Discovery of new biosynthetic pathways: the lipid A story

Abstract

The outer monolayer of the outer membrane of Gram-negative bacteria consists of the lipid A component of lipopolysaccharide (LPS), a glucosamine-based saccharolipid that is assembled on the inner surface of the inner membrane. The first six enzymes of the lipid A pathway are required for bacterial growth and are excellent targets for the development of new antibiotics. Following assembly, the ABC transporter MsbA flips nascent LPS to the periplasmic side of the inner membrane, whereupon additional transport proteins direct it to the outer surface of the outer membrane. Depending on the bacterium, various covalent modifications of the lipid A moiety may occur during the transit of LPS to the outer membrane. These extra-cytoplasmic modification enzymes are therefore useful as reporters for monitoring LPS trafficking. Because of its conserved structure in diverse Gram-negative pathogens, lipid A is recognized as foreign by the TLR4/MD2 receptor of the mammalian innate immune system, resulting in rapid macrophage activation and robust cytokine production. The outer monolayer of the outer membrane of Gram-negative bacteria consists of the lipid A component of lipopolysaccharide (LPS), a glucosamine-based saccharolipid that is assembled on the inner surface of the inner membrane. The first six enzymes of the lipid A pathway are required for bacterial growth and are excellent targets for the development of new antibiotics. Following assembly, the ABC transporter MsbA flips nascent LPS to the periplasmic side of the inner membrane, whereupon additional transport proteins direct it to the outer surface of the outer membrane. Depending on the bacterium, various covalent modifications of the lipid A moiety may occur during the transit of LPS to the outer membrane. These extra-cytoplasmic modification enzymes are therefore useful as reporters for monitoring LPS trafficking. Because of its conserved structure in diverse Gram-negative pathogens, lipid A is recognized as foreign by the TLR4/MD2 receptor of the mammalian innate immune system, resulting in rapid macrophage activation and robust cytokine production. Although lipid A was recognized over 50 years ago as the hydrophobic moiety of lipopolysaccharide (LPS) (Fig. 1), the elucidation of lipid A biosynthesis was delayed until 1983. A key breakthrough was the discovery and structural characterization of a novel Escherichia coli lipid, termed 2,3-diacylglucosamine 1-phosphate or “lipid X” (Fig. 2) (1Takayama K. Qureshi N. Mascagni P. Nashed M.A. Anderson L. Raetz C.R.H. Fatty acyl derivatives of glucosamine 1-phosphate in Escherichia coli and their relation to lipid A: complete structure of a diacyl GlcN-1-P found in a phosphatidylglycerol-deficient mutant.J. Biol. Chem. 1983; 258: 7379-7385Abstract Full Text PDF PubMed Google Scholar). This substance was overlooked in earlier work with E. coli because of its low levels in wild-type cells, but it accumulates in certain phosphatidylglycerol-deficient mutants (2Nishijima M. Raetz C.R.H. Membrane lipid biogenesis in Escherichia coli: identification of genetic loci for phosphatidylglycerophosphate synthetase and construction of mutants lacking phosphatidylglycerol.J. Biol. Chem. 1979; 254: 7837-7844Abstract Full Text PDF PubMed Google Scholar). The recognition of lipid X coincided with the determination of the correct structure of lipid A (1Takayama K. Qureshi N. Mascagni P. Nashed M.A. Anderson L. Raetz C.R.H. Fatty acyl derivatives of glucosamine 1-phosphate in Escherichia coli and their relation to lipid A: complete structure of a diacyl GlcN-1-P found in a phosphatidylglycerol-deficient mutant.J. Biol. Chem. 1983; 258: 7379-7385Abstract Full Text PDF PubMed Google Scholar, 3Raetz C.R.H. Biochemistry of endotoxins.Annu. Rev. Biochem. 