we suggest that the capacity to swarm depends less on the motility engine employed by individual cells, but more on the behavioral algorithm that enhances the flow of densely packed cells near the swarming edge.

  7. Identification of individual biofilm-forming bacterial cells using Raman tweezers

    NASA Astrophysics Data System (ADS)

    Samek, Ota; Bernatová, Silvie; Ježek, Jan; Šiler, Martin; Šerý, Mojmir; Krzyžánek, Vladislav; Hrubanová, Kamila; Zemánek, Pavel; Holá, Veronika; Růžička, Filip

    2015-05-01

    A method for in vitro identification of individual bacterial cells is presented. The method is based on a combination of optical tweezers for spatial trapping of individual bacterial cells and Raman microspectroscopy for acquisition of spectral "Raman fingerprints" obtained from the trapped cell. Here, Raman spectra were taken from the biofilm-forming cells without the influence of an extracellular matrix and were compared with biofilm-negative cells. Results of principal component analyses of Raman spectra enabled us to distinguish between the two strains of Staphylococcus epidermidis. Thus, we propose that Raman tweezers can become the technique of choice for a clearer understanding of the processes involved in bacterial biofilms which constitute a highly privileged way of life for bacteria, protected from the external environment.

  8. Identification of individual biofilm-forming bacterial cells using Raman tweezers.

    PubMed

    Samek, Ota; Bernatová, Silvie; Ježek, Jan; Šiler, Martin; Šerý, Mojmir; Krzyžánek, Vladislav; Hrubanová, Kamila; Zemánek, Pavel; Holá, Veronika; Růžička, Filip

    2015-05-01

    A method for in vitro identification of individual bacterial cells is presented. The method is based on a combination of optical tweezers for spatial trapping of individual bacterial cells and Raman microspectroscopy for acquisition of spectral “Raman fingerprints” obtained from the trapped cell. Here, Raman spectra were taken from the biofilm-forming cells without the influence of an extracellular matrix and were compared with biofilm-negative cells. Results of principal component analyses of Raman spectra enabled us to distinguish between the two strains of Staphylococcus epidermidis. Thus, we propose that Raman tweezers can become the technique of choice for a clearer understanding of the processes involved in bacterial biofilms which constitute a highly privileged way of life for bacteria, protected from the external environment. PMID:25734616

  9. Lectin microarray reveals binding profiles of Lactobacillus casei strains in a comprehensive analysis of bacterial cell wall polysaccharides.

    PubMed

    Yasuda, Emi; Tateno, Hiroaki; Hirabayashi, Jun; Hirabarashi, Jun; Iino, Tohru; Sako, Tomoyuki

    2011-07-01

    We previously showed a pivotal role of the polysaccharide (PS) moiety in the cell wall of the Lactobacillus casei strain Shirota (YIT 9029) as a possible immune modulator (E. Yasuda M. Serata, and T. Sako, Appl. Environ. Microbiol. 74:4746-4755, 2008). To distinguish PS structures on the bacterial cell surface of individual strains in relation to their activities, it would be useful to have a rapid and high-throughput methodology. Recently, a new technique called lectin microarray was developed for rapid profiling of glycosylation in eukaryotic polymers and cell surfaces. Here, we report on the development of a simple and sensitive method based on this technology for direct analysis of intact bacterial cell surface glycomes. The method involves labeling bacterial cells with SYTOX Orange before incubation with the lectin microarray. After washing, bound cells are directly detected using an evanescent-field fluorescence scanner in a liquid phase. Using this method, we compared the cell surface glycomes from 16 different strains of L. casei. The patterns of lectin-binding affinity of most strains were found to be unique. There appears to be two types of lectin-binding profiles: the first is characterized by a few lectins, and the other is characterized by multiple lectins with different specificities. We also showed a dramatic change in the lectin-binding profile of a YIT 9029 derivative with a mutation in the cps1C gene, encoding a putative glycosyltransferase. In conclusion, the developed technique provided a novel strategy for rapid profiling and, more importantly, differentiating numerous bacterial strains with relevance to the biological functions of PS. PMID:21602390

  10. Lectin Microarray Reveals Binding Profiles of Lactobacillus casei Strains in a Comprehensive Analysis of Bacterial Cell Wall Polysaccharides▿†

    PubMed Central

    Yasuda, Emi; Tateno, Hiroaki; Hirabarashi, Jun; Iino, Tohru; Sako, Tomoyuki

    2011-01-01

    We previously showed a pivotal role of the polysaccharide (PS) moiety in the cell wall of the Lactobacillus casei strain Shirota (YIT 9029) as a possible immune modulator (E. Yasuda M. Serata, and T. Sako, Appl. Environ. Microbiol. 74:4746-4755, 2008). To distinguish PS structures on the bacterial cell surface of individual strains in relation to their activities, it would be useful to have a rapid and high-throughput methodology. Recently, a new technique called lectin microarray was developed for rapid profiling of glycosylation in eukaryotic polymers and cell surfaces. Here, we report on the development of a simple and sensitive method based on this technology for direct analysis of intact bacterial cell surface glycomes. The method involves labeling bacterial cells with SYTOX Orange before incubation with the lectin microarray. After washing, bound cells are directly detected using an evanescent-field fluorescence scanner in a liquid phase. Using this method, we compared the cell surface glycomes from 16 different strains of L. casei. The patterns of lectin-binding affinity of most strains were found to be unique. There appears to be two types of lectin-binding profiles: the first is characterized by a few lectins, and the other is characterized by multiple lectins with different specificities. We also showed a dramatic change in the lectin-binding profile of a YIT 9029 derivative with a mutation in the cps1C gene, encoding a putative glycosyltransferase. In conclusion, the developed technique provided a novel strategy for rapid profiling and, more importantly, differentiating numerous bacterial strains with relevance to the biological functions of PS. PMID:21602390

  11. Bacterial β-(1,3)-glucan prevents DSS-induced IBD by restoring the reduced population of regulatory T cells.

    PubMed

    Lee, Kwang-Ho; Park, Min; Ji, Kon-Young; Lee, Hwa-Youn; Jang, Ji-Hun; Yoon, Il-Joo; Oh, Seung-Su; Kim, Su-Man; Jeong, Yun-Hwa; Yun, Chul-Ho; Kim, Mi-Kyoung; Lee, In-Young; Choi, Ha-Rim; Ko, Ki-sung; Kang, Hyung-Sik

    2014-10-01

    Bacterial β-(1,3)-glucan has more advantages in terms of cost, yield and efficiency than that derived from mushrooms, plants, yeasts and fungi. We have previously developed a novel and high-yield β-(1,3)-glucan produced by Agrobacterium sp. R259. This study aimed to elucidate the functional mechanism and therapeutic efficacy of bacterial β-(1,3)-glucan in dextran sulfate sodium (DSS)-induced inflammatory bowel disease (IBD).Mice were orally pretreated with bacterial β-(1,3)-glucan at daily doses of 2.5 or 5mg/kg for 2 weeks. After 6 days of DSS treatment, clinical assessment of IBD severity and expression of pro-inflammatory cytokines were evaluated. In vivo cell proliferation was examined by immunohistochemistry using Ki-67 and ER-TR7 antibodies. The frequency of regulatory T cells (Tregs) was analyzed by flow cytometry. Natural killer (NK) activity and IgA level were evaluated using NK cytotoxicity assay and ELISA.The deterioration of body weight gain, colonic architecture, disease score and histological score was recovered in DSS-induced IBD mice when pretreated with bacterial β-(1,3)-glucan. The recruitment of macrophages and the gene expression of proinflammatory cytokines, such as IL-1β, IL-6 and IL-17A/F, were markedly decreased in the colon of β-(1,3)-glucan-pretreated mice. β-(1,3)-Glucan induced the recovery of Tregs in terms of their frequency in DSS-induced IBD mice. Intriguingly, β-(1,3)-glucan reversed the functional defects of NK cells and excessive IgA production in DSS-induced IBD mice.We conclude that bacterial β-(1,3)-glucan prevented the progression of DSS-induced IBD by recovering the reduction of Tregs, functional defect of NK cells and excessive IgA production. PMID:25092569

  12. Regulation of bacterial communities through antimicrobial activity by the coral holobiont.

    PubMed

    Kvennefors, E Charlotte E; Sampayo, Eugenia; Kerr, Caroline; Vieira, Genyess; Roff, George; Barnes, Andrew C

    2012-04-01

    Interactions between corals and associated bacteria and amongst these bacterial groups are likely to play a key role in coral health. However, the complexity of these interactions is poorly understood. We investigated the functional role of specific coral-associated bacteria in maintaining microbial communities on the coral Acropora millepora (Ehrenberg 1834) and the ability of coral mucus to support or inhibit bacterial growth. Culture-independent techniques were used to assess bacterial community structures whilst bacterial culture was employed to assess intra- and inter-specific antimicrobial activities of bacteria. Members of Pseudoalteromonas and ribotypes closely related to Vibrio coralliilyticus displayed potent antimicrobial activity against a range of other cultured isolates and grew readily on detached coral mucus. Although such bacterial ribotypes would be expected to have a competitive advantage, they were rare or absent on intact and healthy coral colonies growing in situ (analysed using denaturing gradient gel electrophoresis and 16S rRNA gene sequencing). The most abundant bacterial ribotypes found on healthy corals were Gammaproteobacteria, previously defined as type A coral associates. Our results indicate that this group of bacteria and specific members of the Alphaproteobacteria described here as 'type B associates' may be important functional groups for coral health. We suggest that bacterial communities on coral are kept in check by a combination of host-derived and microbial interactions and that the type A associates in particular may play a key role in maintaining stability of microbial communities on healthy coral colonies. PMID:21984347

  13. Electro-active hybrid actuators based on freeze-dried bacterial cellulose and PEDOT:PSS

    NASA Astrophysics Data System (ADS)

    Kim, Si-Seup; Jeon, Jin-Han; Kee, Chang-Doo; Oh, Il-Kwon

    2013-08-01

    We report a high-performance electro-active hybrid actuator based on freeze-dried bacterial cellulose and conducting polymer electrodes. The freeze-dried bacterial cellulose, which has a sponge form, can absorb a much greater amount of ionic liquid, which is a prerequisite for dry-type and high-performance electro-active polymers. In addition, the poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT:PSS) conducting layers are deposited on the top and bottom surfaces of the freeze-dried bacterial cellulose using a simple dipping and drying method. The results show that the freeze-dried bacterial cellulose actuator with conducting polymer electrodes has a much larger tip displacement under electrical stimuli than pure bacterial cellulose actuators with metallic electrodes. The large bending displacement of the freeze-dried bacterial cellulose actuator under low input voltage is due to the synergistic effects of the ion migration of the dissociated ionic liquids inside the bacterial cellulose and the electrochemical doping processes of the PEDOT:PSS electrode layers.

  14. Measuring bacterial adhesion at environmental interfaces with single-cell and single-molecule techniques

    NASA Astrophysics Data System (ADS)

    Camesano, Terri A.; Liu, Yatao; Datta, Meera

    2007-06-01

    A synopsis is provided of techniques currently used to quantify the interactions between bacterial cells and surfaces. Focus is placed on techniques which allow for direct probing of nano, pico, or femto-scale interaction forces between bacteria and surfaces of relevance for environmental science and engineering. We focus on bacterial adhesion measurements and surface characterizations via techniques that measure forces on individual bacterial cells or cellular macromolecules, particularly atomic force microscopy (AFM) and related force spectroscopy. However, we also include overviews of other techniques useful for evaluating cellular forces, such as optical tweezers, evanescent wave scattering-based techniques (i.e. total internal reflection microscopy (TIRM) and total internal reflection aqueous fluorescence (TIRAF) microscopy) and the quartz crystal microbalance (QCM). These latter techniques, while most are not providing direct measurements of forces of adhesion, can be used to explain adhesion and interaction forces in bacterial systems. We review the operating principles, advantages and limitations of each technique, and key bacterial adhesion studies from each area are presented. Qualitative and quantitative methodologies for relating force measurements to bacterial attachment, particularly to bacterial retention in porous media, are discussed.

  15. Innate immunity: Bacterial cell-wall muramyl peptide targets the conserved transcription factor YB-1.

    PubMed

    Laman, A G; Lathe, R; Savinov, G V; Shepelyakovskaya, A O; Boziev, Kh M; Baidakova, L K; Chulin, A N; Brovko, F A; Svirshchevskaya, E V; Kotelevtsev, Y; Eliseeva, I A; Guryanov, S G; Lyabin, D N; Ovchinnikov, L P; Ivanov, V T

    2015-07-01

    The bacterial cell wall muramyl dipeptides MDP and glucosaminyl-MDP (GMDP) are powerful immunostimulators but their binding target remains controversial. We previously reported expression cloning of GMDP-binding polypeptides and identification of Y-box protein 1 (YB-1) as their sole target. Here we show specific binding of GMDP to recombinant YB-1 protein and subcellular colocalization of YB-1 and GMDP. GMDP binding to YB-1 upregulated gene expression levels of NF-κB2, a mediator of innate immunity. Furthermore, YB-1 knockdown abolished GMDP-induced Nfkb2 expression. GMDP/YB-1 stimulation led to NF-κB2 cleavage, transport of activated NF-κB2 p52 to the nucleus, and upregulation of NF-κB2-dependent chemokine Cxcr4 gene expression. Therefore, our findings identify YB-1 as new target for muramyl peptide signaling. PMID:26026270

  16. Disruption of bacterial cell-to-cell communication by marine organisms and its relevance to aquaculture.

    PubMed

    Natrah, F M I; Defoirdt, Tom; Sorgeloos, Patrick; Bossier, Peter

    2011-04-01

    Bacterial disease is one of the most critical problems in commercial aquaculture. Although various methods and treatments have been developed to curb the problem, yet they still have significant drawbacks. A novel and environmental-friendly approach in solving this problem is through the disruption of bacterial communication or quorum sensing (QS). In this communication scheme, bacteria regulate their own gene expression by producing, releasing, and sensing chemical signals from the environment. There seems to be a link between QS and diseases through the regulation of certain phenotypes and the induction of virulence factors responsible for pathogen-host association. Several findings have reported that numerous aquatic organisms such as micro-algae, macro-algae, invertebrates, or even other bacteria have the potential to disrupt QS. The mechanism of action varies from degradation of signals through enzymatic or chemical inactivation to antagonistic as well as agonistic activities. This review focuses on the existing marine organisms that are able to interfere with QS with potential application for aquaculture as bacterial control. PMID:21246235

  17. Polymer-Ag nanocomposites with enhanced antimicrobial activity against bacterial infection.

    PubMed

    Mei, Lin; Lu, Zhentan; Zhang, Xinge; Li, Chaoxing; Jia, Yanxia

    2014-09-24

    Herein, a nontoxic nanocomposite is synthesized by reduction of silver nitrate in the presence of a cationic polymer displaying strong antimicrobial activity against bacterial infection. These nanocomposites with a large concentration of positive charge promote their adsorption to bacterial membranes through electrostatic interaction. Moreover, the synthesized nanocomposites with polyvalent and synergistic antimicrobial effects can effectively kill both Gram-positive and Gram-negative bacteria without the emergence of bacterial resistance. Morphological changes obtained by transmission electron microscope observation show that these nanocomposites can cause leakage and chaos of intracellular contents. Analysis of the antimicrobial mechanism confirms that the lethal action of nanocomposites against the bacteria started with disruption of the bacterial membrane, subsequent cellular internalization of the nanoparticles, and inhibition of intracellular enzymatic activity. This novel antimicrobial material with good cytocompatibility promotes healing of infected wounds in diabetic rats, and has a promising future in the treatment of other infectious diseases. PMID:25170799

  18. Bacterial Population Dynamics in a Laboratory Activated Sludge Reactor Monitored by Pyrosequencing of 16S rRNA

    PubMed Central

    Satoh, Hiroyasu; Oshima, Kenshiro; Suda, Wataru; Ranasinghe, Purnika; Li, Ning; Gunawardana, Egodaha Gedara Wasana; Hattori, Masahira; Mino, Takashi

    2013-01-01

    The microbial population in a laboratory activated sludge reactor was monitored for 245 d at 75 time points by pyrosequencing of 16S rRNA. Synthetic wastewater was used as the influent, and the reactor was operated under the same conditions throughout the experiment. The behaviors of different bacterial operational taxonomic units (OTUs) were observed. Multiple OTUs showed periodic propagation and recession. One of the OTUs showed sharp recession, which suggests that cells in the OTU were selectively killed. The behaviors of different phylogenetic lineages of Candidatus ‘Accumulibacter phosphatis’ were also visualized. It was clearly demonstrated that pyrosequencing with barcoded primers is a very effective tool to clarify the dynamics of the bacterial population in activated sludge. PMID:23100021

  19. Effects of zinc oxide nanoparticles on Kupffer cell phagosomal motility, bacterial clearance, and liver function

    PubMed Central

    Watson, Christa Y; Molina, Ramon M; Louzada, Andressa; Murdaugh, Kimberly M; Donaghey, Thomas C; Brain, Joseph D

    2015-01-01

    Background Zinc oxide engineered nanoparticles (ZnO ENPs) have potential as nanomedicines due to their inherent properties. Studies have described their pulmonary impact, but less is known about the consequences of ZnO ENP interactions with the liver. This study was designed to describe the effects of ZnO ENPs on the liver and Kupffer cells after intravenous (IV) administration. Materials and methods First, pharmacokinetic studies were conducted to determine the tissue distribution of neutron-activated 65ZnO ENPs post-IV injection in Wistar Han rats. Then, a noninvasive in vivo method to assess Kupffer cell phagosomal motility was employed using ferromagnetic iron particles and magnetometry. We also examined whether prior IV injection of ZnO ENPs altered Kupffer cell bactericidal activity on circulating Pseudomonas aeruginosa. Serum and liver tissues were collected to assess liver-injury biomarkers and histological changes, respectively. Results We found that the liver was the major site of initial uptake of 65ZnO ENPs. There was a time-dependent decrease in tissue levels of 65Zn in all organs examined, refecting particle dissolution. In vivo magnetometry showed a time-dependent and transient reduction in Kupffer cell phagosomal motility. Animals challenged with P. aeruginosa 24 hours post-ZnO ENP injection showed an initial (30 minutes) delay in vascular bacterial clearance. However, by 4 hours, IV-injected bacteria were cleared from the blood, liver, spleen, lungs, and kidneys. Seven days post-ZnO ENP injection, creatine phosphokinase and aspartate aminotransferase levels in serum were significantly increased. Histological evidence of hepatocyte damage and marginated neutrophils were observed in the liver. Conclusion Administration of ZnO ENPs transiently inhibited Kupffer cell phagosomal motility and later induced hepatocyte injury, but did not alter bacterial clearance from the blood or killing in the liver, spleen, lungs, or kidneys. Our data show that

  20. MAIT cells are activated during human viral infections

    PubMed Central

    van Wilgenburg, Bonnie; Scherwitzl, Iris; Hutchinson, Edward C.; Leng, Tianqi; Kurioka, Ayako; Kulicke, Corinna; de Lara, Catherine; Cole, Suzanne; Vasanawathana, Sirijitt; Limpitikul, Wannee; Malasit, Prida; Young, Duncan; Denney, Laura; Barnes, Eleanor; Ball, Jonathan; Burgess, Gary; Cooke, Graham; Dillon, John; Gore, Charles; Foster, Graham; Guha, Neil; Halford, Rachel; Herath, Cham; Holmes, Chris; Howe, Anita; Hudson, Emma; Irving, William; Khakoo, Salim; Koletzki, Diana; Martin, Natasha; Mbisa, Tamyo; McKeating, Jane; McLauchlan, John; Miners, Alec; Murray, Andrea; Shaw, Peter; Simmonds, Peter; Spencer, Chris; Targett-Adams, Paul; Thomson, Emma; Vickerman, Peter; Zitzmann, Nicole; Moore, Michael D.; Fabris, Paolo; Giordani, Maria Teresa; Oo, Ye Htun; Laidlaw, Stephen M.; Dustin, Lynn B.; Ho, Ling-Pei; Thompson, Fiona M.; Ramamurthy, Narayan; Mongkolsapaya, Juthathip; Willberg, Christian B.; Screaton, Gavin R.; Klenerman, Paul

    2016-01-01

    Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize bacterial ligands. Here, we demonstrate that MAIT cells are also activated during human viral infections in vivo. MAIT cells activation was observed during infection with dengue virus, hepatitis C virus and influenza virus. This activation—driving cytokine release and Granzyme B upregulation—is TCR-independent but dependent on IL-18 in synergy with IL-12, IL-15 and/or interferon-α/β. IL-18 levels and MAIT cell activation correlate with disease severity in acute dengue infection. Furthermore, HCV treatment with interferon-α leads to specific MAIT cell activation in vivo in parallel with an enhanced therapeutic response. Moreover, TCR-independent activation of MAIT cells leads to a reduction of HCV replication in vitro mediated by IFN-γ. Together these data demonstrate MAIT cells are activated following viral infections, and suggest a potential role in both host defence and immunopathology. PMID:27337592

  1. Predominance of single bacterial cells in composting bioaerosols

    NASA Astrophysics Data System (ADS)

    Galès, Amandine; Bru-Adan, Valérie; Godon, Jean-Jacques; Delabre, Karine; Catala, Philippe; Ponthieux, Arnaud; Chevallier, Michel; Birot, Emmanuel; Steyer, Jean-Philippe; Wéry, Nathalie

    2015-04-01

    Bioaerosols emitted from composting plants have become an issue because of their potential harmful impact on public or workers' health. Accurate knowledge of the particle-size distribution in bioaerosols emitted from open-air composting facilities during operational activity is a requirement for improved modeling of air dispersal. In order to investigate the aerodynamic diameter of bacteria in composting bioaerosols this study used an Electrical Low Pressure Impactor for sampling and quantitative real-time PCR for quantification. Quantitative PCR results show that the size of bacteria peaked between 0.95 μm and 2.4 μm and that the geometric mean diameter of the bacteria was 1.3 μm. In addition, total microbial cells were counted by flow cytometry and revealed that these qPCR results corresponded to single whole bacteria. Finally, the enumeration of cultivable thermophilic microorganisms allowed us to set the upper size limit for fragments at an aerodynamic diameter of ∼0.3 μm. Particle-size distributions of microbial groups previously used to monitor composting bioaerosols were also investigated. In collected the bioaerosols, the aerodynamic diameter of the actinomycetes Saccharopolyspora rectivirgula-and-relatives and also of the fungus Aspergillus fumigatus, appeared to be consistent with a majority of individual cells. Together, this study provides the first culture-independent data on particle-size distribution of composting bioaerosols and reveals that airborne single bacteria were emitted predominantly from open-air composting facilities.

  2. A miniature porous aluminum oxide-based flow-cell for online water quality monitoring using bacterial sensor cells.

    PubMed

    Yagur-Kroll, Sharon; Schreuder, Erik; Ingham, Colin J; Heideman, René; Rosen, Rachel; Belkin, Shimshon

    2015-02-15

    The use of live bacterial reporters as sensing entities in whole-cell biosensors allows the investigation of the biological effects of a tested sample, as well as the bioavailability of its components. Here we present a proof of concept for a new design for online continuous water monitoring flow-cell biosensor, incorporating recombinant reporter bacteria, engineered to generate an optical signal (fluorescent or bioluminescent) in the presence of the target compound(s). At the heart of the flow-cell is a disposable chip made of porous aluminum oxide (PAO), which retains the sensor microorganisms on its rigid planar surface, while its high porosity allows an undisturbed access both to the sample and to essential nutrients. The ability of the bacterial reporters to detect model toxic chemicals was first demonstrated using a "naked" PAO chip placed on solid agar, and later in a chip encased in a specially designed flow-through configuration which enables continuous on-line monitoring. The applicability of the PAO chip to simultaneous online detection of diverse groups of chemicals was demonstrated by the incorporation of a 6-member sensor array into the flow-through chip. The selective response of the array was also confirmed in spiked municipal wastewater effluents. Sensing activity was retained by the bacteria after 12-weeks storage of freeze-dried biochips, demonstrating the biochip potential as a simple minimal maintenance "plug-in" cartridge. This low-cost and easy to handle PAO-based flow-cell biosensor may serve as a basis for a future platform for water quality monitoring. PMID:25441411

  3. New method for estimating bacterial cell abundances in natural samples by use of sublimation

    NASA Technical Reports Server (NTRS)

    Glavin, Daniel P.; Cleaves, H. James; Schubert, Michael; Aubrey, Andrew; Bada, Jeffrey L.

    2004-01-01

    We have developed a new method based on the sublimation of adenine from Escherichia coli to estimate bacterial cell counts in natural samples. To demonstrate this technique, several types of natural samples, including beach sand, seawater, deep-sea sediment, and two soil samples from the Atacama Desert, were heated to a temperature of 500 degrees C for several seconds under reduced pressure. The sublimate was collected on a cold finger, and the amount of adenine released from the samples was then determined by high-performance liquid chromatography with UV absorbance detection. Based on the total amount of adenine recovered from DNA and RNA in these samples, we estimated bacterial cell counts ranging from approximately 10(5) to 10(9) E. coli cell equivalents per gram. For most of these samples, the sublimation-based cell counts were in agreement with total bacterial counts obtained by traditional DAPI (4,6-diamidino-2-phenylindole) staining.

  4. A microfluidic device for physical trapping and electrical lysis of bacterial cells

    NASA Astrophysics Data System (ADS)

    Bao, Ning; Lu, Chang

    2008-05-01

    In this letter, we report a simple microfluidic device that integrates the capture of bacterial cells using a microscale bead array and the rapid electrical lysis for release of intracellular materials. We study the retention of Escherichia coli cells with different concentrations in this type of bead array and the optimal electrical parameters for the electroporative release of intracellular proteins. Our design provides a simple solution to the extraction of intracellular materials from a bacterial cell population based entirely on physical methods without applying chemical or biological reagents.

