No abstract

Helicobacter pylori infection causes gastric pathology such as ulcer and carcinoma. Because H. pylori is auxotrophic for cholesterol, we have explored the assimilation of cholesterol by H. pylori in infection. Here we show that H. pylori follows a cholesterol gradient and extracts the lipid from plasma membranes of epithelial cells for subsequent glucosylation. Excessive cholesterol promotes phagocytosis of H. pylori by antigen-presenting cells, such as macrophages and dendritic cells, and enhances antigen-specific T cell responses. A cholesterol-rich diet during bacterial challenge leads to T cell-dependent reduction of the H. pylori burden in the stomach. Intrinsic alpha-glucosylation of cholesterol abrogates phagocytosis of H. pylori and subsequent T cell activation. We identify the gene hp0421 as encoding the enzyme cholesterol-alpha-glucosyltransferase responsible for cholesterol glucosylation. Generation of knockout mutants lacking hp0421 corroborates the importance of cholesteryl glucosides for escaping phagocytosis, T cell activation and bacterial clearance in vivo. Thus, we propose a mechanism regulating the host-pathogen interaction whereby glucosylation of a lipid tips the scales towards immune evasion or response.

Background:The mechanism of coordination between LPS synthesis and translocation is unknown. Results: Two new proteins, LapA and LapB, co-purify with LPS transport proteins. lapB mutants display defects in lipid A and core assembly. Conclusion: lapB mutants accumulate precursor LPS core species and exhibit elevated levels of LpxC. Significance: Coordinated assembly of LPS is a critical step for targeting to the outer membrane.

Yersinia pestis strain Yreka was grown at 27 or 37°C, and the lipid A structures (lipid A-27°C and lipid A-37°C) of the respective lipopolysaccharides (LPS) were investigated by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Lipid A-27°C consisted of a mixture of tri-acyl, tetra-acyl, penta-acyl, and hexa-acyl lipid A's, of which tetra-acyl lipid A was most abundant. Lipid A-37°C consisted predominantly of tri-and tetra-acylated molecules, with only small amounts of penta-acyl lipid A; no hexa-acyl lipid A was detected. Furthermore, the amount of 4-amino-arabinose was substantially higher in lipid A-27°C than in lipid A-37°C. By use of mouse and human macrophage cell lines, the biological activities of the LPS and lipid A preparations were measured via their abilities to induce production of tumor necrosis factor alpha (TNF-␣). In both cell lines the LPS and the lipid A from bacteria grown at 27°C were stronger inducers of TNF-␣ than those from bacteria grown at 37°C. However, the difference in activity was more prominent in human macrophage cells. These results suggest that in order to reduce the activation of human macrophages, it may be more advantageous for Y. pestis to produce less-acylated lipid A at 37°C.Yersinia pestis was isolated as a causative agent of plague in Hong-Kong in 1894 independently by A. Yersin and S. Kitasato (3). Since then, this highly pathogenic bacterium has been investigated, and many virulence factors have been identified, including fraction 1 antigen, murine toxin, Yop proteins, pH 6 antigen, and iron acquisition systems (5, 28). Lipopolysaccharide (LPS) has also been studied for many decades as one of the virulence factors of Y. pestis, and its composition and endotoxic activity have been examined in earlier studies (1,40,41). By use of modern analytical methods, the LPS of Y. pestis was proven to be a rough type LPS without O-antigenic polysaccharide (7,8,25,29,30,35) that contains 3-hydroxy-myristic acid (3-OH-C 14:0 ) as a main fatty acid in the lipid A portion. However, there was a discrepancy in the amounts of the nonhydroxy-fatty acids found. Hartley et al. reported that nearly 90% of fatty acids in the lipid A of Y. pestis strain 195/R (a virulent strain) consisted of 3-OH-C 14:0 (14). On the other hand, considerable amounts of lauric acid (C 12:0 ), palmitic acid (C 16:0 ), and palmitoleic acid (C 16:1 ) were detected in the lipid A of Y. pestis strain EV40 (an avirulent strain) (4, 39). Using the same strain, Aussel et al. (2) proposed a hexa-acylated lipid A structure (four molecules of 3-OH-C 14:0 , one of C 12:0 , and one of C 16:1 ). In our preliminary experiment we isolated the lipid A from Y. pestis strain Yreka (virulent strain) grown at 37°C and found that it contained 3-OH-C 14:0 and only trace amounts of other fatty acids. A possible explanation of the controversy on fatty acids may be differences in strains or culture conditions. Darveau et al. (8) reported that the molecular size of the LPS from Y. pestis strain EV76 ...

For the elucidation of the very early steps of immune cell activation by endotoxins (lipopolysaccharide, LPS) leading to the production and release of proinflammatory cytokines the question concerning the biologically active unit of endotoxins has to be addressed: are monomeric endotoxin molecules able to activate cells or is the active unit represented by larger endotoxin aggregates? This question has been answered controversially in the past. Inspired by the observation that natural isolates of lipid A, the lipid moiety of LPS harboring its endotoxic principle, from Escherichia coli express a higher endotoxic activity than the same amounts of the synthetic E. coli-like hexaacylated lipid A (compound 506), we looked closer at the chemical composition of natural isolates. We found in these isolates that the largest fraction was hexaacylated, but also significant amounts of penta-and tetraacylated molecules were present that, when administered to human mononuclear cells, may antagonize the induction of cytokines by biologically active hexaacylated endotoxins. We prepared separate aggregates of either compound 506 or 406 (tetraacylated precursor IVa), mixed at different molar ratios, and mixed aggregates containing both compounds in the same ratios. Surprisingly, the latter mixtures showed higher endotoxic activity than that of the pure compound 506 up to an admixture of 20% of compound 406. Similar results were obtained when using various phospholipids instead of compound 406. These observations can only be understood by assuming that the active unit of endotoxins is the aggregate. We further confirmed this result by preparing monomeric lipid A and LPS by a dialysis procedure and found that, at the same concentrations, only the aggregates were biologically active, whereas the monomers showed no activity. Bacterial lipopolysaccharide (LPS)1 is one of the most potent activators of the immune system in mammals. During cell growth or as a result of the action of antibacterial host factors or antimicrobial peptides, LPS is released from the outer leaflet of the cell wall of Gram-negative bacteria. Manifold interactions of LPS with host factors have been described, such as the activation of the complement system, activation of immune cells, and interaction with a variety of serum proteins, to name only a few. The most prominent activity of LPS is its immunostimulatory potency leading to the complex clinical syndrome of Gram-negative sepsis when the initial host response to an infection becomes dysregulated. The clinical manifestation of sepsis is characterized by fever, hypotension, respiratory and renal failure, and intravascular disseminated coagulation (1). These effects are not the result of LPS toxicity but are rather a consequence of cell activation by LPS and a subsequent dysregulation of the inflammatory host response. The biological activity of LPS is harbored in the lipid anchor of the molecule, termed lipid A or "the endotoxic principle" of LPS (2).A variety of investigations in the structural prerequisites ...