Determination of Keto-deoxy-d-manno-8-octanoic acid (KDO) from Lipopolysaccharide of Escherichia coli

Sunayana, M. R.; Reddy, Manjula

doi:10.21769/bioprotoc.1688

Cited by 5 publications

(4 citation statements)

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“…For cell breakage, the Precellys tissue homogenizer and the Precellys glass kit 0.1 mm (Peqlab Biotechnologie GmbH, Erlangen, Germany) were used instead of a French pressure cell. The yield of LPS obtained was quantified by the amount of 2-keto-3-deoxyoctonate (KDO) recovered relative to the cell dry weight . KDO purchased from Sigma was used for a standard curve.…”

Section: Methodsmentioning

confidence: 99%

“…The yield of LPS obtained was quantified by the amount of 2-keto-3-deoxyoctonate (KDO) recovered relative to the cell dry weight. 47 KDO purchased from Sigma was used for a standard curve. For visualization, 5 μg of LPS was loaded on SDS-polyacrylamide gel electrophoresis (PAGE) (15% resolving gel, 4% stacking gel) and stained using the sensitive silver staining method of Tsai and Frasch.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

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Opposing Effects of PhoPQ and PmrAB on the Properties of Salmonella enterica serovar Typhimurium: Implications on Resistance to Antimicrobial Peptides

et al. 2021

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The increasing number of resistant bacteria is a major threat worldwide, leading to the search for new antibiotic agents. One of the leading strategies is the use of antimicrobial peptides (AMPs), cationic and hydrophobic innate immune defense peptides. A major target of AMPs is the bacterial membrane. Notably, accumulating data suggest that AMPs can activate the twocomponent systems (TCSs) of Gram-negative bacteria. These include PhoP-PhoQ (PhoPQ) and PmrA-PmrB (PmrAB), responsible for remodeling of the bacterial cell surface. To better understand this mechanism, we utilized bacteria deficient either in one system alone or in both and biophysical tools including fluorescence spectroscopy, single-cell atomic force microscopy, electron microscopy, and mass spectrometry (

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Section: Methodsmentioning

confidence: 99%

Section: ■ Materials and Methodsmentioning

confidence: 99%

Opposing Effects of PhoPQ and PmrAB on the Properties of Salmonella enterica serovar Typhimurium: Implications on Resistance to Antimicrobial Peptides

et al. 2021

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“…Kdo content was quantified in isolated OMs as previously described [53] with slight modifications. Briefly, 50 µL of OM sample was mixed with 50 µL of 0.25 M H 2 SO 4 and boiled for 20 min.…”

Section: Kdo Assaymentioning

confidence: 99%

Biosynthesis of the Inner Core of Bordetella pertussis Lipopolysaccharides: Effect of Mutations on LPS Structure, Cell Division, and Toll-like Receptor 4 Activation

Pérez-Ortega,

van Boxtel,

Plisnier

et al. 2023

IJMS

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Previously developed whole-cell vaccines against Bordetella pertussis, the causative agent of whooping cough, appeared to be too reactogenic due to their endotoxin content. Reduction in endotoxicity can generally be achieved through structural modifications in the lipid A moiety of lipopolysaccharides (LPS). In this study, we found that dephosphorylation of lipid A in B. pertussis through the heterologous production of the phosphatase LpxE from Francisella novicida did, unexpectedly, not affect Toll-like receptor 4 (TLR4)-stimulating activity. We then focused on the inner core of LPS, whose synthesis has so far not been studied in B. pertussis. The kdtA and kdkA genes, responsible for the incorporation of a single 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residue in the inner core and its phosphorylation, respectively, appeared to be essential. However, the Kdo-bound phosphate could be replaced by a second Kdo after the heterologous production of Escherichia coli kdtA. This structural change in the inner core affected outer-core and lipid A structures and also bacterial physiology, as reflected in cell filamentation and a switch in virulence phase. Furthermore, the eptB gene responsible for the non-stoichiometric substitution of Kdo-bound phosphate with phosphoethanolamine was identified and inactivated. Interestingly, the constructed inner-core modifications affected TLR4-stimulating activity. Whereas endotoxicity studies generally focus on the lipid A moiety, our data demonstrate that structural changes in the inner core can also affect TLR4-stimulating activity.