  5. Bacterial porin disrupts mitochondrial membrane potential and sensitizes host cells to apoptosis.

    PubMed

    Kozjak-Pavlovic, Vera; Dian-Lothrop, Elke A; Meinecke, Michael; Kepp, Oliver; Ross, Katharina; Rajalingam, Krishnaraj; Harsman, Anke; Hauf, Eva; Brinkmann, Volker; Günther, Dirk; Herrmann, Ines; Hurwitz, Robert; Rassow, Joachim; Wagner, Richard; Rudel, Thomas

    2009-10-01

    The bacterial PorB porin, an ATP-binding beta-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (DeltaPsi(m)). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of beta-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of DeltaPsi(m). The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce DeltaPsi(m) loss and apoptosis, demonstrating that dissipation of DeltaPsi(m) is a requirement for cell death caused by neisserial infection. PMID:19851451

  6. Bacterial Ortholog of Mammalian Translocator Protein (TSPO) with Virulence Regulating Activity

    PubMed Central

    Chapalain, Annelise; Chevalier, Sylvie; Orange, Nicole; Murillo, Laurence; Papadopoulos, Vassilios; Feuilloley, Marc G. J.

    2009-01-01

    The translocator protein (TSPO), previously designated as peripheral-type benzodiazepine receptor, is a protein mainly located in the outer mitochondrial membrane of eukaryotic cells. TSPO is implicated in major physiological functions and functionally associated with other proteins such as the voltage-dependent anionic channel, also designated as mitochondrial porin. Surprisingly, a TSPO-related protein was identified in the photosynthetic bacterium Rhodobacter sphaeroides but it was initially considered as a relict of evolution. In the present study we cloned a tspO gene in Pseudomonas fluorescens MF37, a non-photosynthetic eubacterium and we used bioinformatics tools to identify TSPO in the genome of 97 other bacteria. P. fluorescens TSPO was recognized by antibodies against mouse protein and by PK 11195, an artificial ligand of mitochondrial TSPO. As in eukaryotes, bacterial TSPO appears functionally organized as a dimer and the apparent Kd for PK 11195 is in the same range than for its eukaryotic counterpart. When P. fluorescens MF37 was treated with PK 11195 (10−5 M) adhesion to living or artificial surfaces and biofilm formation activity were increased. Conversely, the apoptotic potential of bacteria on eukaryotic cells was significantly reduced. This effect of PK11195 was abolished in a mutant of P. fluorescens MF37 deficient for its major outer membrane porin, OprF. The present results demonstrate the existence of a bacterial TSPO that shares common structural and functional characteristics with its mammalian counterpart. This protein, apparently involved in adhesion and virulence, reveals the existence of a possible new inter kingdom signalling system and suggests that the human microbiome should be involuntarily exposed to the evolutionary pressure of benzodiazepines and related molecules. This discovery also represents a promising opportunity for the development of alternative antibacterial strategies. PMID:19564920

  7. Bacterial ortholog of mammalian translocator protein (TSPO) with virulence regulating activity.

    PubMed

    Chapalain, Annelise; Chevalier, Sylvie; Orange, Nicole; Murillo, Laurence; Papadopoulos, Vassilios; Feuilloley, Marc G J

    2009-01-01

    The translocator protein (TSPO), previously designated as peripheral-type benzodiazepine receptor, is a protein mainly located in the outer mitochondrial membrane of eukaryotic cells. TSPO is implicated in major physiological functions and functionally associated with other proteins such as the voltage-dependent anionic channel, also designated as mitochondrial porin. Surprisingly, a TSPO-related protein was identified in the photosynthetic bacterium Rhodobacter sphaeroides but it was initially considered as a relict of evolution. In the present study we cloned a tspO gene in Pseudomonas fluorescens MF37, a non-photosynthetic eubacterium and we used bioinformatics tools to identify TSPO in the genome of 97 other bacteria. P. fluorescens TSPO was recognized by antibodies against mouse protein and by PK 11195, an artificial ligand of mitochondrial TSPO. As in eukaryotes, bacterial TSPO appears functionally organized as a dimer and the apparent Kd for PK 11195 is in the same range than for its eukaryotic counterpart. When P. fluorescens MF37 was treated with PK 11195 (10(-5) M) adhesion to living or artificial surfaces and biofilm formation activity were increased. Conversely, the apoptotic potential of bacteria on eukaryotic cells was significantly reduced. This effect of PK11195 was abolished in a mutant of P. fluorescens MF37 deficient for its major outer membrane porin, OprF. The present results demonstrate the existence of a bacterial TSPO that shares common structural and functional characteristics with its mammalian counterpart. This protein, apparently involved in adhesion and virulence, reveals the existence of a possible new inter kingdom signalling system and suggests that the human microbiome should be involuntarily exposed to the evolutionary pressure of benzodiazepines and related molecules. This discovery also represents a promising opportunity for the development of alternative antibacterial strategies. PMID:19564920

  8. In vitro inhibitory properties of ferrocene-substituted chalcones and aurones on bacterial and human cell cultures.

    PubMed

    Tiwari, Keshri Nath; Monserrat, Jean-Philippe; Hequet, Arnaud; Ganem-Elbaz, Carine; Cresteil, Thierry; Jaouen, Gérard; Vessières, Anne; Hillard, Elizabeth A; Jolivalt, Claude

    2012-06-01

    Two series of ten chalcones and ten aurones, where ferrocene replaces the C ring and with diverse substituents on the A ring were synthesized. The compounds were tested against two antibiotic-sensitive bacterial strains, E. coli ATCC 25922 and S. aureus ATCC 25923, and two antibiotic-resistant strains, S. aureus SA-1199B and S. epidermidis IPF896. The unsubstituted compound and those with methoxy substitution showed an inhibitory effect on all bacterial strains at minimum inhibitory concentrations ranging between 2 and 32 mg L(-1). For four of these compounds, the effect was bactericidal, as opposed to bacteriostatic. The corresponding organic aurones did not show growth inhibition, underscoring the role of the ferrocene group. The methoxy-substituted aurones and the unsubstituted aurone also showed low micromolar (IC(50)) activity against MRC-5 non-tumoral lung cells and MDA-MB-231 breast cancer cells, suggesting non-specific toxicity. PMID:22240736

  9. Bacterial diversity and active biomass in full-scale granular activated carbon filters operated at low water temperatures.

    PubMed

    Kaarela, Outi E; Härkki, Heli A; Palmroth, Marja R T; Tuhkanen, Tuula A

    2015-01-01

    Granular activated carbon (GAC) filtration enhances the removal of natural organic matter and micropollutants in drinking water treatment. Microbial communities in GAC filters contribute to the removal of the biodegradable part of organic matter, and thus help to control microbial regrowth in the distribution system. Our objectives were to investigate bacterial community dynamics, identify the major bacterial groups, and determine the concentration of active bacterial biomass in full-scale GAC filters treating cold (3.7-9.5°C), physicochemically pretreated, and ozonated lake water. Three sampling rounds were conducted to study six GAC filters of different operation times and flow modes in winter, spring, and summer. Total organic carbon results indicated that both the first-step and second-step filters contributed to the removal of organic matter. Length heterogeneity analysis of amplified 16S rRNA genes illustrated that bacterial communities were diverse and considerably stable over time. α-Proteobacteria, β-Proteobacteria, and Nitrospira dominated in all of the GAC filters, although the relative proportion of dominant phylogenetic groups in individual filters differed. The active bacterial biomass accumulation, measured as adenosine triphosphate, was limited due to low temperature, low flux of nutrients, and frequent backwashing. The concentration of active bacterial biomass was not affected by the moderate seasonal temperature variation. In summary, the results provided an insight into the biological component of GAC filtration in cold water temperatures and the operational parameters affecting it. PMID:25242545

  10. Natural Killer Cells and Helicobacter pylori Infection: Bacterial Antigens and Interleukin-12 Act Synergistically To Induce Gamma Interferon Production

    PubMed Central

    Yun, Cheol H.; Lundgren, Anna; Azem, Josef; Sjöling, Åsa; Holmgren, Jan; Svennerholm, Ann-Mari; Lundin, B. Samuel

    2005-01-01

    Helicobacter pylori is known to induce a local immune response, which is characterized by activation of lymphocytes and the production of IFN-γ in the stomach mucosa. Since not only T cells, but also natural killer (NK) cells, are potent producers of gamma interferon (IFN-γ), we investigated whether NK cells play a role in the immune response to H. pylori infection. Our results showed that NK cells were present in both the gastric and duodenal mucosae but that H. pylori infection did not affect the infiltration of NK cells into the gastrointestinal area. Furthermore, we could show that NK cells could be activated directly by H. pylori antigens, as H. pylori bacteria, as well as lysate from H. pylori, induced the secretion of IFN-γ by NK cells. NK cells were also activated without direct contact when separated from the bacteria by an epithelial cell layer, indicating that the activation of NK cells by H. pylori can also occur in vivo, in the infected stomach mucosa. Moreover, the production of IFN-γ by NK cells was greatly enhanced when a small amount of interleukin-12 (IL-12) was added, and this synergistic effect was associated with increased expression of the IL-12 receptor β2. It was further evident that bacterial lysate alone was sufficient to induce the activation of cytotoxicity-related molecules. In conclusion, we demonstrated that NK cells are present in the gastroduodenal mucosa of humans and that NK cells produce high levels of IFN-γ when stimulated with a combination of H. pylori antigen and IL-12. We propose that NK cells play an active role in the local immune response to H. pylori infection. PMID:15731046

  11. Non-canonical inflammasome activation of caspase-4/caspase-11 mediates epithelial defenses against enteric bacterial pathogens

    PubMed Central

    Knodler, Leigh A.; Crowley, Shauna M.; Sham, Ho Pan; Yang, Hyungjun; Wrande, Marie; Ma, Caixia; Ernst, Robert K.; Steele-Mortimer, Olivia; Celli, Jean; Vallance, Bruce A.

    2014-01-01

    Summary Inflammasome-mediated host defenses have been extensively studied in innate immune cells. Whether inflammasomes function for innate defense in intestinal epithelial cells, which represent the first line of defense against enteric pathogens, remains unknown. We observed enhanced Salmonella enterica serovar Typhimurium colonization in the intestinal epithelium of caspase-11 deficient mice, but not at systemic sites. In polarized epithelial monolayers, siRNA-mediated depletion of caspase-4, a human orthologue of caspase-11, also led to increased bacterial colonization. Decreased rates of pyroptotic cell death, a host defense mechanism that extrudes S. Typhimurium infected cells from the polarized epithelium, accounted for increased pathogen burdens. The caspase-4 inflammasome also governs activation of the proinflammatory cytokine, interleukin (IL)-18, in response to intracellular (S. Typhimurium) and extracellular (enteropathogenic Escherichia coli) enteric pathogens, via intracellular LPS sensing. Therefore an epithelial cell intrinsic non-canonical inflammasome plays a critical role in antimicrobial defense at the intestinal mucosal surface. PMID:25121752

  12. Bacterial pathogens activate plasminogen to breach tissue barriers and escape from innate immunity.

    PubMed

    Peetermans, Marijke; Vanassche, Thomas; Liesenborghs, Laurens; Lijnen, Roger H; Verhamme, Peter

    2016-11-01

    Both coagulation and fibrinolysis are tightly connected with the innate immune system. Infection and inflammation cause profound alterations in the otherwise well-controlled balance between coagulation and fibrinolysis. Many pathogenic bacteria directly exploit the host's hemostatic system to increase their virulence. Here, we review the capacity of bacteria to activate plasminogen. The resulting proteolytic activity allows them to breach tissue barriers and evade innate immune defense, thus promoting bacterial spreading. Yersinia pestis, streptococci of group A, C and G and Staphylococcus aureus produce a specific bacterial plasminogen activator. Moreover, surface plasminogen receptors play an established role in pneumococcal, borrelial and group B streptococcal infections. This review summarizes the mechanisms of bacterial activation of host plasminogen and the role of the fibrinolytic system in infections caused by these pathogens. PMID:26485450

  13. Anti-inflammatory, anti-bacterial, and cytotoxic activity of fibrous clays.

    PubMed

    Cervini-Silva, Javiera; Nieto-Camacho, Antonio-; Ramírez-Apan, María Teresa; Gómez-Vidales, Virginia; Palacios, Eduardo; Montoya, Ascención; Ronquillo de Jesús, Elba

    2015-05-01

    Produced worldwide at 1.2m tons per year, fibrous clays are used in the production of pet litter, animal feed stuff to roof parcels, construction and rheological additives, and other applications needing to replace long-fiber length asbestos. To the authors' knowledge, however, information on the beneficial effects of fibrous clays on health remains scarce. This paper reports on the anti-inflammatory, anti-bacterial, and cytotoxic activity by sepiolite (Vallecas, Spain) and palygorskite (Torrejon El Rubio, Spain). The anti-inflammatory activity was determined using the 12-O-tetradecanoylphorbol-13-acetate (TPA) and myeloperoxidase (MPO) methods. Histological cuts were obtained for quantifying leukocytes found in the epidermis. Palygorkite and sepiolite caused edema inhibition and migration of neutrophils ca. 68.64 and 45.54%, and 80 and 65%, respectively. Fibrous clays yielded high rates of infiltration, explained by cleavage of polysomes and exposure of silanol groups. Also, fibrous clays showed high inhibition of myeloperoxidase contents shortly after exposure, but decreased sharply afterwards. In contrast, tubular clays caused an increasing inhibition of myeloperoxidase with time. Thus, clay structure restricted the kinetics and mechanism of myeloperoxidase inhibition. Fibrous clays were screened in vitro against human cancer cell lines. Cytotoxicity was determined using the protein-binding dye sulforhodamine B (SRB). Exposing cancer human cells to sepiolite or palygorskite showed growth inhibition varying with cell line. This study shows that fibrous clays served as an effective anti-inflammatory, limited by chemical transfer and cellular-level signals responding exclusively to an early exposure to clay, and cell viability decreasing significantly only after exposure to high concentrations of sepiolite. PMID:25819359

  14. Inhibition of Bacterial Toxin Activity by the Nuclear Stain, DRAQ5™.

    PubMed

    Webb, Joshua N; Koufos, Evan; Brown, Angela C

    2016-08-01

    The repeats-in-toxin family of toxins includes proteins produced by Gram negative bacteria such as Escherichia coli (α-hemolysin), Bordetella pertussis (adenylate cyclase toxin), and Aggregatibacter actinomycetemcomitans (LtxA), which contribute to the pathogenesis of these organisms by killing host cells. In the case of LtxA produced by A. actinomycetemcomitans, white blood cells are targeted, allowing the bacteria to avoid clearance by the host immune system. In its association with target cells, LtxA binds to a receptor, lymphocyte function-associated antigen-1, as well as membrane lipids and cholesterol, before being internalized via a lysosomal-mediated pathway. The motivation for this project comes from our discovery that DRAQ5™, a membrane-permeable nuclear stain, prevents the internalization of LtxA in a Jurkat T cell line. We hypothesized that DRAQ5™, in crossing the plasma membrane, alters the properties of the membrane to inhibit LtxA internalization. To investigate how DRAQ5™ interacts with the lipid membrane to prevent LtxA internalization, we used studied DRAQ5™-mediated membrane changes in model membranes using a variety of techniques, including differential scanning calorimetry and fluorescence spectroscopy. Our results suggest that DRAQ5™ inhibits the activity of LtxA by decreasing the fluidity of the cellular lipid membrane, which decreases LtxA binding. These results present an interesting possible anti-virulence strategy; by altering bacterial toxin activity by modifying membrane fluidity, it may be possible to inhibit the pathogenicity of A. actinomycetemcomitans. PMID:27039399

  15. Novel osmotically induced antifungal chitinases and bacterial expression of an active recombinant isoform.

    PubMed Central

    Yun, D J; D'Urzo, M P; Abad, L; Takeda, S; Salzman, R; Chen, Z; Lee, H; Hasegawa, P M; Bressan, R A

    1996-01-01

    NaCl (428 mM)-adapted tobacco (Nicotiana tabacum L. var Wisconsin 38) cells accumulate and secrete several antifungal chitinases. The predominant protein secreted to the culture medium was a 29-kD peptide that, based on internal amino acid sequence, was determined to be a class II acidic chitinase with similarity to PR-Q. The four predominant chitinases (T1, T2, T3, and T4) that accumulated intracellularly in 428 mM NaCl-adapted cells were purified. Based on N-terminal sequence analyses, two of these were identified as class I chitinase isoforms, one similar to the N. tomentosiformis (H. Shinshi, J.M. Neuhaus, J. Ryals, F. Meins [1990] Plant Mol Biol 14:357-368) protein (T1) and the other homologous to the N. sylvestris (Y. Fukuda, M. Ohme, H. Shinshi [1991] Plant Mol Biol 16:1-10) protein (T2). The other two proteins (T3 and T4) were determined to be novel chitinases that have sequence similarity with class I chitinases, but each lacks a chitin-binding domain. All four chitinases inhibited Fusarium oxysporum f. sp. lycopersici and Trichoderma longibrachiatum hyphal growth in vitro, although the isoforms containing a chitin-binding domain were somewhat more active. Conditions were established for the successful expression of soluble and active bacterial recombinant T2. Expression of soluble recombinant T2 was achieved when isopropyl beta-D-thiogalactopyranoside induction occurred at 18 degrees C but not at 25 or 37 degrees C. The purified recombinant protein exhibited antifungal activity comparable to a class I chitinase purified from NaCl-adapted tobacco cells. PMID:8756502

  16. Blockage of protease-activated receptor 1 ameliorates heat-stress induced intestinal high permeability and bacterial translocation.

    PubMed

    Xu, Qiu-lin; Guo, Xiao-hua; Liu, Jing-xian; Chen, Bin; Liu, Zhi-feng; Su, Lei

    2015-04-01

    Accumulated evidences indicate intestinal lesions play an important role in the pathogenesis of heatstroke. However, the underlying mechanisms by which heat stress causes intestinal barrier dysfunction and bacterial translocation remain unclear. In this study, we investigated the role of protease-activated receptor 1 (PAR1) in heat stress-induced intestinal hyper-permeability and bacterial translocation. Intestinal permeability in heat stressed mouse was evaluated by determining plasma endotoxin concentration and urinal lactulose/mannitol (L/M) ratio with gastric administration of L/M solution. Venous blood, liver, spleen and mesenteric lymph node tissues were collected for bacterial load test. Real time PCR was used to determine ileum PAR1 mRNA expression. In vitro study, permeability was assessed by determining trans-epithelial electrical resistance (TEER) in human intestinal Caco-2 cell line. RWJ-58259, a selective antagonist of PAR1, was used both in vivo and in vitro studies. The results showed that heat stress could increase ileum PAR1 mRNA level, urinal L/M ratio, plasma endotoxin concentration and bacterial load in the blood, spleen and mesenteric lymph nodes. Blocking PAR1 with RWJ-58259 (10 mg/kg) pretreatment could significantly reduce heat stress-induced above changes, but have no role to PAR1 mRNA level. In Caco-2 cells, heat stress-induced high permeability could also be reduced by RWJ-58259 (5-20 µmol/L). In summary, our results demonstrated that PAR1 signaling pathway may play an important role in the heat stress-induced elevation of intestinal permeability, bacterial translocation and the occurrence of endotoxemia. PMID:25492552

  17. Mechanisms of ion-bombardment-induced DNA transfer into bacterial E. coli cells

    NASA Astrophysics Data System (ADS)

    Yu, L. D.; Sangwijit, K.; Prakrajang, K.; Phanchaisri, B.; Thongkumkoon, P.; Thopan, P.; Singkarat, S.; Anuntalabhochai, S.

    2014-05-01

    As a useful ion beam biotechnology, ion-bombardment-induced DNA transfer into bacterial Escherichia coli (E. coli) cells has been successfully operated using argon ions. In the process ion bombardment of the bacterial cells modifies the cell envelope materials to favor the exogenous DNA molecules to pass through the envelope to enter the cell. The occurrence of the DNA transfer induction was found ion energy and fluence dependent in a complex manner. At ion energy of a few keV and a few tens of keV to moderate fluences the DNA transfer could be induced by ion bombardment of the bacterial cells, while at the same ion energy but to high fluences DNA transfer could not be induced. On the other hand, when the ion energy was medium, about 10-20 keV, the DNA transfer could not be induced by ion bombardment of the cells. The complexity of the experimental results indicated a complex mechanism which should be related to the complex structure of the bacterial E. coli cell envelope. A phase diagram was proposed to interpret different mechanisms involved as functions of the ion energy and fluence.

  18. Cytotoxicity in bacterial cultures: interaction and cell-specificity, possible factors in periodontal disease.

    PubMed

    Johansson, A; Bergenholtz, A; Holm, S E

    1994-09-01

    Cytotoxicity in culture media of various growing bacterial strains was estimated by Cr-51 release of labelled target-cells. Interaction studies were made by adding each of the different UV-killed bacteria to the medium with viable bacteria. The reference oral bacterial strains were: Actinobacillus actinomycetemcomitans Y4, Porphyromonas gingivalis, Fusobacterium nucleatum and Streptococcus mitis, which were compared with the reference bacteria Staphylococcus aureus 209 and Staphylococcus epidermidis. The target cells were: gingival fibroblasts (GF), periodontal membrane fibroblasts (PMF), pulpal fibroblasts (PF), HeLa-cells (HeLa), and lymphoid neoplasm cells (LN). Synergistic, as well as antagonistic, effects on target cells were observed. The cytotoxicity of A. actinomycetemcomitans in presence of P. gingivalis is neutralized while in presence of S. aureus it was increased. Bacterial interactions with F. nucleatum and P. gingivalis cytotoxicity were observed. The cytotoxicity of F. nucleatum was increased when cultured together with A. actinomycetemcomitans. Each cell type reacted differently to the toxicity of the supernatant of growth medium in which the same bacterial strain had been cultivated, which indicates cell specificity of the toxins. PMID:7799211

  19. Dynamics of phenotypic reversibility of bacterial cells with oscillating hydrostatic pressure

    NASA Astrophysics Data System (ADS)

    Nepal, Sudip; Kumar, Pradeep

    Bacterial cells encounter and respond to physiochemical fluctuations. The response depends on the extent and type of the stresses applied. The response of bacterial cells to the fluctuating stress is relatively unknown. Here, we have studied the response of wild type Escherichia coli (E. coli) under fluctuating hydrostatic pressures ranging from 1 atm to 500 atm. High pressure acts as a stress to E. coli since these bacteria are adapted to grow optimally at atmospheric pressure. Cell division of E. coli is inhibited at high pressures resulting in increase in the length of the cells. Cell-length is reversible in nature and bacterial cells revert back to normal size on a time scale that is proportional to the strength and time of continuous pressure applied upon relaxing the high pressure condition. We have studied the dynamics of cellular reversibility of E. coli under the conditions in which continuous pressure is applied and subsequently relaxed over different time scales. We have quantified the dynamics of cellular reversibility with different relaxation times. Furthermore, we propose a model to describe the reversibility of the bacterial cell with the relaxation time. Our theoretical model fits well to the experimental data. We further

  20. Bacterial cell wall-induced arthritis: chemical composition and tissue distribution of four Lactobacillus strains.

    PubMed

    Simelyte, E; Rimpiläinen, M; Lehtonen, L; Zhang, X; Toivanen, P

    2000-06-01

    To study what determines the arthritogenicity of bacterial cell walls, cell wall-induced arthritis in the rat was applied, using four strains of Lactobacillus. Three of the strains used proved to induce chronic arthritis in the rat; all were Lactobacillus casei. The cell wall of Lactobacillus fermentum did not induce chronic arthritis. All arthritogenic bacterial cell walls had the same peptidoglycan structure, whereas that of L. fermentum was different. Likewise, all arthritogenic cell walls were resistant to lysozyme degradation, whereas the L. fermentum cell wall was lysozyme sensitive. Muramic acid was observed in the liver, spleen, and lymph nodes in considerably larger amounts after injection of an arthritogenic L. casei cell wall than following injection of a nonarthritogenic L. fermentum cell wall. The L. casei cell wall also persisted in the tissues longer than the L. fermentum cell wall. The present results, taken together with those published previously, underline the possibility that the chemical structure of peptidoglycan is important in determining the arthritogenicity of the bacterial cell wall. PMID:10816508

  1. Measuring bacterial activity and community composition at high hydrostatic pressure using a novel experimental approach: a pilot study.

