Identification of seven surface-exposed Brucella outer membrane proteins by use of monoclonal antibodies: immunogold labeling for electron microscopy and enzyme-linked immunosorbent assay

Abstract

A panel of monoclonal antibodies (MAbs) to seven Brucella outer membrane proteins were characterized. These antibodies were obtained by immunizing mice with sodium dodecyl sulfate-insoluble (SDS-I) fractions, cell walls, or whole bacterial cells of Brucella abortus or B. melitensis. Enzyme-linked immunosorbent assays were used to screen the hybridoma supernatants and to determine their binding at the surface of rough and smooth B. abortus and B. melitensis cells. The outer membrane proteins (OMPs) recognized by these antibodies were the proteins with molecular masses of 25 to 27 kDa and 36 to 38 kDa (porin) (major proteins) and the proteins with molecular masses of 10, 16.5, 19, 31 to 34, and 89 kDa (minor proteins). Surface exposure of these OMPs was visualized by electron microscopy by using the MAbs and immunogold labeling. Binding of the MAbs on whole rough bacterial cells indicates that the 10-, 16.5-, 19-, 25to 27-, 31to 34-, 36to 38-, and 89-kDa OMPs are exposed at the cell surface. However, enzyme-linked immunosorbent assay results indicate a much better binding of the anti-OMP MAbs on rough strains than on the corresponding smooth strains except for the anti-19-kDa MAb. Immunoelectron microscopy showed that on smooth B. abortus cells only the 89-and 31to 34-kDa OMPs were not accessible to the MAbs tested. Binding of the anti-31to 34-kDa MAb at the cell surface was observed for the rough B. abortus cells and for the rough and smooth B. melitensis cells. These results indicate the importance of steric hindrance due to the presence of the long lipopolysaccharide 0 side chains in the accessibility of OMPs on smooth Brucella strains and should be considered when undertaking vaccine development.

Major outer membrane proteins of Vibrio cholerae and their role in induction of protective immunity through inhibition of intestinal colonization

“…Thus, it is tempting to speculate that the 40to 43-and 20-kDa proteins are relatively more accessible to antibodies than the other three MOMPs (48 to 50, 35 to 36, and 27 to 28 kDa). This is also consistent with the idea that gram-negative bacteria, including V. cholerae, have fewer surface-exposed proteins (5,36,49), probably as a result of a shielding effect of the long LPS 0 side chain. Interestingly, all the anti-MOMP sera showed comparable ELISA titers against in vivo-and in vitro-grown cells of V. cholerae, thereby suggesting no marked differences in the expression or surface exposition of MOMPs in V. cholerae cells grown in vitro and in vivo.…”

Localized incorporation of outer membrane components in the pathogen Brucella abortus

“…During the analysis of the localization of OM components by IF, Omp25, one of the major Omp of B. abortus (Dubray & Bezard, 1980), was found to have a homogenous distribution [83.7 AE 8.2% (mean AE SD), n = 479 bacteria, five biological replicates] on the cell surface ( Fig 4A, panel 0 h), suggesting that it is continuously produced during the cell cycle like previously reported (Francis et al, 2017). The specificity of the anti-Omp25 mAb (Cloeckaert et al, 1990) was additionally confirmed by IF and Western blot with A Roughness measurements on R-LPS labeled (green) WT and Dgmd cells. AFM images of whole bacteria and of the separated areas (colored squares in first images, 0.4 × 0.4 lm 2 ) are shown.…”

“…This was also found in the rough strain Dgmd, suggesting that the patchy labeling is not an artifact due to the steric hindrance of mAb binding to Omp2b by S-LPS. Labeling with another mAb (A68/25G05/A05) also directed against Omp2b (Cloeckaert et al, 1990) showed the same heterogeneity in localization. We thus wondered whether Omp2b and R-LPS could be co-localized.…”

A quorum-sensing regulator controls expression of both the type IV secretion system and the flagellar apparatus of Brucella melitensis

“…After standardizing for total protein content, samples were subjected to electrophoresis on 12% SDS‐PAGE and transferred to Hybond‐C nitrocellulose membranes (Amersham Pharmacia Biotech) by the semi‐dry transfer technique. Immunodetection of proteins was performed with polyclonal anti‐VirB8 (Rouot et al ., 2003) or anti‐FlgE (Fretin et al ., 2005) antisera (diluted 2500 times) and with monoclonal anti‐Omp1 antibody A53 10B2 (Cloeckaert et al ., 1990) (diluted 1000 times) as a loading control. Bound antibodies were detected using chemiluminescence with peroxydase‐conjugated secondary antibodies and the ECL Western blotting reagents RPN2209 as recommended by the manufacturer (Amersham Pharmacia Biotech).…”