Antibodies against bacterial membrane proteins

Abstract

Prior studies have shown that antiserum raised to the Escherichia coli J5 vaccine binds to three bacterial outer membrane proteins (OMPs) that are present on serum-exposed smooth Enterobacteriaceae and in LPS-containing fragments that are released by bacteria incubated in human serum. The present studies were performed to further analyze release of the three OMPs into human serum. Normal human serum was incubated with E. coli O18K⁺ bacteria and then filtered to remove intact bacteria. Lipopolysaccharide (LPS) was affinity-purified from sterile filtrates using murine monoclonal IgG directed against the O-polysaccharide chain of E. coli O18 LPS (monoclonal anti-O IgG). Components of sterile filtrates were also separated by ultracentrifugation on sequential potassium bromide and cesium chloride gradients. Samples were analyzed by immunoblotting, using anti-J5 IgG and polyclonal anti-O IgG as primary antibodies. Three OMPs, of MWs 5—9, 18, and 35 kDa were detected in samples that were affinity-purified using monoclonal anti-O IgG, indicating that complexes containing at least these three OMPs and LPS (OMP/LPS complexes) are released into human serum. The 18 kDa OMP was detected in low density fractions of the density gradients, indicating that this OMP is also released separately from the OMP/LPS complexes in a form that floats at the same density as serum lipoproteins.

References

Chedid L. , Parant M. , Boyer F. A proposed mechanism for natural immunity to enterobacterial pathogens. J Immunol 1968; 100: 292—301.

Ziegler EJ , McCutchan JA , Fierer J. et al. Treatment of Gram-negative bacteremia and shock with human antiserum to a mutant Escherichia coli. N Engl J Med 1982; 307: 1225—1230.

Siber GR , Kania SA , Warren HS Cross-reactivity of rabbit antibodies to lipopolysaccharides of Escherichia coli J5 and other Gram-negative bacteria. J Infect Dis 1985; 152: 954—964.

Hellman J. , Zanzot EM , Loiselle PM et al. Antiserum against Escherichia coli J5 contains antibodies reactive with outer membrane proteins of heterologous Gram-negative bacteria. J Infect Dis 1997; 176: 1260—1268.

Cryz Jr SJ , Cross AS , Sadoff JC , Furer E. Synthesis and characterization of Escherichia coli O18 O-polysaccharide conjugate vaccines. Infect Immun 1990; 58: 373—377.

Kim KS , Kang JH , Cross AS , Kaufman B. , Zollinger W. , Sadoff J. Functional activities of monoclonal antibodies to the O side chain of Escherichia coli lipopolysaccharides in vitro and in vivo. J Infect Dis 1988; 157: 47—53.

Warren HS , Glennon M. , de Deckker FA , Tello D. Role of normal serum in the binding of lipopolysaccharide to IgG fractions from rabbit antisera to Escherichia coli J5 and other Gram-negative bacteria . J Infect Dis 1991; 163: 1256—1266.

Warren HS , Knights CV , Siber GR Neutralization and lipoprotein binding of lipopolysaccharides in tolerant rabbit serum. J Infect Dis 1986; 154:784—791.

Freudenberg MA , Meier-Dieter U. , Staehelin T. , Galanos C. Analysis of LPS released from Salmonella abortus equi in human serum. Microb Pathog 1992; 10: 93—104.

10.

Munford RS , Andersen JM , Dietschy JM Sites of tissue binding and uptake in vivo of bacterial lipopolysaccharide-high density lipoprotein complexes: studies in the rat and squirrel monkey. J Clin Invest 1981; 68: 1503—1513.

11.

Greisman SE , Johnston CA Evidence against the hypothesis that antibodies to the inner core of lipopolysaccharides in antisera raised by immunization with enterobacterial deep-rough mutants confer broad-spectrum protection during Gram-negative bacterial sepsis. J Endotoxin Res 1997; 4: 123—153.