    PubMed

    Wannicke, Nicola; Frindte, Katharina; Gust, Giselher; Liskow, Iris; Wacker, Alexander; Meyer, Andreas; Grossart, Hans-Peter

    2015-05-01

    In this pilot study, we describe a high-pressure incubation system allowing multiple subsampling of a pressurized culture without decompression. The system was tested using one piezophilic (Photobacterium profundum), one piezotolerant (Colwellia maris) bacterial strain and a decompressed sample from the Mediterranean deep sea (3044 m) determining bacterial community composition, protein production (BPP) and cell multiplication rates (BCM) up to 27 MPa. The results showed elevation of BPP at high pressure was by a factor of 1.5 ± 1.4 and 3.9 ± 2.3 for P. profundum and C. maris, respectively, compared to ambient-pressure treatments and by a factor of 6.9 ± 3.8 fold in the field samples. In P. profundum and C. maris, BCM at high pressure was elevated (3.1 ± 1.5 and 2.9 ± 1.7 fold, respectively) compared to the ambient-pressure treatments. After 3 days of incubation at 27 MPa, the natural bacterial deep-sea community was dominated by one phylum of the genus Exiguobacterium, indicating the rapid selection of piezotolerant bacteria. In future studies, our novel incubation system could be part of an isopiestic pressure chain, allowing more accurate measurement of bacterial activity rates which is important both for modeling and for predicting the efficiency of the oceanic carbon pump. PMID:25873457

  2. Transcriptional Activation of c3 and hsp70 as Part of the Immune Response of Acropora millepora to Bacterial Challenges

    PubMed Central

    Brown, Tanya; Bourne, David; Rodriguez-Lanetty, Mauricio

    2013-01-01

    The impact of disease outbreaks on coral physiology represents an increasing concern for the fitness and resilience of reef ecosystems. Predicting the tolerance of corals to disease relies on an understanding of the coral immune response to pathogenic interactions. This study explored the transcriptional response of two putative immune genes (c3 and c-type lectin) and one stress response gene (hsp70) in the reef building coral, Acropora millepora challenged for 48 hours with bacterial strains, Vibrio coralliilyticus and Alteromonas sp. at concentrations of 106 cells ml-1. Coral fragments challenged with V. coralliilyticus appeared healthy while fragments challenged with Alteromonas sp. showed signs of tissue lesions after 48 hr. Coral-associated bacterial community profiles assessed using denaturing gradient gel electrophoresis changed after challenge by both bacterial strains with the Alteromonas sp. treatment demonstrating the greatest community shift. Transcriptional profiles of c3 and hsp70 increased at 24 hours and correlated with disease signs in the Alteromonas sp. treatment. The expression of hsp70 also showed a significant increase in V. coralliilyticus inoculated corals at 24 h suggesting that even in the absence of disease signs, the microbial inoculum activated a stress response in the coral. C-type lectin did not show a response to any of the bacterial treatments. Increase in gene expression of c3 and hsp70 in corals showing signs of disease indicates their potential involvement in immune and stress response to microbial challenges. PMID:23861754

  3. Transcriptional activation of c3 and hsp70 as part of the immune response of Acropora millepora to bacterial challenges.

    PubMed

    Brown, Tanya; Bourne, David; Rodriguez-Lanetty, Mauricio

    2013-01-01

    The impact of disease outbreaks on coral physiology represents an increasing concern for the fitness and resilience of reef ecosystems. Predicting the tolerance of corals to disease relies on an understanding of the coral immune response to pathogenic interactions. This study explored the transcriptional response of two putative immune genes (c3 and c-type lectin) and one stress response gene (hsp70) in the reef building coral, Acropora millepora challenged for 48 hours with bacterial strains, Vibrio coralliilyticus and Alteromonas sp. at concentrations of 10(6) cells ml(-1). Coral fragments challenged with V. coralliilyticus appeared healthy while fragments challenged with Alteromonas sp. showed signs of tissue lesions after 48 hr. Coral-associated bacterial community profiles assessed using denaturing gradient gel electrophoresis changed after challenge by both bacterial strains with the Alteromonas sp. treatment demonstrating the greatest community shift. Transcriptional profiles of c3 and hsp70 increased at 24 hours and correlated with disease signs in the Alteromonas sp. treatment. The expression of hsp70 also showed a significant increase in V. coralliilyticus inoculated corals at 24 h suggesting that even in the absence of disease signs, the microbial inoculum activated a stress response in the coral. C-type lectin did not show a response to any of the bacterial treatments. Increase in gene expression of c3 and hsp70 in corals showing signs of disease indicates their potential involvement in immune and stress response to microbial challenges. PMID:23861754

  4. Microspectrometric insights on the uptake of antibiotics at the single bacterial cell level.

    PubMed

    Cinquin, Bertrand; Maigre, Laure; Pinet, Elizabeth; Chevalier, Jacqueline; Stavenger, Robert A; Mills, Scott; Réfrégiers, Matthieu; Pagès, Jean-Marie

    2015-01-01

    Bacterial multidrug resistance is a significant health issue. A key challenge, particularly in Gram-negative antibacterial research, is to better understand membrane permeation of antibiotics in clinically relevant bacterial pathogens. Passing through the membrane barrier to reach the required concentration inside the bacterium is a pivotal step for most antibacterials. Spectrometric methodology has been developed to detect drugs inside bacteria and recent studies have focused on bacterial cell imaging. Ultimately, we seek to use this method to identify pharmacophoric groups which improve penetration, and therefore accumulation, of small-molecule antibiotics inside bacteria. We developed a method to quantify the time scale of antibiotic accumulation in living bacterial cells. Tunable ultraviolet excitation provided by DISCO beamline (synchrotron Soleil) combined with microscopy allows spectroscopic analysis of the antibiotic signal in individual bacterial cells. Robust controls and measurement of the crosstalk between fluorescence channels can provide real time quantification of drug. This technique represents a new method to assay drug translocation inside the cell and therefore incorporate rational drug design to impact antibiotic uptake. PMID:26656111

  5. Microspectrometric insights on the uptake of antibiotics at the single bacterial cell level

    PubMed Central

    Cinquin, Bertrand; Maigre, Laure; Pinet, Elizabeth; Chevalier, Jacqueline; Stavenger, Robert A.; Mills, Scott; Réfrégiers, Matthieu; Pagès, Jean-Marie

    2015-01-01

    Bacterial multidrug resistance is a significant health issue. A key challenge, particularly in Gram-negative antibacterial research, is to better understand membrane permeation of antibiotics in clinically relevant bacterial pathogens. Passing through the membrane barrier to reach the required concentration inside the bacterium is a pivotal step for most antibacterials. Spectrometric methodology has been developed to detect drugs inside bacteria and recent studies have focused on bacterial cell imaging. Ultimately, we seek to use this method to identify pharmacophoric groups which improve penetration, and therefore accumulation, of small-molecule antibiotics inside bacteria. We developed a method to quantify the time scale of antibiotic accumulation in living bacterial cells. Tunable ultraviolet excitation provided by DISCO beamline (synchrotron Soleil) combined with microscopy allows spectroscopic analysis of the antibiotic signal in individual bacterial cells. Robust controls and measurement of the crosstalk between fluorescence channels can provide real time quantification of drug. This technique represents a new method to assay drug translocation inside the cell and therefore incorporate rational drug design to impact antibiotic uptake. PMID:26656111

  6. Mechanisms of bacterial morphogenesis: Evolutionary cell biology approaches provide new insights

    PubMed Central

    Jiang, Chao; Caccamo, Paul D.; Brun, Yves V.

    2015-01-01

    How Darwin’s “endless forms most beautiful” have evolved remains one of the most exciting questions in biology. The significant variety of bacterial shapes is most likely due to the specific advantages they confer with respect to the diverse environments they occupy. While our understanding of the mechanisms generating relatively simple shapes has improved tremendously in the last few years, the molecular mechanisms underlying the generation of complex shapes and the evolution of shape diversity are largely unknown. The emerging field of bacterial evolutionary cell biology provides a novel strategy to answer this question in a comparative phylogenetic framework. This relatively novel approach provides hypotheses and insights into cell biological mechanisms, such as morphogenesis, and their evolution that would have been difficult to obtain by studying only model organisms. We discuss the necessary steps, challenges, and impact of integrating “evolutionary thinking” into bacterial cell biology in the genomic era. PMID:25664446

  7. Single-bacterium nanomechanics in biomedicine: unravelling the dynamics of bacterial cells

    NASA Astrophysics Data System (ADS)

    Aguayo, S.; Donos, N.; Spratt, D.; Bozec, L.

    2015-02-01

    The use of the atomic force microscope (AFM) in microbiology has progressed significantly throughout the years since its first application as a high-resolution imaging instrument. Modern AFM setups are capable of characterizing the nanomechanical behaviour of bacterial cells at both the cellular and molecular levels, where elastic properties and adhesion forces of single bacterium cells can be examined under different experimental conditions. Considering that bacterial and biofilm-mediated infections continue to challenge the biomedical field, it is important to understand the biophysical events leading towards bacterial adhesion and colonization on both biological and non-biological substrates. The purpose of this review is to present the latest findings concerning the field of single-bacterium nanomechanics, and discuss future trends and applications of nanoindentation and single-cell force spectroscopy techniques in biomedicine.

  8. Antimicrobial susceptibility testing in 90 min by bacterial cell count monitoring

    PubMed Central

    Broeren, M A C; Maas, Y; Retera, E; Arents, N L A

    2013-01-01

    The rise in antimicrobial resistance has become a serious global health problem. Restrictive use of antibiotics seems the only option to temper this accession since research in new antibiotics has halted. Antimicrobial stewardship programmes rely on quick access to susceptibility data. This study evaluated the concept of bacterial cell count monitoring as a fast method to determine susceptibility. Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus strains were tested for amoxicillin/piperacillin and gentamicin by three conventional methods (VITEK2®, Etest® and broth-macrodilution). Bacterial cell count monitoring reliably predicted susceptibility after 90 min for Escherichia coli and after 120 min for Pseudomonas aeruginosa and Staphylococcus aureus without any minor, major or very major discrepancies. Time-to-result was reduced by 74%, 83% and 76%, respectively. Bacterial cell count monitoring shows great potential for rapid susceptibility testing. PMID:22390723

  9. Antimicrobial susceptibility testing in 90 min by bacterial cell count monitoring.

    PubMed

    Broeren, M A C; Maas, Y; Retera, E; Arents, N L A

    2013-03-01

    The rise in antimicrobial resistance has become a serious global health problem. Restrictive use of antibiotics seems the only option to temper this accession since research in new antibiotics has halted. Antimicrobial stewardship programmes rely on quick access to susceptibility data. This study evaluated the concept of bacterial cell count monitoring as a fast method to determine susceptibility. Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus strains were tested for amoxicillin/piperacillin and gentamicin by three conventional methods (VITEK2(®) , Etest(®) and broth-macrodilution). Bacterial cell count monitoring reliably predicted susceptibility after 90 min for Escherichia coli and after 120 min for Pseudomonas aeruginosa and Staphylococcus aureus without any minor, major or very major discrepancies. Time-to-result was reduced by 74%, 83% and 76%, respectively. Bacterial cell count monitoring shows great potential for rapid susceptibility testing. PMID:22390723

  10. Search for MicroRNAs Expressed by Intracellular Bacterial Pathogens in Infected Mammalian Cells

    PubMed Central

    Furuse, Yuki; Finethy, Ryan; Saka, Hector A.; Xet-Mull, Ana M.; Sisk, Dana M.; Smith, Kristen L. Jurcic; Lee, Sunhee; Coers, Jörn; Valdivia, Raphael H.; Tobin, David M.; Cullen, Bryan R.

    2014-01-01

    MicroRNAs are expressed by all multicellular organisms and play a critical role as post-transcriptional regulators of gene expression. Moreover, different microRNA species are known to influence the progression of a range of different diseases, including cancer and microbial infections. A number of different human viruses also encode microRNAs that can attenuate cellular innate immune responses and promote viral replication, and a fungal pathogen that infects plants has recently been shown to express microRNAs in infected cells that repress host cell immune responses and promote fungal pathogenesis. Here, we have used deep sequencing of total expressed small RNAs, as well as small RNAs associated with the cellular RNA-induced silencing complex RISC, to search for microRNAs that are potentially expressed by intracellular bacterial pathogens and translocated into infected animal cells. In the case of Legionella and Chlamydia and the two mycobacterial species M. smegmatis and M. tuberculosis, we failed to detect any bacterial small RNAs that had the characteristics expected for authentic microRNAs, although large numbers of small RNAs of bacterial origin could be recovered. However, a third mycobacterial species, M. marinum, did express an ∼23-nt small RNA that was bound by RISC and derived from an RNA stem-loop with the characteristics expected for a pre-microRNA. While intracellular expression of this candidate bacterial microRNA was too low to effectively repress target mRNA species in infected cultured cells in vitro, artificial overexpression of this potential bacterial pre-microRNA did result in the efficient repression of a target mRNA. This bacterial small RNA therefore represents the first candidate microRNA of bacterial origin. PMID:25184567

  11. A silicon cell cycle in a bacterial model of calcium phosphate mineralogenesis.

    PubMed

    Linton, Kathryn M; Tapping, Charles R; Adams, David G; CarterR, D Howard; Shore, Roger C; Aaron, Jean E

    2013-01-01

    The prokaryote Corynebacterium matruchotii produces calcium phosphate (bone salt) and may serve as a convenient model for examining individual factors relevant to vertebrate calcification. A factor of current clinical uncertainty is silicon. To investigate its possible role in biomineralisation advanced optical (digital deconvolution and 3D fluorescent image rendering) and electron microscopy (EDX microanalysis and elemental mapping) were applied to calcifying microbial colonies grown in graded Si concentrations (0-60mM). Cell viability was confirmed throughout by TO-PRO-3-iodide and SYTO-9 nucleic acid staining. It was observed that calcium accumulated in dense intracellular microspherical objects (types i-iii) as nanoparticles (5 nm, type i), nanospheres (30-50 nm, type ii) and filamentous clusters (0.1-0.5 μm, type iii), with a regular transitory Si content evident. With bacterial colony development (7-28 days) the P content increased from 5 to 60%, while Si was displaced from 60 to 5%, distinguishing the phenomenon from random contamination, and with a significant relationship (p<0.001) found between calcified object number and Si supplementation (optimum 0.01mM). The Si-containing, intracellular calcified objects (also positive for Mg and negative with Lysensor blue DND-167 for acidocalcisomes) were extruded naturally in bubble-like chains to complete the cycle by coating the cell surface with discrete mineral particles. These could be harvested by lysis, French press and density fractionation when Si was confirmed in a proportion. It was concluded that the unexplained orthopaedic activity of Si may derive from its special property to facilitate calcium phosphorylation in biological systems, thereby recapitulating an ancient and conserved bacterial cycle of calcification via silicification. PMID:23098642

  12. Ankyrin-mediated self-protection during cell invasion by the bacterial predator Bdellovibrio bacteriovorus

    PubMed Central

    Lambert, Carey; Cadby, Ian T.; Till, Rob; Bui, Nhat Khai; Lerner, Thomas R.; Hughes, William S.; Lee, David J.; Alderwick, Luke J.; Vollmer, Waldemar; Sockett, Elizabeth R.; Lovering, Andrew L.

    2015-01-01

    Predatory Bdellovibrio bacteriovorus are natural antimicrobial organisms, killing other bacteria by whole-cell invasion. Self-protection against prey-metabolizing enzymes is important for the evolution of predation. Initial prey entry involves the predator's peptidoglycan DD-endopeptidases, which decrosslink cell walls and prevent wasteful entry by a second predator. Here we identify and characterize a self-protection protein from B. bacteriovorus, Bd3460, which displays an ankyrin-based fold common to intracellular pathogens of eukaryotes. Co-crystal structures reveal Bd3460 complexation of dual targets, binding a conserved epitope of each of the Bd3459 and Bd0816 endopeptidases. Complexation inhibits endopeptidase activity and cell wall decrosslinking in vitro. Self-protection is vital — ΔBd3460 Bdellovibrio deleteriously decrosslink self-peptidoglycan upon invasion, adopt a round morphology, and lose predatory capacity and cellular integrity. Our analysis provides the first mechanistic examination of self-protection in Bdellovibrio, documents protection-multiplicity for products of two different genomic loci, and reveals an important evolutionary adaptation to an invasive predatory bacterial lifestyle. PMID:26626559

  13. Desialylation of Spermatozoa and Epithelial Cell Glycocalyx Is a Consequence of Bacterial Infection of the Epididymis.

    PubMed

    Khosravi, Farhad; Michel, Vera; Galuska, Christina E; Bhushan, Sudhanshu; Christian, Philipp; Schuppe, Hans-Christian; Pilatz, Adrian; Galuska, Sebastian P; Meinhardt, Andreas

    2016-08-19

    Urinary tract infections caused by uropathogenic Escherichia coli (UPEC) pathovars belong to the most frequent infections in humans. In men, pathogens can also spread to the genital tract via the continuous ductal system, eliciting bacterial prostatitis and/or epididymo-orchitis. Antibiotic treatment usually clears pathogens in acute epididymitis; however, the fertility of patients can be permanently impaired. Because a premature acrosome reaction was observed in an UPEC epididymitis mouse model, and sialidases on the sperm surface are considered to be activated via proteases of the acrosome, we aimed to investigate whether alterations of the sialome of epididymal spermatozoa and surrounding epithelial cells occur during UPEC infection. In UPEC-elicited acute epididymitis in mice, a substantial loss of N-acetylneuraminic acid residues was detected in epididymal spermatozoa and epithelial cells using combined laser microdissection/HPLC-ESI-MS analysis. In support, a substantial reduction of sialic acid residues bound to the surface of spermatozoa was documented in men with a recent history of E. coli-associated epididymitis. In vitro, such an UPEC induced N-acetylneuraminic acid release from human spermatozoa was effectively counteracted by a sialidase inhibitor. These findings strongly suggest a substantial remodeling of the glycocalyx of spermatozoa and epididymal epithelial cells by endogenous sialidases after a premature acrosome reaction during acute epididymitis. PMID:27339898

  14. [Disinfectants - bacterial cells interactions in the view of hygiene and public health].

    PubMed

    Książczyk, Marta; Krzyżewska, Eva; Futoma-Kołoch, Bożena; Bugla-Płoskońska, Gabriela

    2015-01-01

    In recent years, the use of biocides has increased rapidly. One common example is triclosan, with wide application in households as well as medical and industrial fields, especially food industry and animal husbandry. Chemical disinfection is a major mean to control and eliminate pathogenic bacteria, particularly those with multidrug resistance (MDR) phenotype. However, exposition to biocides results in an adaptive response in microorganisms, causing them to display a wide range of resistance mechanisms. Numerous microorganisms are characterized by either natural resistance to chemical compounds or an ability to adapt to biocides using various strategies, such as: modification of cell surface structures (lipopolisaccharide), membrane fatty acids), over-expression of efflux pumps (a system for active transport of toxic compounds out of bacterial cell), enzymatic inactivation of biocides or altering biocide targets. For instance, it was shown that in vitro exposition of Salmonella Typhimurium to subinhibitory concentration of biocides (triclosan, quaternary ammonium compounds [QACs]) resulted in selection of variants resistant to tested biocides and, additionally, to acridine dyes and antibiotics. Bacillus subtilis and Micrococcus luteus strains isolated from chlorine dioxide containing disinfection devices were found to be resistant to chlorine dioxide and also to other oxidizing compounds, such as peracetic acid and hydrogen peroxide. Interaction between chemical compounds, including disinfectants and microbial cells, can create a serious threat to public health and sanitary-hygienic security. This phenomenon is connected with factor risk that intensify the probability of selection and dissemination of multidrug resistance among pathogenic bacteria. PMID:26400890

  15. Ankyrin-mediated self-protection during cell invasion by the bacterial predator Bdellovibrio bacteriovorus.

    PubMed

    Lambert, Carey; Cadby, Ian T; Till, Rob; Bui, Nhat Khai; Lerner, Thomas R; Hughes, William S; Lee, David J; Alderwick, Luke J; Vollmer, Waldemar; Sockett, R Elizabeth; Sockett, Elizabeth R; Lovering, Andrew L

    2015-01-01

    Predatory Bdellovibrio bacteriovorus are natural antimicrobial organisms, killing other bacteria by whole-cell invasion. Self-protection against prey-metabolizing enzymes is important for the evolution of predation. Initial prey entry involves the predator's peptidoglycan DD-endopeptidases, which decrosslink cell walls and prevent wasteful entry by a second predator. Here we identify and characterize a self-protection protein from B. bacteriovorus, Bd3460, which displays an ankyrin-based fold common to intracellular pathogens of eukaryotes. Co-crystal structures reveal Bd3460 complexation of dual targets, binding a conserved epitope of each of the Bd3459 and Bd0816 endopeptidases. Complexation inhibits endopeptidase activity and cell wall decrosslinking in vitro. Self-protection is vital - ΔBd3460 Bdellovibrio deleteriously decrosslink self-peptidoglycan upon invasion, adopt a round morphology, and lose predatory capacity and cellular integrity. Our analysis provides the first mechanistic examination of self-protection in Bdellovibrio, documents protection-multiplicity for products of two different genomic loci, and reveals an important evolutionary adaptation to an invasive predatory bacterial lifestyle. PMID:26626559

  16. Inhibitory activity of cranberry extract on the bacterial adhesiveness in the urine of women: an ex-vivo study.

    PubMed

    Tempera, G; Corsello, S; Genovese, C; Caruso, F E; Nicolosi, D

    2010-01-01

    Strains of uropathogenic E. coli are responsible for approximately 90% of community-acquired, uncomplicated cystitis, and fimbriae represent the adhesive factors enabling E. coli to be anchored to uroepithelial cells in the first step of the infectious process. Recently, a few studies have shown that a correlation between the consumption of cranberry (Vaccinium macrocarpon) and prevention of UTI is related to the ability of proanthocyanidins to reduce the bacterial adhesion to uroepithelial cells. In this study we evaluate the inhibitory activity of urine of healthy women treated with tablets containing cranberry extract on the adhesiveness of E. coli to uroepithelial human cells. Two groups of 12 female volunteers each, aged between 18 and 65 years, were enrolled, one group with negative history and one group with positive history of recurrent cystitis. Subjects were treated with the active product or placebo in a random, cross-over, double-blinded sequence for one week in each of the two treatment sequences. Urine samples were collected at the beginning and the end of each study period. Tests of bacterial adhesiveness were performed with two strains of E. coli (ATCC 25922 and ATCC 35218) on HT1376 human bladder carcinoma cells. Significant reductions of bacterial adhesiveness were observed in women who received cranberry extract (-50.9%; p less than 0.0001), regardless of their medical history and the treatment period in the cross-over sequence. No changes were observed with placebo (-0.29%; n.s.). This ex-vivo study showed that the assumption of cranberry extract in suitable amounts can have an anti-adhesive activity on uropathogenic E. coli. PMID:20646356

  17. Disturbance of the bacterial cell wall specifically interferes with biofilm formation.

    PubMed

    Bucher, Tabitha; Oppenheimer-Shaanan, Yaara; Savidor, Alon; Bloom-Ackermann, Zohar; Kolodkin-Gal, Ilana

    2015-12-01

    In nature, bacteria communicate via chemical cues and establish complex communities referred to as biofilms, wherein cells are held together by an extracellular matrix. Much research is focusing on small molecules that manipulate and prevent biofilm assembly by modifying cellular signalling pathways. However, the bacterial cell envelope, presenting the interface between bacterial cells and their surroundings, is largely overlooked. In our study, we identified specific targets within the biosynthesis pathways of the different cell wall components (peptidoglycan, wall teichoic acids and teichuronic acids) hampering biofilm formation and the anchoring of the extracellular matrix with a minimal effect on planktonic growth. In addition, we provide convincing evidence that biofilm hampering by transglycosylation inhibitors and D-Leucine triggers a highly specific response without changing the overall protein levels within the biofilm cells or the overall levels of the extracellular matrix components. The presented results emphasize the central role of the Gram-positive cell wall in biofilm development, resistance and sustainment. PMID:26472159

  18. Fluorescence activated cell sorting.

    NASA Technical Reports Server (NTRS)

    Bonner, W. A.; Hulett, H. R.; Sweet, R. G.; Herzenberg, L. A.

    1972-01-01

    An instrument has been developed for sorting biological cells. The cells are rendered differentially fluorescent and incorporated into a small liquid stream illuminated by a laser beam. The cells pass sequentially through the beam, and fluorescent light from the cells gives rise to electrical signals. The stream is broken into a series of uniform size drops downstream of the laser. The cell signals are used to give appropriate electrostatic charges to drops containing the cells. The drops then pass between two charged plates and are deflected to appropriate containers. The system has proved capable of providing fractions containing large numbers of viable cells highly enriched in a particular functional type.

  19. Synthesis of bacterial magnetic particles during cell cycle of Magnetospirillum magneticum AMB-1.

    PubMed

    Yang, C D; Takeyama, H; Tanaka, T; Hasegawa, A; Matsunaga, T

    2001-01-01

    We investigated the relationship between the synthesis of bacterial magnetic particles (BMPs) and the transcription of magA gene-encoding iron transport protein using synchronous culture of Magnetospirillum magneticum AMB-1. Synchronously cultured cells were subjected to transmission electron microscopic observation and fluorescence in situ hybridization. The average number of BMPs slowly increased in the cell with increasing cell size. A sharp increase in BMPs occurred just before cell division and resulted in maximum BMP production of 30 particles/cell. The transcription of magA was regulated immediately before and after cell division. PMID:11963844

  20. Active viscoelastic matter: from bacterial drag reduction to turbulent solids.

    PubMed

    Hemingway, E J; Maitra, A; Banerjee, S; Marchetti, M C; Ramaswamy, S; Fielding, S M; Cates, M E

    2015-03-01

    A paradigm for internally driven matter is the active nematic liquid crystal, whereby the equations of a conventional nematic are supplemented by a minimal active stress that violates time-reversal symmetry. In practice, active fluids may have not only liquid-crystalline but also viscoelastic polymer degrees of freedom. Here we explore the resulting interplay by coupling an active nematic to a minimal model of polymer rheology. We find that adding a polymer can greatly increase the complexity of spontaneous flow, but can also have calming effects, thereby increasing the net throughput of spontaneous flow along a pipe (a "drag-reduction" effect). Remarkably, active turbulence can also arise after switching on activity in a sufficiently soft elastomeric solid. PMID:25793858

  1. Modelling and analysis of bacterial tracks suggest an active reorientation mechanism in Rhodobacter sphaeroides

    PubMed Central

    Rosser, Gabriel; Baker, Ruth E.; Armitage, Judith P.; Fletcher, Alexander G.

