Surface Physicochemical Properties At The Micro And Nano Length Scales: Role On Bacterial Adhesion And Xylella Fastidiosa Biofilm Development.

The phytopathogen Xylella fastidiosa grows as a biofilm causing vascular occlusion and consequently nutrient and water stress in different plant hosts by adhesion on xylem vessel surfaces composed of cellulose, hemicellulose, pectin and proteins. Understanding the factors which influence bacterial adhesion and biofilm development is a key issue in identifying mechanisms for preventing biofilm formation in infected plants. In this study, we show that X. fastidiosa biofilm development and architecture correlate well with physicochemical surface properties after interaction with the culture medium. Different biotic and abiotic substrates such as silicon (Si) and derivatized cellulose films were studied. Both biofilms and substrates were characterized at the micro- and nanoscale, which corresponds to the actual bacterial cell and membrane/ protein length scales, respectively. Our experimental results clearly indicate that the presence of surfaces with different chemical composition affect X. fastidiosa behavior from the point of view of gene expression and adhesion functionality. Bacterial adhesion is facilitated on more hydrophilic surfaces with higher surface potentials; XadA1 adhesin reveals different strengths of interaction on these surfaces. Nonetheless, despite different architectural biofilm geometries and rates of development, the colonization process occurs on all investigated surfaces. Our results univocally support the hypothesis that different adhesion mechanisms are active along the biofilm life cycle representing an adaptation mechanism for variations on the specific xylem vessel composition, which the bacterium encounters within the infected plant.8e7524

Strained In1-xGaxAsyP1-y/InP quantum well heterostructures grown by low-pressure metalorganic vapor phase epitaxy

We have investigated the optical and the structural properties of strained In1-xGaxAsyP1-y/InP and strain compensated In1-xGaxAsyP1-y/In1-zGazAsqP1-q/InP multi-quantum well heterostructures grown by low-pressure metalorganic vapor phase epitaxy at different growth conditions. Our results indicate an increase of the compositional fluctuation of quaternary materials as the alloy composition moves from the outer spinodal isotherm into the miscibility gap region. In1-xGaxAsyP1-y layers grown at high tensile strained values exhibit a three-dimensional-like growth mode. Strain compensated structures revealed the presence of a broad photoluminescence emission band below the fundamental quantum well transition, well defined elongated features along the [011] direction and interface undulations. All these effects were found to be strongly dependent on the growth temperature and the number of wells.495

The antitoxin protein of a toxin-antitoxin system from Xylella fastidiosa is secreted via outer membrane vesicles.

The Xylella fastidiosa subsp pauca strain 9a5c is a Gram-negative, xylem-limited bacterium that is able to form a biofilm and affects citrus crops in Brazil. Some genes are considered to be involved in biofilm formation, but the specific mechanisms involved in this process remain unknown. This limited understanding of how some bacteria form biofilms is a major barrier to our comprehension of the progression of diseases caused by biofilm-producing bacteria. Several investigations have shown that the toxin-antitoxin (TA) operon is related to biofilm formation. This operon is composed of a toxin with RNAse activity and its cognate antitoxin. Previous reports have indicated that the antitoxin is able to inhibit toxin activity and modulate the expression of the operon as well as other target genes involved in oxidative stress and mobility. In this study, we characterize a toxin-antitoxin system consisting of XfMqsR and XfYgiT, respectively, from X. fastidiosa subsp pauca strain 9a5c. These proteins display a high similarity to their homologues in X. fastidiosa strain Temecula and a predicted tridimensional structure that is similar to MqsR-YgiT from Escherichia coli. The characterization was performed using in vitro assays such as analytical ultracentrifugation (AUC), size exclusion chromatography, isothermal titration calorimetry and western blotting. Using a fluorometric assay to detect RNAses, we demonstrated that XfMqsR is thermostable and can degrade RNA. XfMqsR is inhibited by XfYgiT, which interacts with its own promoter. XfYgiT is known to be localized in the intracellular compartment; however, we provide strong evidence that X. fastidiosa secretes wild-type XfYgiT into the extracellular environment via outer membrane vesicles, as confirmed by western blotting and specific immunofluorescence labeling visualized by fluorescence microscopy. Taken together, our results characterize the TA system from X. fastidiosa strain 9a5c, and we also discuss the possible influence of wild-type XfYgiT in the cell

Stiffness signatures along early stages of Xylella fastidiosa biofilm formation

FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPCONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESThe pathogenicity of Xylella fastidiosa is associated with its systematic colonization of the plant xylem, forming bacterial biofilms. Mechanisms of bacterial transport among different xylem vessels, however, are not completely understood yet, but are strongly influenced by the presence of extracellular polymeric substances (EPS), which surrounds the assembly of cells forming the biofilm. In this work, we show quantitative measurements on the elastic properties of the system composed by EPS and bacterial cell. In order to investigate the mechanical properties of this system, force spectroscopy and confocal Raman measurements were carried out during Xylella fastidiosa subsp. pauca initial stages of adhesion and cluster formation. We show that stiffness progressively decreases with increasing culture growth time, from two to five days. For early adhesion samples, stiffness values are quite different at the bacterial polar and body regions. Lower stiffness values at the cell pole suggest a flexible mechanical, response at this region, associated with first cell adhesion to a surface. These results correlate very well with our observations of cell motion within microchannels, under conditions simulating xylem flow. Both the oscillatory movement of vertically attached single cells, as well as the transport of cell clusters within the biofilm matrix can be explained by the presence of softer materials at the cell pole and EPS matrix. Our results may thus add to a more detailed understanding of mechanisms used by cells to migrate among vessels in plant xylem.159174182FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPCONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPESFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPCONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQCOORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPES08/57906-32010/5174872013/109570573913/2008-0479486/2012-3Sem informaçãoThe authors acknowledge technical assistance from Vitor Pelegatti, from INFABIC/UNICAMP, Maria H. Piazetta and A.Gobbi, from LNNano/CNPEM. The authors are also greatly indebted to Dr. Richard Janissen, from Kavli Institute of Nanoscience, Delft University of Technology, and M.Sc. Duber Murillo from Applied Physics Department at the State University of Campinas, for fruitful discussions during this work

