Infection causes disturbances in lipid metabolism that may be mediated by cytokines. Therefore we studied plasma lipids, lipoproteins, triglyceride (TG) metabolism, and serum cytokines in three groups: patients with the acquired immunodeficiency syndrome (AIDS) without active secondary infection, patients with evidence of human immunodeficiency virus infection but without clinical AIDS (HIV+), and controls. Plasma TGs and FFA were increased in AIDS, while plasma cholesterol, high density lipoprotein (HDL) cholesterol, apolipoprotein-A-1 (Apo-A-1), low density lipoprotein (LDL) cholesterol, and Apo-B-100 levels were decreased. Increased TG levels in AIDS were primarily due to increases in very low density lipoprotein of normal composition; in addition, LDL and HDL were TG enriched. In HIV+, TGs and FFA were not increased, but total cholesterol, HDL cholesterol, Apo-A-1, and Apo-B-100 were significantly decreased. Interferon-alpha (IFN alpha) and C-reactive protein levels were increased in AIDS, but tumor necrosis factor and haptoglobin levels were not. There was a significant correlation between plasma TGs and IFN alpha levels (r = 0.477; P less than 0.01), but not between TGs and tumor necrosis factor, C-reactive protein, haptoglobin, or P-24 antigen. In addition, there was no relationship between circulating IFN alpha levels and plasma cholesterol, HDL cholesterol, Apo-A-1, LDL cholesterol, Apo-B-100, or FFA. TG clearance time and postheparin lipase were significantly decreased in AIDS and HIV+. There was a strong correlation between serum IFN alpha levels and TG clearance time in AIDS and HIV+ (r = 0.783; P less than 0.001). In summary, decreases in cholesterol and cholesterol containing lipoproteins (including HDL) in both AIDS and HIV+ precede the appearance of hypertriglyceridemia and are not related to IFN alpha or TG levels. Our data raise the possibility that with development of AIDS, subsequent increases in IFN alpha may contribute to increases in plasma TG levels in part by decreasing the clearance of TG.

Escherichia coli phospholipids and lipopolysaccharide, made on the inner surface of the inner membrane, are rapidly transported to the outer membrane by mechanisms that are not well characterized. We now report a temperature-sensitive mutant (WD2) with an A270T substitution in a trans-membrane region of the ABC transporter MsbA. As shown by 32 P i and 14 C-acetate labeling, export of all major lipids to the outer membrane is inhibited by ϳ90% in WD2 after 30 min at 44°C. Transport of newly synthesized proteins is not impaired. Electron microscopy shows reduplicated inner membranes in WD2 at 44°C, consistent with a key role for MsbA in lipid trafficking.The envelope of Gram-negative bacteria consists of an inner membrane, the peptidoglycan cell wall, and an outer membrane ( Fig. 1A) (1). The latter is an asymmetric bilayer with glycerophospholipids ( Fig. 1B) on its inner surface and lipid A (Fig. 1B), the hydrophobic anchor of lipopolysaccharide (2, 3), on the outside (1). The lipid A moiety is a hexa-acylated disaccharide of glucosamine unique to Gram-negative bacteria (4) and is a potent activator of innate immunity in animals via the receptor TLR-4 (5-7). The enzymes that make phospholipids and lipid A are well characterized in Escherichia coli (3,8,9). They are located in the cytoplasm or inner membrane and are targets for the design of novel antibacterial agents (10,11).How E. coli lipids cross the inner membrane and are transported to the outer membrane is unknown (Fig. 1A). A clue to lipopolysaccharide transport has recently emerged from studies of E. coli htrB mutants (12) and their suppression by msbA (13-15). HtrB is a lauroyl transferase that functions late in lipid A biosynthesis ( Fig. 1A) (12). Lipopolysaccharides bearing tetra-acylated lipid A species accumulate in the inner membrane of htrB mutants at 44°C, inhibiting growth (15). MsbA is an essential ABC transporter (Fig. 2), closely related to eucaryotic Mdr proteins (13-15). MsbA overexpression restores growth of htrB mutants at 44°C without restoring laurate incorporation, resulting in export of lipopolysaccharide with tetra-acylated lipid A anchors to the outer membrane (15).E. coli msbA knockouts are lethal. However, their biochemical analysis is complicated by long times (4 -8 h) needed to dilute out pre-existing MsbA supplied in trans from a temperaturesensitive plasmid (15) and by the fact that the lpxK gene, which is immediately downstream in an operon with MsbA, is also essential for cell growth (16).We now report the isolation and characterization of a novel temperature-sensitive point mutant of E. coli that harbors a single amino acid substitution in MsbA. Rapid inhibition of MsbA function in vivo following a shift of mid log phase cells from 30 to 44°C results in the immediate arrest of the export of all newly made lipopolysaccharide and phospholipids, demonstrating that MsbA is an essential component of a general lipid transport system in E. coli. EXPERIMENTAL PROCEDURESIsolation of Mutant WD2 and Growth Conditions-WD2 was isolated...