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“…The OMVs from these three conditions were selected for further characterization in terms of protein profiling by SDS-PAGE (Sambrook and Russel, 2001) as recommended by Klimentová and Stulík, (2015) and lipopolysaccharide concentration and zeta size. The estimation of the lipopolysaccharide (LPS) content of OMVs was done by Kdo method (Sunayana and Reddy, 2015). The zeta size of the outer membrane vesicles were measured using the dyanamic light scattering in Institute of Advanced Study in Science and Technology, Guwahati, Assam.…”

Section: Culture and Isolation Of Outer Membrane Vesiclesmentioning

confidence: 99%

Optimization of Cultural Conditions for Maximum Production of Outer Membrane Vesicles from Salmonella Typhimurium

Solo

Tamuly

Barkalita

et al. 2021

IJAR

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Background: The non-typhoidal Salmonella causes gastroenteritis in humans that makes its way to the food chain mainly through the animal products. The multiple drug resistance imposes one of the major hurdle in the treatment of the disease. The vaccination appears to be the most important method for prevention of the disease. Unfortunately, there is no liscenced vaccine available against non-typhoidal Salmonellae. The use of outer membrane vesicles (OMVs) of Salmonella as a vaccine candidate has attained significant centre-stage in the recent years given to its protective immunogenicity. However, the large scale production of OMVs is difficult owing to low yield per liter of culture. Methods: In the present study, we have optimized the culture conditions viz. pH, phase of growth and presence of oxidative stress for maximum production of OMVs from Salmonella Typhimurium. The OMVs were characterized based on yield based on protein concentration, lipopolysaccharide concentration and zeta size. Result: In the present study, it was found that incubation of Salmonella Typhimurium up to peak of the growth phase at pH 7 in presence of oxidative stress was found to be the most suitable condition for maximum production of OMVs.

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Determination of Keto-deoxy-d-manno-8-octanoic acid (KDO) from Lipopolysaccharide of Escherichia coli

Cited by 5 publications

References 0 publications

Opposing Effects of PhoPQ and PmrAB on the Properties of Salmonella enterica serovar Typhimurium: Implications on Resistance to Antimicrobial Peptides

Opposing Effects of PhoPQ and PmrAB on the Properties of Salmonella enterica serovar Typhimurium: Implications on Resistance to Antimicrobial Peptides

Biosynthesis of the Inner Core of Bordetella pertussis Lipopolysaccharides: Effect of Mutations on LPS Structure, Cell Division, and Toll-like Receptor 4 Activation

Optimization of Cultural Conditions for Maximum Production of Outer Membrane Vesicles from Salmonella Typhimurium

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Determination of Keto-deoxy-d-manno-8-octanoic acid (KDO) from Lipopolysaccharide of Escherichia coli

Cited by 5 publications

References 0 publications

Opposing Effects of PhoPQ and PmrAB on the Properties of Salmonella enterica serovar Typhimurium: Implications on Resistance to Antimicrobial Peptides

Opposing Effects of PhoPQ and PmrAB on the Properties of Salmonella enterica serovar Typhimurium: Implications on Resistance to Antimicrobial Peptides

Biosynthesis of the Inner Core of Bordetella pertussis Lipopolysaccharides: Effect of Mutations on LPS Structure, Cell Division, and Toll-like Receptor 4 Activation

​Optimization of Cultural Conditions for Maximum Production of Outer Membrane Vesicles from Salmonella Typhimurium

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Product

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Optimization of Cultural Conditions for Maximum Production of Outer Membrane Vesicles from Salmonella Typhimurium