Antigenic Relationships between Pore Proteins of Escherichia coli K12

Abstract

Antisera were raised against the purified Esckerichia coli K 12 outer membrane proteins ompA-, ompC-and ompF proteins and protein e. Several immunological methods were used to investigate the specificity of the antisera and the immunological relationship between the major outer membrane proteins. Although the antisera had been raised against highly purified proteins, several of them contained activity against lipopolysaccharide and lipoprotein due to minor impurities in the immunogens. The three general porins ompF protein, ompC protein and protein e were shown to be cross-reactive. Anti-(ompA protein) serum only reacted with the homologous protein. None of these antisera reacted with the phage lambda receptor protein or with protein 111. Pore protein preparations isolated from SaImonella typhimurium, Klebsiella aerogenes, Enterobacter cloaceae and Proteus mirabilis were found to be structurally related to the E. coli K12 porins as they reacted with the antisera raised against E. coli K12 porins.The outer membrane of Enterobacteriaceae contains phospholipids, which are mainly or exclusively present in the inner monolayer [1,2], lipopolysaccharide, which is exclusively located in the outer monolayer [3] and proteins [4-71, many of which transverse the lipid bilayer [8]. The most abundant protein in the outer membrane of Escherichia coli is the lipoprotein (7 x lo5 copies per cell) [9, lo]. Wildtype cells of E. coli K12 contain several other major proteins. For the proteins designated as b, c and d in our laboratory [5], other nomenclatures were also used [4,7,11], but it is now agreed that the proteins will be named according to the designation of their structural genes [12], which are ompF, ompC, and ompA respectively. Although these proteins are often called 'major outer membrane proteins', this term can be misleading, as the relative amounts of these proteins are dependent on the growth conditions [ 13 -181.The ompA protein has a function in the F-pilusmediated conjugation [19,20] and might play a role in the uptake of colicin L [21] and ferrichrome iron [22]. The ompF and ompC proteins form tight complexes with the peptidoglycan layer, which resists incubation in 2 % sodium dodecyl sulphate at 60°C [15,23,24]. These proteins form aqueous pores through which small molecules up to a molecular weight of about 700 can pass the outer membrane [25-291. can be present in pseudo revertants of mutants lacking the ompF and ompC proteins [21,26,30,31]. We have recently observed that this protein is induced in wildtype E. coli K12 cells by phosphate limitation [17] and is coregulated with alkaline phosphatase [32]. Also protein e is peptidoglycan-associated and has porin properties [31]. The ompF and ompC proteins and protein e are very similar with respect to their total amino acid composition [31,33,34] and their amino termini [30], but considerable differences were found in the fragment patterns obtained by incubation of the purified proteins with cyanogen bromide and proteolytic enzymes [31,33,34]. Protein I11 usuall...

Structural relatedness of enteric bacterial porins assessed with monoclonal antibodies to Salmonella typhimurium OmpD and OmpC

“…The six antibodies that bind only homologous trimer (Table 1) demonstrate that sequence variability exists in the cell-surface-exposed parts of the three related Salmonella pore proteins. This is consistent with findings of several other studies which have shown that MAbs directed against native porins fail to detect common antigenic sites among, PhoE, OmpC, and OmpF pore proteins (2,45,57,61), despite high sequence homology and cross-reactivity with polyclonal antisera (43). Since surface domains of OM proteins act as receptors for phages and colicins, the variability in their constituent residues may arise from selective pressure to evade such toxic agents and antibodies (4).…”

“…Purified porins are immunogenic in mice and rabbits (19,43) as trimers or monomers. Although antisera to trimers usually do not recognize monomers and vice versa (19,43,46), MAbs raised to monomers can sometimes recognize epitopes on trimers (2).…”

Increased efficiency of the outer membrane PhoE protein pore in escherichia coli K-12 mutants with heptose-deficient lipopolysaccharide

“…PhoE protein shares many properties with OmpF and OmpC protein. It can be isolated associated with the peptidoglycan layer [8,13] and it is immunologically related to OmpF and OmpC protein [14], the latter being consistent with the recently observed strong homology in primary structure between OmpC protein, OmpF protein and PhoE protein [15,16]. PhoE protein functions as a general pore like OmpF and OmpC protein [6,8,13,17,18].…”

Effect of lipopolysaccharide structure on reactivity of antiporin monoclonal antibodies with the bacterial cell surface

“…Bacteria (3 x 109 per gel) were pelleted by centrifugation at 10,000 x g, washed in 10 mM Tris (pH 8.0), and subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (31). Proteins were electrophoretically transferred to nitrocellulose at 10 V for 16 h (44).…”