Role of Normal Serum in the Binding of Lipopolysaccharide to IgG Fractions from Rabbit Antisera to Escherichia coli J5 and Other Gram-Negative Bacteria

Warren, H. Shaw; Glennon, M.; Deckker, F A de; Tello, Diana

doi:10.1093/infdis/163.6.1256

Cited by 10 publications

(9 citation statements)

References 43 publications

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“…IgG was purified from hyperimmune serum, from artificial capillary cell culture supernatants, and from ammonium sulfate-precipitated ascites fluid by passage over a protein G-Sepharose 4 fast-flow column (Pharmacia, Piscataway, N.J.) as described elsewhere (27,64). Bound IgG was eluted from the column with 0.1 M glycine (pH 2.7) and was immediately neutralized with 1 M Tris buffer (pH 9.0).…”

mentioning

confidence: 99%

Outer Membrane Protein A, Peptidoglycan-Associated Lipoprotein, and Murein Lipoprotein Are Released byEscherichia coliBacteria into Serum

Hellman

Loiselle²,

Tehan

et al. 2000

Infect Immun

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4Complexes containing lipopolysaccharide (LPS) and three outer membrane proteins (OMPs) are released by gram-negative bacteria incubated in human serum and into the circulation in an experimental model of sepsis. The same OMPs are bound by immunoglobulin G (IgG) in the cross-protective antiserum raised to Escherichia coli J5 (anti-J5 IgG). This study was performed to identify the three OMPs. The 35-kDa OMP was identified as outer membrane protein A (OmpA) by immunoblotting studies using OmpA-deficient bacteria and recombinant OmpA protein. The 18-kDa OMP was identified as peptidoglycan-associated lipoprotein (PAL) based on peptide sequences from the purified protein and immunoblotting studies using PAL-deficient bacteria. The 5-to 9-kDa OMP was identified as murein lipoprotein (MLP) based on immunoblotting studies using MLPdeficient bacteria. The studies identify the OMPs released into human serum and into the circulation in an experimental model of sepsis as OmpA, PAL, and MLP.Bacterial cell wall components released into the bloodstream are believed to be important in the pathogenesis of gram-negative sepsis. Although prior investigators have reported that bacteria release lipopolysaccharide (LPS) into serum (62, 63) and into the circulation (4,18,56,66), the full composition of released bacterial products has not been established. Very little is known about release of non-LPS gramnegative outer membrane components such as outer membrane proteins (OMPs) in sepsis. Fragments containing LPS, OmpA, and another faintly staining protein, of 17 kDa, were affinity purified from filtrates of human serum incubated with Salmonella enterica serovar Abortus equi bacteria using Ochain-specific anti-LPS immunoglobulin G (IgG) (20). Similarly, we have affinity purified complexes containing LPS and at least three OMPs, with estimated molecular masses of 35, 18, and 5 to 9 kDa, from filtrates of normal human serum incubated with Escherichia coli bacteria, using O-chain-specific anti-LPS IgG (29,30).Previous studies indicated that passive and active immunity directed to rough mutant bacteria such as S. enterica serovar Minnesota Re595 and E. coli J5 protect in experimental and clinical gram-negative sepsis (1,5,11,42,43,68). Protection has been attributed to antibodies directed to conserved core components of LPS (lipid A and core oligosaccharide). However, it has been difficult to prove that antisera to rough strains of bacteria contain cross-reactive anti-lipid A or anti-core oligosaccharide IgGs (15, 57), and the exact mechanism of protection remains unclear and controversial.We have demonstrated that IgG in antiserum raised to heatkilled E. coli J5 (J5 antiserum) binds to the same three gramnegative bacterial OMPs that are released into serum in the OMP-LPS complexes described above (30). OMP-LPS complexes are also released into the bloodstream of burned rats with E. coli O18K ϩ sepsis (29). In addition, at least one OMP, with an estimated molecular mass of 18 kDa, is released from bacteria separately from the OMP-LPS comple...

