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Role of lipopolysaccharide phase variation in susceptibility of Haemophilus influenzae to bactericidal immunoglobulin M antibodies in rabbit sera.

ABSTRACT: The effect of phase variation of lipopolysaccharide (LPS) structure on the susceptibility of Haemophilus influenzae to complement-dependent killing by normal human sera and normal rat sera has been described previously. The phase-variable structure phosphorylcholine (ChoP) confers susceptibility to human serum, since ChoP on the bacterial cell surface binds to serum C-reactive protein and activates complement. In contrast, expression of galalpha1,4gal, a second phase-variable epitope that is also found on human glycoconjugates, confers resistance to human serum. We studied the role of phase variation of these structures in the susceptibilities of H. influenzae KW20 (Rd) and a clinical isolate of nontypeable H. influenzae to killing by rabbit sera, which often possess naturally acquired complement-dependent bactericidal activity for unencapsulated H. influenzae. Expression of ChoP increased the resistance of strain KW20 to killing by bactericidal rabbit sera. In contrast, the serum resistance of a clinical isolate, H233, was unaffected by ChoP expression but was reduced by galalpha1,4gal expression. The rabbit sera with bactericidal activity (but not the nonbactericidal sera) all contained immunoglobulin M (IgM) antibodies able to bind to the surface of H. influenzae bacteria, as detected by flow cytometry, and contained IgM antibodies to LPS purified from strain KW20. Preincubation of sera with LPS reduced their bactericidal activity. Bactericidal activity was recovered quantitatively in an IgM-enriched fraction of sera. It is concluded that naturally occurring bactericidal activity for unencapsulated H. influenzae is largely due to IgM antibodies directed against phase-variable structures of the LPS.

SUBMITTER: Erwin AL

PROVIDER: S-EPMC97491 | biostudies-literature | 2000 May

REPOSITORIES: biostudies-literature

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Role of lipopolysaccharide phase variation in susceptibility of Haemophilus influenzae to bactericidal immunoglobulin M antibodies in rabbit sera.

Erwin A L AL Brewah Y A YA Couchenour D A DA Barren P R PR Burke S J SJ Choi G H GH Lathigra R R Hanson M S MS Weiser J N JN

Infection and immunity 20000501 5

The effect of phase variation of lipopolysaccharide (LPS) structure on the susceptibility of Haemophilus influenzae to complement-dependent killing by normal human sera and normal rat sera has been described previously. The phase-variable structure phosphorylcholine (ChoP) confers susceptibility to human serum, since ChoP on the bacterial cell surface binds to serum C-reactive protein and activates complement. In contrast, expression of galalpha1,4gal, a second phase-variable epitope that is als ...[more]

PMID: 10768976

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Project description:Non-typeable Haemophilus influenzae (NTHi), a commensal organism in pre-school children, is an opportunistic pathogen causing respiratory tract infections including acute otitis media. Adults suffering from chronic obstructive pulmonary disease (COPD) are persistently colonized by NTHi. Previous research has suggested that, in some bacterial species, the intracellular elongation factor thermo-unstable (EF-Tu) can moonlight as a surface protein upon host encounter. The aim of this study was to determine whether EF-Tu localizes to the surface of H. influenzae, and if such surface-associated EF-Tu is a target for bactericidal antibodies. Using flow cytometry, transmission immunoelectron microscopy, and epitope mapping, we demonstrated that EF-Tu is exposed at the surface of NTHi, and identified immunodominant epitopes of this protein. Rabbits immunized with whole-cell NTHi produced significantly more immunoglobulin G (IgG) directed against EF-Tu than against the NTHi outer membrane proteins D and F as revealed by enzyme-linked immunosorbent assays. Chemical cleavage of NTHi EF-Tu by cyanogen bromide (CNBr) followed by immunoblotting showed that the immunodominant epitopes were located within the central and C-terminal regions of the protein. Peptide epitope mapping by dot blot analysis further revealed four different immunodominant peptide sequences; EF-Tu41-65, EF-Tu161-185, EF-Tu221-245, and EF-Tu281-305. These epitopes were confirmed to be surface-exposed and accessible by peptide-specific antibodies in flow cytometry. We also analyzed whether antibodies raised against NTHi EF-Tu cross-react with other respiratory tract pathogens. Anti-EF-Tu IgG significantly detected EF-Tu on unencapsulated bacteria, including the Gram-negative H. parainfluenzae, H. haemolyticus, Moraxella catarrhalis and various Gram-positive Streptococci of the oral microbiome. In contrast, considerably less EF-Tu was observed at the surface of encapsulated bacteria including H. influenzae serotype b (Hib) and Streptococcus pneumoniae (e.g., serotype 3 and 4). Removal of the capsule, as exemplified by Hib RM804, resulted in increased EF-Tu surface density. Finally, anti-NTHi EF-Tu IgG promoted complement-dependent bacterial killing of NTHi and other unencapsulated Gram-negative bacteria as well as opsonophagocytosis of Gram-positive bacteria. In conclusion, our data demonstrate that NTHi EF-Tu is surface-exposed and recognized by antibodies mediating host innate immunity against NTHi in addition to other unencapsulated respiratory tract bacteria.

Dataset Information

Role of lipopolysaccharide phase variation in susceptibility of Haemophilus influenzae to bactericidal immunoglobulin M antibodies in rabbit sera.

Publications

Role of lipopolysaccharide phase variation in susceptibility of Haemophilus influenzae to bactericidal immunoglobulin M antibodies in rabbit sera.

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