    2014-01-01

    Most free-swimming bacteria move in approximately straight lines, interspersed with random reorientation phases. A key open question concerns varying mechanisms by which reorientation occurs. We combine mathematical modelling with analysis of a large tracking dataset to study the poorly understood reorientation mechanism in the monoflagellate species Rhodobacter sphaeroides. The flagellum on this species rotates counterclockwise to propel the bacterium, periodically ceasing rotation to enable reorientation. When rotation restarts the cell body usually points in a new direction. It has been assumed that the new direction is simply the result of Brownian rotation. We consider three variants of a self-propelled particle model of bacterial motility. The first considers rotational diffusion only, corresponding to a non-chemotactic mutant strain. Two further models incorporate stochastic reorientations, describing ‘run-and-tumble’ motility. We derive expressions for key summary statistics and simulate each model using a stochastic computational algorithm. We also discuss the effect of cell geometry on rotational diffusion. Working with a previously published tracking dataset, we compare predictions of the models with data on individual stopping events in R. sphaeroides. This provides strong evidence that this species undergoes some form of active reorientation rather than simple reorientation by Brownian rotation. PMID:24872500

  2. Bacterial Cell-Cell Communication in the Host via RRNPP Peptide-Binding Regulators.

    PubMed

    Perez-Pascual, David; Monnet, Véronique; Gardan, Rozenn

    2016-01-01

    Human microbiomes are composed of complex and dense bacterial consortia. In these environments, bacteria are able to react quickly to change by coordinating their gene expression at the population level via small signaling molecules. In Gram-positive bacteria, cell-cell communication is mostly mediated by peptides that are released into the extracellular environment. Cell-cell communication based on these peptides is especially widespread in the group Firmicutes, in which they regulate a wide array of biological processes, including functions related to host-microbe interactions. Among the different agents of communication, the RRNPP family of cytoplasmic transcriptional regulators, together with their cognate re-internalized signaling peptides, represents a group of emerging importance. RRNPP members that have been studied so far are found mainly in species of bacilli, streptococci, and enterococci. These bacteria are characterized as both human commensal and pathogenic, and share different niches in the human body with other microorganisms. The goal of this mini-review is to present the current state of research on the biological relevance of RRNPP mechanisms in the context of the host, highlighting their specific roles in commensalism or virulence. PMID:27242728

  3. In Vitro Activity of Gepotidacin, a Novel Triazaacenaphthylene Bacterial Topoisomerase Inhibitor, against a Broad Spectrum of Bacterial Pathogens.

    PubMed

    Biedenbach, D J; Bouchillon, S K; Hackel, M; Miller, L A; Scangarella-Oman, N E; Jakielaszek, C; Sahm, D F

    2016-01-01

    Gepotidacin inhibits bacterial DNA replication through a mode different from that of fluoroquinolones. Gepotidacin and comparators were tested by broth and agar dilution against clinical isolates. The in vitro activities of gepotidacin were comparable against methicillin-susceptible and -resistant Staphylococcus aureus (MSSA and MRSA, respectively) isolates (MIC90, 0.5 μg/ml). The gepotidacin MIC90s were as follows (in micrograms per milliliter) for the indicated bacteria: Streptococcus pyogenes, 0.25; Escherichia coli, 2; Moraxella catarrhalis, ≤0.06; Streptococcus pneumoniae (0.25), Haemophilus influenzae, 1; Clostridium perfringens, 0.5; and Shigella spp., 1, including levofloxacin-resistant subsets. Gepotidacin warrants further investigation for clinical development. PMID:26729499

  4. Slight Pro-Inflammatory Immunomodulation Properties of Dendritic Cells by Gardnerella vaginalis: The “Invisible Man” of Bacterial Vaginosis?

    PubMed Central

    Bertran, Thomas; Brachet, Patrick; Vareille-Delarbre, Marjolaine; Falenta, Julie; Dosgilbert, Annie; Vasson, Marie-Paule; Forestier, Christiane; Tridon, Arlette; Evrard, Bertrand

    2016-01-01

    Bacterial vaginosis (BV), the most common genital infection in reproductive-aged women, is associated with increased risk of sexually transmitted infections. Its etiology remains unclear, especially the role of Gardnerella (G.) vaginalis, an anaerobic bacterium characteristic of the BV-alteration of the vaginal ecosystem. In the genital mucosa, dendritic cells (DCs) sense bacteria of the microenvironment via receptors and then orchestrate the immune response by induction of different T cell subtypes. We investigated the interactions between G. vaginalis and human monocyte-derived DCs using a wide range of bacterial concentrations (multiplicity of infection from 0.01 to 100), and the effects of this pathogen on PHA-induced lymphocyte proliferation. As observed by electron microscopy and cytometry, G. vaginalis reduced the internalization ability of DCs by forming extracellular clusters and induced neither DC maturation, nor DC secretion of cytokines, except at the highest dose with a very early DC maturation state. The same profile was observed on lymphocytes with significant increases of proliferation and cytokine secretion only at the highest bacterial concentration. Our findings indicate that G. vaginalis possesses slight immune-stimulating activities against DCs and T cells, reflecting thus a defective inflammatory response and giving rise to the atypical, non- or low-grade, inflammatory clinical disease profile. PMID:26989700

  5. Anopheles gambiae Ag-STAT, a new insect member of the STAT family, is activated in response to bacterial infection.

    PubMed

    Barillas-Mury, C; Han, Y S; Seeley, D; Kafatos, F C

    1999-02-15

    A new insect member of the STAT family of transcription factors (Ag-STAT) has been cloned from the human malaria vector Anopheles gambiae. The domain involved in DNA interaction and the SH2 domain are well conserved. Ag-STAT is most similar to Drosophila D-STAT and to vertebrate STATs 5 and 6, constituting a proposed ancient class A of the STAT family. The mRNA is expressed at all developmental stages, and the protein is present in hemocytes, pericardial cells, midgut, skeletal muscle and fat body cells. There is no evidence of transcriptional activation following bacterial challenge. However, bacterial challenge results in nuclear translocation of Ag-STAT protein in fat body cells and induction of DNA-binding activity that recognizes a STAT target site. In vitro treatment with pervanadate (vanadate and H2O2) translocates Ag-STAT to the nucleus in midgut epithelial cells. This is the first evidence of direct participation of the STAT pathway in immune responses in insects. PMID:10022838

  6. Microfluidic pretreatment of bacterial cells for analysis of intracellular contents

    NASA Astrophysics Data System (ADS)

    Wang, Hsiang-Yu; Lu, Chang; Banada, Padmapriya P.; Jagadeesan, Balamurugan; Bhunia, Arun K.

    2005-11-01

    Electrical lysis of biological cells on a microfluidic platform has been raising a lot of interests due to its applications in rapid recovering intracellular contents without introducing lytic agents. In this study, we demonstrated a simple microfluidic device which lysed green fluorescent protein (GFP) expressing E. coli cells under continuous DC voltage while cells flowed through. The cell lysis only happened in a defined section of a microfluidic channel due to the local field amplification by geometric modification. The geometric modification also effectively decreased the required voltage for lysis by several folds. We found that a local field strength of 1500V/cm was required for lysis of nearly 100% of E. coli cells. This lysis field strength was considerably lower than the value reported in the literature, possibly due to the longer duration of the field. The lysis was witnessed by plate count and fluorescence spectroscopy. The devices were fabricated using low-cost soft lithography with channel widths considerably larger than the cell size to avoid clogging and ensure stable performance. Our tool will be ideal for high throughput processing of a large number of cells. Furthermore, the application of continuous DC field makes it straightforward to couple our cell lysis device with on-chip electrophoresis to realize the integration of cell pretreatment and chemical analysis. In principle, the same approach can also be applied for the lysis of mammalian cells and for the electroporation and transfection.

  7. Effect of cell physicochemical characteristics and motility on bacterial transport in groundwater

    USGS Publications Warehouse

    Becker, M.W.; Collins, S.A.; Metge, D.W.; Harvey, R.W.; Shapiro, A.M.

    2004-01-01

    The influence of physicochemical characteristics and motility on bacterial transport in groundwater were examined in flow-through columns. Four strains of bacteria isolated from a crystalline rock groundwater system were investigated, with carboxylate-modified and amidine-modified latex microspheres and bromide as reference tracers. The bacterial isolates included a gram-positive rod (ML1), a gram-negative motile rod (ML2), a nonmotile mutant of ML2 (ML2m), and a gram-positive coccoid (ML3). Experiments were repeated at two flow velocities, in a glass column packed with glass beads, and in another packed with iron-oxyhydroxide coated glass beads. Bacteria breakthrough curves were interpreted using a transport equation that incorporates a sorption model from microscopic observation of bacterial deposition in flow-cell experiments. The model predicts that bacterial desorption rate will decrease exponentially with the amount of time the cell is attached to the solid surface. Desorption kinetics appeared to influence transport at the lower flow rate, but were not discernable at the higher flow rate. Iron-oxyhydroxide coatings had a lower-than-expected effect on bacterial breakthrough and no effect on the microsphere recovery in the column experiments. Cell wall type and shape also had minor effects on breakthrough. Motility tended to increase the adsorption rate, and decrease the desorption rate. The transport model predicts that at field scale, desorption rate kinetics may be important to the prediction of bacteria transport rates. ?? 2003 Elsevier B.V. All rights reserved.

  8. Re-Evaluation of a Bacterial Antifreeze Protein as an Adhesin with Ice-Binding Activity

    PubMed Central

    Guo, Shuaiqi; Garnham, Christopher P.; Whitney, John C.; Graham, Laurie A.; Davies, Peter L.

    2012-01-01

    A novel role for antifreeze proteins (AFPs) may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, Marinomonas primoryensis. MpAFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII) and region IV (RIV), divide MpAFP into five distinct regions, all of which require mM Ca2+ levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca2+-bound beta-helix containing thirteen Repeats-In-Toxin (RTX)-like repeats. RII accounts for approximately 90% of the mass of MpAFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2) server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice. PMID:23144980

  9. Re-evaluation of a bacterial antifreeze protein as an adhesin with ice-binding activity.

    PubMed

    Guo, Shuaiqi; Garnham, Christopher P; Whitney, John C; Graham, Laurie A; Davies, Peter L

    2012-01-01

    A novel role for antifreeze proteins (AFPs) may reside in an exceptionally large 1.5-MDa adhesin isolated from an Antarctic Gram-negative bacterium, Marinomonas primoryensis. MpAFP was purified from bacterial lysates by ice adsorption and gel electrophoresis. We have previously reported that two highly repetitive sequences, region II (RII) and region IV (RIV), divide MpAFP into five distinct regions, all of which require mM Ca(2+) levels for correct folding. Also, the antifreeze activity is confined to the 322-residue RIV, which forms a Ca(2+)-bound beta-helix containing thirteen Repeats-In-Toxin (RTX)-like repeats. RII accounts for approximately 90% of the mass of MpAFP and is made up of ∼120 tandem 104-residue repeats. Because these repeats are identical in DNA sequence, their number was estimated here by pulsed-field gel electrophoresis. Structural homology analysis by the Protein Homology/analogY Recognition Engine (Phyre2) server indicates that the 104-residue RII repeat adopts an immunoglobulin beta-sandwich fold that is typical of many secreted adhesion proteins. Additional RTX-like repeats in RV may serve as a non-cleavable signal sequence for the type I secretion pathway. Immunodetection shows both repeated regions are uniformly distributed over the cell surface. We suggest that the development of an AFP-like domain within this adhesin attached to the bacterial outer surface serves to transiently bind the host bacteria to ice. This association would keep the bacteria within the upper reaches of the water column where oxygen and nutrients are potentially more abundant. This novel envirotactic role would give AFPs a third function, after freeze avoidance and freeze tolerance: that of transiently binding an organism to ice. PMID:23144980

  10. Bacterial growth, detachment and cell size control on polyethylene terephthalate surfaces

    PubMed Central

    Wang, Liyun; Fan, Daming; Chen, Wei; Terentjev, Eugene M.

    2015-01-01

    In medicine and food industry, bacterial colonisation on surfaces is a common cause of infections and severe illnesses. However, the detailed quantitative information about the dynamics and the mechanisms involved in bacterial proliferation on solid substrates is still lacking. In this study we investigated the adhesion and detachment, the individual growth and colonisation, and the cell size control of Escherichia coli (E. coli) MG1655 on polyethylene terephthalate (PET) surfaces. The results show that the bacterial growth curve on PET exhibits the distinct lag and log phases, but the generation time is more than twice longer than in bulk medium. Single cells in the lag phase are more likely to detach than clustered ones in the log phase; clustered bacteria in micro-colonies have stronger adhesive bonds with surfaces and their neighbours with the progressing colonisation. We show that the cell size is under the density-dependent pathway control: when the adherent cells are at low density, the culture medium is responsible for coordinating cell division and cell size; when the clustered cells are at high population density, we demonstrate that the effect of quorum sensing causes the cell size decrease as the cell density on surfaces increases. PMID:26464114

  11. Effect of Lewis blood group antigen expression on bacterial adherence to COS-1 cells.

    PubMed Central

    Gaffney, R A; Schaeffer, A J; Anderson, B E; Duncan, J L

    1994-01-01

    Epithelial cells from secretor individuals demonstrate decreased bacterial adherence compared with cells from nonsecretors. Lewis blood group antigen expression is one component of the secretor/nonsecretor phenotype and several epidemiologic studies have suggested a link between Lewis blood group antigen phenotype and susceptibility to urinary tract infections. In this study, we examined the possibility that the expression of the difucosylated Lewis blood group determinants, Leb and Ley (associated with the secretor phenotype), made cells less susceptible to Escherichia coli adherence by masking receptors for pili. COS-1 cells, which do not produce Lewis (Lea, Leb, Le(x), and Ley) blood group antigens, were used as target cells for bacterial adherence. The surface blood group antigen expression pattern of the cells was then modified by cotransfection with plasmids containing DNA inserts encoding alpha (1,2)-fucosyltransferase and alpha (1,3)- and alpha (1,4)-fucosyltransferases, resulting in the expression of Leb and Ley. E. coli HB101 expressing various adhesins (type 1, PapJ96, PapIA2, PapAD110, Prs, and S) from recombinant plasmids bound equally well to untransfected cells and transfected cells expressing Lea and Le(x) (nonsecretor phenotype) and Leb and Ley (secretor phenotype) antigens. We conclude that the presence of Leb and Ley antigens on cells from secretors does not alone mask receptors for E. coli pili or hinder bacterial adherence. PMID:8005692

  12. Relationship between Milk Microbiota, Bacterial Load, Macronutrients, and Human Cells during Lactation.

    PubMed

    Boix-Amorós, Alba; Collado, Maria C; Mira, Alex

    2016-01-01

    Human breast milk is considered the optimal nutrition for infants, providing essential nutrients and a broad range of bioactive compounds, as well as its own microbiota. However, the interaction among those components and the biological role of milk microorganisms is still uncovered. Thus, our aim was to identify the relationships between milk microbiota composition, bacterial load, macronutrients, and human cells during lactation. Bacterial load was estimated in milk samples from a total of 21 healthy mothers through lactation time by bacteria-specific qPCR targeted to the single-copy gene fusA. Milk microbiome composition and diversity was estimated by 16S-pyrosequencing and the structure of these bacteria in the fluid was studied by flow cytometry, qPCR, and microscopy. Fat, protein, lactose, and dry extract of milk as well as the number of somatic cells were also analyzed. We observed that milk bacterial communities were generally complex, and showed individual-specific profiles. Milk microbiota was dominated by Staphylococcus, Pseudomonas, Streptococcus, and Acinetobacter. Staphylococcus aureus was not detected in any of these samples from healthy mothers. There was high variability in composition and number of bacteria per milliliter among mothers and in some cases even within mothers at different time points. The median bacterial load was 10(6) bacterial cells/ml through time, higher than those numbers reported by 16S gene PCR and culture methods. Furthermore, milk bacteria were present in a free-living, "planktonic" state, but also in equal proportion associated to human immune cells. There was no correlation between bacterial load and the amount of immune cells in milk, strengthening the idea that milk bacteria are not sensed as an infection by the immune system. PMID:27148183

  13. Relationship between Milk Microbiota, Bacterial Load, Macronutrients, and Human Cells during Lactation

    PubMed Central

    Boix-Amorós, Alba; Collado, Maria C.; Mira, Alex

    2016-01-01

    Human breast milk is considered the optimal nutrition for infants, providing essential nutrients and a broad range of bioactive compounds, as well as its own microbiota. However, the interaction among those components and the biological role of milk microorganisms is still uncovered. Thus, our aim was to identify the relationships between milk microbiota composition, bacterial load, macronutrients, and human cells during lactation. Bacterial load was estimated in milk samples from a total of 21 healthy mothers through lactation time by bacteria-specific qPCR targeted to the single-copy gene fusA. Milk microbiome composition and diversity was estimated by 16S-pyrosequencing and the structure of these bacteria in the fluid was studied by flow cytometry, qPCR, and microscopy. Fat, protein, lactose, and dry extract of milk as well as the number of somatic cells were also analyzed. We observed that milk bacterial communities were generally complex, and showed individual-specific profiles. Milk microbiota was dominated by Staphylococcus, Pseudomonas, Streptococcus, and Acinetobacter. Staphylococcus aureus was not detected in any of these samples from healthy mothers. There was high variability in composition and number of bacteria per milliliter among mothers and in some cases even within mothers at different time points. The median bacterial load was 106 bacterial cells/ml through time, higher than those numbers reported by 16S gene PCR and culture methods. Furthermore, milk bacteria were present in a free-living, “planktonic” state, but also in equal proportion associated to human immune cells. There was no correlation between bacterial load and the amount of immune cells in milk, strengthening the idea that milk bacteria are not sensed as an infection by the immune system. PMID:27148183

  14. Differences in activity profile of bacterial cultures studied by dynamic speckle patterns

    NASA Astrophysics Data System (ADS)

    Ramírez-Miquet, E. E.; Otero, I.; Rodríguez, D.; Darias, J. G.; Combarro, A. M.; Contreras, O. R.

    2013-02-01

    We outline the main differences in the activity profile of bacterial cultures studied by dynamic laser speckle (or biospeckle) patterns. The activity is detected in two sorts of culture mediums. The optical setup and the experimental procedure are presented. The experimentally obtained images are processed by the temporal difference method and a qualitative assessment is made with the time history of speckle patterns of the sample. The main differences are studied after changing the culture medium composition. We conclude that the EC medium is suitable to detect the E. coli bacterial presence in early hours and that Mueller Hinton agar delays some additional hours to make possible the assessment of bacteria in time.

  15. Active bacterial community structure along vertical redox gradients in Baltic Sea sediment

    SciTech Connect

    Jansson, Janet; Edlund, Anna; Hardeman, Fredrik; Jansson, Janet K.; Sjoling, Sara

    2008-05-15

    Community structures of active bacterial populations were investigated along a vertical redox profile in coastal Baltic Sea sediments by terminal-restriction fragment length polymorphism (T-RFLP) and clone library analysis. According to correspondence analysis of T-RFLP results and sequencing of cloned 16S rRNA genes, the microbial community structures at three redox depths (179 mV, -64 mV and -337 mV) differed significantly. The bacterial communities in the community DNA differed from those in bromodeoxyuridine (BrdU)-labeled DNA, indicating that the growing members of the community that incorporated BrdU were not necessarily the most dominant members. The structures of the actively growing bacterial communities were most strongly correlated to organic carbon followed by total nitrogen and redox potentials. Bacterial identification by sequencing of 16S rRNA genes from clones of BrdU-labeled DNA and DNA from reverse transcription PCR (rt-PCR) showed that bacterial taxa involved in nitrogen and sulfur cycling were metabolically active along the redox profiles. Several sequences had low similarities to previously detected sequences indicating that novel lineages of bacteria are present in Baltic Sea sediments. Also, a high number of different 16S rRNA gene sequences representing different phyla were detected at all sampling depths.

  16. Anaerobic ammonium oxidation (anammox) bacterial diversity, abundance, and activity in marsh sediments of the Yangtze Estuary

    NASA Astrophysics Data System (ADS)

    Hou, Lijun; Zheng, Yanling; Liu, Min; Gong, Jun; Zhang, Xiaoli; Yin, Guoyu; You, Li

    2013-07-01

    ammonium oxidation (anammox) as an important process of nitrogen cycle has been studied in estuarine environments. However, knowledge about the dynamics of anammox bacteria and their interactions with associated activity remains scarce in these environments. Here we report the anammox bacterial diversity, abundance, and activity in the Yangtze Estuary, using molecular and isotope-tracing techniques. The phylogenetic analysis of 16S rRNA indicated that high anammox bacterial diversity occurred in this estuary, including Scalindua, Brocadia, Kuenenia, and two novel clusters. The patterns of community composition and diversity of anammox bacteria differed across the estuary. Salinity was a key environmental factor defining the geographical distribution and diversity of the anammox bacterial community at the estuarine ecosystem. Temperature and organic carbon also had significant influences on anammox bacterial biodiversity. The abundance of anammox bacteria ranged from 2.63 × 106 and 1.56 × 107 gene copies g-1, and its spatiotemporal variations were related significantly to salinity, temperature, and nitrite content. The anammox activity was related to temperature, nitrite, and anammox bacterial abundance, with values of 0.94-6.61 nmol N g-1 h-1. The tight link between the anammox and denitrification processes implied that denitrifying bacteria may be a primary source of nitrite for the anammox bacteria in the estuarine marshes. On the basis of the 15N tracing experiments, the anammox process was estimated to contribute 6.6%-12.9% to the total nitrogen loss whereas the remainder was attributed to denitrification.

  17. Comparison of bacterial communities of conventional and A-stage activated sludge systems

    PubMed Central

    Gonzalez-Martinez, Alejandro; Rodriguez-Sanchez, Alejandro; Lotti, Tommaso; Garcia-Ruiz, Maria-Jesus; Osorio, Francisco; Gonzalez-Lopez, Jesus; van Loosdrecht, Mark C. M.

    2016-01-01

    The bacterial community structure of 10 different wastewater treatment systems and their influents has been investigated through pyrosequencing, yielding a total of 283486 reads. These bioreactors had different technological configurations: conventional activated sludge (CAS) systems and very highly loaded A-stage systems. A-stage processes are proposed as the first step in an energy producing municipal wastewater treatment process. Pyrosequencing analysis indicated that bacterial community structure of all influents was similar. Also the bacterial community of all CAS bioreactors was similar. Bacterial community structure of A-stage bioreactors showed a more case-specific pattern. A core of genera was consistently found for all influents, all CAS bioreactors and all A-stage bioreactors, respectively, showing that different geographical locations in The Netherlands and Spain did not affect the functional bacterial communities in these technologies. The ecological roles of these bacteria were discussed. Influents and A-stage bioreactors shared several core genera, while none of these were shared with CAS bioreactors communities. This difference is thought to reside in the different operational conditions of the two technologies. This study shows that bacterial community structure of CAS and A-stage bioreactors are mostly driven by solids retention time (SRT) and hydraulic retention time (HRT), as suggested by multivariate redundancy analysis. PMID:26728449

  18. Effect of Condensed Tannins on Bacterial Diversity and Metabolic Activity in the Rat Gastrointestinal Tract

    PubMed Central

    Smith, Alexandra H.; Mackie, Roderick I.

    2004-01-01

    The effect of dietary condensed tannins (proanthocyanidins) on rat fecal bacterial populations was ascertained in order to determine whether the proportion on tannin-resistant bacteria increased and if there was a change in the predominant bacterial populations. After 3 weeks of tannin diets the proportion of tannin-resistant bacteria increased significantly (P < 0.05) from 0.3% ± 5.5% to 25.3% ± 8.3% with a 0.7% tannin diet and to 47.2% ± 5.1% with a 2% tannin diet. The proportion of tannin-resistant bacteria returned to preexposure levels in the absence of dietary tannins. A shift in bacterial populations was confirmed by molecular fingerprinting of fecal bacterial populations by denaturing gradient gel electrophoresis (DGGE). Posttreatment samples were generally still distinguishable from controls after 3.5 weeks. Sequence analysis of DGGE bands and characterization of tannin-resistant isolates indicated that tannins selected for Enterobacteriaceae and Bacteroides species. Dot blot quantification confirmed that these gram-negative bacterial groups predominated in the presence of dietary tannins and that there was a corresponding decrease in the gram-positive Clostridium leptum group and other groups. Metabolic fingerprint patterns revealed that functional activities of culturable fecal bacteria were affected by the presence of tannins. Condensed tannins of Acacia angustissima altered fecal bacterial populations in the rat gastrointestinal tract, resulting in a shift in the predominant bacteria towards tannin-resistant gram-negative Enterobacteriaceae and Bacteroides species. PMID:14766594

  19. Cell motility and antibiotic tolerance of bacterial swarms

    NASA Astrophysics Data System (ADS)

    Zuo, Wenlong

    Many bacteria species can move across moist surfaces in a coordinated manner known as swarming. It is reported that swarm cells show higher tolerance to a wide variety of antibiotics than planktonic cells. We used the model bacterium E. coli to study how motility affects the antibiotic tolerance of swarm cells. Our results provide new insights for the control of pathogenic invasion via regulating cell motility. Mailing address: Room 306 Science Centre North Block, The Chinese University of Hong Kong, Shatin, N.T. Hong Kong SAR. Phone: +852-3943-6354. Fax: +852-2603-5204. E-mail: zwlong@live.com.

  20. Production Model Press for the Preparation of Bacterial Cell Walls

    PubMed Central

    Perrine, T. D.; Ribi, E.; Maki, W.; Miller, B.; Oertli, E.