1990; 59: 129-170Crossref PubMed Scopus (1040) Google Scholar, 4Rietschel E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Seydel U.Za¨hringer, U. Padova F.Di al et Bacterial endotoxin: molecular relationships of structure to activity and function.FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1326) Google Scholar). The identification of 2,3-diacylglucosamine 1-phosphate as a precursor of the proximal (right) subunit of lipid A (Fig. 2) was critical for developing testable hypotheses regarding the enzymology and genetics of lipid A assembly (5Raetz C.R.H. Reynolds C.M. Trent M.S. Bishop R.E. Lipid A modification systems in gram-negative bacteria.Annu. Rev. Biochem. 2007; 76: 295-329Crossref PubMed Scopus (933) Google Scholar).Fig. 2The constitutive pathway of lipid A biosynthesis in E. coli.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Given the importance of structure determination for the discovery of new pathways, as illustrated by the lipid A story (5Raetz C.R.H. Reynolds C.M. Trent M.S. Bishop R.E. Lipid A modification systems in gram-negative bacteria.Annu. Rev. Biochem. 2007; 76: 295-329Crossref PubMed Scopus (933) Google Scholar), the LIPID MAPS Consortium is attempting to identify additional novel lipids, using mass spectrometry as the starting point (6Fahy E. Subramaniam S. Brown H.A. Glass C.K. Merrill Jr., A.H. Murphy R.C. Raetz C.R.H. Russell D.W. Seyama Y. Shaw W. al et A comprehensive classification system for lipids.J. Lipid Res. 2005; 46: 839-862Abstract Full Text Full Text PDF PubMed Scopus (1141) Google Scholar). Many as yet unknown lipid pathways probably exist in all organisms with possible roles in signal transduction, regulation, and membrane assembly. Lipid A, the hydrophobic anchor of LPS, is a glucosamine-based lipid that makes up the outer monolayer of the outer membrane (OM) (Fig. 1) of Gram-negative bacteria (5Raetz C.R.H. Reynolds C.M. Trent M.S. Bishop R.E. Lipid A modification systems in gram-negative bacteria.Annu. Rev. Biochem. 2007; 76: 295-329Crossref PubMed Scopus (933) Google Scholar, 7Raetz C.R.H. Whitfield C. Lipopolysaccharide endotoxins.Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3382) Google Scholar). An E. coli cell contains ∼106 lipid A residues and ∼107 glycerophospholipids (7Raetz C.R.H. Whitfield C. Lipopolysaccharide endotoxins.Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3382) Google Scholar). The lipid A and Kdo domains of LPS (Figs. 1 and 2) are usually required for growth (7Raetz C.R.H. Whitfield C. Lipopolysaccharide endotoxins.Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3382) Google Scholar), but Kdo can be eliminated in the presence of suppressors (8Meredith T.C. Aggarwal P. Mamat U. Lindner B. Woodard R.W. Redefining the requisite lipopolysaccharide structure in Escherichia coli.ACS Chem. Biol. 2006; 1: 33-42Crossref PubMed Scopus (115) Google Scholar). Wild-type LPS contains additional core and O-antigen sugars, which are not needed for growth, but protect against antibiotics and complement (7Raetz C.R.H. Whitfield C. Lipopolysaccharide endotoxins.Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3382) Google Scholar). The constitutive enzymes of the lipid A pathway are conserved (7Raetz C.R.H. Whitfield C. Lipopolysaccharide endotoxins.Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3382) Google Scholar). They are located in the cytoplasm or on the inner surface of the inner membrane (IM) (7Raetz C.R.H. Whitfield C. Lipopolysaccharide endotoxins.Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3382) Google Scholar). The first step is the acylation of UDP-GlcNAc (Fig. 2), catalyzed by LpxA. In E. coli LpxA is selective for β-hydroxymyristoyl acyl carrier protein (9Anderson M.S. Raetz C.R.H. Biosynthesis of lipid A precursors in Escherichia coli: a cytoplasmic acyltransferase that converts UDP-N-acetylglucosamine to UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine.J. Biol. Chem. 1987; 262: 5159-5169Abstract Full Text PDF PubMed Google Scholar). The active site of E. coli LpxA functions as an accurate hydrocarbon ruler that incorporates a β-hydroxymyristoyl chain two orders of magnitude faster than a β-hydroxylauroyl or a β-hydroxypalmitoyl chain (10Wyckoff T.J.O. Lin S. Cotter R.J. Dotson G.D. Raetz C.R.H. Hydrocarbon rulers in UDP-N-acetylglucosamine acyltransferases.J. Biol. Chem. 1998; 273: 32369-32372Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Other LpxA orthologs are designed to incorporate longer or shorter β-hydroxyacyl chains (7Raetz C.R.H. Whitfield C. Lipopolysaccharide endotoxins.Annu. Rev. Biochem. 