Antibodies from chronically infected cystic fibrosis patients react with lipopolysaccharides extracted by new micromethods from all serotypes of Pseudomonas aeruginosa.
Micromethods were developed to extract lipopolysaccharide (LPS, endotoxin) from single bacterial colonies of the 20 recognized Pseudomonas aeruginosa type strains. The appearance of these LPSs in polyacrylamide gel electrophoresis (PAGE) and their reactivity with serum of cystic fibrosis (CF) patients chronically infected with P. aeruginosa was studied. Silver staining of LPS after PAGE showed that 13 of the P. aeruginosa LPSs had high numbers of O-repeating units arranged in 1-4 clusters of banding. Low-molecular-weight LPS fractions were more prominent in six of the serotype strains, of which O:7 and O:14 appeared semi-rough. Corresponding immunoblots using the CF sera showed LPS patterns very similar to the silver-stained appearance, indicating an immune reaction to all P. aeruginosa LPS including that from the newly discovered O:18, O:19 and O:20. This was unexpected since only a few serotype strains (mostly O:3, O:6 and O:9) had been isolated from the patients. Absorption experiments using purified and chemically defined P. aeruginosa rough LPS and smooth LPS suggested these immune reactions were due to antibodies cross-reactive to core/lipid A as well as to lower molecular weight O-polysaccharides or "A-bands". However, in some cases O:3, O:6, and O:9 LPSs were also found to contain additional distinct O-epitopes. Immune recognition of various polyagglutinable P. aeruginosa LPSs seemed also to be caused by cross-reactive antibodies. The described microextraction methods, followed by PAGE and silver staining or immunoblotting, are easy and convenient techniques with which to study antibodies against LPS epitopes and to screen for LPS phenotypic appearance using only a few bacterial colonies from larger numbers of Gram-negative bacterial strains.
Fomsgaard
A
A
Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark.
Shand
G H
GH
Freudenberg
M A
MA
Galanos
C
C
Conrad
R S
RS
Kronborg
G
G
Høiby
N
N
D000906:Antibodies / Q000032:analysis / Q000276:immunology* / Q000378:metabolism; D003429:Cross Reactions; D003550:Cystic Fibrosis / Q000097:blood / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D006801:Humans; D015151:Immunoblotting; D008070:Lipopolysaccharides / Q000276:immunology* / Q000378:metabolism; D008722:Methods; D010641:Phenotype; D011550:Pseudomonas aeruginosa / Q000145:classification / Q000378:metabolism*; D012703:Serotyping; D016622:Silver Staining
D000906:Antibodies; D000939:Epitopes; D008070:Lipopolysaccharides