title: Use of a bacteriophage-encoded glycanase enzyme in the generation of lipopolysaccharide O side chain deficient mutants of Escherichia coli O9:K30 and Klebsiella O1:K20: role of O and K antigens in resistance to complement-mediated serum killing.
abstract: Coliphage K30 lysates contain free and phage-associated forms of a bacteriophage-encoded capsule depolymerase (glycanase) enzyme, active against the serotype K30 capsular polysaccharide of Escherichia coli. The free glycanase has been purified to apparent homogeneity. The molecular weight of the enzyme was estimated at 450,000, and when heated in SDS at 100 degrees C, the enzyme dissociated into two subunits of 90,000 and 52,000. The glycanase enzyme was used as a reagent to reversibly degrade the capsular layers on cells of Escherichia coli O9:K30 and Klebsiella O1:K20. This treatment rendered these bacteria sensitive to their respective lipopolysaccharide-specific bacteriophages, coliphage O9-1 and Klebsiella phage O1-3. This novel approach facilitated isolation of lipopolysaccharide O antigen side chain deficient mutants which retained the ability to synthesize the capsule. The response of defined mutants, O+:K-, O-:K+, and O-:K-, to exposure to nonimmune rabbit serum was measured. Results showed that the primary barrier against complement-mediated serum killing in both Escherichia coli O9:K30 and Klebsiella O1:K20 was the O antigen side chains of the lipopolysaccharide molecules. In both strains, the capsule played no role in the determination of serum resistance.
authors: Department of Microbiology, College of Biological Sciences, University of Guelph, Ont., Canada.
authors: K L
authors: KL
authors: McCallum
authors: D H
authors: DH
authors: Laakso
authors: C
authors: C
authors: Whitfield
mesh_terms: D000942:Antigens, Bacterial / Q000276:immunology* / Q000302:isolation & purification; D000954:Antigens, Surface / Q000276:immunology / Q000302:isolation & purification; D001435:Bacteriophages; D001770:Blood Bactericidal Activity; D003090:Coliphages; D003165:Complement System Proteins / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology*; D006026:Glycoside Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007709:Klebsiella / Q000235:genetics / Q000276:immunology*; D008070:Lipopolysaccharides / Q000276:immunology* / Q000302:isolation & purification; D009154:Mutation; D019081:O Antigens
chemical_list: D000942:Antigens, Bacterial; D000954:Antigens, Surface; C030659:K antigens; D008070:Lipopolysaccharides; D019081:O Antigens; D003165:Complement System Proteins; D006026:Glycoside Hydrolases; C020668:glycanase