scholarly journals The Type II Secreted Lipase/Esterase LesA is a Key Virulence Factor Required for Xylella fastidiosa Pathogenesis in Grapevines

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Rafael Nascimento ◽  
Hossein Gouran ◽  
Sandeep Chakraborty ◽  
Hyrum W. Gillespie ◽  
Hebréia O. Almeida-Souza ◽  
...  
Keyword(s):  
Culture Supernatant ◽  
Bacterial Culture ◽  
Xylella Fastidiosa ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Type Ii ◽  
Symptom Development ◽  
Bacterial Titer ◽  
Bacterial Culture Supernatant ◽  
Xylem Elements

Abstract Pierce’s disease (PD) of grapevines is caused by Xylella fastidiosa (Xf), a xylem-limited gamma-proteobacterium that is responsible for several economically important crop diseases. The occlusion of xylem elements and interference with water transport by Xf and its associated biofilm have been posited as the main cause of PD symptom development; however, Xf virulence mechanisms have not been described. Analysis of the Xf secretome revealed a putative lipase/esterase (LesA) that was abundantly secreted in bacterial culture supernatant and was characterized as a protein ortholog of the cell wall-degrading enzyme LipA of Xanthomonas strains. LesA was secreted by Xf and associated with a biofilm filamentous network. Additional proteomic analysis revealed its abundant presence in outer membrane vesicles (OMVs). Accumulation of LesA in leaf regions associated positively with PD symptoms and inversely with bacterial titer. The lipase/esterase also elicited a hypersensitive response in grapevine. Xf lesA mutants were significantly deficient for virulence when mechanically inoculated into grapevines. We propose that Xf pathogenesis is caused by LesA secretion mediated by OMV cargos and that its release and accumulation in leaf margins leads to early stages of observed PD symptoms.

scholarly journals The Xylella fastidiosa PD1063 Protein Is Secreted in Association with Outer Membrane Vesicles

PLoS ONE ◽  
2014 ◽  
Vol 9 (11) ◽  
pp. e113504 ◽  
Author(s):  
Brittany K. Pierce ◽  
Tanja Voegel ◽  
Bruce C. Kirkpatrick
Keyword(s):  
Outer Membrane ◽  
Xylella Fastidiosa ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles

In Situ, Real-Time FT-IR/CIR/ATR Study of the Biocorrosion of Copper by Gum Arabic, Alginic Acid, Bacterial Culture Supernatant and Pseudomonas atlantica Exopolymer

1989 ◽  
Vol 43 (6) ◽  
pp. 1062-1067 ◽  
Author(s):  
John G. Jolley ◽  
Gill G. Geesey ◽  
Michael R. Hankins ◽  
Randy B. Wright ◽  
Paul L. Wichlacz
Keyword(s):  
Aqueous Solution ◽  
Real Time ◽  
Culture Supernatant ◽  
Bacterial Culture ◽  
Alginic Acid ◽  
Gum Arabic ◽  
Bacterial Culture Supernatant ◽  
Ft Ir ◽  
Simulated Seawater

Thin films (2.0 nm) of copper on germanium internal reflection elements (IREs) were exposed to 10% gum arabic (aqueous solution), 2% alginic acid (aqueous solution), 1% bacterial culture supernatant (BCS, simulated seawater solution), and 0.5% Pseudomonas atlantica exopolymer (simulated seawater solution) and monitored in situ, real time, with the use of Fourier transform infrared/cylindrical internal reflection/attenuated total reflection spectroscopy as a function of time at ambient conditions. Ancillary graphite furnace atomic absorption spectroscopy was used to monitor the removal process of the copper thin film from the germanium IREs. Results indicate that some of the copper was removed from the Cu/Ge interface by all four polymers and incorporated into the polymer matrix. Thus, biocorrosion of copper was exhibited by the four polymers in the order of alginic acid < gum arabic < BCS > Pseudomonas atlantica exopolymer. The FT-IR/CIR/ATR technique can be successfully used to monitor biocorrosion systems in in situ, real-time settings.

scholarly journals Induction of iron regulated proteins during normal growth of Neisseria meningitidis in a chemically defined medium

1994 ◽  
Vol 36 (4) ◽  
pp. 301-310 ◽  
Author(s):  
Maria Cristina de Cunto Brandileone ◽  
Rosemeire Cobo Zanella ◽  
Vera Simonsen Dias Vieira ◽  
Claudio Tavares Sacciii ◽  
Lucimar Gonçalves Milagres ◽  
...  
Keyword(s):  
Bacterial Growth ◽  
Culture Supernatant ◽  
Culture Media ◽  
Membrane Vesicles ◽  
Normal Growth ◽  
Growth Conditions ◽  
Defined Medium ◽  
Outer Membrane Vesicles ◽  
Iron Deficient

