Antigenic Determinants of Murein Lipoprotein and Its Exposure at the Surface of Enterobacteriaceae

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Abstract
Murein lipoprotein from the outer membrane of Escherichia coli could be fixed to erythrocytes without pretreatment of the erythrocytes. Passive hemagglutination of immune hemolysis could be used as sensitive assays to determine antibodies against lipoprotein. In rabbit antisera prepared against whole cells of E. coli, Salmonella, Arizona and Shigella antibodies against lipoprotein were present. The respective titers were lowest in encapsulated smooth strains and highest in rough mutants. Antisera against deep rough mutants showed even higher anti-lipoprotein titers than anti-R-lipopolysaccharide titers. Correspondingly, absorption of lipoprotein antibodies with enterobacterial strains was most pronounced with deep rough mutants and lowest with smooth strains. Lipoprotein becomes increasingly an immunogen as well as an antigen the more sugar residues are missing in the lipopolysaccharide on the cell surface. In wild-type cells lipoprotein is buried in the outer membrane; its exposure in mutant cells is related to defects at the cell surface. Antisera were prepared in rabbits against erythrocytes coated with various lipoprotein samples which differ at the C-terminal end of the polypeptide chain. Antibodies against lipoprotein containing 2 attached muropeptides (called lipoprotein I) are directed against the muropeptide attachment region. Antibodies against lipoprotein ending with lysine-55 (lipoprotein II) were directed specifically against this terminal lycine residue. No serological reaction was observable after splitting off this lysine residue by carboxypeptidase B. This antiserum against lipoprotein II neither reacted with the free lipoprotein ending with lysine-58 (lipoprotein IV) nor with the muropeptide containing lipoprotein I. Antisera against the lipoprotein lacking lysine-55 (lipoprotein III) reacted with all lipoprotein samples as did the antibodies in antisera prepared against whole heat-killed cells, indicating a common binding site located in the polypeptide chain towards the N-terminal end. No indication of an antigenic reactivity of the covalently linked lipoid was obtained. Lipoprotein treated with boiling 4% sodium dodecylsulfate and acetone reacted strongly with antisera against the native lipoprotein. The recovery of the antigenic sites demonstrates the remarkable stability of the native conformation. The lipoprotein is a new common antigen for closely related Enterobacteriaceae. Lipoprotein antisera might be useful for characterization of outer membrane mutants of Enterobacteriaceae.