The genetics, biosynthesis and translocation of group 1 capsules in gram-negative bacteria

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Drummelsmith, Jolyne

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University of Guelph

Abstract

Group 1 capsules are a major virulence factor of numerous bacteria, including pathogens of plants, livestock and humans (including some enterotoxigenic strains of 'Escherichia coli' and 'Klebsiella pneumoniae '). This work was initiated with the objective of resolving the genetics and biosynthesis of group 1 capsules, using 'E. coli' O9a:K30 as a prototype. Cloning and sequencing revealed that the 'cps' gene cluster is found between the genes 'dcd' (encoding dCTP-deaminase) and 'gnd' (encoding 6-phosphogluconate dehydrogenase) at a position equivalent to approximately 45 min on the 'E. coli' K-12 chromosomal map. The 'cps' cluster contains 12 genes within approximately 16 kb of DNA. Four of these genes, namely 'wbaP, wbaZ, wcaO' and 'wcaN', are predicted to encode glycosyltransferases responsible for the biosynthesis of the K30 repeating unit. Specific chromosomal mutants were constructed to elucidate the mode of polymerization and assembly of group 1 capsules. It was determined that group 1 capsules are polymerized by a Wzy-dependent mechanism similar to that used for the synthesis of many LPS O antigens, including those of 'Salmonella enterica'. One gene found in the 'E. coli' group 1 capsule cluster, ' wza'CPS, encodes a protein with homologues found in many polysaccharide biosynthetic systems, including the group 2 capsules. These capsules are virulence factors of the significant human pathogens ' Neisseria meningitidis, Haemophilus influenzae', and invasive strains of 'E. coli'. Their biosynthesis and assembly differ from those of the group 1 capsules, suggesting that Wza might function independently of these steps. One such function could be in export, although little data existed to support this assignment. Analysis of WzaCPS showed that it is required for capsule formation, and that it exists as ring-shaped multimeric complexes visible by electron microscopy. Together, these observations suggest that WzaCPS may form a pore through which the capsular polysaccharide exits the cell. This ring-like structure is reminiscent of those formed by secretins, proteins involved in the translocation of secreted proteins across the outer membrane of Gram-negative bacteria. The presence of a structural homologue indicates that mechanistic aspects of translocation may be conserved among cell-surface polysaccharides and other macromolecules, lending a broad significance to this work.

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genetics, biosynthesis, translocation, group 1 capsules, gram-negative bacteria

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