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Characterization of lipopolysaccharide of Haemophilus influenzae
Abstract:
Lipopolysaccharide from strains of Haemophilus influenzae was extracted and isolated by the hot phenol-water procedure. The preparations were relatively insoluble in water but could be solubilized with surface-active agents. The preparations contained carbohydrate (30%), fatty acid (29%), and phosphate (4.7%); protein content was less than 1%. Thin-layer chromatography, gas-liquid chromatography, and colorimetric assays detected glucose, galactose, glucosamine, heptose, and a 2-keto-3-deoxy-octonate-like molecule (less than 1%). Neither methylpentose nor dideoxyhexose was detected. The lipid portion was composed of fatty acids common to lipopolysaccharide of Salmonella. The preparations provoked positive dermal Shwartzman reactions and biphasic febrile responses in rabbits, responses typical of endotoxic activity. The 50% lethal dose for mice was decreased from 16.5 microgram/g to 0.015 microgram/g by concomitant administration of actinomycin D. The preparations were shown to be polyclonal activators of bone marrow-derived (B) cells. Limulus lysate gelation was seen with 8.0 ng of lipopolysaccharide. Preliminary hemagglutination data suggested at least three different antigenic factors associated with the lipopolysaccharide of H. influenzae type b. The H. influenzae lipopolysaccharide appeared biologically similar to that of enterobacteria but chemically different.
Insights
Lipopolysaccharide from Haemophilus influenzae was isolated and characterized, revealing endotoxic activity and B-cell activation. Its chemical structure differs from enterobacteria, despite biological similarities.
Area of Science:
- Microbiology
- Immunology
- Biochemistry
Background:
- Lipopolysaccharides (LPS) are critical components of Gram-negative bacterial outer membranes.
- Haemophilus influenzae LPS structure and biological activity are not fully elucidated.
- Understanding LPS is crucial for developing vaccines and therapeutics.
Purpose of the Study:
- To extract, isolate, and characterize LPS from Haemophilus influenzae strains.
- To assess the biological activities, including endotoxicity and B-cell activation, of the isolated LPS.
- To compare the chemical and biological properties of H. influenzae LPS with those of enterobacteria.
Main Methods:
- Hot phenol-water extraction for LPS isolation.
- Thin-layer chromatography (TLC), gas-liquid chromatography (GLC), and colorimetric assays for chemical composition analysis.
- Dermal Shwartzman reactions, febrile responses in rabbits, and LD50 assays in mice for endotoxicity assessment.
- Actinomycin D co-administration to evaluate LPS toxicity.
- Bone marrow-derived B-cell activation assays.
- Limulus lysate assay for endotoxin detection.
- Hemagglutination assays for antigenic factor analysis.
Main Results:
- LPS preparations contained carbohydrate (30%), fatty acid (29%), and phosphate (4.7%), with minimal protein (<1%).
- Detected sugars included glucose, galactose, glucosamine, heptose, and a 2-keto-3-deoxy-octonate-like molecule; methylpentose and dideoxyhexose were absent.
- The lipid portion comprised fatty acids common to Salmonella LPS.
- Preparations exhibited typical endotoxic activity, including positive Shwartzman reactions and febrile responses in rabbits, and were highly toxic to mice (LD50 0.015 μg/g with actinomycin D).
- H. influenzae LPS acted as a polyclonal activator of B cells and showed potent Limulus lysate gelation (8.0 ng).
- Preliminary hemagglutination suggested at least three antigenic factors.
Conclusions:
- Haemophilus influenzae LPS possesses significant endotoxic and B-cell activating properties, similar to enterobacterial LPS.
- Chemically, H. influenzae LPS differs from enterobacterial LPS, particularly in its sugar composition.
- The findings provide a foundation for understanding H. influenzae pathogenesis and developing targeted interventions.