    1962-01-01

    A modification of the apparatus previously described permits the preparation of cell walls in quantity. This consists of a heavy duty, double-acting hydraulic press with motor-driven pump, and a superstrength alloy steel pressure cell which is corrosion resistant. Liquid cooling of the jet is substituted for the previously used gas cooling to minimize aerosol formation and to facilitate subsequent treatment of the products. The device produces cell walls of excellent quality in good yield. The pressure cell has been used satisfactorily up to about 60,000 psi. Design details are given. Images FIG. 1 FIG. 2 FIG. 6 PMID:14485524

  1. Modulation of Toll-Like Receptor Activity by Leukocyte Ig-Like Receptors and Their Effects during Bacterial Infection

    PubMed Central

    Pilsbury, Louise E.; Allen, Rachel L.; Vordermeier, Martin

    2010-01-01

    Toll-like receptors (TLRs) are a potent trigger for inflammatory immune responses. Without tight regulation their activation could lead to pathology, so it is imperative to extend our understanding of the regulatory mechanisms that govern TLR expression and function. One family of immunoregulatory proteins which can provide a balancing effect on TLR activity are the Leukocyte Ig-like receptors (LILRs), which act as innate immune receptors for self-proteins. Here we describe the LILR family, their inhibitory effect on TLR activity in cells of the monocytic lineage, their signalling pathway, and their antimicrobial effects during bacterial infection. Agents have already been identified which enhances or inhibits LILR activity raising the future possibility that modulation of LILR function could be used as a means to modulate TLR activity. PMID:20634939

  2. Degradation of endogenous bacterial cell wall polymers by the muralytic enzyme mutanolysin prevents hepatobiliary injury in genetically susceptible rats with experimental intestinal bacterial overgrowth.

    PubMed Central

    Lichtman, S N; Okoruwa, E E; Keku, J; Schwab, J H; Sartor, R B

    1992-01-01

    Jejunal self-filling blind loops with subsequent small bowel bacterial overgrowth (SBBO) induce hepatobiliary injury in genetically susceptible Lewis rats. Lesions consist of portal tract inflammation, bile duct proliferation, and destruction. To determine the pathogenesis of SBBO-induced hepatobiliary injury, we treated Lewis rats with SBBO by using several agents with different mechanisms of activity. Buffer treatment, ursodeoxycholic acid, prednisone, methotrexate, and cyclosporin A failed to prevent SBBO-induced injury as demonstrated by increased plasma aspartate aminotransferase (AST) and elevated histology scores. However, hepatic injury was prevented by mutanolysin, a muralytic enzyme whose only known activity is to split the beta 1-4 N-acetylmuramyl-N-acetylglucosamine linkage of peptidoglycan-polysaccharide (PG-PS), a bacterial cell wall polymer with potent inflammatory and immunoregulatory properties. Mutanolysin therapy started on the day blind loops were surgically created and continued for 8 wk significantly diminished AST (101 +/- 37 U/liter) and liver histology scores (2.2 +/- 2.7) compared to buffer-treated rats (228 +/- 146 U/liter, P < 0.05, 8.2 +/- 1.9, P < 0.001 respectively). Mutanolysin treatment started during the early phase of hepatic injury, 16-21 d after surgery, decreased AST in 7 of 11 rats from 142 +/- 80 to 103 +/- 24 U/liter contrasted to increased AST in 9 of 11 buffer-treated rats from 108 +/- 52 to 247 +/- 142 U/liter, P < 0.05. Mutanolysin did not change total bacterial numbers within the loop, eliminate Bacteroides sp., have in vitro antibiotic effects, or diminish mucosal PG-PS transport. However, mutanolysin treatment prevented elevation of plasma anti-PG antibodies and tumor necrosis factor-alpha (TNF alpha) levels which occurred in buffer treated rats with SBBO and decreased TNF alpha production in isolated Kupffer cells stimulated in vitro with PG-PS. Based on the preventive and therapeutic activity of this highly specific

  3. Nanoscale Electric Permittivity of Single Bacterial Cells at Gigahertz Frequencies by Scanning Microwave Microscopy.

    PubMed

    Biagi, Maria Chiara; Fabregas, Rene; Gramse, Georg; Van Der Hofstadt, Marc; Juárez, Antonio; Kienberger, Ferry; Fumagalli, Laura; Gomila, Gabriel

    2016-01-26

    We quantified the electric permittivity of single bacterial cells at microwave frequencies and nanoscale spatial resolution by means of near-field scanning microwave microscopy. To this end, calibrated complex admittance images have been obtained at ∼19 GHz and analyzed with a methodology that removes the nonlocal topographic cross-talk contributions and thus provides quantifiable intrinsic dielectric images of the bacterial cells. Results for single Escherichia coli cells provide a relative electric permittivity of ∼4 in dry conditions and ∼20 in humid conditions, with no significant loss contributions. Present findings, together with the ability of microwaves to penetrate the cell membrane, open an important avenue in the microwave label-free imaging of single cells with nanoscale spatial resolution. PMID:26643251

  4. Fluorescence imaging for bacterial cell biology: from localization to dynamics, from ensembles to single molecules.

    PubMed

    Yao, Zhizhong; Carballido-López, Rut

    2014-01-01

    Fluorescent proteins and developments in superresolution (nanoscopy) and single-molecule techniques bring high sensitivity, speed, and one order of magnitude gain in spatial resolution to live-cell imaging. These technologies have only recently been applied to prokaryotic cell biology, revealing the exquisite subcellular organization of bacterial cells. Here, we review the parallel evolution of fluorescence microscopy methods and their application to bacteria, mainly drawing examples from visualizing actin-like MreB proteins in the model bacterium Bacillus subtilis. We describe the basic principles of nanoscopy and conventional techniques and their advantages and limitations to help microbiologists choose the most suitable technique for their biological question. Looking ahead, multidimensional live-cell nanoscopy combined with computational image analysis tools, systems biology approaches, and mathematical modeling will provide movie-like, mechanistic, and quantitative description of molecular events in bacterial cells. PMID:25002084

  5. Effect of Micro- and Nanoscale Topography on the Adhesion of Bacterial Cells to Solid Surfaces

    PubMed Central

    Hsu, Lillian C.; Fang, Jean; Borca-Tasciuc, Diana A.; Worobo, Randy W.

    2013-01-01

    Attachment and biofilm formation by bacterial pathogens on surfaces in natural, industrial, and hospital settings lead to infections and illnesses and even death. Minimizing bacterial attachment to surfaces using controlled topography could reduce the spreading of pathogens and, thus, the incidence of illnesses and subsequent human and financial losses. In this context, the attachment of key microorganisms, including Escherichia coli, Listeria innocua, and Pseudomonas fluorescens, to silica and alumina surfaces with micron and nanoscale topography was investigated. The results suggest that orientation of the attached cells occurs preferentially such as to maximize their contact area with the surface. Moreover, the bacterial cells exhibited different morphologies, including different number and size of cellular appendages, depending on the topographical details of the surface to which they attached. This suggests that bacteria may utilize different mechanisms of attachment in response to surface topography. These results are important for the design of novel microbe-repellant materials. PMID:23416997

  6. Histamine induces Toll-like receptor 2 and 4 expression in endothelial cells and enhances sensitivity to Gram-positive and Gram-negative bacterial cell wall components

    PubMed Central

    Talreja, Jaya; Kabir, Mohammad H; Filla, Michael B; Stechschulte, Daniel J; Dileepan, Kottarappat N

    2004-01-01

    Histamine is a major inflammatory molecule released from the mast cell, and is known to activate endothelial cells. However, its ability to modulate endothelial responses to bacterial products has not been evaluated. In this study we determined the ability of histamine to modulate inflammatory responses of endothelial cells to Gram-negative and Gram-positive bacterial cell wall components and assessed the role of Toll-like receptors (TLR) 2 and 4 in the co-operation between histamine and bacterial pathogens. Human umbilical vein endothelial cells (HUVEC) were incubated with lipopolysaccharide (LPS), lipoteichoic acid (LTA), or peptidoglycan (PGN) in the presence or absence of histamine, and the expression and release of interleukin-6 (IL-6), and NF-κB translocation were determined. The effect of histamine on the expression of mRNA and proteins for TLR2 and TLR4 was also evaluated. Incubation of HUVEC with LPS, LTA and PGN resulted in marked enhancement of IL-6 mRNA expression and IL-6 secretion. Histamine alone markedly enhanced IL-6 mRNA expression in HUVEC, but it did not stimulate proportional IL-6 release. When HUVEC were incubated with LPS, LTA, or PGN in the presence of histamine marked amplification of both IL-6 production and mRNA expression was noted. HUVEC constitutively expressed TLR2 and TLR4 mRNA and proteins, and these were further enhanced by histamine. The expression of mRNAs encoding MD-2 and MyD88, the accessory molecules associated with TLR signalling, were unchanged by histamine treatment. These results demonstrate that histamine up-regulates the expression of TLR2 and TLR4 and amplifies endothelial cell inflammatory responses to Gram-negative and Gram-positive bacterial components. PMID:15379983

  7. A mechanistic stochastic framework for regulating bacterial cell division

    PubMed Central

    Ghusinga, Khem Raj; Vargas-Garcia, Cesar A.; Singh, Abhyudai

    2016-01-01

    How exponentially growing cells maintain size homeostasis is an important fundamental problem. Recent single-cell studies in prokaryotes have uncovered the adder principle, where cells add a fixed size (volume) from birth to division, irrespective of their size at birth. To mechanistically explain the adder principle, we consider a timekeeper protein that begins to get stochastically expressed after cell birth at a rate proportional to the volume. Cell-division time is formulated as the first-passage time for protein copy numbers to hit a fixed threshold. Consistent with data, the model predicts that the noise in division timing increases with size at birth. Intriguingly, our results show that the distribution of the volume added between successive cell-division events is independent of the newborn cell size. This was dramatically seen in experimental studies, where histograms of the added volume corresponding to different newborn sizes collapsed on top of each other. The model provides further insights consistent with experimental observations: the distribution of the added volume when scaled by its mean becomes invariant of the growth rate. In summary, our simple yet elegant model explains key experimental findings and suggests a mechanism for regulating both the mean and fluctuations in cell-division timing for controlling size. PMID:27456660

  8. A mechanistic stochastic framework for regulating bacterial cell division.

    PubMed

    Ghusinga, Khem Raj; Vargas-Garcia, Cesar A; Singh, Abhyudai

    2016-01-01

    How exponentially growing cells maintain size homeostasis is an important fundamental problem. Recent single-cell studies in prokaryotes have uncovered the adder principle, where cells add a fixed size (volume) from birth to division, irrespective of their size at birth. To mechanistically explain the adder principle, we consider a timekeeper protein that begins to get stochastically expressed after cell birth at a rate proportional to the volume. Cell-division time is formulated as the first-passage time for protein copy numbers to hit a fixed threshold. Consistent with data, the model predicts that the noise in division timing increases with size at birth. Intriguingly, our results show that the distribution of the volume added between successive cell-division events is independent of the newborn cell size. This was dramatically seen in experimental studies, where histograms of the added volume corresponding to different newborn sizes collapsed on top of each other. The model provides further insights consistent with experimental observations: the distribution of the added volume when scaled by its mean becomes invariant of the growth rate. In summary, our simple yet elegant model explains key experimental findings and suggests a mechanism for regulating both the mean and fluctuations in cell-division timing for controlling size. PMID:27456660

  9. Temporal and spatial trigger of post-exponential virulence-associated regulatory cascades by Legionella pneumophila after bacterial escape into the host cell cytosol.

    PubMed

    Molmeret, Maëlle; Jones, Snake; Santic, Marina; Habyarimana, Fabien; Esteban, Maria Teresa Garcia; Kwaik, Yousef Abu

    2010-03-01

    During late stages of infection and prior to lysis of the infected macrophages or amoeba, the Legionella pneumophila-containing phagosome becomes disrupted, followed by bacterial escape into the host cell cytosol, where the last few rounds of bacterial proliferation occur prior to lysis of the plasma membrane. This coincides with growth transition into the post-exponential (PE) phase, which is controlled by regulatory cascades including RpoS and the LetA/S two-component regulator. Whether the temporal expression of flagella by the regulatory cascades at the PE phase is exhibited within the phagosome or after bacterial escape into the host cell cytosol is not known. We have utilized fluorescence microscopy-based phagosome integrity assay to differentiate between vacuolar and cytosolic bacteria/or bacteria within disrupted phagosomes. Our data show that during late stages of infection, expression of FlaA is triggered after bacterial escape into the macrophage cytosol and the peak of FlaA expression is delayed for few hours after cytosolic residence of the bacteria. Importantly, bacterial escape into the host cell cytosol is independent of flagella, RpoS and the two-component regulator LetA/S, which are all triggered by L. pneumophila upon growth transition into the PE phase. Disruption of the phagosome and bacterial escape into the cytosol of macrophages is independent of the bacterial pore-forming activity, and occurs prior to the induction of apoptosis during late stages of infection. We conclude that the temporal and spatial engagement of virulence-associated regulatory cascades by L. pneumophila at the PE phase is temporally and spatially triggered after phagosomal escape and bacterial residence in the host cell cytosol. PMID:19958381

  10. Temporal and spatial trigger of post-exponential virulence-associated regulatory cascades by Legionella pneumophila after bacterial escape into the host cell cytosol

    PubMed Central

    Molmeret, Maëlle; Jones, Snake; Santic, Marina; Habyarimana, Fabien; Esteban, Maria Teresa Garcia; Kwaik, Yousef Abu

    2009-01-01

    Summary During late stages of infection and prior to lysis of the infected macrophages or amoeba, the Legionella pneumophila-containing phagosome becomes disrupted, followed by bacterial escape into the host cell cytosol, where the last few rounds of bacterial proliferation occur prior to lysis of the plasma membrane. This coincides with growth transition into the post-exponential (PE) phase, which is controlled by regulatory cascades including RpoS and the LetA/S two component regulator. Whether the temporal expression of flagella by the regulatory cascades at the PE phase is exhibited within the phagosome or after bacterial escape into the host cell cytosol is not known. We have utilized fluorescence microscopy-based phagosome integrity assay to differentiate between vacuolar and cytosolic bacteria/ or bacteria within disrupted phagosomes. Our data show that during late stages of infection, expression of FlaA is triggered after bacterial escape into the macrophage cytosol and the peak of FlaA expression is delayed for few hours after cytosolic residence of the bacteria. Importantly, bacterial escape into the host cell cytosol is independent of flagella, RpoS, and the two component regulator LetA/S, which are all triggered by L. pneumophila upon growth transition into the PE phase. Disruption of the phagosome and bacterial escape into the cytosol of macrophages is independent of the bacterial pore-forming activity, and occurs prior to the induction of apoptosis during late stages of infection. We conclude that the temporal and spatial engagement of virulence-associated regulatory cascades by L. pneumophila at the PE phase is temporally and spatially triggered after phagosomal escape and bacterial residence in the host cell cytosol. PMID:19958381

  11. Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing.

    PubMed

    Riba, J; Gleichmann, T; Zimmermann, S; Zengerle, R; Koltay, P

    2016-01-01

    The isolation and analysis of single prokaryotic cells down to 1 μm and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35 pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20 μm in size. Further, the magnification of the optical system used for cell detection was increased. Redesign of the optical path allows for collision-free addressing of any flat substrate since no compartment protrudes below the nozzle of the dispenser chip anymore. The improved system allows for deterministic isolation of individual bacterial cells. A single-cell printing efficiency of 93% was obtained as shown by printing fluorescent labeled E. coli. A 96-well plate filled with growth medium is inoculated with single bacteria cells on average within about 8 min. Finally, individual bacterial cells from a heterogeneous sample of E. coli and E. faecalis were isolated for clonal culturing directly on agar plates in user-defined array geometry. PMID:27596612

  12. The percentage of living bacterial cells related to organic carbon release from senescent oceanic phytoplankton

    NASA Astrophysics Data System (ADS)

    Lasternas, S.; Agustí, S.

    2014-11-01

    Bacteria recycle vast amounts of organic carbon, playing key biogeochemical and ecological roles in the ocean. Bacterioplankton dynamics are expected to be dependent on phytoplankton primary production, but there is a high diversity of processes (e.g., sloppy feeding, cell exudation, viral lysis) involved in the transfer of primary production to dissolved organic carbon available to bacteria. Here, we show the percentage of living heterotrophic bacterioplankton in the subtropical NE Atlantic Ocean in relation to phytoplankton extracellular carbon release (PER). PER represents the fraction of primary production released as dissolved organic carbon. PER variability was explained by phytoplankton cell death, with communities experiencing higher phytoplankton cell mortality showing a larger proportion of phytoplankton extracellular carbon release. Both PER and the percentage of dead phytoplankton cells increased from eutrophic to oligotrophic waters, while abundance of heterotrophic bacteria was highest in the intermediate waters. The percentage of living heterotrophic bacterial cells (range: 60-95%) increased with increasing phytoplankton extracellular carbon release from productive to oligotrophic waters in the subtropical NE Atlantic. The lower PERs, observed at the upwelling waters, have resulted in a decrease in the flux of phytoplankton dissolved organic carbon (DOC) per bacterial cell. The results highlight phytoplankton cell death as a process influencing the flow of dissolved photosynthetic carbon in this region of the subtropical NE Atlantic Ocean, and suggest a close coupling between the fraction of primary production released and heterotrophic bacterial cell survival.

  13. Vehicles, Replicators, and Intercellular Movement of Genetic Information: Evolutionary Dissection of a Bacterial Cell

    PubMed Central

    Jalasvuori, Matti

    2012-01-01

    Prokaryotic biosphere is vastly diverse in many respects. Any given bacterial cell may harbor in different combinations viruses, plasmids, transposons, and other genetic elements along with their chromosome(s). These agents interact in complex environments in various ways causing multitude of phenotypic effects on their hosting cells. In this discussion I perform a dissection for a bacterial cell in order to simplify the diversity into components that may help approach the ocean of details in evolving microbial worlds. The cell itself is separated from all the genetic replicators that use the cell vehicle for preservation and propagation. I introduce a classification that groups different replicators according to their horizontal movement potential between cells and according to their effects on the fitness of their present host cells. The classification is used to discuss and improve the means by which we approach general evolutionary tendencies in microbial communities. Moreover, the classification is utilized as a tool to help formulating evolutionary hypotheses and to discuss emerging bacterial pathogens as well as to promote understanding on the average phenotypes of different replicators in general. It is also discussed that any given biosphere comprising prokaryotic cell vehicles and genetic replicators may naturally evolve to have horizontally moving replicators of various types. PMID:22567533

  14. Label-free isolation and deposition of single bacterial cells from heterogeneous samples for clonal culturing

    PubMed Central

    Riba, J.; Gleichmann, T.; Zimmermann, S.; Zengerle, R.; Koltay, P.

    2016-01-01

    The isolation and analysis of single prokaryotic cells down to 1 μm and less in size poses a special challenge and requires micro-engineered devices to handle volumes in the picoliter to nanoliter range. Here, an advanced Single-Cell Printer (SCP) was applied for automated and label-free isolation and deposition of bacterial cells encapsulated in 35 pl droplets by inkjet-like printing. To achieve this, dispenser chips to generate micro droplets have been fabricated with nozzles 20 μm in size. Further, the magnification of the optical system used for cell detection was increased. Redesign of the optical path allows for collision-free addressing of any flat substrate since no compartment protrudes below the nozzle of the dispenser chip anymore. The improved system allows for deterministic isolation of individual bacterial cells. A single-cell printing efficiency of 93% was obtained as shown by printing fluorescent labeled E. coli. A 96-well plate filled with growth medium is inoculated with single bacteria cells on average within about 8 min. Finally, individual bacterial cells from a heterogeneous sample of E. coli and E. faecalis were isolated for clonal culturing directly on agar plates in user-defined array geometry. PMID:27596612

  15. Do bacterial cell numbers follow a theoretical Poisson distribution? Comparison of experimentally obtained numbers of single cells with random number generation via computer simulation.

    PubMed

    Koyama, Kento; Hokunan, Hidekazu; Hasegawa, Mayumi; Kawamura, Shuso; Koseki, Shigenobu

    2016-12-01

    We investigated a bacterial sample preparation procedure for single-cell studies. In the present study, we examined whether single bacterial cells obtained via 10-fold dilution followed a theoretical Poisson distribution. Four serotypes of Salmonella enterica, three serotypes of enterohaemorrhagic Escherichia coli and one serotype of Listeria monocytogenes were used as sample bacteria. An inoculum of each serotype was prepared via a 10-fold dilution series to obtain bacterial cell counts with mean values of one or two. To determine whether the experimentally obtained bacterial cell counts follow a theoretical Poisson distribution, a likelihood ratio test between the experimentally obtained cell counts and Poisson distribution which parameter estimated by maximum likelihood estimation (MLE) was conducted. The bacterial cell counts of each serotype sufficiently followed a Poisson distribution. Furthermore, to examine the validity of the parameters of Poisson distribution from experimentally obtained bacterial cell counts, we compared these with the parameters of a Poisson distribution that were estimated using random number generation via computer simulation. The Poisson distribution parameters experimentally obtained from bacterial cell counts were within the range of the parameters estimated using a computer simulation. These results demonstrate that the bacterial cell counts of each serotype obtained via 10-fold dilution followed a Poisson distribution. The fact that the frequency of bacterial cell counts follows a Poisson distribution at low number would be applied to some single-cell studies with a few bacterial cells. In particular, the procedure presented in this study enables us to develop an inactivation model at the single-cell level that can estimate the variability of survival bacterial numbers during the bacterial death process. PMID:27554145

  16. Synchronization of Caulobacter crescentus for investigation of the bacterial cell cycle.

    PubMed

    Schrader, Jared M; Shapiro, Lucy

    2015-01-01

    The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle. PMID:25938623

  17. Modulation of pokeweed mitogen-induced B cell differentiation by polymorphonuclear cells: effects of bacterial lipopolysaccharides.

    PubMed

    Tortorella, C; Ottolenghi, A; Testa, A; Decandia, P; Jirillo, E; Antonaci, S

    1994-01-01

    The capacity of polymorphonuclear (PMN) cells to release several cytokines stresses the potential immunomodulatory role of these cells. The effects mediated by purified PMN cell suspensions on pokeweed mitogen (PWM)-driven B cell differentiation was investigated. Results showed that the addition of increasing concentrations of resting PMN cells to peripheral blood mononuclear cell (PBMC) cultures gave rise to inhibition of immunoglobulin (Ig) production. At the same time, similar results were obtained using lipopolysaccharide (LPS)-pretreated PMN cells. In contrast, when LPS, at different concentrations, and PMN cells were both added to PBMC cultures an enhancement of IgG or IgM release in comparison with cultures treated with PMN cells only occurred at low PMN cell/PBMC ratios (1:20 and 1:10), which was maximal in the presence of 10 or 100 ng/ml LPS. This effect was probably mediated by LPS-induced monocyte stimulation, since the supplementation of LPS-activated monocyte supernatants to PMN cell/PBMC cocultures led to an Ig synthesis which mimicked that seen in similarly-treated PBMC cultures. These data suggest the occurrence of various in vitro modulatory effects in the interactions between PMN, LPS and lymphocytes in a PWM-induced B cell polyclonal responsiveness system. PMID:8047026

  18. Interactions of bacterial pathogens with dendritic cells during invasion of mucosal surfaces.

    PubMed

    Granucci, Francesca; Ricciardi-Castagnoli, Paola

    2003-02-01

    Recent studies of mucosal immunity suggest a key role for dendritic cells in the regulation of gut immune responses, in both physiological and pathological conditions. Dendritic cells are widely distributed in the lamina propria of the gut and are involved in direct bacterial uptake across mucosal surfaces, which questions the role of dendritic cells in innate mucosal responses. Approximately 400 commensal microbial species are present in the gut lumen. So how do dendritic cells distinguish pathogens from luminal microflora? Are the cytokines and chemokines induced in dendritic cells tailored to the class of microbes being recognized? Several very important questions still need to be addressed. PMID:12615223

  19. The Escherichia coli baby cell column: a novel cell synchronization method provides new insight into the bacterial cell cycle

    PubMed Central

    Bates, David; Epstein, Jessica; Boye, Erik; Fahrner, Karen; Berg, Howard; Kleckner, Nancy

    2010-01-01

    Summary We describe a new method for synchronizing bacterial cells. Cells that have transiently expressed an inducible mutant ‘sticky’ flagellin are adhered to a volume of glass beads suspended in a chromatography column though which growth medium is pumped. Following repression of flagellin synthesis, newborn cells are eluted from the column in large quantities exceeding that of current baby machine techniques by approximately 10-fold. Eluted cultures of ‘baby cells’ are highly synchronous as determined by analysis of DNA replication, cell division and other events, over time after elution from the column. We also show that use of ‘minutes after elution’ as a time metric permits much greater temporal resolution among sequential chromosomal events than the commonly used metric of cell size (length). The former approach reveals the existence of transient intermediate stages that are missed by the latter approach. This finding has two important implications. First, at a practical level, the baby cell column is a particularly powerful method for temporal analysis. Second, at a conceptual level, replication-related events are more tightly linked to cell birth (i.e. cell division) than to absolute cell mass. PMID:15978072

  20. Soil-Borne Bacterial Structure and Diversity Does Not Reflect Community Activity in Pampa Biome

    PubMed Central

    Lupatini, Manoeli; Suleiman, Afnan Khalil Ahmad; Jacques, Rodrigo Josemar Seminoti; Antoniolli, Zaida Inês; Kuramae, Eiko Eurya; de Oliveira Camargo, Flávio Anastácio; Roesch, Luiz Fernando Würdig

    2013-01-01

    The Pampa biome is considered one of the main hotspots of the world’s biodiversity and it is estimated that half of its original vegetation was removed and converted to agricultural land and tree plantations. Although an increasing amount of knowledge is being assembled regarding the response of soil bacterial communities to land use change, to the associated plant community and to soil properties, our understanding about how these interactions affect the microbial community from the Brazilian Pampa is still poor and incomplete. In this study, we hypothesized that the same soil type from the same geographic region but under distinct land use present dissimilar soil bacterial communities. To test this hypothesis, we assessed the soil bacterial communities from four land-uses within the same soil type by 454-pyrosequencing of 16S rRNA gene and by soil microbial activity analyzes. We found that the same soil type under different land uses harbor similar (but not equal) bacterial communities and the differences were controlled by many microbial taxa. No differences regarding diversity and richness between natural areas and areas under anthropogenic disturbance were detected. However, the measures of microbial activity did not converge with the 16S rRNA data supporting the idea that the coupling between functioning and composition of bacterial communities is not necessarily correlated. PMID:24146873

  1. The bacterial cytoplasm has glass-like properties and is fluidized by metabolic activity

    NASA Astrophysics Data System (ADS)

    Parry, Brad; Surovtsev, Ivan; Cabeen, Matthew; O'Hern, Corey; Dufresne, Eric; Jacobs-Wagner, Christine

    2014-03-01

    In eukaryotes, active transport involves motor proteins and cytoskeletal filaments. In contrast, bacteria (which lack cytoskeletal motor proteins) are thought to rely on diffusion for molecular transport, though the physical properties of the bacterial cytoplasm are poorly understood. Through single particle tracking of foreign particles of different sizes, we have found that the bacterial cytoplasm exhibits striking similarities to glass-forming liquids. Glass-forming liquids are noted for their metastability near the glass transition where their behavior changes from liquid-like to amorphous solid with even small perturbations. Particles of different sizes exhibit distinct dynamics and their mobility changes from fluid-like to glassy with increasing size. This size dependency provides an explanation for previous reports of both normal and anomalous diffusion in the bacterial cytoplasm. Moreover, we find that cellular metabolism attenuates the glassy properties of the bacterial cytoplasm. As a result, components that would otherwise be caged in narrow regions of confinement are able to explore the cytoplasmic space under metabolically active conditions. These findings have broad implications for our understanding of bacterial physiology as the glassy behavior of the cytoplasm impacts all intracellular processes involving large cellular components. Supported by the Howard Hughes Medical Institute.