2002; 71: 635-700Crossref PubMed Scopus (3382) Google Scholar). The equilibrium constant (∼0.01) for UDP-GlcNAc acylation is unfavorable (11Anderson M.S. Bull H.S. Galloway S.M. Kelly T.M. Mohan S. Radika K. Raetz C.R.H. UDP-N-acetylglucosamine acyltransferase of Escherichia coli: the first step of endotoxin biosynthesis is thermodynamically unfavorable.J. Biol. Chem. 1993; 268: 19858-19865Abstract Full Text PDF PubMed Google Scholar). The deacetylation of UDP-3-O-(acyl)-GlcNAc by LpxC is therefore the committed step. LpxC is a Zn2+-dependent enzyme (12Jackman J.E. Raetz C.R.H. Fierke C.A. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase of Escherichia coli is a zinc metalloenzyme.Biochemistry. 1999; 38: 1902-1911Crossref PubMed Scopus (117) Google Scholar), conserved in diverse Gram-negative bacteria, and is an excellent target for new antibiotics (13Onishi H.R. Pelak B.A. Gerckens L.S. Silver L.L. Kahan F.M. Chen M.H. Patchett A.A. Galloway S.M. Hyland S.A. Anderson M.S. al et Antibacterial agents that inhibit lipid A biosynthesis.Science. 1996; 274: 980-982Crossref PubMed Scopus (357) Google Scholar, 14McClerren A.L. Endsley S. Bowman J.L. Andersen N.H. Guan Z. Rudolph J. Raetz C.R.H. A slow, tight-binding inhibitor of the zinc-dependent deacetylase LpxC of lipid A biosynthesis with antibiotic activity comparable to ciprofloxacin.Biochemistry. 2005; 44: 16574-16583Crossref PubMed Scopus (128) Google Scholar). Slow, tight-binding inhibitors of LpxC, such as CHIR-090, have antibiotic activity comparable to ciprofloxacin and are effective against almost all Gram-negatives (14McClerren A.L. Endsley S. Bowman J.L. Andersen N.H. Guan Z. Rudolph J. Raetz C.R.H. A slow, tight-binding inhibitor of the zinc-dependent deacetylase LpxC of lipid A biosynthesis with antibiotic activity comparable to ciprofloxacin.Biochemistry. 2005; 44: 16574-16583Crossref PubMed Scopus (128) Google Scholar), including E. coli, Pseudomonas aeruginosa, and Francisella novicida. Following deacetylation, a second β-hydroxymyristoyl chain is added by E. coli LpxD to make UDP-2,3-diacyl-GlcN (Fig. 2) (15Bartling C.M. Raetz C.R.H. Steady-state kinetics and mechanism of LpxD, the N-acyltransferase of lipid A biosynthesis.Biochemistry. 2008; 47: 5290-5302Crossref PubMed Scopus (45) Google Scholar), which is cleaved by LpxH to form 2,3-diacyl-GlcN-1-phosphate (lipid X) (16Babinski K.J. Ribeiro A.A. Raetz C.R.H. The Escherichia coli gene encoding the UDP-2,3-diacylglucosamine pyrophosphatase of lipid A biosynthesis.J. Biol. Chem. 2002; 277: 25937-25946Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). A β,1′-6 linked disaccharide is then generated by LpxB, which condenses another molecule of UDP-2,3-diacyl-GlcN with lipid X (17Radika K. Raetz C.R.H. Purification and properties of lipid A disaccharide synthase of Escherichia coli.J. Biol. Chem. 1988; 263: 14859-14867Abstract Full Text PDF PubMed Google Scholar). LpxA, LpxC, and LpxD are soluble proteins with known structures (18Raetz C.R.H. Roderick S.L. A left-handed parallel β helix in the structure of UDP-N-acetylglucosamine acyltransferase.Science. 1995; 270: 997-1000Crossref PubMed Scopus (297) Google Scholar, 19Coggins B.E. McClerren A.L. Jiang L. Li X. Rudolph J. Hindsgaul O. Raetz C.R.H. Zhou P. 