Lipopolysaccharides (LPSs) are remarkable glycolipids that comprise the outer surfaces of Gram-negative bacteria, including the symbiotic organism, Rhizobium leguminosarum. In Escherichia coli, the lipid A anchor of LPS is a hexa-acylated disaccharide of glucosamine, bearing phosphate moieties at positions 1 and 4'. The minimal LPS required for a growth of E.coli consists of lipid A and two extra sugars. Emerging genomic sequences indicate that the enzymes that make lipid A in E. coli are present in most other Gram-negative bacteria. Lipid A (often termed endotoxin) is also the active component of LPS responsible for the clinical complications of Gram-negative sepsis. Minor modifications in the structure of lipid A can have profound effects on pathogenesis. Some lipid A analogs are actually potent endotoxin antagonists. Compared to E. coli, the chemical structures of the lipid A and core domains of R. leguminosarum LPS are very unusual. R. leguminosarum lipid A lacks the and 4'phosphates, but is modified with galacturonic acid at position 4'. It is acylated with a peculiar 28 carbon fatty acid, and contains 2-deoxy-2-aminogluconate in place of the proximal glucosamine. The structure of R. leguminosarum LPS indicates the existence of novel enzymes for generating diverse lipid A and core species. It is now established that the first seven enzymes of lipid A biosynthesis are in fact the same in E. coli and R. leguminosarum. The differences arise in the later stages of the pathway. To date, enzymes identified as unique to R. leguminosarum include a 4'-phosphatase that is also a phosphotransferase, a 1-phosphatase, a long chain acyltransferase with its own acyl carrier protein, and three distinct core glycosyltransferases. Characterization of the R. leguminosarum system should provide insights into the function of lipid A-like molecules, including special roles during symbiosis in plants, and affords the opportunity to create novel lipid A hybrids that may have interesting adjuvant or antagonist activities. Some structural features of R. leguminosarum lipid A are seen in human pathogens. Legionella pneumophila lipid A contains a C28 chain, while Porphyromonas gingivalis and Helicobacter pylori lipid A lack the 4' phosphate. In the coming grant period, the specific aims are: I) cloning of the C28 acyltransferase of R. leguminosarum; II) analysis of the lipid A 4'-phosphatase/phosphotransferase, especially its ability to synthesize PtdIns-4-P; III) determination of the enzymatic basis for proximal unit diversity in R. leguminosarum lipid A; and IV) characterization of enzymes that incorporate the unique inner core sugars of R. leguminosarum LPS.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM051796-09
Application #
6603842
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Marino, Pamela
Project Start
1995-07-01
Project End
2004-06-30
Budget Start
2003-07-01
Budget End
2004-06-30
Support Year
9
Fiscal Year
2003
Total Cost
$305,494
Indirect Cost
Name
Duke University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
Sohlenkamp, Christian; Raetz, Christian R H; Ingram, Brian O (2013) The calcium-stimulated lipid A 3-O deacylase from Rhizobium etli is not essential for plant nodulation. Biochim Biophys Acta 1831:1250-9
Sohlenkamp, Christian; Raetz, Christian R H; Ingram, Brian O (2012) The calcium-stimulated lipid A 3-O deacylase from Rhizobium etli is not essential for plant nodulation. Biochim Biophys Acta 1831:1250-9
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Kong, Qingke; Six, David A; Liu, Qing et al. (2011) Palmitoylation state impacts induction of innate and acquired immunity by the Salmonella enterica serovar typhimurium msbB mutant. Infect Immun 79:5027-38
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Ingram, Brian O; Masoudi, Ali; Raetz, Christian R H (2010) Escherichia coli mutants that synthesize dephosphorylated lipid A molecules. Biochemistry 49:8325-37

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