  2. Induction of apoptosis in cancer cell lines by the Red Sea brine pool bacterial extracts

    PubMed Central

    2013-01-01

    Background Marine microorganisms are considered to be an important source of bioactive molecules against various diseases and have great potential to increase the number of lead molecules in clinical trials. Progress in novel microbial culturing techniques as well as greater accessibility to unique oceanic habitats has placed the marine environment as a new frontier in the field of natural product drug discovery. Methods A total of 24 microbial extracts from deep-sea brine pools in the Red Sea have been evaluated for their anticancer potential against three human cancer cell lines. Downstream analysis of these six most potent extracts was done using various biological assays, such as Caspase-3/7 activity, mitochondrial membrane potential (MMP), PARP-1 cleavage and expression of γH2Ax, Caspase-8 and -9 using western blotting. Results In general, most of the microbial extracts were found to be cytotoxic against one or more cancer cell lines with cell line specific activities. Out of the 13 most active microbial extracts, six extracts were able to induce significantly higher apoptosis (>70%) in cancer cells. Mechanism level studies revealed that extracts from Chromohalobacter salexigens (P3-86A and P3-86B(2)) followed the sequence of events of apoptotic pathway involving MMP disruption, caspase-3/7 activity, caspase-8 cleavage, PARP-1 cleavage and Phosphatidylserine (PS) exposure, whereas another Chromohalobacter salexigens extract (K30) induced caspase-9 mediated apoptosis. The extracts from Halomonas meridiana (P3-37B), Chromohalobacter israelensis (K18) and Idiomarina loihiensis (P3-37C) were unable to induce any change in MMP in HeLa cancer cells, and thus suggested mitochondria-independent apoptosis induction. However, further detection of a PARP-1 cleavage product, and the observed changes in caspase-8 and -9 suggested the involvement of caspase-mediated apoptotic pathways. Conclusion Altogether, the study offers novel findings regarding the anticancer

  3. Effects of bacterial cells and two types of extracellular polymers on bioclogging of sand columns

    NASA Astrophysics Data System (ADS)

    Xia, Lu; Zheng, Xilai; Shao, Haibing; Xin, Jia; Sun, Zhaoyue; Wang, Leyun

    2016-04-01

    Microbially induced reductions in the saturated hydraulic conductivity, Ks, of natural porous media, conventionally called bioclogging, occurs frequently in natural and engineered subsurface systems. Bioclogging can affect artificial groundwater recharge, in situ bioremediation of contaminated aquifers, or permeable reactive barriers. In this study, we designed a series of percolation experiments to simulate the growth and metabolism of bacteria in sand columns. The experimental results showed that the bacterial cell amount gradually increased to a maximum of 8.91 log10 CFU/g sand at 144 h during the bioclogging process, followed by a decrease to 7.89 log10 CFU/g sand until 336 h. The same variation pattern was found for the concentration of tightly bound extracellular polymeric substances (TB-EPS), which had a peak value of 220.76 μg/g sand at 144 h. In the same experiments, the concentration of loosely bound extracellular polymeric substances (LB-EPS) increased sharply from 54.45 to 575.57 μg/g sand in 192 h, followed by a slight decline to 505.04 μg/g sand. The increase of the bacterial cell amount along with the other two concentrations could reduce the Ks of porous media, but their relative contributions varied to a large degree during different percolation stages. At the beginning of the tests (e.g., 48 h before), bacterial cells were likely responsible for the Ks reduction of porous media because no increase was found for the other two concentrations. With the accumulation of cells and EPS production from 48 to 144 h, both were important for the reduction of Ks. However, in the late period of percolation tests from 144 to 192 h, LB-EPS was probably responsible for the further reduction of Ks, as the bacterial cell amount and TB-EPS concentration decreased. Quantitative contributions of bacterial cell amount and the two types of extracellular polymers to Ks reductions were also evaluated.

  4. Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA.

    PubMed

    Belcourt, M F; Penketh, P G; Hodnick, W F; Johnson, D A; Sherman, D H; Rockwell, S; Sartorelli, A C

    1999-08-31

    The mitomycin C-resistance gene, mcrA, of Streptomyces lavendulae produces MCRA, a protein that protects this microorganism from its own antibiotic, the antitumor drug mitomycin C. Expression of the bacterial mcrA gene in mammalian Chinese hamster ovary cells causes profound resistance to mitomycin C and to its structurally related analog porfiromycin under aerobic conditions but produces little change in drug sensitivity under hypoxia. The mitomycins are prodrugs that are enzymatically reduced and activated intracellularly, producing cytotoxic semiquinone anion radical and hydroquinone reduction intermediates. In vitro, MCRA protects DNA from cross-linking by the hydroquinone reduction intermediate of these mitomycins by oxidizing the hydroquinone back to the parent molecule; thus, MCRA acts as a hydroquinone oxidase. These findings suggest potential therapeutic applications for MCRA in the treatment of cancer with the mitomycins and imply that intrinsic or selected mitomycin C resistance in mammalian cells may not be due solely to decreased bioactivation, as has been hypothesized previously, but instead could involve an MCRA-like mechanism. PMID:10468636

  5. Mitomycin resistance in mammalian cells expressing the bacterial mitomycin C resistance protein MCRA

    PubMed Central

    Belcourt, Michael F.; Penketh, Philip G.; Hodnick, William F.; Johnson, David A.; Sherman, David H.; Rockwell, Sara; Sartorelli, Alan C.

    1999-01-01

    The mitomycin C-resistance gene, mcrA, of Streptomyces lavendulae produces MCRA, a protein that protects this microorganism from its own antibiotic, the antitumor drug mitomycin C. Expression of the bacterial mcrA gene in mammalian Chinese hamster ovary cells causes profound resistance to mitomycin C and to its structurally related analog porfiromycin under aerobic conditions but produces little change in drug sensitivity under hypoxia. The mitomycins are prodrugs that are enzymatically reduced and activated intracellularly, producing cytotoxic semiquinone anion radical and hydroquinone reduction intermediates. In vitro, MCRA protects DNA from cross-linking by the hydroquinone reduction intermediate of these mitomycins by oxidizing the hydroquinone back to the parent molecule; thus, MCRA acts as a hydroquinone oxidase. These findings suggest potential therapeutic applications for MCRA in the treatment of cancer with the mitomycins and imply that intrinsic or selected mitomycin C resistance in mammalian cells may not be due solely to decreased bioactivation, as has been hypothesized previously, but instead could involve an MCRA-like mechanism. PMID:10468636

  6. Life without a cell membrane: Challenging the specificity of bacterial endophytes within Bryopsis (Bryopsidales, Chlorophyta)

    PubMed Central

    2011-01-01

    Background The siphonous green macroalga Bryopsis has some remarkable characteristics. Besides hosting a rich endophytic bacterial flora, Bryopsis also displays extraordinary wound repair and propagation mechanisms. This latter feature includes the formation of protoplasts which can survive in the absence of a cell membrane for several minutes before regenerating into new individuals. This transient 'life without a membrane' state, however, challenges the specificity of the endophytic bacterial communities present and raises the question whether these bacteria are generalists, which are repeatedly acquired from the environment, or if there is some specificity towards the Bryopsis host. Results To answer this question, we examined the temporal stability and the uniqueness of endobiotic bacterial communities within Bryopsis samples from the Mexican west coast after prolonged cultivation. DGGE analysis revealed that Bryopsis endophytic bacterial communities are rather stable and clearly distinct from the epiphytic and surrounding cultivation water bacterial communities. Although these endogenous communities consist of both facultative and obligate bacteria, results suggest that Bryopsis owns some intrinsic mechanisms to selectively maintain and/or attract specific bacteria after repeated wounding events in culture. Conclusions This suggests that Bryopsis algae seem to master transient stages of life without a cell membrane well as they harbor specific - and possibly ecological significant - endophytic bacteria. PMID:22103477

  7. Antibacterial Activity of Cinnamaldehyde and Estragole Extracted from Plant Essential Oils against Pseudomonas syringae pv. actinidiae Causing Bacterial Canker Disease in Kiwifruit

    PubMed Central

    Song, Yu-Rim; Choi, Min-Seon; Choi, Geun-Won; Park, Il-Kwon; Oh, Chang-Sik

    2016-01-01

    Pseudomonas syringae pv. actinidiae (Psa) causes bacterial canker disease in kiwifruit. Antibacterial activity of plant essential oils (PEOs) originating from 49 plant species were tested against Psa by a vapor diffusion and a liquid culture assays. The five PEOs from Pimenta racemosa, P. dioica, Melaleuca linariifolia, M. cajuputii, and Cinnamomum cassia efficiently inhibited Psa growth by either assays. Among their major components, estragole, eugenol, and methyl eugenol showed significant antibacterial activity by only the liquid culture assay, while cinnamaldehyde exhibited antibacterial activity by both assays. The minimum inhibitory concentrations (MICs) of estragole and cinnamaldehyde by the liquid culture assay were 1,250 and 2,500 ppm, respectively. The MIC of cinnamaldehyde by the vapor diffusion assay was 5,000 ppm. Based on the formation of clear zones or the decrease of optical density caused by these compounds, they might kill the bacterial cells and this feature might be useful for managing the bacterial canker disease in kiwifruit. PMID:27493612

  8. Antibacterial Activity of Cinnamaldehyde and Estragole Extracted from Plant Essential Oils against Pseudomonas syringae pv. actinidiae Causing Bacterial Canker Disease in Kiwifruit.

    PubMed

    Song, Yu-Rim; Choi, Min-Seon; Choi, Geun-Won; Park, Il-Kwon; Oh, Chang-Sik

    2016-08-01

    Pseudomonas syringae pv. actinidiae (Psa) causes bacterial canker disease in kiwifruit. Antibacterial activity of plant essential oils (PEOs) originating from 49 plant species were tested against Psa by a vapor diffusion and a liquid culture assays. The five PEOs from Pimenta racemosa, P. dioica, Melaleuca linariifolia, M. cajuputii, and Cinnamomum cassia efficiently inhibited Psa growth by either assays. Among their major components, estragole, eugenol, and methyl eugenol showed significant antibacterial activity by only the liquid culture assay, while cinnamaldehyde exhibited antibacterial activity by both assays. The minimum inhibitory concentrations (MICs) of estragole and cinnamaldehyde by the liquid culture assay were 1,250 and 2,500 ppm, respectively. The MIC of cinnamaldehyde by the vapor diffusion assay was 5,000 ppm. Based on the formation of clear zones or the decrease of optical density caused by these compounds, they might kill the bacterial cells and this feature might be useful for managing the bacterial canker disease in kiwifruit. PMID:27493612

  9. Biosynthesis of a Fully Functional Cyclotide inside Living Bacterial Cells

    SciTech Connect

    Camarero, J A; Kimura, R H; Woo, Y; Cantor, J; Shekhtman, A

    2007-04-05

    The cyclotide MCoTI-II is a powerful trypsin inhibitor recently isolated from the seeds of Momordica cochinchinensis, a plant member of cucurbitaceae family. We report for the first time the in vivo biosynthesis of natively-folded MCoTI-II inside live E. coli cells. Our biomimetic approach involves the intracellular backbone cyclization of a linear cyclotide-intein fusion precursor mediated by a modified protein splicing domain. The cyclized peptide then spontaneously folds into its native conformation. The use of genetically engineered E. coli cells containing mutations in the glutathione and thioredoxin reductase genes considerably improves the production of folded MCoTI-II in vivo. Biochemical and structural characterization of the recombinant MCoTI-II confirmed its identity. Biosynthetic access to correctly-folded cyclotides allows the possibility of generating cell-based combinatorial libraries that can be screened inside living cells for their ability to modulate or inhibit cellular processes.

  10. Increased electrical output when a bacterial ABTS oxidizer is used in a microbial fuel cell

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microbial fuel cells (MFCs) are a technology that provides electrical energy from the microbial oxidation of organic compounds. Most MFCs use oxygen as the oxidant in the cathode chamber. The present study examined the formation in culture of an unidentified bacterial oxidant and investigated the ...

  11. A portable immunomagnetic cell capture system to accelerate culture diagnosis of bacterial infections.

    PubMed

    Singh, Saurabh; Upadhyay, Mohita; Sharma, Jyoti; Gupta, Shalini; Vivekanandan, Perumal; Elangovan, Ravikrishnan

    2016-05-23

    Bacterial infections continue to be a major cause of deaths globally, particularly in resource-poor settings. In the absence of rapid and affordable diagnostic solutions, patients are mostly administered broad spectrum antibiotics leading to antibiotics resistance and poor recovery. Culture diagnosis continues to be a gold standard for diagnosis of bacterial infection, despite its long turnaround time of 24 to 48 h. We have developed a portable immunomagnetic cell capture (iMC(2)) system that allows rapid culture diagnosis of bacterial pathogens. Our approach involves the culture growth of the blood samples in broth media for 6 to 8 h, followed by immunomagnetic enrichment of the target cells using the iMC(2) device. The device comprises a disposable capture chip that has two chambers of 5 ml and 50 μl volume connected through a channel with a manual valve. Bacterial cells bound to antibody coated magnetic nanoparticles are swept from the 5 ml sample chamber into the 50 μl recovery chamber by moving an external magnetic field with respect to the capture chip using a linear positioner. This enables specific isolation and up to 100× enrichment of the target cells. The presence of bacteria in the recovered sample is confirmed visually using a lateral flow immunoassay. The system is demonstrated in buffer and blood samples spiked with S. typhi. The method has high sensitivity (10 CFU ml(-1)), specificity and a rapid turnaround time of less than 7 h, a significant improvement over conventional methods. PMID:27118505

  12. The antimicrobial polymer PHMB enters cells and selectively condenses bacterial chromosomes

    PubMed Central

    Chindera, Kantaraja; Mahato, Manohar; Kumar Sharma, Ashwani; Horsley, Harry; Kloc-Muniak, Klaudia; Kamaruzzaman, Nor Fadhilah; Kumar, Satish; McFarlane, Alexander; Stach, Jem; Bentin, Thomas; Good, Liam

    2016-01-01

    To combat infection and antimicrobial resistance, it is helpful to elucidate drug mechanism(s) of action. Here we examined how the widely used antimicrobial polyhexamethylene biguanide (PHMB) kills bacteria selectively over host cells. Contrary to the accepted model of microbial membrane disruption by PHMB, we observed cell entry into a range of bacterial species, and treated bacteria displayed cell division arrest and chromosome condensation, suggesting DNA binding as an alternative antimicrobial mechanism. A DNA-level mechanism was confirmed by observations that PHMB formed nanoparticles when mixed with isolated bacterial chromosomal DNA and its effects on growth were suppressed by pairwise combination with the DNA binding ligand Hoechst 33258. PHMB also entered mammalian cells, but was trapped within endosomes and excluded from nuclei. Therefore, PHMB displays differential access to bacterial and mammalian cellular DNA and selectively binds and condenses bacterial chromosomes. Because acquired resistance to PHMB has not been reported, selective chromosome condensation provides an unanticipated paradigm for antimicrobial action that may not succumb to resistance. PMID:26996206

  13. The antimicrobial polymer PHMB enters cells and selectively condenses bacterial chromosomes.

    PubMed

    Chindera, Kantaraja; Mahato, Manohar; Kumar Sharma, Ashwani; Horsley, Harry; Kloc-Muniak, Klaudia; Kamaruzzaman, Nor Fadhilah; Kumar, Satish; McFarlane, Alexander; Stach, Jem; Bentin, Thomas; Good, Liam

    2016-01-01

    To combat infection and antimicrobial resistance, it is helpful to elucidate drug mechanism(s) of action. Here we examined how the widely used antimicrobial polyhexamethylene biguanide (PHMB) kills bacteria selectively over host cells. Contrary to the accepted model of microbial membrane disruption by PHMB, we observed cell entry into a range of bacterial species, and treated bacteria displayed cell division arrest and chromosome condensation, suggesting DNA binding as an alternative antimicrobial mechanism. A DNA-level mechanism was confirmed by observations that PHMB formed nanoparticles when mixed with isolated bacterial chromosomal DNA and its effects on growth were suppressed by pairwise combination with the DNA binding ligand Hoechst 33258. PHMB also entered mammalian cells, but was trapped within endosomes and excluded from nuclei. Therefore, PHMB displays differential access to bacterial and mammalian cellular DNA and selectively binds and condenses bacterial chromosomes. Because acquired resistance to PHMB has not been reported, selective chromosome condensation provides an unanticipated paradigm for antimicrobial action that may not succumb to resistance. PMID:26996206

  14. [Detection of cell death markers as a tool for bacterial antimicrobial susceptibility testing].

    PubMed

    Mlynárčik, P; Kolář, M

    2016-01-01

    Antimicrobial resistance among nosocomial pathogens has emerged as one of the most important health care problems in the new millennium. In this review, we present new methods for bacterial antimicrobial susceptibility testing, based on the detection of antibiotic-mediated cell death markers that could provide valuable alternatives to existing phenotypic approaches in the very near future. PMID:27467325

  15. Practical Synthesis of PC190723, An Inhibitor of the Bacterial Cell Division Protein FtsZ

    PubMed Central

    Sorto, Nohemy A.; Olmstead, Marilyn M.; Shaw, Jared T.

    2010-01-01

    A high-yielding and practical synthesis of the bacterial cell division inhibitor PC190723 is described. The synthesis is completed in a longest linear sequence of five steps from commercially available starting materials and can be readily executed on a multi-gram scale. PMID:21033691

  16. CELL DENSITY AND NON-EQUILIBRIUM SORPTION EFFECTS ON BACTERIAL DISPERSAL IN GROUNDWATER MICROCOSMS

    EPA Science Inventory

    The relative importance of dispersion, physical straining, non-equilibrium sorption, and cell density on the dispersal of bacteria was examined in saturated, flow-dynamic sand columns. The bacterial breakthrough as a was followed by measuring the effluent concentration of 3H-aden...

  17. Spring constants and adhesive properties of native bacterial biofilm cells measured by atomic force microscopy.

    PubMed

    Volle, C B; Ferguson, M A; Aidala, K E; Spain, E M; Núñez, M E

    2008-11-15

    Bacterial biofilms were imaged by atomic force microscopy (AFM), and their elasticity and adhesion to the AFM tip were determined from a series of tip extension and retraction cycles. Though the five bacterial strains studied included both Gram-negative and -positive bacteria and both environmental and laboratory strains, all formed simple biofilms on glass surfaces. Cellular spring constants, determined from the extension portion of the force cycle, varied between 0.16+/-0.01 and 0.41+/-0.01 N/m, where larger spring constants were measured for Gram-positive cells than for Gram-negative cells. The nonlinear regime in the extension curve depended upon the biomolecules on the cell surface: the extension curves for the smooth Gram-negative bacterial strains with the longest lipopolysaccharides on their surface had a larger nonlinear region than the rough bacterial strain with shorter lipopolysaccharides on the surface. Adhesive forces between the retracting silicon nitride tip and the cells varied between cell types in terms of the force components, the distance components, and the number of adhesion events. The Gram-negative cells' adhesion to the tip showed the longest distance components, sometimes more than 1 microm, whereas the shortest distance adhesion events were measured between the two Gram-positive cell types and the tip. Fixation of free-swimming planktonic cells by NHS and EDC perturbed both the elasticity and the adhesive properties of the cells. Here we consider the biochemical meaning of the measured physical properties of simple biofilms and implications to the colonization of surfaces in the first stages of biofilm formation. PMID:18815013

  18. The Active Bacterial Community in a Pristine Confined Aquifer

    EPA Science Inventory

    This study of the active bacteria residing in a pristine confined aquifer provides unexpected insights into the ecology of iron-reducing and sulfate-reducing bacteria in the subsurface. At 18 wells in east-central Illinois, we trapped the microbes that attached to aquifer sedimen...

  19. Origin of optical activity in the purple bacterial photoreaction center

    SciTech Connect

    Mar, T.; Gingras, G.

    1995-07-18

    The photoreaction center (RC) of purple bacteria contains four bacteriochlorophyll (Bph) and two bacteriopheophytin (Bph) molecules as prosthetic groups. Their optical activity, as measured by circular dichroism (CD) spectroscopy, is largely increased in situ as compared to organic solutions. The all-exciton hypothesis posits that this enhanced optical activity is entirely due to excitonic interactions between the electronic transitions of all six bacteriochlorin molecules. Using the simple exciton theory, this model predicts that the near-infrared CD spectra should be conservative. The fact that they are not, whether the special pair of Bch (SP) that constitutes the primary electron donor is reduced or oxidized, has been explained by hyperchromic effects. The present work tests this hypothesis by successively eliminating the absorption and, therefore, the optical activity of the Bphs and of the non-special-pair (non-SP) Bchs. This was accomplished by trapping these pigments in their reduced state. RC preparations with the four non-SP bacteriochlorins trapped in their reduced state and, therefore, with an intact SP displayed conservative CD spectra. RC preparations with only the electronic transitions of SP and of one non-SP Bch also showed conservative CD spectra. These conservative CD spectra and their corresponding absorption spectra were simulated using simple exciton theory without assuming hyperchromic effects. Bleaching half of the 755-nm absorption band by phototrapping one of the two Bph molecules led to the complete disappearance of the corresponding CD band. This cannot be explained by the all-exciton hypothesis. These results suggest that the optical activity of the SP alone, or with one non-SP Bch, is due to excitonic interactions. They also suggest that the optical activity of the other three bacteriochlorins is due to other factors, such as pigment-protein interaction. 32 refs., 9 figs., 2 tabs.

  20. Exploiting Bacterial Peptide Display Technology to Engineer Biomaterials for Neural Stem Cell Culture

    PubMed Central

    Little, Lauren; Dane, Karen; Daugherty, Patrick; Healy, Kevin; Schaffer, David

    2010-01-01

    Stem cells are often cultured on substrates that present extracellular matrix (ECM) proteins; however, the heterogeneous and poorly defined nature of ECM proteins presents challenges both for basic biological investigation of cell-matrix investigations and translational applications of stem cells. Therefore, fully synthetic, defined materials conjugated with bioactive ligands, such as adhesive peptides, are preferable for stem cell biology and engineering. However, identifying novel ligands that engage cellular receptors can be challenging, and we have thus developed a high throughput approach to identify new adhesive ligands. We selected an unbiased bacterial peptide display library for the ability to bind adult neural stem cells (NSCs), and 44 bacterial clones expressing peptides were identified and found to bind to NSCs with high avidity. Of these clones, four contained RGD motifs commonly found in integrin binding domains, and three exhibited homology to ECM proteins. Three peptide clones were chosen for further analysis, and their synthetic analogs were adsorbed on tissue culture polystyrene (TCPS) or grafted onto an interpenetrating polymer network (IPN) for cell culture. These three peptides were found to support neural stem cell self-renewal in defined medium as well as multi-lineage differentiation. Therefore, bacterial peptide display offers unique advantages to isolate bioactive peptides from large, unbiased libraries for applications in biomaterials engineering. PMID:21129772

  1. In situ probing the interior of single bacterial cells at nanometer scale

    NASA Astrophysics Data System (ADS)

    Liu, Boyin; Hemayet Uddin, Md; Ng, Tuck Wah; Paterson, David L.; Velkov, Tony; Li, Jian; Fu, Jing

    2014-10-01

    We report a novel approach to probe the interior of single bacterial cells at nanometre resolution by combining focused ion beam (FIB) and atomic force microscopy (AFM). After removing layers of pre-defined thickness in the order of 100 nm on the target bacterial cells with FIB milling, AFM of different modes can be employed to probe the cellular interior under both ambient and aqueous environments. Our initial investigations focused on the surface topology induced by FIB milling and the hydration effects on AFM measurements, followed by assessment of the sample protocols. With fine-tuning of the process parameters, in situ AFM probing beneath the bacterial cell wall was achieved for the first time. We further demonstrate the proposed method by performing a spatial mapping of intracellular elasticity and chemistry of the multi-drug resistant strain Klebsiella pneumoniae cells prior to and after it was exposed to the ‘last-line’ antibiotic polymyxin B. Our results revealed increased stiffness occurring in both surface and interior regions of the treated cells, suggesting loss of integrity of the outer membrane from polymyxin treatments. In addition, the hydrophobicity measurement using a functionalized AFM tip was able to highlight the evident hydrophobic portion of the cell such as the regions containing cell membrane. We expect that the proposed FIB-AFM platform will help in gaining deeper insights of bacteria-drug interactions to develop potential strategies for combating multi-drug resistance.

