Lipopolysaccharides (LPSs) are remarkable glycolipids that comprise the outer surfaces of Gram-negative bacteria, including the symbiotic organism, Rhizobium leguminosamm. 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 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/?, leguminosarum LPS are very unusual. R. leguminosarum lipid A lacks the 1 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/?, 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 I biosynthesis are in fact the same in E. coli and /?. leguminosarum. The differences arise in the later stages of the pathway. To date, enzymes identified as unique to /?. 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 /?. leguminosarum system should provide insights into the functions 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/?, leguminosarum lipid A are seen in human pathogens. Legionella pneumophila lipid A i 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/?, leguminosarum; i 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 /?. leguminosarum lipid A; and IV) i characterization of enzymes that incorporate the unique inner core sugars of/?, 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 #
5R37GM051796-14
Application #
7459771
Study Section
Special Emphasis Panel (NSS)
Program Officer
Marino, Pamela
Project Start
1998-07-01
Project End
2009-06-30
Budget Start
2008-07-01
Budget End
2009-06-30
Support Year
14
Fiscal Year
2008
Total Cost
$365,050
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
Kong, Qingke; Six, David A; Liu, Qing et al. (2012) Phosphate groups of lipid A are essential for Salmonella enterica serovar Typhimurium virulence and affect innate and adaptive immunity. Infect Immun 80:3215-24
Kanistanon, Duangjit; Powell, Daniel A; Hajjar, Adeline M et al. (2012) Role of Francisella lipid A phosphate modification in virulence and long-term protective immune responses. Infect Immun 80:943-51
Kong, Qingke; Six, David A; Roland, Kenneth L et al. (2011) Salmonella synthesizing 1-dephosphorylated [corrected] lipopolysaccharide exhibits low endotoxic activity while retaining its immunogenicity. J Immunol 187:412-23
Chung, Hak Suk; Raetz, Christian R H (2011) Dioxygenases in Burkholderia ambifaria and Yersinia pestis that hydroxylate the outer Kdo unit of lipopolysaccharide. Proc Natl Acad Sci U S A 108:510-5
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
Lu, Yi-Hsueh; Guan, Ziqiang; Zhao, Jinshi et al. (2011) Three phosphatidylglycerol-phosphate phosphatases in the inner membrane of Escherichia coli. J Biol Chem 286:5506-18
Stead, Christopher M; Zhao, Jinshi; Raetz, Christian R H et al. (2010) Removal of the outer Kdo from Helicobacter pylori lipopolysaccharide and its impact on the bacterial surface. Mol Microbiol 78:837-52
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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