Size, charge and structural heterogeneity of Brucella abortus lipopolysaccharides demonstrated by two‐dimensional gel electrophoresis

Abstract

Health 3Department of Veterinary Microbiology and Parasitology, Texas Agricultural Experiment Station Texas A&M University, College Station, TX Size, charge and structural heterogeneity of Brucella abortus lipopolysaccharides demonstrated by twodimensional gel electrophoresis Phenol extracted, alkali-treated lipopolysaccharide (aLPS)fromvaccine strain (S 19)Brucella abortus was demonstrated by two-dimensional gel electrophoresis to consist of at least ten silver staining, polydisperse analogues having different pZs. When tested on nitrocellulose immunoblots, all ten were antigenically reactive with bovine anti-B. abortus polyclonal sera, but only six reacted with anti-B. abortus 0-antigen murine monoclonal antibody. Analogues focusing at different pZs were concluded to arise from differences in either core or 0-antigen side chain structure or because of covalently bound protein. While not qualitatively different, aLPS from pathogenic B. abortus strain 2308 had lesser amounts of analogues 1,2,5,6, and 8 than did aLPS from strain 19 (vaccine). The 2-D gel electrophoresis method was demonstrated to be of value in the analysis of aLPS from B. abortus and may be useful in the study of lipopolysaccharides from other sources. Abbreviations: LPS, lipopolysaccharide; aLPS, alkali-treated lipopolysaccharide; SDS-PAGE, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis; IEF, isoelectric focusing; 2-D, two-dimensional; CBB, Coomassie Brilliant Blue R-250; NMR, nuclear magnetic resonance; ELISA, enzyme-linked immuno-sorbent assay: CFU, colony forming unit; HRPO, horseradish peroxidase; C/M, chargdmass ratio 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1986

A 36-kilodalton Brucella abortus cell envelope protein is encoded by repeated sequences closely linked in the genomic DNA

“…Following irradiation with 60Co, cell envelopes were prepared essentially as described by Sowa et al (54,55) with modifications described elsewhere (B. A.…”

“…3. This activity would appear as a heterogeneous mixture of bands of various molecular weights distributed along the entire length of the gel (54).…”

“…The major OMPs of B. abortus represent potential candidates as broad-range immunogens because of their conservation between strains (13,49,61,62) and their capacity to elicit both humoral and cell-mediated immune responses during natural infection (4, 50, 51;' L. G. Adams, unpublished results). However, examination of the protective immunity induced by B. abortus cell envelope (outer and cytoplasmic membranes) proteins is complicated by the presence of large amounts of immunogenic lipopolysaccharide (LPS) which contaminates these cell fractions (49,54,61,62). Recent vaccine trials have indicated that a substantial level of protective immunity against abortion and infection is stimulated by rough-cell envelopes lacking extensive LPS (Adams, unpublished).…”

Protection against Brucella abortus in mice with O-polysaccharide-specific monoclonal antibodies

“…Serumfree medium supernatants were concentrated to 10.0 mg of immunoglobulin per ml and isotyped with anti-mouse isotype-specific ELISA procedures (Table 1). In nitrocellulose electroblots of sodium dodecyl sulfate-polyacrylamide gels of a whole-cell extract of strain 2308 MAbs 144 and 145 reacted with a single band corresponding to the porin (24), whereas MAbs 34 and 47 reacted with the typical multiple bands of LPS (22), and MAb 135 failed to react with any antigen (Table 1). Inhibition tests were performed with purified 0 antigen (25), which had been established to be a perosamine homopolymer by nuclear magnetic resonance analysis in confirmation of a previous report (6), and purified porin electroeluted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, which migrated as a single band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in accordance with published reports (24).…”

Comprehensive Identification of Immunodominant Proteins of Brucella abortus and Brucella melitensis Using Antibodies in the Sera from Naturally Infected Hosts

“…, and Y. enterocolitica [ 7 , 21 , 31 ]. LPS has been shown to bind to sodium dodecyl sulfate (SDS), and may mimic protein spots in SDS polyacrylamide gels [ 32 , 33 ]. LPS closely associates with the protein and traces of LPS are expected to be present in the whole cell protein extract irrespective of the extraction method used.…”