  2. Antagonistic activity of Bacillus subtilis SB1 and its biocontrol effect on tomato bacterial wilt

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A potential biocontrol agent of bacterial wilt, Bacillus subtilis SB1, isolated from tomato roots, showed a broad-spectrum of antimicrobial activity in in vitro experiments. It inhibited the growth of many plant pathogens, including Ralstonia solanacearum, Xanthomonas oryzae pv. oryzae, Fusarium ox...

  3. Induction of apoptotic cell death leads to the development of bacterial rot caused by Pseudomonas cichorii.

    PubMed

    Kiba, Akinori; Sangawa, Yasutaka; Ohnishi, Kouhei; Yao, Nan; Park, Pyoyun; Nakayashiki, Hitoshi; Tosa, Yukio; Mayama, Shigeyuki; Hikichi, Yasufumi

    2006-02-01

    Pseudomonas cichorii is the major causal agent of bacterial rot of lettuce. Collapse and browning symptoms were observed in lettuce leaf tissue from 15 to 24 h after inoculation (HAI) with P. cichorii; superoxide anion generation was detected at 1 to 6 HAI; and cell death was induced at 6 HAI, reaching a maximum at approximately 9 and 12 HAI. Heterochromatin condensation and DNA laddering also were observed within 3 HAI. Pharmacological studies showed that induction of cell death and DNA laddering was closely associated with de novo protein synthesis, protein kinase, intracellular reactive oxygen species, DNase, serine protease, and caspase III-like protease. Moreover, chemicals, which inhibited the induction of cell death and DNA laddering, also suppressed the development of disease symptoms. These results suggest that apoptotic cell death might be closely associated with the development of bacterial rot caused by P. cichorii. PMID:16529373

  4. Bacterial Ghosts of Escherichia coli Drive Efficient Maturation of Bovine Monocyte-Derived Dendritic Cells

    PubMed Central

    Hajam, Irshad Ahmed; Dar, Pervaiz Ahmad; Appavoo, Elamurugan; Kishore, Subodh; Bhanuprakash, Veerakyathappa; Ganesh, Kondabattula

    2015-01-01

    Bacterial ghosts (BGs) are empty cell envelopes derived from Gram-negative bacteria. They not only represent a potential platform for development of novel vaccines but also provide a tool for efficient adjuvant and antigen delivery system. In the present study, we investigated the interaction between BGs of Escherichia coli (E. coli) and bovine monocyte-derived dendritic cells (MoDCs). MoDCs are highly potent antigen-presenting cells and have the potential to act as a powerful tool for manipulating the immune system. We generated bovine MoDCs in vitro from blood monocytes using E. coli expressed bovine GM-CSF and IL-4 cytokines. These MoDCs displayed typical morphology and functions similar to DCs. We further investigated the E. coli BGs to induce maturation of bovine MoDCs in comparison to E. coli lipopolysaccharide (LPS). We observed the maturation marker molecules such as MHC-II, CD80 and CD86 were induced early and at higher levels in BG stimulated MoDCs as compared to the LPS stimulated MoDCs. BG mediated stimulation induced significantly higher levels of cytokine expression in bovine MoDCs than LPS. Both pro-inflammatory (IL-12 and TNF-α) and anti-inflammatory (IL-10) cytokines were induced in MoDCs after BGs stimulation. We further analysed the effects of BGs on the bovine MoDCs in an allogenic mixed lymphocyte reaction (MLR). We found the BG-treated bovine MoDCs had significantly (p<0.05) higher capacity to stimulate allogenic T cell proliferation in MLR as compared to the LPS. Taken together, these findings demonstrate the E. coli BGs induce a strong activation and maturation of bovine MoDCs. PMID:26669936

  5. Deciphering the Metabolism of Undecaprenyl-Phosphate: The Bacterial Cell-Wall Unit Carrier at the Membrane Frontier

    PubMed Central

    Manat, Guillaume; Roure, Sophie; Auger, Rodolphe; Bouhss, Ahmed; Barreteau, Hélène; Mengin-Lecreulx, Dominique

    2014-01-01

    During the biogenesis of bacterial cell-wall polysaccharides, such as peptidoglycan, cytoplasmic synthesized precursors should be trafficked across the plasma membrane. This essential process requires a dedicated lipid, undecaprenyl-phosphate that is used as a glycan lipid carrier. The sugar is linked to the lipid carrier at the inner face of the membrane and is translocated toward the periplasm, where the glycan moiety is transferred to the growing polymer. Undecaprenyl-phosphate originates from the dephosphorylation of its precursor undecaprenyl-diphosphate, with itself generated by de novo synthesis or by recycling after the final glycan transfer. Undecaprenyl-diphosphate is de novo synthesized by the cytosolic cis-prenyltransferase undecaprenyl-diphosphate synthase, which has been structurally and mechanistically characterized in great detail highlighting the condensation process. In contrast, the next step toward the formation of the lipid carrier, the dephosphorylation step, which has been overlooked for many years, has only started revealing surprising features. In contrast to the previous step, two unrelated families of integral membrane proteins exhibit undecaprenyl-diphosphate phosphatase activity: BacA and members of the phosphatidic acid phosphatase type 2 super-family, raising the question of the significance of this multiplicity. Moreover, these enzymes establish an unexpected link between the synthesis of bacterial cell-wall polymers and other biological processes. In the present review, the current knowledge in the field of the bacterial lipid carrier, its mechanism of action, biogenesis, recycling, regulation, and future perspective works are presented. PMID:24799078

  6. Note: An automated image analysis method for high-throughput classification of surface-bound bacterial cell motions

    NASA Astrophysics Data System (ADS)

    Shen, Simon; Syal, Karan; Tao, Nongjian; Wang, Shaopeng

    2015-12-01

    We present a Single-Cell Motion Characterization System (SiCMoCS) to automatically extract bacterial cell morphological features from microscope images and use those features to automatically classify cell motion for rod shaped motile bacterial cells. In some imaging based studies, bacteria cells need to be attached to the surface for time-lapse observation of cellular processes such as cell membrane-protein interactions and membrane elasticity. These studies often generate large volumes of images. Extracting accurate bacterial cell morphology features from these images is critical for quantitative assessment. Using SiCMoCS, we demonstrated simultaneous and automated motion tracking and classification of hundreds of individual cells in an image sequence of several hundred frames. This is a significant improvement from traditional manual and semi-automated approaches to segmenting bacterial cells based on empirical thresholds, and a first attempt to automatically classify bacterial motion types for motile rod shaped bacterial cells, which enables rapid and quantitative analysis of various types of bacterial motion.

  7. Note: An automated image analysis method for high-throughput classification of surface-bound bacterial cell motions.

    PubMed

    Shen, Simon; Syal, Karan; Tao, Nongjian; Wang, Shaopeng

    2015-12-01

    We present a Single-Cell Motion Characterization System (SiCMoCS) to automatically extract bacterial cell morphological features from microscope images and use those features to automatically classify cell motion for rod shaped motile bacterial cells. In some imaging based studies, bacteria cells need to be attached to the surface for time-lapse observation of cellular processes such as cell membrane-protein interactions and membrane elasticity. These studies often generate large volumes of images. Extracting accurate bacterial cell morphology features from these images is critical for quantitative assessment. Using SiCMoCS, we demonstrated simultaneous and automated motion tracking and classification of hundreds of individual cells in an image sequence of several hundred frames. This is a significant improvement from traditional manual and semi-automated approaches to segmenting bacterial cells based on empirical thresholds, and a first attempt to automatically classify bacterial motion types for motile rod shaped bacterial cells, which enables rapid and quantitative analysis of various types of bacterial motion. PMID:26724085

  8. Bacterial differentiation via gradual activation of global regulators.

    PubMed

    Kovács, Ákos T

    2016-02-01

    Bacteria have evolved to adapt to various conditions and respond to certain stress conditions. The ability to sense and efficiently reply to these environmental effects involve versatile array of sensors and global or specific regulators. Interestingly, modulation of the levels of active global regulators enables bacteria to respond to diverse signals via a single central transcriptional regulator and to activate or repress certain differentiation pathways at a spatio-temporal manner. The Gram-positive Bacillus subtilis is an ideal bacterium to study how membrane bound and cytoplasmic sensor kinases affect the level of phosphorylated global regulator, Spo0A which in response activates genes related to sliding, biofilm formation, and sporulation. In addition, other global regulators, including the two-component system DegS-DegU, modulate overlapping and complementary genes in B. subtilis related to surface colonization and biofilm formation. The intertwinement of global regulatory systems also allows the accurate modulation of differentiation pathways. Studies in the last decade enable us to get a deeper insight into the role of global regulators on the smooth transition of developmental processes in B. subtilis. PMID:26458398

  9. Phase Resetting Reveals Network Dynamics Underlying a Bacterial Cell Cycle

    PubMed Central

    Lin, Yihan; Li, Ying; Crosson, Sean; Dinner, Aaron R.; Scherer, Norbert F.

    2012-01-01

    Genomic and proteomic methods yield networks of biological regulatory interactions but do not provide direct insight into how those interactions are organized into functional modules, or how information flows from one module to another. In this work we introduce an approach that provides this complementary information and apply it to the bacterium Caulobacter crescentus, a paradigm for cell-cycle control. Operationally, we use an inducible promoter to express the essential transcriptional regulatory gene ctrA in a periodic, pulsed fashion. This chemical perturbation causes the population of cells to divide synchronously, and we use the resulting advance or delay of the division times of single cells to construct a phase resetting curve. We find that delay is strongly favored over advance. This finding is surprising since it does not follow from the temporal expression profile of CtrA and, in turn, simulations of existing network models. We propose a phenomenological model that suggests that the cell-cycle network comprises two distinct functional modules that oscillate autonomously and couple in a highly asymmetric fashion. These features collectively provide a new mechanism for tight temporal control of the cell cycle in C. crescentus. We discuss how the procedure can serve as the basis for a general approach for probing network dynamics, which we term chemical perturbation spectroscopy (CPS). PMID:23209388

  10. Bacterial Cell Surface Adsorption of Rare Earth Elements

    NASA Astrophysics Data System (ADS)

    Jiao, Y.; Park, D.; Reed, D.; Fujita, Y.; Yung, M.; Anderko, A.; Eslamimanesh, A.

    2015-12-01

    Rare earth elements (REE) play a critical role in many emerging clean energy technologies, including high-power magnets, wind turbines, solar panels, hybrid/electric vehicle batteries and lamp phosphors. In order to sustain demand for such technologies given current domestic REE shortages, there is a need to develop new approaches for ore processing/refining and recycling of REE-containing materials. To this end, we have developed a microbially-mediated bioadsorption strategy with application towards enrichment of REE from complex mixtures. Specifically, the bacterium Caulobacter crescentus was genetically engineered to display lanthanide binding tags (LBTs), short peptides that possess high affinity and specificity for rare earth elements, on its cell surface S-layer protein. Under optimal conditions, LBT-displayed cells adsorbed greater than 5-fold more REE than control cells lacking LBTs. Competition binding experiments with a selection of REEs demonstrated that our engineered cells could facilitate separation of light- from heavy- REE. Importantly, binding of REE onto our engineered strains was much more favorable compared to non-REE metals. Finally, REE bound to the cell surface could be stripped off using citrate, providing an effective and non-toxic REE recovery method. Together, this data highlights the potential of our approach for selective REE enrichment from REE containing mixtures.

  11. Response of Bacterial Metabolic Activity to Riverine Dissolved Organic Carbon and Exogenous Viruses in Estuarine and Coastal Waters: Implications for CO2 Emission

    PubMed Central

    Xu, Jie; Sun, Mingming; Shi, Zhen; Harrison, Paul J.; Liu, Hongbin

    2014-01-01

    A cross-transplant experiment between estuarine water and seawater was conducted to examine the response of bacterial metabolic activity to riverine dissolved organic carbon (DOC) input under virus-rich and virus-free conditions, as well as to exogenous viruses. Riverine DOC input increased bacterial production significantly, but not bacterial respiration (BR) because of its high lability. The bioavailable riverine DOC influenced bulk bacterial respiration in two contrasting ways; it enhanced the bulk BR by stimulating bacterial growth, but simultaneously reduced the cell-specific BR due to its high lability. As a result, there was little stimulation of the bulk BR by riverine DOC. This might be partly responsible for lower CO2 degassing fluxes in estuaries receiving high sewage-DOC that is highly labile. Viruses restricted microbial decomposition of riverine DOC dramatically by repressing the growth of metabolically active bacteria. Bacterial carbon demand in the presence of viruses only accounted for 7–12% of that in the absence of viruses. Consequently, a large fraction of riverine DOC was likely transported offshore to the shelf. In addition, marine bacteria and estuarine bacteria responded distinctly to exogenous viruses. Marine viruses were able to infect estuarine bacteria, but not as efficiently as estuarine viruses, while estuarine viruses infected marine bacteria as efficiently as marine viruses. We speculate that the rapid changes in the viral community due to freshwater input destroyed the existing bacteria-virus relationship, which would change the bacterial community composition and affect the bacterial metabolic activity and carbon cycling in this estuary. PMID:25036641

  12. Uranium Biomineralization As a Result of Bacterial Phosphatase Activity: Insights From Bacterial Isolates From a Contaminated Subsurface

    SciTech Connect

    Beazley, M.J.; Martinez, R.J.; Sobecky, P.A.; Webb, S.M.; Taillefert, M.; /Georgia Tech /SLAC, SSRL

    2007-10-31

    Uranium contamination is an environmental concern at the Department of Energy's Field Research Center in Oak Ridge, Tennessee. In this study, we investigated whether phosphate biomineralization, or the aerobic precipitation of U(VI)-phosphate phases facilitated by the enzymatic activities of microorganisms, offers an alternative to the more extensively studied anaerobic U(VI) bioreduction. Three heterotrophic bacteria isolated from FRC soils were studied for their ability to grow and liberate phosphate in the presence of U(VI) and an organophosphate between pH 4.5 and 7.0. The objectives were to determine whether the strains hydrolyzed sufficient phosphate to precipitate uranium, to determine whether low pH might have an effect on U(VI) precipitation, and to identify the uranium solid phase formed during biomineralization. Two bacterial strains hydrolyzed sufficient organophosphate to precipitate 73-95% total uranium after 120 h of incubation in simulated groundwater. The highest rates of uranium precipitation and phosphatase activity were observed between pH 5.0 and 7.0. EXAFS spectra identified the uranyl phosphate precipitate as an autunite/meta-autunite group mineral. The results of this study indicate that aerobic heterotrophic bacteria within a uranium-contaminated environment that can hydrolyze organophosphate, especially in low pH conditions, may play an important role in the bioremediation of uranium.

  13. Plasmonic imaging of protein interactions with single bacterial cells.

    PubMed

    Syal, Karan; Wang, Wei; Shan, Xiaonan; Wang, Shaopeng; Chen, Hong-Yuan; Tao, Nongjian

    2015-01-15

    Quantifying the interactions of bacteria with external ligands is fundamental to the understanding of pathogenesis, antibiotic resistance, immune evasion, and mechanism of antimicrobial action. Due to inherent cell-to-cell heterogeneity in a microbial population, each bacterium interacts differently with its environment. This large variability is washed out in bulk assays, and there is a need of techniques that can quantify interactions of bacteria with ligands at the single bacterium level. In this work, we present a label-free and real-time plasmonic imaging technique to measure the binding kinetics of ligand interactions with single bacteria, and perform statistical analysis of the heterogeneity. Using the technique, we have studied interactions of antibodies with single Escherichia coli O157:H7 cells and demonstrated a capability of determining the binding kinetic constants of single live bacteria with ligands, and quantify heterogeneity in a microbial population. PMID:25064821

  14. Drivers shaping the diversity and biogeography of total and active bacterial communities in the South China Sea

    PubMed Central

    Zhang, Yao; Zhao, Zihao; Dai, Minhan; Jiao, Nianzhi; Herndl, Gerhard J

    2014-01-01

    To test the hypothesis that different drivers shape the diversity and biogeography of the total and active bacterial community, we examined the bacterial community composition along two transects, one from the inner Pearl River estuary to the open waters of the South China Sea (SCS) and the other from the Luzon Strait to the SCS basin, using 454 pyrosequencing of the 16S rRNA and 16S rRNA gene (V1-3 regions) and thereby characterizing the active and total bacterial community, respectively. The diversity and biogeographic patterns differed substantially between the active and total bacterial communities. Although the composition of both the total and active bacterial community was strongly correlated with environmental factors and weakly correlated with geographic distance, the active bacterial community displayed higher environmental sensitivity than the total community and particularly a greater distance effect largely caused by the active assemblage from deep waters. The 16S rRNA vs. rDNA relationships indicated that the active bacteria were low in relative abundance in the SCS. This might be due to a high competition between active bacterial taxa as indicated by our community network models. Based on these analyses, we speculate that high competition could cause some dispersal limitation of the active bacterial community resulting in a distinct distance-decay relationship. Altogether, our results indicated that the biogeographic distribution of bacteria in the SCS is the result of both environmental control and distance decay. PMID:24684298

  15. A Novel Mechanism of Bacterial Toxin Transfer within Host Blood Cell-Derived Microvesicles

    PubMed Central

    Ståhl, Anne-lie; Arvidsson, Ida; Johansson, Karl E.; Chromek, Milan; Rebetz, Johan; Loos, Sebastian; Kristoffersson, Ann-Charlotte; Békássy, Zivile D.; Mörgelin, Matthias; Karpman, Diana

    2015-01-01

    Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS), associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system. PMID:25719452

  16. Spatial Patterning of Newly-Inserted Material during Bacterial Cell Growth

    NASA Astrophysics Data System (ADS)

    Ursell, Tristan

    2012-02-01

    In the life cycle of a bacterium, rudimentary microscopy demonstrates that cell growth and elongation are essential characteristics of cellular reproduction. The peptidoglycan cell wall is the main load-bearing structure that determines both cell shape and overall size. However, simple imaging of cellular growth gives no indication of the spatial patterning nor mechanism by which material is being incorporated into the pre-existing cell wall. We employ a combination of high-resolution pulse-chase fluorescence microscopy, 3D computational microscopy, and detailed mechanistic simulations to explore how spatial patterning results in uniform growth and maintenance of cell shape. We show that growth is happening in discrete bursts randomly distributed over the cell surface, with a well-defined mean size and average rate. We further use these techniques to explore the effects of division and cell wall disrupting antibiotics, like cephalexin and A22, respectively, on the patterning of cell wall growth in E. coli. Finally, we explore the spatial correlation between presence of the bacterial actin-like cytoskeletal protein, MreB, and local cell wall growth. Together these techniques form a powerful method for exploring the detailed dynamics and involvement of antibiotics and cell wall-associated proteins in bacterial cell growth.[4pt] In collaboration with Kerwyn Huang, Stanford University.

  17. An evolutionarily-conserved role for murine Ly-1 B cells in protection against bacterial infections.

    PubMed

    Lalor, P A

    1991-01-01

    The murine Ly-1 B cell lineage, although comprising only a minority of peripheral IgM+ B cells, secretes a major proportion of the IgM antibodies occurring naturally in serum. Ly-1 B cells also seed a large number of IgA+ plasma cells to the gut walls, thereby contributing significantly to production of natural IgA antibodies in response to chronic stimulation by the normal gut flora. Apart from these naturally-produced antibodies, Ly-1 B cells also produce specific antibodies following deliberate immunisation with the bacterial cell wall antigens, phosphorylcholine and dextran. The inability of the X-linked immunodeficient CBA/N mice to produce antibody responses to these two antigens is overcome by reconstitution with normal Ly-1 B cells from the parental CBA strain. Ly-1 B cells therefore appear to play a dominant role in natural immunity and protection against bacterial infections. The compartmentalisation of development and function within murine B cells is suggestive of an evolutionary structuring of the murine immune system, with Ly-1 B cells representing a conserved, primitive B cell lineage and retaining key, associated functions. PMID:1742426

  18. Dual 4- and 5-phosphatase activities regulate SopB-dependent phosphoinositide dynamics to promote bacterial entry.

    PubMed

    Piscatelli, Heather L; Li, Menghan; Zhou, Daoguo

    2016-05-01

    Salmonella are able to invade non-phagocytic cells such as intestinal epithelial cells by modulating the host actin cytoskeleton to produce membrane ruffles. Two type III effector proteins SopB and SopE play key roles to this modulation. SopE is a known guanine nucleotide exchange factor (GEF) capable of activating Rac1 and CDC42. SopB is a phosphatidylinositol 4-phosphatase and 5-phosphatase promoting membrane ruffles and invasion of Salmonella through undefined mechanisms. Previous studies have demonstrated that the 4-phosphatase activity of SopB is required for PtdIns-3-phosphate (PtdIns(3)P) accumulation and SopB-mediated invasion. We show here that both the 4-phosphatase as well as the 5-phosphatase activities of SopB are essential in ruffle formation and subsequent invasion. We found that the 5-phosphatase activity of SopB is likely responsible for generating PtdIns-3,4-bisphosphate (PtdIns(3,4)P2 ) and subsequent recruitment of sorting nexin 9 (SNX9), an actin modulating protein. Intriguingly, the 4-phosphatase activity is responsible for the dephosphorylation of PtdIns(3,4)P2 into PtdIns(3)P. Alone, neither activity is sufficient for ruffling but when acting in conjunction with one another, the 4-phosphatase and 5-phosphatase activities led to SNX9-mediated ruffling and Salmonella invasion. This work reveals the unique ability of bacterial effector protein SopB to utilize both its 4- and 5-phosphatase activities to regulate phosphoinositide dynamics to promote bacterial entry. PMID:26537021

  19. Internalin must be on the bacterial surface to mediate entry of Listeria monocytogenes into epithelial cells.

    PubMed

    Lebrun, M; Mengaud, J; Ohayon, H; Nato, F; Cossart, P

    1996-08-01

    Entry of Listeria monocytogenes into cultured epithelial cells requires production of internalin, a protein with features characteristic of some Gram-positive bacterial surface proteins, in particular an LPXTG motif preceding a hydrophobic sequence and a few basic residues at its C-terminal end. By immunofluorescence and immunogold labelling, we show that in wild-type L. monocytogenes, internalin is present on the cell surface and has a polarized distribution similar to that of ActA, another surface protein of L. monocytogenes involved in actin assembly. Through a genetic analysis, we establish that the C-terminal region of internalin is necessary for cell-surface association, and that although internalin is partially released in the culture medium, its location on the bacterial surface is required to promote entry. Finally, using a 'domain-swapping' strategy-replacement of the cell wall anchor of IniA by the membrane anchor of ActA- we show that the reduced ability to adhere and enter cells of strains expressing IniA-ActA correlates with a lower amount of surface-exposed internalin. Taken together, these results suggest that internalin exposed on the bacterial surface mediates direct contact between the bacterium and the host cell. PMID:8866480

  20. Antimicrobial Activity of Monoramnholipids Produced by Bacterial Strains Isolated from the Ross Sea (Antarctica).

    PubMed

    Tedesco, Pietro; Maida, Isabel; Palma Esposito, Fortunato; Tortorella, Emiliana; Subko, Karolina; Ezeofor, Chidinma Christiana; Zhang, Ying; Tabudravu, Jioji; Jaspars, Marcel; Fani, Renato; de Pascale, Donatella

    2016-05-01

    Microorganisms living in extreme environments represent a huge reservoir of novel antimicrobial compounds and possibly of novel chemical families. Antarctica is one of the most extraordinary places on Earth and exhibits many distinctive features. Antarctic microorganisms are well known producers of valuable secondary metabolites. Specifically, several Antarctic strains have been reported to inhibit opportunistic human pathogens strains belonging to Burkholderia cepacia complex (Bcc). Herein, we applied a biodiscovery pipeline for the identification of anti-Bcc compounds. Antarctic sub-sea sediments were collected from the Ross Sea, and used to isolate 25 microorganisms, which were phylogenetically affiliated to three bacterial genera (Psychrobacter, Arthrobacter, and Pseudomonas) via sequencing and analysis of 16S rRNA genes. They were then subjected to a primary cell-based screening to determine their bioactivity against Bcc strains. Positive isolates were used to produce crude extracts from microbial spent culture media, to perform the secondary screening. Strain Pseudomonas BNT1 was then selected for bioassay-guided purification employing SPE and HPLC. Finally, LC-MS and NMR structurally resolved the purified bioactive compounds. With this strategy, we achieved the isolation of three rhamnolipids, two of which were new, endowed with high (MIC < 1 μg/mL) and unreported antimicrobial activity against Bcc strains. PMID:27128927

  1. Characterization of airborne ice-nucleation-active bacteria and bacterial fragments

    NASA Astrophysics Data System (ADS)

    Šantl-Temkiv, Tina; Sahyoun, Maher; Finster, Kai; Hartmann, Susan; Augustin-Bauditz, Stefanie; Stratmann, Frank; Wex, Heike; Clauss, Tina; Nielsen, Niels Woetmann; Sørensen, Jens Havskov; Korsholm, Ulrik Smith; Wick, Lukas Y.; Karlson, Ulrich Gosewinkel

    2015-05-01

    Some bacteria have the unique capacity of synthesising ice-nucleation-active (INA) proteins and exposing them at their outer membrane surface. As INA bacteria enter the atmosphere, they may impact the formation of clouds and precipitation. We studied members of airborne bacterial communities for their capacity to catalyse ice formation and we report on the excretion of INA proteins by airborne Pseudomonas sp. We also observed for the first time that INA biological fragments <220 nm were present in precipitation samples (199 and 482 INA fragments per L of precipitation), which confirms the presence of submicron INA biological fragments in the atmosphere. During 14 precipitation events, strains affiliated with the genus Pseudomonas, which are known to carry ina genes, were dominant. A screening for INA properties revealed that ∼12% of the cultivable bacteria caused ice formation at ≤-7 °C. They had likely been emitted to the atmosphere from terrestrial surfaces, e.g. by convective transport. We tested the ability of isolated INA strains to produce outer membrane vesicles and found that two isolates could do so. However, only very few INA vesicles were released per INA cell. Thus, the source of the submicron INA proteinaceous particles that we detected in the atmosphere remains to be elucidated.

