Abstract
The core oligosaccharide region of Klebsiella pneumoniae lipopolysaccharide contains some novel features that distinguish it from the corresponding lipopolysaccharide region in other members of the Enterobacteriaceae family, such as Escherichia coli and Salmonella. The conserved Klebsiella outer core contains the unusual trisaccharide 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo)-(2,6)-GlcN-(1,4)-GalUA. In general, Kdo residues are normally found in the inner core, but in K. pneumoniae, this Kdo residue provides the ligation site for O polysaccharide. The outer core Kdo residue can also be non-stoichiometrically substituted with an l-glycero-d-manno-heptopyranose (Hep) residue, another component more frequently found in the inner core. To understand the genetics and biosynthesis of core oligosaccharide synthesis in Klebsiella, the gene products involved in the addition of the outer core GlcN (WabH), Kdo (WabI), and Hep (WabJ) residues as well as the inner core HepIII residue (WaaQ) were identified. Non-polar mutations were created in each of the genes, and the resulting mutant lipopolysaccharide was analyzed by mass spectrometry. The in vitro glycosyltransferase activity of WabI and WabH was verified. WabI transferred a Kdo residue from CMP-Kdo onto the acceptor lipopolysaccharide. The activated precursor required for GlcN addition has not been identified. However, lysates overexpressing WabH were able to transfer a GlcNAc residue from UDP-GlcNAc onto the acceptor GalUA residue in the outer core.
Highlights
Klebsiella pneumoniae is an important nosocomial pathogen and is implicated in diseases including urinary tract infections, pneumonia, and bacteremia
WabI Is Required for Addition of the Outer Core Kdo Residue—The wabI gene product is encoded in the waa gene cluster of K. pneumoniae
As an initial step to determine the function of WabI in K. pneumoniae core OS biosynthesis, a non-polar aphA-3 cassette was inserted into the wabI gene of K. pneumoniae CWK2
Summary
Klebsiella pneumoniae is an important nosocomial pathogen and is implicated in diseases including urinary tract infections, pneumonia, and bacteremia (reviewed in Ref. 1). It has been shown that isolates of serotypes O1 and O2 during growth release an extracellular toxic complex composed of LPS, capsule, and a small amount of protein. As might be expected for a member of the Enterobacteriaceae, K. pneumoniae LPS shares significant similarity with the well characterized LPS structures of E. coli and Salmonella [5, 6]. The outer core region shows more diversity and is usually composed of hexose and acetamidohexose sugars in E. coli and Salmonella. The inner core of K. pneumoniae LPS is made up of the Hep-Kdo backbone seen in other members of the Enterobacteriaceae. The negative charge provided by these residues plays an important role in maintenance of the barrier function of the outer membrane by providing sites for cross-linking of adjacent LPS molecules with divalent cations (reviewed in Ref. 6). The gene required for the addition of this GalUA residue (wabG)
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