An improved silver stain for the visualization of lipopolysaccharides on polyacrylamide gels

Zhu, Zhongxin; Cong, Weitao; Ni, Maowei; Wang, Xi; Ma, Weide; Ye, Wei-Jian; Jin, Litai; Li, Xiaokun

doi:10.1002/elps.201100492

Cited by 22 publications

(20 citation statements)

References 13 publications

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“…SDS‐PAGE was performed with 15% acrylamide gels and the peqGOLD pre‐stained Protein Marker V (PEQLAB Biotechnologie GmbH, Ehrlangen, Germany) was used as molecular mass standard. Silver staining of the gels was performed as described previously (Zhu et al ., ).…”

Section: Methodsmentioning

confidence: 97%

Specific surface glycan decorations enable antimicrobial peptide resistance in plant‐beneficial pseudomonads with insect‐pathogenic properties

Kupferschmied

Chai

Flury

et al. 2016

Environmental Microbiology

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Summary Some plant‐beneficial pseudomonads can invade and kill pest insects in addition to their ability to protect plants from phytopathogens. We explored the genetic basis of O‐polysaccharide (O‐PS, O‐antigen) biosynthesis in the representative insecticidal strains Pseudomonas protegens CHA0 and Pseudomonas chlororaphis PCL1391 and investigated its role in insect pathogenicity. Both strains produce two distinct forms of O‐PS, but differ in the organization of their O‐PS biosynthesis clusters. Biosynthesis of the dominant O‐PS in both strains depends on a gene cluster similar to the O‐specific antigen (OSA) cluster of Pseudomonas aeruginosa. In CHA0 and other P. protegens strains, the OSA cluster is extensively reduced and new clusters were acquired, resulting in high diversity of O‐PS structures, possibly reflecting adaptation to different hosts. CHA0 mutants lacking the short OSA form of O‐PS were significantly impaired in insect virulence in Galleria injection and Plutella feeding assays. CHA0, PCL1391, and other insecticidal pseudomonads exhibited high resistance to antimicrobial peptides, including cecropins that are central to insect immune defense. Resistance of both model strains depended on the dominant OSA‐type O‐PS. Our results suggest that O‐antigen is essential for successful insect infection and illustrate, for the first time, its importance in resistance of Pseudomonas to antimicrobial peptides.

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Section: Methodsmentioning

confidence: 97%

Specific surface glycan decorations enable antimicrobial peptide resistance in plant‐beneficial pseudomonads with insect‐pathogenic properties

Kupferschmied

Chai

Flury

et al. 2016

Environmental Microbiology

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“…Subsurface twitching motility tests were performed as described [23]. LPS was extracted using the iNTRON LPS extraction kit, resolved on NuPAGE 4%–12% BisTris gels, and silver stained as previously described [24]. Details are provided in the Supplementary Materials.…”

Section: Methodsmentioning

confidence: 99%

Synergistic interaction between phage therapy and antibiotics clears Pseudomonas aeruginosa infection in endocarditis and reduces virulence

Oechslin

Piccardi

Mancini

et al. 2016

INFDIS

241

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Background.Increasing antibiotic resistance warrants therapeutic alternatives. Here we investigated the efficacy of bacteriophage-therapy (phage) alone or combined with antibiotics against experimental endocarditis (EE) due to Pseudomonas aeruginosa, an archetype of difficult-to-treat infection.Methods.In vitro fibrin clots and rats with aortic EE were treated with an antipseudomonas phage cocktail alone or combined with ciprofloxacin. Phage pharmacology, therapeutic efficacy, and resistance were determined.Results.In vitro, single-dose phage therapy killed 7 log colony-forming units (CFUs)/g of fibrin clots in 6 hours. Phage-resistant mutants regrew after 24 hours but were prevented by combination with ciprofloxacin (2.5 × minimum inhibitory concentration). In vivo, single-dose phage therapy killed 2.5 log CFUs/g of vegetations in 6 hours (P < .001 vs untreated controls) and was comparable with ciprofloxacin monotherapy. Moreover, phage/ciprofloxacin combinations were highly synergistic, killing >6 log CFUs/g of vegetations in 6 hours and successfully treating 64% (n = 7/11) of rats. Phage-resistant mutants emerged in vitro but not in vivo, most likely because resistant mutations affected bacterial surface determinants important for infectivity (eg, the pilT and galU genes involved in pilus motility and LPS formation).Conclusions.Single-dose phage therapy was active against P. aeruginosa EE and highly synergistic with ciprofloxacin. Phage-resistant mutants had impaired infectivity. Phage-therapy alone or combined with antibiotics merits further clinical consideration.

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“…Visualization was achieved by SDS‐PAGE (12%) resolution of 20 μg purified LPS and consequent staining by the improved silver stain protocol described by Zhu et al . ().…”

Section: Methodsmentioning

confidence: 97%

“…Purified LPS, without any residual phenol, were lyophilized and its dry weight was determined, and then it was resuspended in PBS to a final concentration of 2 mg mL À1 and kept at À20°C until used. Visualization was achieved by SDS-PAGE (12%) resolution of 20 lg purified LPS and consequent staining by the improved silver stain protocol described by Zhu et al (2012).…”

Section: Bacterial Culture Optimizationmentioning

confidence: 99%

Tenacibaculosis induction in the Senegalese sole (Solea senegalensis) and studies of Tenacibaculum maritimum survival against host mucus and plasma

Mabrok

Machado

Serra

et al. 2016

Journal of Fish Diseases

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Tenacibaculum maritimum, the aetiological agent for marine tenacibaculosis, is one of the most significant pathogens that threaten Senegalese sole, Solea senegalensis (Kaup), aquaculture. Because no immersion challenge with T. maritimum has been reported previously for this flatfish species, this study aimed to optimize bacterial yields as well as to establish a challenge model for tenacibaculosis induction. Several approaches were performed to optimize bacterial culture conditions, including treatment with non-ionic surfactants, detergents, cellulase hydrolysis and strong shaking. A prolonged bath challenge was performed for 24 h under two different temperatures, 16 and 23 °C. Moreover, mucus and plasma bactericidal activities against T. maritimum were also assessed. Culturing bacteria with strong shaking and continuous shaking provided suitable culture conditions to obtain higher bacterial yields without aggregation and fluctuation, contrary to most other treatments that showed a huge amount of bacterial aggregates. A prolonged bath method for 24 h, without skin or gill scarification, was considered suitable for disease induction with high mortality rates. Moreover, data regarding mucus and plasma bactericidal activities suggested that there is a lack of host innate immune response against T. maritimum or that this particular pathogen presents evading strategies against Senegalese sole.

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An improved silver stain for the visualization of lipopolysaccharides on polyacrylamide gels

Cited by 22 publications

References 13 publications

Specific surface glycan decorations enable antimicrobial peptide resistance in plant‐beneficial pseudomonads with insect‐pathogenic properties

Specific surface glycan decorations enable antimicrobial peptide resistance in plant‐beneficial pseudomonads with insect‐pathogenic properties

Synergistic interaction between phage therapy and antibiotics clears Pseudomonas aeruginosa infection in endocarditis and reduces virulence

Tenacibaculosis induction in the Senegalese sole (Solea senegalensis) and studies of Tenacibaculum maritimum survival against host mucus and plasma

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