Outer membrane proteins of Brucella abortus: isolation and characterization

Abstract

Outer membrane proteins were derived from one rough and four smooth strains of Brucella abortus by sequential extraction of physically disrupted cells with N-lauroylsarcosinate and dipolar ionic detergent. Extraction of outer membrane proteins was ineffective, however, without predigestion with lysozyme. Three groups of proteins were present and could be separated in their native state by sequential anion-exchange chromatography and gel filtration. Membrane proteins contained substantial quantities of tightly adherent lipopolysaccharide which could be reduced but not eliminated by extraction of cells with trichloroacetic acid before disruption. Group 2 proteins, apparently trimers in their native state, gave rise to 43,000- and 41,000-molecular-weight bands after complete denaturation in sodium dodecyl sulfate. They were antigenically identical among all the strains, showed close resemblance in amino acid composition to each other and a general similarity to OmpF of Escherichia coli, and are proposed to be the porins of B. abortus. Group 3 proteins occurred as 30,000-molecular-weight bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, although additional bands were frequently observed in this region. In none of the strains did group 3 proteins manifest heat-modifiable characteristics. Proteins of different strains bore a high degree of similarity to each other in amino acid composition, except in methionine, isoleucine, tyrosine, and histidine. Differences occurred consistently in amino acid composition between group 2 and 3 proteins, and some of these correspond to differences between OmpF and OmpA. Group 2 and 3 proteins were antigenically distinct from each other, but the principal group 3 antigens were shared among all the strains. Despite the lack of heat modifiability, perhaps influenced by adherent lipopolysaccharide, group 3 proteins are proposed as counterparts to OmpA. Most of the group 1 proteins, minor components, were physically associated with those of group 3 unless in sodium dodecyl sulfate. Group 1 proteins produced a major band at 94,000 and exhibited heat modifiability. No evidence was found of a low-molecular-weight lipoprotein in the outer membrane of B. abortus, but this is not taken to exclude its occurrence.

Brucella group 3 outer membrane proteins contain a heat-modifiable protein

“…The upshifl in apparent molecular mass of OmpA is due to the unusually high 3-structure content of this protein that makes the native OmpA bind amounts of SDS larger than those bound by the SDS-denatured form [13]. Due to the overall similarity in amino acid composition between Brucella group 3 and OmpA [2], this same explanation can be advanced for the heat modifiability of the polypeptide present in Brucella OM blebs. This is also supported by the cross-reactivity detected by immunoblot between OmpA and the Brucella OM blebs protein.…”

“…It has been shown by sequential Sarkosyl-Zwittergent extraction that the outer membrane (OM) of Brucella contains three major groups of OM proteins, each one appearing in SDS-PAGE as a cluster of polypeptides of close apparent molecular mass [1,2]. The characterization of the Brucella OM proteins of groups 2 and 3 has been achieved in part.…”

“…The characterization of the Brucella OM proteins of groups 2 and 3 has been achieved in part. Group 2 has been shown to have porin activity [3], and the aminoacid composition of the cluster of polypeptides included in group 3 [2] suggests that they could be the Bru-Correspondence to: C. Gamazo, Departamento de Microbiologla, Facultad de Medicina, Universidad de Navarra, Aptdo. 273, 31080, Pamplona, Spain.…”

Immunoblot studies in the differential diagnosis of porcine brucellosis: an immunodominant 62 kDa protein is related to the mycobacterial 65 kDa heat shock protein (HSP-65)

“…This problem was resolved by using a rough strain of B. melitensis, a strategy previously used by others . This was adopted in view of the high DNA relatedness of all Brucella species (Verger et al, 1985 ) ; the antigenic similarity of proteins of the different species ofBrucella (Diaz et al, 1967;Berman et al, 1975;Verstreate et al, 1982;Santos et al, 1984;Holman et al, 1985;and Gamazo et al, 1989); and improved sensitivity of B. melitensis outer membrane proteins in the detection of human infection by all species of Brucella (Hunter et al, 1986).…”

Urban outbreak of a Brucella melitensis infection in an Argentine family: Clinical and diagnostic aspects

“…After washing 3 times with PBS, the plates were blocked by 1 h incubation with 200 Fzl/well of a solution of PBS containing 3% skim milk. After blocking, the plates were washed with PBS-Tween 20 0.05% (PBS-T) and incubated with 10 txg/well of the cytoplasmic fraction of Brucella abortus (CYT) which was prepared according to the protocol of Verstreate et al [11]. After incubating for 1 h at room temperature, the plates were washed with PBS-T and serial dilutions of the sera under study were dispensed.…”