Temperature-sensitive mutants of Mycoplasma pneumoniae. II. Response of hamsters.
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Biomedical subjects
Publications and source records attributed to R M Chanock.
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Eight Mycoplasma species of human origin were successfully cultivated on glass. Complement-fixing (CF) antigens prepared from glass-adherent mycoplasmas were potent, specific, and free from anticomplementary activity. PPLO broth medium supplemented with 1 to 5% PPLO serum fraction (bovine), 2.5% fresh yeast extract, and 1% glucose (glycolytic species) or 1% arginine (arginine-utilizing species) supported moderate to luxuriant growth of mycoplasmas on glass. The potency of CF antigens prepared from glass-adherent mycoplasmas varied with the species of Mycoplasma tested and the duration of incubation. When the potency of CF antigens prepared from glass-adherent mycoplasmas was compared with that material sedimented from the broth phase of the same culture, three patterns of growth were observed: M. hominis and M. orale type 2 grew preferentially in the broth phase; M. salivarium, M. orale types 1 and 3, M. pneumoniae, and M. lipophilum preferentially adhered to the glass; and M. fermentans was biphasic. The growth of mycoplasmas on glass provides a simple means of concentrating and purifying such organisms for immunological and biochemical studies.
The glycolipid haptens of Mycoplasma pneumoniae became immunogenic when bound to membrane proteins of Acholeplasma laidlawii by reaggregation. This process consisted of the solubilization of lipid-depleted A. laidlawii membranes and M. pneumoniae glycolipids in 20 mm sodium dodecyl sulfate and dialysis of the mixed solutions against 20 mm Mg(2+). The antibodies produced in rabbits to the reaggregated glycolipids inhibited the metabolism of M. pneumoniae, fixed complement with M. pneumoniae glycolipids or whole cells, precipitated M. pneumoniae glycolipids, and agglutinated M. pneumoniae cells. All these antibody activities could be blocked or absorbed by the purified glycolipids but not by a series of carbohydrates containing glucose and galactose. It was concluded that the antiserum to the reaggregated glycolipids may be regarded as a specific serum to membrane glycolipids of M. pneumoniae, since the antibodies to A. laidlawii membrane proteins, present in this serum, did not react with the glycolipids or with any other cell component of M. pneumoniae.
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The glycolipid haptens of Mycoplasma pneumoniae were bound to membrane proteins of acholeplasma laidlawii (formerly Mycoplasma laidlawii) by reaggregation. This process consisted of the solubilization of lipid-depleted A. laidlawii membranes and M. pneumoniae glycolipids in 20 mM sodium dodecyl sulfate, and dialysis of the mixed solutions against 20 mM Mg(2+). The hybrid reaggregate, collected by centrifugation, was highly immunogenic in rabbits, eliciting the production of a high titer of antibodies that fixed complement with the purified glycolipids and inhibited the metabolism of M. pneumoniae cells. The free glycolipids, or their mixture with A. laidlawii proteins, were much less effective in stimulating these antibodies. The antibodies to the hybrid reaggregate agglutinated M. pneumoniae cells and inhibited their ability to absorb erythrocytes, which indicates that at least some of the serologically-active glycolipids are exposed at the outer membrane surface. The ability to bind selected membrane lipids to membrane proteins of a serologically unrelated species provides a new tool for producing antibodies to lipid haptens.
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