  2. Antimicrobial Activity of Monoramnholipids Produced by Bacterial Strains Isolated from the Ross Sea (Antarctica) †

    PubMed Central

    Tedesco, Pietro; Maida, Isabel; Palma Esposito, Fortunato; Tortorella, Emiliana; Subko, Karolina; Ezeofor, Chidinma Christiana; Zhang, Ying; Tabudravu, Jioji; Jaspars, Marcel; Fani, Renato; de Pascale, Donatella

    2016-01-01

    Microorganisms living in extreme environments represent a huge reservoir of novel antimicrobial compounds and possibly of novel chemical families. Antarctica is one of the most extraordinary places on Earth and exhibits many distinctive features. Antarctic microorganisms are well known producers of valuable secondary metabolites. Specifically, several Antarctic strains have been reported to inhibit opportunistic human pathogens strains belonging to Burkholderia cepacia complex (Bcc). Herein, we applied a biodiscovery pipeline for the identification of anti-Bcc compounds. Antarctic sub-sea sediments were collected from the Ross Sea, and used to isolate 25 microorganisms, which were phylogenetically affiliated to three bacterial genera (Psychrobacter, Arthrobacter, and Pseudomonas) via sequencing and analysis of 16S rRNA genes. They were then subjected to a primary cell-based screening to determine their bioactivity against Bcc strains. Positive isolates were used to produce crude extracts from microbial spent culture media, to perform the secondary screening. Strain Pseudomonas BNT1 was then selected for bioassay-guided purification employing SPE and HPLC. Finally, LC-MS and NMR structurally resolved the purified bioactive compounds. With this strategy, we achieved the isolation of three rhamnolipids, two of which were new, endowed with high (MIC < 1 μg/mL) and unreported antimicrobial activity against Bcc strains. PMID:27128927

  3. Cultured C2C12 cell lines as a model for assessment of bacterial attachment to bovine primary muscle cells.

    PubMed

    Zulfakar, Siti Shahara; White, Jason D; Ross, Tom; Tamplin, Mark L

    2013-06-01

    The mechanisms of bacterial attachment to meat tissues need to be understood to enhance meat safety interventions. However, little is known about attachment of foodborne pathogens to meat muscle cells. In this study, attachment of six Escherichia coli and two Salmonella strains to primary bovine muscle cells and a cultured muscle cell line, C2C12, was measured, including the effect of temperature. At 37°C, all but one strain (EC623) attached to C2C12 cells, whereas only five of eight strains (M23Sr, H10407, EC473, Sal1729a and Sal691) attached to primary cells. At 10 °C, two strains (H10407 and EC473) attached to C2C12 cells, compared to four strains (M23Sr, EC614, H10407 and Sal1729a) of primary cells. Comparing all strains at both temperatures, EC614 displayed the highest CFU per C2C12 cell (4.60±2.02CFU/muscle cell at 37 °C), whereas greater numbers of M23Sr attached per primary cell (51.88±39.43CFU/muscle cell at 37 °C). This study indicates that primary bovine muscle cells may provide a more relevant model system to study bacterial attachment to beef carcasses compared to cell lines such as C2C12. PMID:23501253

  4. Low-frequency dielectric dispersion of bacterial cell suspensions.

    PubMed

    Asami, Koji

    2014-07-01

    Dielectric spectra of Escherichia coli cells suspended in 0.1-10 mM NaCl were measured over a frequency range of 10 Hz to 10 MHz. Low-frequency dielectric dispersion, so-called the α-dispersion, was found below 10 kHz in addition to the β-dispersion, due to interfacial polarization, appearing above 100 kHz. When the cells were killed by heating at 60°C for 30 min, the β-dispersion disappeared completely, whereas the α-dispersion was little influenced. This suggests that the plasma (or inner) membranes of the dead cells are no longer the permeability barrier to small ions, and that the α-dispersion is not related to the membrane potential due to selective membrane permeability of ions. The intensity of the α-dispersion depended on both of the pH and ionic strength of the external medium, supporting the model that the α-dispersion results from the deformation of the ion clouds formed outside and inside the cell wall containing charged residues. PMID:24835050

  5. Anti-bacterial activity of Euphorbia fusiformis--a rare medicinal herb.

    PubMed

    Natarajan, D; Britto, S John; Srinivasan, K; Nagamurugan, N; Mohanasundari, C; Perumal, G

    2005-10-31

    Euphorbia fusiformis Buch.-Ham. ex. D.Don (Euphorbiaceae) is a rare medicinal herb. Aqueous and organic solvent extracts of the leaves and rootstocks were investigated for anti-bacterial properties by using disc diffusion and well-in agar methods, against pathogenic strains of Gram positive (Bacillus subtilis and Staphylococcus aureus) and Gram negative bacteria (Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella typhii A and Salmonella typhii B). The different extracts differed significantly in their anti-bacterial properties with the methanolic extract being very effective followed by acetone and chloroform extracts. Aqueous and ethanolic extract showed very least activity. The result highlights that rootstock extracts had good anti-bacterial properties than leaf extracts. The results of this study support the use of this plant in traditional medicine to treat fever, wound infections and intestinal disorders. PMID:16159702

  6. Antibacterial activity of alkyl gallates is a combination of direct targeting of FtsZ and permeabilization of bacterial membranes

    PubMed Central

    Król, Ewa; de Sousa Borges, Anabela; da Silva, Isabel; Polaquini, Carlos R.; Regasini, Luis O.; Ferreira, Henrique; Scheffers, Dirk-Jan

    2015-01-01

    Alkyl gallates are compounds with reported antibacterial activity. One of the modes of action is binding of the alkyl gallates to the bacterial membrane and interference with membrane integrity. However, alkyl gallates also cause cell elongation and disruption of cell division in the important plant pathogen Xanthomonas citri subsp. citri, suggesting that cell division proteins may be targeted by alkyl gallates. Here, we use Bacillus subtilis and purified B. subtilis FtsZ to demonstrate that FtsZ is a direct target of alkyl gallates. Alkyl gallates disrupt the FtsZ-ring in vivo, and cause cell elongation. In vitro, alkyl gallates bind with high affinity to FtsZ, causing it to cluster and lose its capacity to polymerize. The activities of a homologous series of alkyl gallates with alkyl side chain lengths ranging from five to eight carbons (C5–C8) were compared and heptyl gallate was found to be the most potent FtsZ inhibitor. Next to the direct effect on FtsZ, alkyl gallates also target B. subtilis membrane integrity—however the observed anti-FtsZ activity is not a secondary effect of the disruption of membrane integrity. We propose that both modes of action, membrane disruption and anti-FtsZ activity, contribute to the antibacterial activity of the alkyl gallates. We propose that heptyl gallate is a promising hit for the further development of antibacterials that specifically target FtsZ. PMID:25972861

  7. Antibacterial activity of alkyl gallates is a combination of direct targeting of FtsZ and permeabilization of bacterial membranes.

    PubMed

    Król, Ewa; de Sousa Borges, Anabela; da Silva, Isabel; Polaquini, Carlos R; Regasini, Luis O; Ferreira, Henrique; Scheffers, Dirk-Jan

    2015-01-01

    Alkyl gallates are compounds with reported antibacterial activity. One of the modes of action is binding of the alkyl gallates to the bacterial membrane and interference with membrane integrity. However, alkyl gallates also cause cell elongation and disruption of cell division in the important plant pathogen Xanthomonas citri subsp. citri, suggesting that cell division proteins may be targeted by alkyl gallates. Here, we use Bacillus subtilis and purified B. subtilis FtsZ to demonstrate that FtsZ is a direct target of alkyl gallates. Alkyl gallates disrupt the FtsZ-ring in vivo, and cause cell elongation. In vitro, alkyl gallates bind with high affinity to FtsZ, causing it to cluster and lose its capacity to polymerize. The activities of a homologous series of alkyl gallates with alkyl side chain lengths ranging from five to eight carbons (C5-C8) were compared and heptyl gallate was found to be the most potent FtsZ inhibitor. Next to the direct effect on FtsZ, alkyl gallates also target B. subtilis membrane integrity-however the observed anti-FtsZ activity is not a secondary effect of the disruption of membrane integrity. We propose that both modes of action, membrane disruption and anti-FtsZ activity, contribute to the antibacterial activity of the alkyl gallates. We propose that heptyl gallate is a promising hit for the further development of antibacterials that specifically target FtsZ. PMID:25972861

  8. Enhancement of aerobic biodegradability potential of municipal waste activated sludge by ultrasonic aided bacterial disintegration.

    PubMed

    Kavitha, S; Jessin Brindha, G M; Sally Gloriana, A; Rajashankar, K; Yeom, Ick Tae; Rajesh Banu, J

    2016-01-01

    An investigation was performed to study the influence of ultrasonic aided bacterial disintegration on the aerobic degradability of sludge. In first phase of the study, effective floc disruption was achieved at an ultrasonic specific energy input of 2.45kJ/kg TS with 44.5mg/L of Extracellular Polymeric Substance (EPS) release including 0.035U/mL and 0.025U/mL protease and amylase activity respectively. In second phase, experimental outcomes revealed bacterial disintegration of floc disrupted-sludge showing a maximum solubilization of about 23% and was observed to be superior to bacterially disintegrated (11%) and control (6%), respectively. The result of aerobic biodegradability of ultrasonic aided bacterially pretreated sludge showed volatile solids (VS) degradation of about 40.2%. The kinetic study of aerobic biodegradability through non linear regression modelling reveals that floc disrupted sludge showed better biodegradability with decay constant of about 0.19d(-1) relatively higher than the control (0.14d(-1)) and bacterially disintegrated (0.17d(-1)) sludges. PMID:26479431

  9. Bacterial β-glucosidase function and metabolic activity depend on soil management in semiarid rainfed agriculture

    PubMed Central

    Cañizares, Rosa; Moreno, Beatriz; Benitez, Emilio

    2012-01-01

    Genomic and transcriptomic approaches were used to gain insights into the relationship between soil management and bacterial-mediated functions in an olive orchard agroecosystem. Four management practices were assessed in a 30-year trial in a semiarid Mediterranean region. Transcriptional activity of bacterial 16S rRNA genes increased in noncovered soils, indicating higher microbial maintenance requirements to thrive in less favorable environmental conditions. The 16S rRNA transcript:gene copy ratio confirmed this assumption and pointed toward a much higher constitutive expression from rRNA operons in noncovered soils and to even higher expression levels when spontaneous vegetation was removed chemically. As described for 16S rRNA, potential transcription did not reveal the real transcription of bacterial β-glucosidase genes, and higher gene expression in noncovered soils plus herbicides was evidenced. Since no relationship between total or soluble organic carbon and bacterial β-glucosidase transcription was found, the above hypothesis could indicate either that soluble organic carbon is not the main pool of enzyme-inducing substrates or that constitutive production of bacterial β-glucosidase enzymes increases as soil conditions worsen. PMID:22837821

  10. Electron microscopy study of antioxidant interaction with bacterial cells

    NASA Astrophysics Data System (ADS)

    Plotnikov, Oleg P.; Novikova, Olga V.; Konnov, Nikolai P.; Korsukov, Vladimir N.; Gunkin, Ivan F.; Volkov, Uryi P.

    2000-10-01

    To maintain native microorganisms genotype and phenotype features a lyophylization technique is widely used. However in this case cells are affected by influences of vacuum and low temperature that cause a part of the cells population to be destruction. Another factor reduced microorganisms vitality is formation of reactive oxygen forms that damage certain biological targets (such as DNA, membranes etc.) Recently to raise microorganism's resistance against adverse condition natural and synthetic antioxidants are used. Antioxidant- are antagonists of free radicals. Introduction of antioxidants in protective medium for lyophylization increase bacteria storage life about 2,0-4,8 fold in comparison with reference samples. In the article the main results of our investigation of antioxidants interaction with microorganism cells is described. As bacteria cells we use vaccine strain yersinia pestis EV, that were grown for 48 h at 28 degree(s)C on the Hottinger agar (pH 7,2). Antioxidants are inserted on the agar surface in specimen under test. To investigate a localization of antioxidants for electron microscopy investigation, thallium organic antioxidants were used. The thallium organic compounds have an antioxidant features if thallium is in low concentration (about 1(mu) g/ml). The localization of the thallium organic antioxidants on bacteria Y. pestis EV is visible in electron microscopy images, thallium being heavy metal with high electron density. The negatively stained bacteria and bacteria thin sections with thallium organic compounds were investigated by means of transmission electron microscopy. The localization of the thallium organic compounds is clearly visible in electron micrographs as small dark spots with size about 10-80nm. Probably mechanisms of interaction of antioxidants with bacteria cells are discussed.

  11. Computer simulation of the processes of inactivation of bacterial cells by dynamic low-coherent speckles

    NASA Astrophysics Data System (ADS)

    Ulianova, Onega V.; Ulyanov, Sergey S.; Sazanova, Elena V.; Zhihong, Zhang; Sibo, Zhou; Luo, Qingming; Zudina, Irina; Bednov, Andrey

    2006-05-01

    Biochemical, biophysical and optical aspects of interaction of low-coherent light with bacterial cells have been discussed. Influence of low-coherent speckles on the colonies grows is investigated. It has been demonstrated that effects of light on the inhibition of cells (Francisella Tularensis) are connected with speckle dynamics. The regimes of illumination of cell suspension with purpose of devitalization of hazard bacteria, caused very dangerous infections, such as tularemia, are found. Mathematical model of interaction of low-coherent laser radiation with bacteria suspension has been proposed. Computer simulations of the processes of laser-cells interaction have been carried out.

  12. Isolation and solubilization of gram-positive bacterial cell wall-associated proteins.

    PubMed

    Cole, Jason N; Djordjevic, Steven P; Walker, Mark J

    2008-01-01

    This chapter describes a simple, rapid and reproducible method to prepare bacterial cell wall extracts for two-dimensional gel electrophoresis (2DE). The extraction process uses mutanolysin, an N-acetylmuramidase, to gently solubilize cell wall-associated proteins from Gram-positive prokaryotes. The cells are first washed with buffer and resuspended in a solution containing mutanolysin. Following incubation at 37 degrees C, the sample is centrifuged and the supernatant containing the soluble cell wall-associated proteins is harvested. Following a brief precipitation step, the pellet is solubilized in sample buffer ready for isoelectric focusing and 2DE analysis. PMID:18369905

  13. Purification of transfection-grade plasmid DNA from bacterial cells with superparamagnetic nanoparticles

    NASA Astrophysics Data System (ADS)

    Chiang, Chen-Li; Sung, Ching-Shan

    2006-07-01

    The functionalized magnetic nanobeads were used to develop a rapid protocol for extracting and purifying transfection-grade plasmid DNA from bacterial culture. Nanosized superparamagnetic nanoparticles (Fe 3O 4) were prepared by chemical coprecipitation method using Fe 2+, Fe 3+ salt, and ammonium hydroxide under a nitrogen atmosphere. The surface of Fe 3O 4 nanoparticles was modified by coating with the multivalent cationic agent, polyethylenimine (PEI). The PEI-modified magnetic nanobeads were employed to simplify the purification of plasmid DNA from bacterial cells. We demonstrated a useful plasmid, pRSETB-EGFP, encoding the green fluorescent protein with T7 promoter, was amplified in DE3 strain of Escherichia coli. The loaded nanobeads are recovered by magnetically driven separation and regenerated by exposure to the elution buffer with optimal ionic strength (1.25 M) and pH (9.0). Up to approximately 819 μg of high-purity (A 260/A 280 ratio=1.86) plasmid DNA was isolated from 100 ml of overnight bacterial culture. The eluted plasmid DNA was used directly for restriction enzyme digestion, bacterial cell transformation and animal cell transfection applications with success. The PEI-modified magnetic nanobead delivers significant time-savings, overall higher yields and better transfection efficiencies compared to anion-exchange and other methods. The results presented in this report show that PEI-modified magnetic nanobeads are suitable for isolation and purification of transfection-grade plasmid DNA.

  14. Label-free mapping of single bacterial cells using surface-enhanced Raman spectroscopy.

    PubMed

    Wang, Panxue; Pang, Shintaro; Chen, Juhong; McLandsborough, Lynne; Nugen, Sam R; Fan, Mingtao; He, Lili

    2016-02-01

    Here we presented a simple, rapid and label-free surface-enhanced Raman spectroscopy (SERS) based mapping method for the detection and discrimination of Salmonella enterica and Escherichia coli on silver dendrites. The sample preparation was first optimized to maximize sensitivity. The mapping method was then used to scan through the bacterial cells adsorbed on the surface of silver dendrites. The intrinsic and distinct SERS signals of bacterial cells were used as the basis for label-free detection and discrimination. The results show the developed method is able to detect single bacterial cells adsorbed on the silver dendrites with a limit of detection as low as 10(4) CFU mL(-1), which is two orders of magnitude lower than the traditional SERS method under the same experimental condition. The time needed for collecting a 225 points map was approximately 24 minutes. Moreover, the developed SERS mapping method can realize simultaneous detection and identification of Salmonella enterica subsp. enterica BAA1045 and Escherichia coli BL21 from a mixture sample using principle component analysis. Our results demonstrate the great potential of the label-free SERS mapping method to detect, identify and quantify bacteria and bacterial mixtures simultaneously. PMID:26750611

  15. Comparative Analysis of UV Irradiation Effects on Escherichia coli and Pseudomonas aeruginosa Bacterial Cells Utilizing Biological and Computational Approaches.

    PubMed

    Margaryan, A; Badalyan, H; Trchounian, A

    2016-09-01

    Microorganisms have a large number of tools to withstand different, and sometimes strong, environmental stresses, including irradiation, but this ability should be further evaluated for certain applications. Growth inhibition and morphological alterations of Escherichia coli M-17 and Pseudomonas aeruginosa GRP3 wild-type cells caused by UV-A irradiation have been detected in the present study. Comparative analysis was carried out using well-established microbiological methods (determination of specific growth rate, growth lag phase duration, and colony-forming unit number-CFU) and computational approaches, employing light microscopy and digital image analysis to evaluate bacterial cell morphology. Decreases in the specific growth rate, prolonged lag-phases, and lowered CFUs were observed after 5 and 10 min of UV irradiation (approx. 40 Gy) compared to the control (nonirradiated) cells. Accordingly, two computational parameters-the average bacterial cell surface area and the bacterial cell perimeter (i.e., of the 2D projection of bacterial cells in microscopy image)-were reduced. The ratio of bacterial cell surface area (S) to the square of the perimeter (p (2) ) was reduced after 5 min of irradiation, but after 10 min of irradiation the studied bacterial cells became flat cylinders. The revealed findings are concluded to be highly useful in developing new, rapid analysis methods to monitor environmental and UV irradiation effects on bacteria and to detect bacterial cell morphology alterations. PMID:27334536

  16. Bacterial diversity is strongly associated with historical penguin activity in an Antarctic lake sediment profile

    PubMed Central

    Zhu, Renbin; Shi, Yu; Ma, Dawei; Wang, Can; Xu, Hua; Chu, Haiyan

    2015-01-01

    Current penguin activity in Antarctica affects the geochemistry of sediments and their microbial communities; the effects of historical penguin activity are less well understood. Here, bacterial diversity in ornithogenic sediment was investigated using high-throughput pyrosequencing. The relative abundances of dominant phyla were controlled by the amount of historical penguin guano deposition. Significant positive correlations were found between both the bacterial richness and diversity, and the relative penguin number (p < 0.01); this indicated that historical penguin activity drove the vertical distribution of the bacterial communities. The lowest relative abundances of individual phyla corresponded to lowest number of penguin population at 1,800–2,300 yr BP during a drier and colder period; the opposite was observed during a moister and warmer climate (1,400–1,800 yr BP). This study shows that changes in the climate over millennia affected penguin populations and the outcomes of these changes affect the sediment bacterial community today. PMID:26601753

  17. Impact of freshwater inflow on bacterial abundance and activity in the estuarine system Ria de Aveiro

    NASA Astrophysics Data System (ADS)

    Santos, Luísa; Vaz, Leandro; Marcial Gomes, Newton C.; Vaz, Nuno; Dias, João Miguel; Cunha, Ângela; Almeida, Adelaide

    2014-02-01

    The influence of freshwater flow on bacterial communities in the estuarine system Ria de Aveiro (Portugal) was investigated at two sites differently impacted by river inputs, representative of the marine and brackish water zones of the estuary. Sampling events were clustered based on hydrological features. The hydrodynamic was simulated with a Lagrangian model and related to microbiological parameters. Estuarine bacteria responded to different freshwater regimes developing distinct patterns of abundance and activity at the marine and brackish water zones. A circulation pattern induced by high river inflow produced vertical stratification in the marine zone, promoting a seaward flux of bacterioplankton, and stimulating the import of riverine phytoplankton and particle-attached bacteria to the brackish water zone. Advective transport and resuspension processes contributed to a 3-times increase in abundance of particle-attached bacteria during intense freshwater inputs. Additionally, bacterial activity in the estuary was controlled by inorganic nitrogen, responding to different freshwater inputs, which, in association with different prevailing sources of organic substrates induced significant changes in bacterial production. The dynamic and main controlling factors of bacterial communities are clearly impacted by freshwater inputs. Therefore, significant changes in the recycling of nutrients by microbial activities can be expected from alterations in freshwater inputs either related to global climate change or regional hydrological regimes.

  18. Analysis of gene expression levels in individual bacterial cells without image segmentation

    SciTech Connect

    Kwak, In Hae; Son, Minjun; Hagen, Stephen J.

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer We present a method for extracting gene expression data from images of bacterial cells. Black-Right-Pointing-Pointer The method does not employ cell segmentation and does not require high magnification. Black-Right-Pointing-Pointer Fluorescence and phase contrast images of the cells are correlated through the physics of phase contrast. Black-Right-Pointing-Pointer We demonstrate the method by characterizing noisy expression of comX in Streptococcus mutans. -- Abstract: Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on a segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analyzing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly.

  19. Mutagenic effect of accelerated heavy ions on bacterial cells

    NASA Astrophysics Data System (ADS)

    Boreyko, A. V.; Krasavin, E. A.

    2011-11-01

    The heavy ion accelerators of the Joint Institute for Nuclear Research were used to study the regularities and mechanisms of formation of different types of mutations in prokaryote cells. The induction of direct (lac-, ton B-, col B) mutations for Esherichia coli cells and reverse his- → His+ mutations of Salmonella typhimurium, Bacillus subtilis cells under the action of radiation in a wide range of linear energy transfer (LET) was studied. The regularities of formation of gene and structural (tonB trp-) mutations for Esherichia coli bacteria under the action of accelerated heavy ions were studied. It was demonstrated that the rate of gene mutations as a function of the dose under the action of Γ rays and accelerated heavy ions is described by linear-quadratic functions. For structural mutations, linear "dose-effect" dependences are typical. The quadratic character of mutagenesis dose curves is determined by the "interaction" of two independent "hitting" events in the course of SOS repair of genetic structures. The conclusion made was that gene mutations under the action of accelerated heavy ions are induced by δ electron regions of charged particle tracks. The methods of SOS chromotest, SOS lux test, and λ prophage induction were used to study the regularities of SOS response of cells under the action of radiations in a wide LET range. The following proposition was substantiated: the molecular basis for formation of gene mutations are cluster single-strand DNA breaks, and that for structural mutations, double-strand DNA breaks. It was found out that the LET dependence of the relative biological efficiency of accelerated ions is described by curves with a local maximum. It was demonstrated that the biological efficiency of ionizing radiations with different physical characteristics on cells with different genotype, estimated by the lethal action, induction of gene and deletion mutations, precision excision of transposons, is determined by the specific

  20. Asynchrony in the growth and motility responses to environmental changes by individual bacterial cells

    SciTech Connect

    Umehara, Senkei; Hattori, Akihiro; Inoue, Ippei; Yasuda, Kenji . E-mail: yasuda.bmi@tmd.ac.jp

    2007-05-04

    Knowing how individual cells respond to environmental changes helps one understand phenotypic diversity in a bacterial cell population, so we simultaneously monitored the growth and motility of isolated motile Escherichia coli cells over several generations by using a method called on-chip single-cell cultivation. Starved cells quickly stopped growing but remained motile for several hours before gradually becoming