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UBC Theses and Dissertations

Structural studies of some bacterial capsular polysaccharides from the family Enterobacteriaceae:Klebsiella serotypes K79 and K35 and Escherichia coli serotype K44 Vee San Lim, Andrew


Klebsiella and Escherichia coli are among the most frequently found pathogenic Enterobacteria. Serological classification of these two Enterobacteria is based mainly on the immunologically dominant capsular polysaccharides (K antigens), due to their location at the outermost surface of the bacterial cell. In order to understand the chemical basis of serological differentiation and the immunochemistry of these antigens, the systematic structural investigation of all the capsular polysaccharides of Klebsiella (77 serotypes) and E. coli (74 serotypes) is currently being undertaken in this and other laboratories. To date, the structure of the capsular polysaccharides of approximately seventy Klebsiella serotypes and thirty E. coli serotypes have been elucidated. These polysaccharides were found to be unique in each case. As a contribution to the immunology of bacterial capsular polysaccharides, the structures of the K antigens from Klebsiella K79 and K35, and E. coli K44 were determined and are presented in this thesis. The techniques of sugar analysis, methylation, chromic acid oxidation, deamination, base-catalyzed uronic acid degradation, Smith degradation and partial hydrolysis were used in the structural elucidation of these polysaccharides. Methods such as gas-liquid chromatography, gas chromatography-mass spectrometry, gel permeation, ion-exchange and paper-chromatography were used to isolate and characterize oligosaccharides obtained from degradative procedures. N.m.r. spectroscopy (¹H and ¹³C) was widely used in the characterization of the polysaccharides and of derived poly- and oligo- saccharides. In a few instances, n.m.r. spectroscopy and mass spectrometry were used to delineate the sequence of the sugars in the structure of the poly- and oligo-saccharides. The sum of these experiments established the following structures. [See Thesis for Diagrams] The structure for the Klebsiella K79 capsular polysaccharide represents the second example of a "5+2" pattern in the Klebsiella capsular polysaccharides series. The "4+1" structural pattern of Klebsiella K35 capsular polysaccharide, with the hexuronic acid in the side chain, is similar to but yet different from those of Klebsiella K9 and K59. The acidic capsular polysaccharide of E. coli K44 represents the first instance of E. coli K antigens (of group A) to contain two different 2-acetamido-2-deoxyhexoses. For almost every strain of bacteria, there exists a bacteriophage (bacterial virus, denoted by Φ) that infects it and replicates within the bacterial cell. Associated with these bacteriophages are enzymes (endoglycanases) that specifically cleave the capsular polysaccharides of the host bacteria to oligosaccharides comprising one or more repeating units of the polysaccharide. These oligosaccharides when coupled to carrier proteins have been used in the production of protective vaccines. In our laboratory, however, these bacteriophage-borne endoglycanases are being used to generate oligosaccharides in the structural elucidation of bacterial capsular polysaccharides. The bacteriophages for Klebsiella K79 and E. coli K44 were isolated from sewage and propagated on their host strains. The bacteriophage-induced depolymer-izations of the capsular polysaccharides of Klebsiella K79 and E. coli K44 are reported in this thesis. The oligosaccharides isolated were characterized using the techniques described. The sum of these experiments demonstrated that the endoglycanase associated with Klebsiella Φ 79 had β-galactosidase activity. The endoglycanase from E. coli Φ 44 exhibited β-N-acetyl-galactosaminidase activity which is novel, in that it is the first reported action of this nature in the bacteriophages isolated for the species E. coli.

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