Lipid A Modifications in Polymyxin-resistant Salmonella typhimurium

Abstract

Salmonella typhimurium and related organisms synthesize lipid A by the same pathway as Escherichia coli K-12 (1, 2), but they usually modify the final product with additional covalent appendages (Fig. 1A), such as 4-amino-4-deoxy-L-arabinose (L-Ara4N) 1 (3-7), phosphoethanolamine (pEtN) (4 -6), (S)-2-hydroxymyristate (8, 9), and palmitate (5, 6, 10 -12). Different combinations of these substituents account for the remarkable heterogeneity of lipid A molecules found in S. typhimurium.The biosynthesis of lipid A modifications is under the control of the PhoP/PhoQ and the PmrA/PmrB two-component signaling systems (13-15). Addition of the L-Ara4N unit is required for resistance to polymyxin (16 -18). Incorporation of the palmitoyl chain confers resistance to certain cationic anti-microbial peptides (11). Modification of lipid A with L-Ara4N, pEtN, and/or palmitate may also occur in E. coli K-12, but only under special circumstances, as in polymyxin-resistant (pmrA constitutive) mutants (17) or in wild type cells exposed to ammonium metavanadate (7,19).With the exception of PagP, the outer membrane enzyme that incorporates palmitate (12, 20), the enzymes responsible for the covalent modifications of lipid A have not been identified. The L-Ara4N residue is attached primarily to the 4Ј-phosphate group of lipid A in wild type S. typhimurium or in metavanadate-treated E. coli, whereas pEtN is usually attached to the 1-phosphate (19). However, in S. typhimurium mutants defective in Kdo biosynthesis, lipid A precursors accumulate in which L-Ara4N is linked exclusively to the 1-phosphate, and pEtN is attached mainly to the 4Ј-phosphate (5,6,19). The enzymatic pathways that account for these interesting and complex structural anomalies are unknown.An important clue to the origin of the L-Ara4N moiety has emerged from the discovery of the pmrE and pmrF genes, which are required for the maintenance of polymyxin resistance and the biosynthesis of L-Ara4N-modified lipid A (18). The pmrE (ugd) gene encodes UDP-glucose dehydrogenase (18), suggesting that L-Ara4N is derived from UDP-glucuronic acid. The pmrF gene encodes a homologue of yeast dolichyl phosphate-mannose synthase and is part of an operon (18) that includes additional open reading frames hypothesized to encode other putative enzymes required for L-Ara4N biosynthesis and attachment to lipid A (7, 21). The operon is regulated directly by PmrA (18), which in turn may be activated by PhoP/PhoQ, low pH, or ferric ions (14,22,23). So far, no in vitro assays have been developed to validate the functions of the proteins encoded by the pmrF operon.Because of its heterogeneity (Fig. 1A), S. typhimurium lipid * This work was supported by National Institutes of Health Grants GM-51310 (to C. R. H.R.), AI-30479 (to S. I. M.), and GM54882 (to R. J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.** To w...

Oxidative Decarboxylation of UDP-Glucuronic Acid in Extracts of Polymyxin-resistant Escherichia coli

“…The covalent modification of the phosphate groups of lipid A with L-Ara4N moieties confers resistance to polymyxin and cationic anti-microbial peptides in E. coli and S. typhimurium (17,18,20,25,27,28). The L-Ara4N unit is thought to arise by a novel pathway from UDP-GlcA.…”

“…Substituents attached to lipid A in various combinations include palmitate, 2-hydroxymyristate, phosphoethanolamine (pEtN), 1 and 4-amino-4-deoxy-L-arabinose (L-Ara4N) ( Fig. 1) (14,24,25). PmrA-PmrB, a two-component regulatory system activated by low pH, PhoP/PhoQ, or certain pmrA mutations (15), is required for the modification of lipid A with pEtN and L-Ara4N moieties (20,24,25), resulting in less anionic lipid A species that do not bind cationic antimicrobial substances as strongly and prevent them from penetrating the outer membrane.…”

Rhizobium etli CE3 Bacteroid Lipopolysaccharides Are Structurally Similar but Not Identical to Those Produced by Cultured CE3 Bacteria

“…These molecules often act via a combination of their positive charge, which enables them to interact with anionic molecules on the bacterial surface, in combination with their hydrophobic character, which results in a disruption of the bacterial membrane (54,55). The LPS is the target molecule of defensins in Gram-negative bacteria, and bacteria that are resistant acquire this resistance by modification to their LPS structures (56 -60); perhaps, one of the best known examples is the resistance acquired through the addition of aminoarabinose and ethanol amine groups to the LPS of S. typhimurium (57)(58)(59)(60). Because of the structural alteration of the LPS during bacteroid formation, bacteria and bacteroids were compared for their resistance to poly-L-lysine, polymyxin B, and melittin.…”

ATPase Activity of the MsbA Lipid Flippase of Escherichia coli

“…Phosphatidylethanolamine was not bound to the column, whereas phosphatidylglycerol and other phospholipids eluted in the 60 -120 mM fractions. The Kdo 2 -lipid A 1,4Ј-bis-phosphate species eluted in the 240 mM fraction, but the Kdo 2 -lipid A 1-pyrophosphate variant (32,33) eluted in the 360 mM fractions. The fractions containing the purified Kdo 2 -lipid A 1,4Ј-bis-phosphate were pooled and converted to a neutral two-phase Bligh-Dyer system by the addition of appropriate amounts of chloroform and water.…”