Pseudomonas aeruginosa Lectins

Extracts of certain strains of Pseudomonas aeruginosa contain two lectins: PAI and PAII. This chapter presents procedure for purification and characterization of PAI and PAII. The PAI purification involves crude extraction from bacteria grown in Grelet's medium, ammonium sulfate precipitation, and chromatography on Sepharose 4B. The yield of the purified PA-I is relatively high and is practically free of PA-II. PA-II is purified from the extracts of the bacteria grown in nutrient broth. The first two steps of its purification are the same as for PA-I: heating to 70 ° and precipitation by ammonium sulfate, as described above. The dissolved ammonium sulfate precipitate is purified by affinity chromatography on D-mannose-bearing Sepharose 4B. Thirty milliliters of the lectin preparation are loaded onto a column (4 x 20 cm) containing the modified Sepharose. The activity of the lectins is determined by measuring their ability to agglutinate neuraminidase or papain-treated human erythrocytes. The hemagglutination assay can also be used as an indirect demonstration of lectin interaction with nonagglutinable cells by determining the decrease in the hemagglutinating activity of the lectin preparations as a result of their exposure to the examined cells. The interaction of PA-II with cells can also be detected by a peroxidase-binding assay.