The expression of iron regulated proteins (IRPs) in vitro has been obtained in the past by adding iron chelators to the culture after bacterial growth, in the presence of an organic iron source. We have investigated aspects concerning full expression of the meningococcal IRPs during normal growth, in defined conditions using Catlin medium, Mueller Hinton and Tryptic Soy Broth (TSB). The expression of IRPs varied between different strains with respect to Ethylenediamine Di-ortho-Hidroxy-phenyl-acetic acid (EDDA) concentrations, and according to culture medium, and also between different lots of TSB. For each strain, a specific set of IRPs were expressed and higher EDDA concentrations, or addition of glucose, or use of different culture media did not resulted in a differential expression of IRPs. We were not able to grow N. meningitidis under normal growth conditions using Desferal. We looked for a good yield of outer membrane vesicles (OMVs) expressing IRPs in iron-deficient Catlin medium containing EDDA and Hemin. Culture for 32 h at 30ºC after growing for 16 h at 37ºC supported good bacterial growth. Bacterial lysis was noted after additional 24 h at 30ºC. Approximately 4 times more OMVs was recoverable from a culture supernatant after 24 h at 30ºC than from the cells after 16 h at 37ºC. The IRP were as well expressed in OMVs from culture supernatant obtained after 24 h at 30ºC as from the cells after 16 h at 37ºC.

scholarly journals Strong Decrease in Invasive Ability and Outer Membrane Vesicle Release in Crohn's Disease-Associated Adherent-Invasive Escherichia coli Strain LF82 with the yfgL Gene Deleted

2005 ◽  
Vol 187 (7) ◽  
pp. 2286-2296 ◽  
Author(s):  
Nathalie Rolhion ◽  
Nicolas Barnich ◽  
Laurent Claret ◽  
Arlette Darfeuille-Michaud
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Outer Membrane ◽  
Culture Supernatant ◽  
Intestinal Epithelial Cells ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Intestinal Epithelial ◽  
Isogenic Mutant

ABSTRACT Adherent-invasive Escherichia coli strain LF82 recovered from a chronic lesion of a patient with Crohn's disease is able to invade cultured intestinal epithelial cells. Three mutants with impaired ability to invade epithelial cells had the Tn5phoA transposon inserted in the yfgL gene encoding the YfgL lipoprotein. A yfgL- negative isogenic mutant showed a marked decrease both in its ability to invade Intestine-407 cells and in the amount of the outer membrane proteins OmpA and OmpC in the culture supernatant, as shown by analysis of the culture supernatant protein contents by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectrometry. Transcomplementation of the LF82-ΔyfgL isogenic mutant with the cloned yfgL gene restored invasion ability and outer membrane protein release in the culture supernatant. The outer membrane proteins in the culture supernatant of strain LF82 resulted from the formation of vesicles. This was shown by Western blot analysis of periplasmic and outer membrane fraction markers typically found in outer membrane vesicles and by transmission electron microscopic analysis of ultracentrifuged cell-free LF82 supernatant pellets, indicating the presence of vesicles with a bilayered structure surrounding a central electron-dense core. Thus, deletion of the yfgL gene in strain LF82 resulted in a decreased ability to invade intestinal epithelial cells and a decreased release of outer membrane vesicles.

scholarly journals Insecticidal Activity Associated with the Outer Membrane Vesicles of Xenorhabdus nematophilus

2003 ◽  
Vol 69 (4) ◽  
pp. 2032-2037 ◽  
Author(s):  
Puneet Khandelwal ◽  
Nirupama Banerjee-Bhatnagar
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Insecticidal Activity ◽  
Culture Supernatant ◽  
Outer Membrane Proteins ◽  
Membrane Vesicles ◽  
Molecular Complexes ◽  
Soluble Proteins ◽  
Outer Membrane Vesicles ◽  
Xenorhabdus Nematophilus