Development of a recombinant fusion protein based on the dynein light chain LC8 for non-viral gene delivery

The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cell's nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells. A DNA binding sequence was fused to the N-terminus of the recombinant human dynein light chain LC8. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. As expected, gel permeation assays found the purified protein mainly present as dimers, the functional oligomeric state of LC8. Gel retardation assays and atomic force microscopy proved the ability of the fusion protein to interact and condense pDNA. Zeta potential measurements indicated that LC8 with DNA binding domain (LD4) has an enhanced capacity to interact and condense pDNA, generating positively charged complexes. Transfection of cultured HeLa cells confirmed the ability of the LD4 to facilitate pDNA uptake and indicate the involvement of the retrograde transport in the intracellular trafficking of pDNA:LD4 complexes. Finally, cytotoxicity studies demonstrated a very low toxicity of the fusion protein vector, indicating the potential for in vivo applications. The study presented here is part of an effort to develop new modular shuttle proteins able to take advantage of strategies used by viruses to infect mammalian cells, aiming to provide new tools for gene therapy and DNA vaccination studies1592222231FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPnão te

Biofilm architecture and quantitative biofilm size distribution.

<p>Size distribution histograms (<b>A</b>-<b>C</b>) and color enhanced SEM images of biofilms architecture (<b>D</b>-<b>I</b>) grown on bare silicon (Si; <b>A, D, E</b>), ethyl cellulose (EC; <b>B, F</b>, <b>G</b>) and cellulose acetate (CA; <b>C, H, I</b>) substrates in PW medium. Images E, G and I show details of the biofilm edges on each substrate at higher magnification. Scale bars correspond to 20µm for D, F, H and 5µm for <b>E</b>, <b>G</b>, <b>I</b>. The insets in <b>A-C</b> show re-scaled histograms to more precisely visualize the presence of larger biofilms.</p

Surface morphology and potential alterations of silicon and cellulose surfaces induced by culture media.

<p>AFM topography (<b>A</b>-<b>F</b>) and surface potential (SP; <b>G-I</b>) images of the substrate surfaces. (<b>A</b>-<b>C</b>) show topography of pristine silicon (Si), ethyl cellulose (EC) and cellulose acetate (CA) surfaces, respectively; (<b>D</b>-<b>F</b>) illustrates topography of Si, EC and CA surfaces after incubation in PW medium for 24h, respectively; (<b>G</b>-<b>I</b>) present SP images of the surfaces shown in (<b>D</b>-<b>F</b>). Scale bars correspond to 1µm.</p

Surface potential of culture media conditioning film formed on pristine silicon.

<p>AFM topography (<b>A</b>) and surface potential (<b>B</b>) images showing the edge of the thin conditioning film caused by periwinkle wilt (PW) medium on silicon (Si) substrate (scale bar 2 µm). The corresponding profiles of height (1) and surface potential differences (2) are shown below.</p

Comparison of biofilm growth on pristine silicon and cellulose acetate surfaces.

<p>AFM topography images of <i>Xylella fastidiosa</i> biofilms grown on bare silicon (Si) and cellulose acetate (CA) surfaces side-by-side in periwinkle wilt (PW) medium (scale bar 5 µm). Images are shown with different height contrasts to illustrate individually the CA topography (<b>A</b>) and biofilm (<b>B</b>) more accurately. As a control image, please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075247#pone.0075247.s002" target="_blank">Figure <b>S2A</b></a> in supplemental information.</p

Interaction forces of bacterial adhesin XadA1 on silicon and cellulose surfaces.

<p>Schematics of the force spectroscopy (<b>A</b>) measurements at five different positions on the surface where force curves were acquired. The force histograms (<b>B</b>-<b>D</b>) for bare silicon (Si, B) ethyl cellulose (EC, <b>C</b>) and cellulose acetate (CA, <b>D</b>), show the total number of events (please notice the different scales for the number of events); multiple rupture events were observed for EC and CA. Typical retraction force-distance curves for AFM tips coated with XadA1 adhesion protein (<b>E</b>) for Si (i), EC (ii) and CA (iii). The inset shows the fluorescence image of the functionalized AFM tip and cantilever visualized using fluorophore labeled second antibodies (scale bar = 40µm). Measured interaction forces (<b>F</b>-<b>H</b>) between XadA1 coated AFM tip and bare Si (<b>F</b>), EC (<b>G</b>) and CA (<b>H</b>) substrates in PBS buffer shown in sequential acquisition order. The colors indicate a different region of the sample probed in each set of force curves. The bin sizes for (<b>B</b>), (<b>C</b>) were adjusted to 22pN and (<b>D</b>) to 5pN to allow accurate visualization of the molecular interaction characteristics.</p