Hierarchical-Clustering, Scaffold-Mining Exercises and Dynamics Simulations for Effectual Inhibitors Against Lipid-A Biosynthesis of Helicobacter pylori

ABSTRACT The lipid A domain anchors lipopolysaccharide (LPS) to the outer membrane and is typically a disaccharide of glucosamine that is both acylated and phosphorylated. The core and O-antigen carbohydrate domains are linked to the lipid A moiety through the eight-carbon sugar 3-deoxy-d-manno-octulosonic acid known as Kdo. Helicobacter pylori LPS has been characterized as having a single Kdo residue attached to lipid A, predicting in vivo a monofunctional Kdo transferase (WaaA). However, using an in vitro assay system we demonstrate that H. pylori WaaA is a bifunctional enzyme transferring two Kdo sugars to the tetra-acylated lipid A precursor lipid IVA. In the present work we report the discovery of a Kdo hydrolase in membranes of H. pylori capable of removing the outer Kdo sugar from Kdo2-lipid A. Enzymatic removal of the Kdo group was dependent upon prior removal of the 1-phosphate group from the lipid A domain, and mass spectrometric analysis of the reaction product confirmed the enzymatic removal of a single Kdo residue by the Kdo-trimming enzyme. This is the first characterization of a Kdo hydrolase involved in the modification of gram-negative bacterial LPS.

Download Full-text

Evolution of the Kdo2-lipid A biosynthesis in bacteria

BMC Evolutionary Biology ◽

10.1186/1471-2148-10-362 ◽

2010 ◽

Vol 10 (1) ◽

pp. 362 ◽

Cited By ~ 23

Author(s):

Stephen O Opiyo ◽

Rosevelt L Pardy ◽

Hideaki Moriyama ◽

Etsuko N Moriyama

Keyword(s):

Lipid A ◽

Lipid A Biosynthesis

Download Full-text

Structural Basis for the Recognition of Peptide RJPXD33 by Acyltransferases in Lipid A Biosynthesis

Journal of Biological Chemistry ◽

10.1074/jbc.m114.564278 ◽

2014 ◽

Vol 289 (22) ◽

pp. 15527-15535 ◽

Cited By ~ 15

Author(s):

Ronald J. Jenkins ◽

Kyle A. Heslip ◽

Jennifer L. Meagher ◽

Jeanne A. Stuckey ◽

Garry D. Dotson

Keyword(s):

Lipid A ◽

Structural Basis ◽

Lipid A Biosynthesis

Download Full-text

Characterization of a waaF mutant of Helicobacter pylori strain 26695 provides evidence that an extended lipopolysaccharide structure has a limited role in the invasion of gastric cancer cells

Biochemistry and Cell Biology ◽

10.1139/o07-056 ◽

2007 ◽

Vol 85 (5) ◽

pp. 582-590 ◽

Cited By ~ 8

Author(s):

Vandana Chandan ◽

Susan M. Logan ◽

Blair A. Harrison ◽

Evgenii Vinogradov ◽

Annie Aubry ◽

...

Keyword(s):

Helicobacter Pylori ◽

Mutant Strain ◽

Lipid A ◽

Inner Core ◽

Cell Model ◽

Critical Factor ◽

Gastric Cancer Cells ◽

Level Of Invasion ◽

Insertional Inactivation

An ld-heptosyltransferase gene, HP1191 (waaF), involved in biosynthesis of the inner-core region of Helicobacter pylori strain 26695 lipopolysaccharide (LPS), has been cloned and its function established by complementation of Salmonella enterica serovar Typhimurium waaF mutant strain, strain 3789. Insertional inactivation of the HP1191 open reading frame in strain 26695 resulted in the formation of a deeply truncated LPS molecule, as observed using SDS–PAGE. Subsequent compositional and fatty acid analyses, followed by capillary electrophoresis – mass spectrometry and nuclear magnetic resonance studies established its structure as the following: PE→7)-l-α-d-Hepp-(1→5)-α-Kdop-(2→6)-Lipid A, where PE represents a phosphoethanolamine group, ld-Hep represents l-glycero-d-manno-heptose, and Kdo represents 3-deoxy-d-manno-oct-2-ulosonic acid. This structural analysis identifies the activity of HP1191 as a heptosyltransferase and a waaF homolog. In vitro invasion assays using human cultured gastric adenocarcinoma cells as a host cell model confirmed that the level of invasion was unaffected for an H. pylori HP1191::Kan deep-rough mutant strain compared with the wild-type strain 26695 expressing the O-chain polysaccharide, providing evidence that LPS is not a critical factor for invasion.

Download Full-text