ABSTRACT Xenorhabdus nematophilus secretes a large number of proteins into the culture supernatant as soluble proteins and also as large molecular complexes associated with the outer membrane. Transmission electron micrographs of X. nematophilus cells showed that there was blebbing of the outer membrane from the surface of the bacterium. The naturally secreted outer membrane vesicles (OMVs) were purified from the culture supernatant of X. nematophilus and analyzed. Electron microscopy revealed a vesicular organization of the large molecular complexes, whose diameters varied from 20 to 100 nm. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of the vesicles showed that in addition to outer membrane proteins, several other polypeptides were also present. The membrane vesicles contained lipopolysaccharide, which appeared to be of the smooth type. Live cells of X. nematophilus and the OMV proteins derived from them exhibited oral insecticidal activity against neonatal larvae of Helicoverpa armigera. The proteins present in the OMVs are apparently responsible for the biological activity of the OMVs. The soluble proteins left after removal of the OMVs and the outer membrane proteins also showed low levels of oral toxicity to H. armigera neonatal larvae. The OMV protein preparations were cytotoxic to Sf-21 cells in an in vitro assay. The OMV proteins showed chitinase activity. This is the first report showing toxicity of outer membrane blebs secreted by the insect pathogen X. nematophilus into the extracellular medium.

scholarly journals Proteomic and Metabolomic Analyses of Xylella fastidiosa OMV-Enriched Fractions Reveal Association with Virulence Factors and Signaling Molecules of the DSF Family

2019 ◽  
Vol 109 (8) ◽  
pp. 1344-1353 ◽  
Author(s):  
Oséias R. Feitosa-Junior ◽  
Eliezer Stefanello ◽  
Paulo A. Zaini ◽  
Rafael Nascimento ◽  
Paulo M. Pierry ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Xylella Fastidiosa ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Bacterial Attachment ◽  
Intercellular Signaling ◽  
Citrus Variegated Chlorosis ◽  
Systemic Dissemination ◽  
First Time

Xylella fastidiosa releases outer membrane vesicles (OMVs) known to play a role in the systemic dissemination of this pathogen. OMVs inhibit bacterial attachment to xylem wall and traffic lipases/esterases that act on the degradation of plant cell wall. Here, we extended the characterization of X. fastidiosa OMVs by identifying proteins and metabolites potentially associated with OMVs produced by Temecula1, a Pierce’s disease strain, and by 9a5c and Fb7, two citrus variegated chlorosis strains. These results strengthen that one of the OMVs multiple functions is to carry determinants of virulence, such as lipases/esterases, adhesins, proteases, porins, and a pectin lyase-like protein. For the first time, we show that the two citrus variegated chlorosis strains produce X. fastidiosa diffusible signaling factor 2 (DSF2) and citrus variegated chlorosis-DSF (likewise, Temecula1) and most importantly, that these compounds of the DSF (X. fastidiosa DSF) family are associated with OMV-enriched fractions. Altogether, our findings widen the potential functions of X. fastidiosa OMVs in intercellular signaling and host–pathogen interactions.

scholarly journals Differential Susceptibility of Xylella fastidiosa Strains to Synthetic Bactericidal Peptides

2020 ◽  
Vol 110 (5) ◽  
pp. 1018-1026
Author(s):  
Aina Baró ◽  
Isabel Mora ◽  
Laura Montesinos ◽  
Emilio Montesinos
Keyword(s):  
Xylella Fastidiosa ◽  
Differential Susceptibility ◽  
Membrane Vesicles ◽  
Bactericidal Effect ◽  
Outer Membrane Vesicles ◽  
Bacterial Cells ◽  
Cell Inactivation ◽  
Threshold Ratio ◽  
Nonculturable State ◽  
Synthetic Antimicrobial Peptides

The kinetics of cell inactivation and the susceptibility of Xylella fastidiosa subspecies fastidiosa, multiplex, and pauca to synthetic antimicrobial peptides from two libraries (CECMEL11 and CYCLO10) were studied. The bactericidal effect was dependent on the relative concentrations of peptide and bacterial cells, and was influenced by the diluent, either buffer or sap. The most bactericidal and lytic peptide was BP178, an enlarged derivative of the amphipathic cationic linear undecapeptide BP100. The maximum reduction in survivors after BP178 treatment occurred within the first 10 to 20 min of contact and at micromolar concentrations (<10 μM), resulting in pore formation in cell membranes, abundant production of outer membrane vesicles, and lysis. A threshold ratio of 109 molecules of peptide per bacterial cell was estimated to be necessary to initiate cell inactivation. There was a differential susceptibility to BP178 among strains, with DD1 being the most resistant and CFBP 8173 the most susceptible. Moreover, strains showed a proportion of cells under the viable but nonculturable state, which was highly variable among strains. These findings may have implications for managing the diseases caused by X. fastidiosa.

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