Polymyxin-resistant mutants of Escherichia coli andPolyisoprene-linked sugars function as donor substrates for many types of glycosyltransferases (1). In eubacteria, undecaprenyl moieties (1, 2) serve as lipid carriers for sugar residues that are transferred to acceptors located outside of the cytoplasm, where sugar nucleotides are not available. Undecaprenyl diphosphate-sugars (generally oligosaccharides) are precursors for polymerization of O-antigen (3-6), enterobacterial common antigen (7,8), and peptidoglycan (1, 9 -11). Undecaprenyl phosphate-sugars (typically monosaccharides) are thought to be donors for processes that include bacteriophagemediated O-antigen conversion (12, 13), glycosylation of teichoic acids (14 -17), and biosynthesis of mycobacterial lipoglycans (18 -21). In eucaryotic cells, the structurally related dolichyl phosphate-sugars and dolichyl diphosphate-sugars (1, 22-24) play important roles in various stages of protein glycosylation and in the assembly of phosphatidylinositol-linked glycans.As demonstrated in the preceding article (25), a membranebound donor, proposed to be undecaprenyl phosphate-␣-LAra4N 1 (Fig. 1) 2 based upon bioinformatic considerations (26, 27), is required for the modification of lipid A with 4-amino-4-deoxy-L-arabinose (L-Ara4N) units in polymyxin-resistant mutants of Escherichia coli and Salmonella typhimurium. A novel L-Ara4N transferase, encoded by arnT (previously designated orf5 or pmrK) (28, 29), catalyzes L-Ara4N transfer to lipid A-like molecules in vitro when membranes of polymyxin-resistant mutants are employed as the source of the L-Ara4N donor (25). The formation of L-Ara4N and its transfer to lipid A are induced by activation of the transcription factor PmrA, which may occur by mutation (30 -32), by activation of PhoP (33, 34), or by exposure of cells to mildly acidic pH, ferric ions, or metavanadate (26,35,36). Attachment of the positively charged L-Ara4N moiety to lipid A is critical for resistance to the antibiotic polymyxin and to certain cationic antimicrobial peptides present inside phagocytic cells (37,38).We now report the purification and structural characterization of a novel, minor lipid that accumulates in polymyxinresistant mutants of E. coli and S. typhimurium. The purified lipid functions as a donor of L-Ara4N residues in the ArnTcatalyzed modification of lipid A in vitro. MALDI/TOF mass spectrometry and high resolution NMR spectroscopy strongly * This work was supported in part by National Institutes of Health Grants GM-51310 (to C. R. H. R.) 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.

MsbA is an essential ABC transporter in Escherichia coli required for exporting newly synthesized lipids from the inner to the outer membrane. It remains uncertain whether or not MsbA catalyzes trans-bilayer lipid movement (i.e. flip-flop) within the inner membrane. We now show that newly synthesized lipid A accumulates on the cytoplasmic side of the inner membrane after shifting an E. coli msbA missense mutant to the non-permissive temperature. This conclusion is based on the selective inhibition of periplasmic, but not cytoplasmic, covalent modifications of lipid A that occur in polymyxin-resistant strains of E. coli. The accessibility of newly synthesized phosphatidylethanolamine to membrane impermeable reagents, like 2,4,6-trinitrobenzene sulfonic acid, is also reduced severalfold. Our data showed that MsbA facilitates the rapid translocation of some lipids from the cytoplasmic to the periplasmic side of the inner membrane in living cells.

Escherichia coli