Identification and partial characterization of a novel bipartite protein antigen associated with the outer membrane of Escherichia coli

Abstract

A study by crossed immunoelectrophoresis performed in conjunction with precipitate excision and polypeptide analysis identified a new antigen complex in the envelope of Escherichia coli ML308-225. This antigen corresponds to antigen 43 in the crossed immunoelectrophoresis profile of membrane vesicles (P. Owen and H. R. Kaback, Proc. Natl. Acad. Sci. USA 75:3148-3152, 1978). Immunoprecipitation experiments conducted with specific antiserum revealed that the complex was expressed on the cell surface and that it contained, in equal stoichiometry, two chemically distinct polypeptides termed a and (Mrs of 60,000 and 53,000, respectively). The polypeptide was heat modifiable, displaying an apparent Mr of 37,000 when solubilized at temperatures below 70°C. Analysis of fractions obtained following cell disruption, isopycnic centrifugation, and detergent extraction indicated that both a and polypeptides were components of the outer membrane. The two polypeptides were not linked by disulfide bonds, and neither was peptidoglycan associated. The complex contained no detectable lipopolysaccharide, enzyme activity, fatty acyl groups, or other cofactors. Neither correlated with E. coli proteins of similar molecular weight which had previously been shown to be associated with the outer membrane. Antibodies were raised to individual a and I1 polypeptides. Each of these sera was shown to be subunit specific when tested against denatured membrane proteins. In contrast, each immunoglobulin preparation coprecipitated both a and polypeptides when tested against undenatured proteins derived from Triton X-100-treated membranes. The results reveal the presence of a novel bipartite protein antigen in the outer membrane of E. coli.The outer membrane of Escherichia coli is known to contain a number of major protein species (1, 15). Several of these are porins involved in the passive accumulation of small hydrophilic solutes (2,19). Others, such as the ironregulated outer membrane proteins and the vitamin B12 receptor, are involved in the TonB-dependent uptake of more specific solutes (4). Other major proteins of the E. coli outer membrane include the Braun lipoprotein (the Lpp protein), peptidoglycan-associated lipoprotein, and the ompA gene product. In part at least, these appear to play a structural role for the cell envelope (1, 15). Several other minor proteins and enzymes have also been identified and studied (10,17,20,35). In addition to the above components, there exist in the outer membratne several additional proteins about which considerably less is known, e.g., protein III, an 83-kilodalton iron-regulated protein, and several proteins in the 60-to 40-kilodalton range (15,26).In an attempt to identify and characterize the major antigens of the cell envelope of aerobically grown E. coli, a crossed immunoelectrophoresis (CIE) reference profile has been established in which over half of the 50 or so resolved antigens have been identified in functional terms or partially characterized (for reviews, see references 21 to 23).

Type V Protein Secretion Pathway: the Autotransporter Story

“…As such, antigen 43 was one of the first autotransporters identified, however, it was not until much later that its mode of biogenesis was uncovered (204). In the intervening period, much time was invested in the biochemical characterization of this protein; Owen and coworkers (54,370) demonstrated that antigen 43 existed as a bipartite complex of two proteins (termed ␣ 43 and ␤ 43 ) which could be resolved under denaturing conditions. These subunit proteins were found to exist in a stoichiometric fashion at a ratio of 1:1, and the interaction between the two proteins was noncovalent.…”

Purification and N-terminal sequence of the alpha subunit of antigen 43, a unique protein complex associated with the outer membrane of Escherichia coli

“…The complex differs from the well-characterized proteins of the outer membrane of E. coli in that it consists of two different subunits (31,41). In addition, the antigen is surface exposed, and its yield varies in a manner which appears to be independent of external growth conditions.…”

“…The polypeptide is heat modifiable and shows an apparent Mr of 37,000 when solubilized at temperatures lower than 70°C before analysis by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Analysis by CIE performed in conjunction with zymogram staining and radiolabeling failed to detect the presence of enzyme activity, fatty acyl groups, or other cofactors for the complex (41).…”

“…Purified outer membranes were isolated from envelopes that had been prepared from spheroplasts generated by the method of Witholt et al (53). The inner and outer membranes were separated by isopycnic centrifugation on 30 to 55% (wt/wt) sucrose gradients and were identified by analyzing the resultant fractions on SDS-polyacrylamide gels (7,41).…”

The cell surface protein Ag43 facilitates phage infection of Escherichia coli in the presence of bile salts and carbohydrates

“…Ag43 is composed of two subunits, α and β, which are encoded by a single gene, agn43 (formerly called flu) in E. coli K-12, M. Gabig and others and maturation requires removal of the N-terminal signal peptide and proteolytic cleavage of the remaining polypeptide (Caffrey & Owen, 1989 ;Henderson et al, 1997). The 43 kDa α subunit is surface-expressed and is attached to the cell through an interaction between its C-terminal domain and the 43 kDa β subunit, an integral outer-membrane protein (Caffrey & Owen, 1989 ;Owen et al, 1987). Presentation of Ag43 on the cell surface is responsible for the so-called ' frizzy ' or form 1 phenotype (Diderichsen, 1980 ;Henderson et al, 1997 ;Warne et al, 1990).…”