Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Lesse, Alan J.; Campagnari, Anthony A.; Bittner, William E.; Apicella, Michael A.

doi:10.1016/0022-1759(90)90018-q

Cited by 317 publications

(235 citation statements)

References 28 publications

Supporting

Mentioning

234

Contrasting

Order By: Relevance

“…LPS were electrophoresed in 7 cm 18% polyacrylamide (PAGE) gels in SDS Tris-HCl-glycine [1,14]. Alternatively, LPS were analyzed in 16 cm 18% PAGE gels in Tris-Tricine-HCl-glycine (TSDS-PAGE) [2,15]. For Western blots, gels were electro transferred onto nitrocellulose sheets (Whatman, Dassel, Germany), blocked with 3% skim milk in PBS with 0.05% Tween 20 (PBST) overnight, and washed with the same buffer.…”

Section: Lps Extraction and Characterizationmentioning

confidence: 99%

The identification of wadB, a new glycosyltransferase gene, confirms the branched structure and the role in virulence of the lipopolysaccharide core of Brucella abortus

Gil-Ramírez

Conde-Álvarez

Palacios-Chaves

et al. 2014

Microbial Pathogenesis

View full text Add to dashboard Cite

Brucellosis is a worldwide extended zoonosis caused by Brucella spp. These gram-negative bacteria are not readily detected by innate immunity, a virulence-related property largely linked to their surface lipopolysaccharide (LPS). The role of the LPS lipid A and O-polysaccharide in virulence is well known. Moreover, mutation of the glycosyltransferase gene wadC of B. abortus, although not affecting O-polysaccharide assembly onto the lipid-A core section causes a core oligosaccharide defect that increases recognition by innate immunity. Here, we report on a second gene (wadB) encoding a LPS core glycosyltransferase not involved in the assembly of the O-polysaccharide-linked core section. As compared to wild-type B. abortus, a wadB mutant was sensitive to bactericidal peptides and non-immune serum, and was attenuated in mice and dendritic cells. These observations show that as WadC, WadB is also involved in the assembly of a branch of Brucella LPS core and support the concept that this LPS section is a virulence-related structure.4

show abstract

Section: Lps Extraction and Characterizationmentioning

confidence: 99%

The identification of wadB, a new glycosyltransferase gene, confirms the branched structure and the role in virulence of the lipopolysaccharide core of Brucella abortus

Gil-Ramírez

Conde-Álvarez

Palacios-Chaves

et al. 2014

Microbial Pathogenesis

View full text Add to dashboard Cite

show abstract

“…The LPS was pelleted by centrifugation for 3 h at 150,000 ϫ g and dissolved in distilled water, after which the LPS concentration was determined by analyzing the 3OH-C 14 content, using an Agilent 6890 gas chromatograph, as described previously (62). Approximately 250 pmol purified LPS was applied to a Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) gel (30). The electrophoresis was run for 17 h at a constant current of 20 mA, after which the gel was fixed with water-ethanol-acetic acid (11:8:1, vol/vol/vol) and subsequently stained with silver as described previously (60).…”

Section: Vol 74 2006 Modulation Of B Pertussis Endotoxic Activity mentioning

confidence: 99%

Expression of the Lipopolysaccharide-Modifying Enzymes PagP and PagL Modulates the Endotoxic Activity ofBordetella pertussis

Geurtsen¹,

Steeghs

Hamstra³

et al. 2006

Infect Immun

View full text Add to dashboard Cite

Lipopolysaccharide (LPS) is one of the major constituents of the gram-negative bacterial cell envelope. Its endotoxic activity causes the relatively high reactogenicity of whole-cell vaccines. Several bacteria harbor LPS-modifying enzymes that modulate the endotoxic activity of the LPS. Here we evaluated whether two such enzymes, i.e., PagP and PagL, could be useful tools for the development of an improved and less reactogenic whole-cell pertussis vaccine. We showed that expression of PagP and PagL in Bordetella pertussis leads to increased and decreased endotoxic activity of the LPS, respectively. As expected, PagP activity also resulted in increased endotoxic activity of whole bacterial cells. However, more unexpectedly, this was also the case for PagL. This paradoxical result may be explained, in part, by an increased release of LPS, which we observed in the PagL-expressing cells.

show abstract

“…The lysates were fractionated by Tricine-sodium dodecyl sulphate polyacrylamide gel electrophoresis (T-SDS-PAGE) (Lesse et al, 1990) and LPS profiles were detected by staining with silver (Quicksilver; Amersham) (Roche et al, 1994). LPS was transferred to nitrocellulose membranes for Western analysis (Towbin et al, 1979) then incubated with mAb 4C4 as described by Weiser et al (1997).…”

Section: Strainmentioning

confidence: 99%

The role of lex2 in lipopolysaccharide biosynthesis in Haemophilus influenzae strains RM7004 and RM153

et al. 2003

View full text Add to dashboard Cite

The locus lex2, comprising lex2A and lex2B, contributes to the phase-variable expression of lipopolysaccharide (LPS) of Haemophilus influenzae and was found to be present in 74 % of strains investigated. lex2A contains 59-GCAA repeats which vary in number from 4 to 46 copies between strains. The locus was cloned from the serotype b strains RM7004 and RM153 and showed >99 % nucleotide sequence identity between these strains and the published lex2 sequence. Disruption of the lex2B gene in strain RM7004 resulted in truncation of some LPS glycoforms, shown by gel fractionation, with only one glycoform reacting with a digalactosidespecific monoclonal antibody, 4C4, compared with four LPS glycoforms in the more elongated LPS of the parent strain. Mass spectrometry and NMR analyses of LPS from the lex2B mutant revealed loss of the terminal digalactoside as well as the second b-glucose extending from the first heptose of the inner core. The authors conclude that Lex2B is the b-(1-4)-glucosyltransferase that adds the second b-glucose to the first b-glucose as part of the oligosaccharide extension from the first heptose of the LPS of strain RM7004. Investigation of the expression of the lex2 locus indicated that the genes are co-transcribed and that both reading frames are required for addition of this second b-glucose in a phase-variable manner.

show abstract

Increased resolution of lipopolysaccharides and lipooligosaccharides utilizing tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Cited by 317 publications

References 28 publications

The identification of wadB, a new glycosyltransferase gene, confirms the branched structure and the role in virulence of the lipopolysaccharide core of Brucella abortus

The identification of wadB, a new glycosyltransferase gene, confirms the branched structure and the role in virulence of the lipopolysaccharide core of Brucella abortus

Expression of the Lipopolysaccharide-Modifying Enzymes PagP and PagL Modulates the Endotoxic Activity ofBordetella pertussis

The role of lex2 in lipopolysaccharide biosynthesis in Haemophilus influenzae strains RM7004 and RM153

Contact Info

Product

Resources

About