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confidence: 99%

Outer Membrane Protein A, Peptidoglycan-Associated Lipoprotein, and Murein Lipoprotein Are Released byEscherichia coliBacteria into Serum

Hellman

Loiselle²,

Tehan

et al. 2000

Infect Immun

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“…IgG was purified from ascites after ammonium sulfate precipitation [12] and from hyperimmune serum [14,15]. In brief, affinity chromatography was performed by passage over a protein G-sepharose 4 fast-flow column (Pharmacia, Piscataway, NJ).…”

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confidence: 99%

Release of Gram‐Negative Outer‐Membrane Proteins into Human Serum and Septic Rat Blood and Their Interactions with Immunoglobulin in Antiserum toEscherichia coliJ5

et al. 2000

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Prior studies indicate that 3 bacterial outer-membrane proteins (OMPs) are released into serum associated with lipopolysaccharide (LPS) and are bound by IgG in antiserum to Escherichia coli J5 (anti-J5 IgG). The present studies analyzed the interaction of the OMPs with anti-J5 IgG and evaluated their release in an infected burn model of gram-negative sepsis. Affinity purification studies were performed on filtrates of bacteria incubated in human serum and plasma from rats with sepsis by use of O chain-specific anti-LPS IgG and anti-J5 IgG. All 3 OMPs were captured from septic rat blood by anti-LPS IgG. Release of OMPs into serum was highest for immature bacterial cultures and was increased by antibiotics in vitro and in vivo. Anti-J5 IgG selectively captured an 18-kDa OMP released into serum and into plasma from septic rats. The results raise the possibility that anti-J5 IgG may, in part, protect via anti-OMP antibodies.

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“…The plates were again washed three times, and peroxidase substrate (1 mg/mL H 2 O 2 Preparation of IgG fractions. IgG was prepared from hyperimmune serum as described [18,19]. Briefly, affinity chromatography in ABTS, citric acid, Na 2 HPO 4 ) was added.…”

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confidence: 99%

Antiserum againstEscherichia coliJ5 Contains Antibodies Reactive with Outer Membrane Proteins of Heterologous Gram‐Negative Bacteria

Hellman¹,

Zanzot²,

Loiselle³

et al. 1997

J INFECT DIS

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The binding of IgG in antiserum to Escherichia coli J5 to the surface of Enterobacteriaceae and to cell wall fragments released from serum-exposed bacteria was studied in a search for potentially protective epitopes other than lipopolysaccharide (LPS). IgG titers to multiple heterologous gram-negative smooth bacteria increased following incubation of the bacteria in serum and decreased following absorption with serum-exposed heterologous bacteria. IgG eluted from absorbing bacteria bound to at least three conserved bacterial outer membrane proteins (OMPs), but not LPS, as assessed by immunoblotting. The same OMPs were present in LPS-containing macromolecular cell wall fragments released by incubation of heterologous gram-negative bacteria in human serum. Part of the protection offered by J5 antiserum could be from binding of IgG to conserved OMPs at the bacterial surface or to OMPs in cell-wall fragments released from dying bacteria.

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Role of Normal Serum in the Binding of Lipopolysaccharide to IgG Fractions from Rabbit Antisera to Escherichia coli J5 and Other Gram-Negative Bacteria

Cited by 10 publications

References 43 publications

Outer Membrane Protein A, Peptidoglycan-Associated Lipoprotein, and Murein Lipoprotein Are Released byEscherichia coliBacteria into Serum

Outer Membrane Protein A, Peptidoglycan-Associated Lipoprotein, and Murein Lipoprotein Are Released byEscherichia coliBacteria into Serum

Release of Gram‐Negative Outer‐Membrane Proteins into Human Serum and Septic Rat Blood and Their Interactions with Immunoglobulin in Antiserum toEscherichia coliJ5

Antiserum againstEscherichia coliJ5 Contains Antibodies Reactive with Outer Membrane Proteins of Heterologous Gram‐Negative Bacteria

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