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MsbA lipid A on the periplasmic surface of the inner of Francisella in Escherichia coli.J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google (Figs. and on the outer surface of the The of is not The have been and in E. coli, their in MsbA IM for X. Cotter R.J. Raetz C.R.H. and periplasmic of the Francisella lipid A Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus (77) Google Scholar, X. Cotter R.J. Raetz C.R.H. MsbA lipid A on the periplasmic surface of the inner of Francisella in Escherichia coli.J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In the is in lipid A X. Ribeiro A.A. Guan Z. S. Cotter Raetz C.R.H. Structure and biosynthesis of lipid A that lipopolysaccharide in Francisella 2006; PubMed Scopus Google and lipid A (Fig. E. analysis of Francisella J. Biochem. 2002; PubMed Scopus Google Scholar, B. M.A. modification of lipid A of Francisella PubMed Scopus Google Scholar). the is (Fig. X. Cotter R.J. Raetz C.R.H. MsbA lipid A on the periplasmic surface of the inner of Francisella in Escherichia coli.J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the 1-phosphate is in lipid A, but is in of the LPS (Fig. E. analysis of Francisella J. Biochem. 2002; PubMed Scopus Google Scholar). of in of the and the chain X. Ribeiro A.A. Guan Z. Raetz C.R.H. of a Francisella lacking the lipid A Natl. Acad. Sci. USA. 2007; 104: PubMed Scopus Google (Fig. (Fig. mutants are X. Ribeiro A.A. Guan Z. Raetz C.R.H. of a Francisella lacking the lipid A Natl. Acad. Sci. USA. 2007; 104: PubMed Scopus Google and can be to against wild-type and C. The moiety (Fig. which is on lipid A and on lipid A (Fig. is by of E. coli on the outer surface of the IM X. Ribeiro A.A. Guan Z. S. Cotter Raetz C.R.H. Structure and biosynthesis of lipid A that lipopolysaccharide in Francisella 2006; PubMed Scopus Google Scholar, T. S.A. L. al et A Francisella in lipid A modification 2008; PubMed Scopus Google Scholar). The is (Fig. which is generated and and C. The of the chain (Fig. probably in the (Fig. as in the with C.M. Ribeiro A.A. Cotter R.J. Raetz C.R.H. Trent M.S. An outer membrane enzyme by that the moiety of lipid A.J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar), but the gene not yet been is and C. but its LPS contains one Kdo E. analysis of Francisella J. Biochem. 2002; PubMed Scopus Google Scholar). This is in by the presence of a Kdo in X. Cotter R.J. Raetz C.R.H. MsbA lipid A on the periplasmic surface of the inner of Francisella in Escherichia coli.J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), which consists of a and a termed and and C. This of is found in C. A. Jr., S. Cotter Trent S. A novel that the outer Kdo of 2005; PubMed Scopus Google Scholar). of in is not but it LPS and of lipid A and C. the constitutive lipid A pathway is the systems for lipid A modification are diverse and are usually not required for growth (5Raetz C.R.H. Reynolds C.M. Trent M.S. Bishop R.E. Lipid A modification systems in gram-negative bacteria.Annu. Rev. Biochem. 2007; 76: 295-329Crossref PubMed Scopus (933) Google Scholar). the modification enzymes have in is their (Fig. (5Raetz C.R.H. Reynolds C.M. Trent M.S. Bishop R.E. Lipid A modification systems in gram-negative bacteria.Annu. Rev. Biochem. 2007; 76: 295-329Crossref PubMed Scopus (933) Google Scholar). (5Raetz C.R.H. Reynolds C.M. Trent M.S. Bishop R.E. Lipid A modification systems in gram-negative bacteria.Annu. Rev. Biochem. 2007; 76: 295-329Crossref PubMed Scopus (933) Google Scholar), or modification of the lipid A on the outer surface of the of acyl chains in the (5Raetz C.R.H. Reynolds C.M. Trent M.S. Bishop R.E. Lipid A modification systems in gram-negative bacteria.Annu. Rev. Biochem. 