Periplasmic Modification of the 1-Phosphate Group of Lipid A in Gram-Negative Bacteria.

<p>Modification of the lipid A domain of lipopolysaccharide (LPS) is important for the pathogenesis and virulence of various Gram-negative bacteria. The major lipid A species of <em>Helicobacter pylori</em> is significantly different from that of <em>Escherichia coli</em>. <em>H. pylori</em> lipid A contains fewer acyl chains and phosphate groups with only one Kdo sugar attached to the disaccharide backbone. However, <em>H. pylori</em> produces a minor lipid A species that resembles <em>E. coli</em> lipid A, suggesting that the major lipid A species results from the action of specific modifying enzymes. This work describes two enzymes, a lipid A phosphatase and a phosphoethanolamine (pEtN) transferase, involved in modifying the 1-position of <em>H. pylori</em> lipid A. <em>H. pylori</em> lipid A contains a pEtN unit directly linked to the 1-position of the disaccharide backbone. This is in contrast to the pEtN units found in other pathogens, which are attached to the lipid A phosphate group to form a pyrophosphate linkage. Using <em>in-vitro</em> assay systems, we demonstrate that the modification of the 1-position of <em>H. pylori</em> lipid A is a two-step process involving the removal of the 1-phosphate group by LpxE_HP followed by the addition of a pEtN residue catalyzed by EptA_HP. As compared to wild-type <em>H. pylori</em>, <em>lpxE_HP<em> mutants are extremely sensitive to the cationic peptide polymyxin, thus, demonstrating the importance of modifying the 1-position of lipid A. Furthermore, this work describes another enzyme, YeiU (renamed LpxT), which specifically utilizes the carrier lipid undecaprenyl pyrophsphate (C₅₅-PP) to modify the 1-position of <em>E. coli</em> lipid A. Typically, <em>E. coli</em> lipid A is a hexa-acylated disaccharide of glucosamine in which monophosphate groups are attached at positions 1 and 4'; however, a small fraction contains a diphosphate moiety at the 1-position (lipid A 1-diphosphate). ³²P-labeled lipid A obtained from <em>lpxT</em> deficient mutants produces only lipid A, and complementation with a plasmid expressing LpxT restores lipid A 1-diphosphate formation. Inhibition of lipid A 1-diphosphate synthesis was demonstrated by sequestering C₅₅-PP with the cyclic polypeptide antibiotic bacitracin. In conclusion, this work describes two novel pathways for lipid A modification at the 1-position in Gram-negative bacteria.</p>

Name thesis:degree_name

PhD (Doctor of Philosophy)

Level thesis:degree_level

Dissertation - restricted

Discipline thesis:degree_discipline

Biomedical Sciences

Year dc:date.issued

2007

Author dc:creator

• Tran, An Xuong

• LPS
• Lipid A
• Endotoxin
• Phosphatase
• Phosphoethanolamine
• Cationic antimicrobial peptides
• Phosphotransferase
• Chemicals and Drugs
• Enzymes and Coenzymes
• Medicine and Health Sciences

Statement dc:rights

• Copyright by the authors.

Repository record dc:identifier

https://dc.etsu.edu/etd/2036

OAI identifier oai:identifier

oai:dc.etsu.edu:etd-3397