title: Expression of a Porphyromonas gingivalis lipid A palmitylacyltransferase in Escherichia coli yields a chimeric lipid A with altered ability to stimulate interleukin-8 secretion.
abstract: In Escherichia coli the gene htrB codes for an acyltransferase that catalyses the incorporation of laurate into lipopolysaccharide (LPS) as a lipid A substituent. We describe the cloning, expression and characterization of a Porphyromonas gingivalis htrB homologue. When the htrB homologue was expressed in wild-type E. coli or a mutant strain deficient in htrB, a chimeric LPS with altered lipid A structure was produced. Compared with wild-type E. coli lipid A, the new lipid A species contained a palmitate (C16) in the position normally occupied by laurate (C12) suggesting that the cloned gene performs the same function as E. coli htrB but preferentially transfers the longer-chain palmitic acid that is known to be present in P. gingivalis LPS. LPS was purified from wild-type E. coli, the E. coli htrB mutant strain and the htrB mutant strain expressing the P. gingivalis acyltransferase. LPS from the palmitate bearing chimeric LPS as well as the htrB mutant exhibited a reduced ability to activate human embryonic kidney 293 (HEK293) cells transfected with TLR4/MD2. LPS from the htrB mutant also had a greatly reduced ability to stimulate interleukin-8 (IL-8) secretion in both endothelial cells and monocytes. In contrast, the activity of LPS from the htrB mutant bacteria expressing the P. gingivalis gene displayed wild-type activity to stimulate IL-8 production from endothelial cells but a reduced ability to stimulate IL-8 secretion from monocytes. The intermediate activation observed in monocytes for the chimeric LPS was similar to the pattern seen in HEK293 cells expressing TLR4/MD2 and CD14. Thus, the presence of a longer-chain fatty acid on E. coli lipid A altered the activity of the LPS in monocytes but not endothelial cell assays and the difference in recognition does not appear to be related to differences in Toll-like receptor utilization.
authors.affiliation: Department of Oral Biology, University of Washington, Seattle, WA 98195, USA.
authors.forename: Brian W
authors.initials: BW
authors.lastname: Bainbridge
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Coats
authors.forename: Thu-Thao T
authors.initials: TT
authors.lastname: Pham
authors.forename: Robert A
authors.initials: RA
authors.lastname: Reife
authors.forename: Richard P
authors.initials: RP
authors.lastname: Darveau
mesh_terms: D000217:Acyltransferases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000235:genetics; D006801:Humans; D016209:Interleukin-8 / Q000378:metabolism*; D007848:Laurates / Q000378:metabolism; D008050:Lipid A / Q000096:biosynthesis* / Q000302:isolation & purification; D018950:Lipopolysaccharide Receptors / Q000235:genetics / Q000378:metabolism; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism; D009154:Mutation; D010168:Palmitates / Q000378:metabolism*; D016966:Porphyromonas gingivalis / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D016209:Interleukin-8; D007848:Laurates; D008050:Lipid A; D018950:Lipopolysaccharide Receptors; D010168:Palmitates; D011994:Recombinant Proteins; D051197:Toll-Like Receptor 4; D000217:Acyltransferases; C492237:LpxL protein, E coli