2007; 76: 295-329Crossref PubMed Scopus (933) Google (Fig. The of the modification in to the constitutive enzymes (Fig. 2), makes the excellent reporters for LPS H.S. Raetz C.R.H. of the inner membrane of Escherichia coli.J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, N. L.S. Identification of two proteins required for the transport of lipopolysaccharide to the outer membrane of Escherichia Natl. Acad. Sci. USA. 2008; PubMed Scopus Google Scholar). Lipid A modification enzymes can be one bacterial to of in E. coli or in of lipid A growth X. Cotter R.J. Raetz C.R.H. MsbA lipid A on the periplasmic surface of the inner of Francisella in Escherichia coli.J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of in E. coli or LpxM mutants lipid in the complete of the X. Cotter R.J. Raetz C.R.H. and periplasmic of the Francisella lipid A Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus (77) Google Scholar). wild-type lipid A X. Cotter R.J. Raetz C.R.H. and periplasmic of the Francisella lipid A Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The of mutants their to because of the by the lipid A (Fig. X. Ribeiro A.A. Guan Z. Raetz C.R.H. of a Francisella lacking the lipid A Natl. Acad. Sci. USA. 2007; 104: PubMed Scopus Google Scholar). it is possible to novel bacterial by or lipid A modification enzymes X. Cotter R.J. Raetz C.R.H. and periplasmic of the Francisella lipid A Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus (77) Google Scholar, X. Ribeiro A.A. Guan Z. Raetz C.R.H. of a Francisella lacking the lipid A Natl. Acad. Sci. USA. 2007; 104: PubMed Scopus Google Scholar). Many Gram-negatives lipid A of E. coli (Fig. 2) (5Raetz C.R.H. Reynolds C.M. Trent M.S. Bishop R.E. Lipid A modification systems in gram-negative bacteria.Annu. Rev. Biochem. 2007; 76: 295-329Crossref PubMed Scopus (933) Google Scholar). lipid A is by the TLR4/MD2 the structure of which was U. K. K. Y. Crystal structures of and its complex with lipid 2007; PubMed Scopus Google Scholar, J. P. N. H. al et Crystal structure of the complex with endotoxin antagonist 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). E. coli lipid A to make of such as and E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Seydel U.Za¨hringer, U. Padova F.Di al et Bacterial endotoxin: molecular relationships of structure to activity and function.FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1326) Google Scholar). These are for but during proteins can and to of J. 2006; PubMed Scopus Google Scholar). The structural of E. coli lipid A (Fig. 2), its two and are needed to TLR4/MD2 activation E.T. Kirikae T. Schade F.U. Mamat U. Schmidt G. Loppnow H. Ulmer A.J. Seydel U.Za¨hringer, U. Padova F.Di al et Bacterial endotoxin: molecular relationships of structure to activity and function.FASEB J. 1994; 8: 217-225Crossref PubMed Scopus (1326) Google Scholar). the of modification enzymes the lipid A moiety of bacteria can be by X. Cotter R.J. Raetz C.R.H. MsbA lipid A on the periplasmic surface of the inner of Francisella in Escherichia coli.J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google to be a TLR4/MD2 its properties its P. C. S. a on receptor as and Biol. PubMed Scopus Google Scholar). The regarding lipid A and to be 1) is the lipid A moiety of LPS required for growth in Gram-negative of N. in which lipid A is not required for growth, the J. of the gram-negative bacterial outer Rev. 2007; PubMed Scopus Google 2) in for LPS and transport be The genetic for the of the MsbA transporter and the protein complex is but it to be with in new antibiotics be by LpxC and enzymes of the lipid A The LpxC inhibitors (14McClerren A.L. Endsley S. Bowman J.L. Andersen N.H. Guan Z. Rudolph J. Raetz C.R.H. A slow, tight-binding inhibitor of the zinc-dependent deacetylase LpxC of lipid A biosynthesis with antibiotic activity comparable to ciprofloxacin.Biochemistry. 2005; 44: 16574-16583Crossref PubMed Scopus (128) Google are for but of and have not been and lipid A modification enzymes be for The by mutants of X. Ribeiro A.A. Guan Z. Raetz C.R.H. of a Francisella lacking the lipid A Natl. Acad. Sci. USA. 2007; 104: PubMed Scopus Google or lacking are H. S. A. A. J. R.J. N. C. encoding acylation of lipid A are required for to and of the 2002; PubMed Scopus (117) Google Scholar). The properties of which the form of lipid A, be of