Structure Determination of Lipid A with Multiple Glycosylation Sites by Tandem MS of Lithium-Adducted Negative Ions.

FLATn is a tandem mass spectrometric technique that can be used to rapidly generate spectral information applicable for structural elucidation of lipids like lipid A from Gram-negative bacterial species from a single bacterial colony. In this study, we extend the scope and capability of FLATn by tandem MS fragmentation of lithium-adducted molecular lipid A anions and fragments (FLATn-Li) that provides additional structural and diagnostic data from FLATn samples allowing for the discrimination of terminal phosphate modifications in a variety of pathogenic and environmental species. Using FLATn-Li, we elucidated the lipid A structure from several bacterial species, including novel structures from arctic bacterioplankton of the Duganella and Massilia genera that favor 4-amino-4-deoxy-l-arabinopyranose (Ara4N) modification at the 1-phosphate position and that demonstrate double glycosylation with Ara4N at the 1 and 4' phosphate positions simultaneously. The structures characterized in this work demonstrate that some environmental psychrophilic species make extensive use of this structural lipid A modification previously characterized as a pathogenic adaptation and the structural basis of resistance to cationic antimicrobial peptides. This observation extends the role of phosphate modification(s) in environmental species adaptation and suggests that Ara4N modification can functionally replace the positive charge of the phosphoethanolamine modification that is more typically found attached to the 1-phosphate position of modified lipid A.

Temperature-Dependence of Lipid A Acyl Structure in Psychrobacter cryohalolentis and Arctic Isolates of Colwellia hornerae and Colwellia piezophila.

Lipid A is a fundamental Gram-negative outer membrane component and the essential element of lipopolysaccharide (endotoxin), a potent immunostimulatory molecule. This work describes the metabolic adaptation of the lipid A acyl structure by Psychrobacter cryohalolentis at various temperatures in its facultative psychrophilic growth range, as characterized by MALDI-TOF MS and FAME GC-MS. It also presents the first elucidation of lipid A structure from the Colwellia genus, describing lipid A from strains of Colwellia hornerae and Colwellia piezophila, which were isolated as primary cultures from Arctic fast sea ice and identified by 16S rDNA sequencing. The Colwellia strains are obligate psychrophiles, with a growth range restricted to 15 °C or less. As such, these organisms have less need for fluidity adaptation in the acyl moiety of the outer membrane, and they do not display alterations in lipid A based on growth temperature. Both Psychrobacter and Colwellia make use of extensive single-methylene variation in the size of their lipid A molecules. Such single-carbon variations in acyl size were thought to be restricted to psychrotolerant (facultative) species, but its presence in these Colwellia species shows that odd-chain acyl units and a single-carbon variation in lipid A structure are present in obligate psychrophiles, as well.

Endotoxin structures in the psychrophiles Psychromonas marina and Psychrobacter cryohalolentis contain distinctive acyl features.

Lipid A is the essential component of endotoxin (Gram-negative lipopolysaccharide), a potent immunostimulatory compound. As the outer surface of the outer membrane, the details of lipid A structure are crucial not only to bacterial pathogenesis but also to membrane integrity. This work characterizes the structure of lipid A in two psychrophiles, Psychromonas marina and Psychrobacter cryohalolentis, and also two mesophiles to which they are related using MALDI-TOF MS and fatty acid methyl ester (FAME) GC-MS. P. marina lipid A is strikingly similar to that of Escherichia coli in organization and total acyl size, but incorporates an unusual doubly unsaturated tetradecadienoyl acyl residue. P. cryohalolentis also shows structural organization similar to a closely related mesophile, Acinetobacter baumannii, however it has generally shorter acyl constituents and shows many acyl variants differing by single methylene (-CH2-) units, a characteristic it shares with the one previously reported psychrotolerant lipid A structure. This work is the first detailed structural characterization of lipid A from an obligate psychrophile and the second from a psychrotolerant species. It reveals distinctive structural features of psychrophilic lipid A in comparison to that of related mesophiles which suggest constitutive adaptations to maintain outer membrane fluidity in cold environments.

YopJ targets TRAF proteins to inhibit TLR-mediated NF-kappaB, MAPK and IRF3 signal transduction.

The Yersinia pestis virulence factor YopJ is a potent inhibitor of the NF-kappaB and MAPK signalling pathways, however, its molecular mechanism and relevance to pathogenesis are the subject of much debate. In this report, we characterize the effects of this type III effector protein on bone fide signalling events downstream of Toll-like receptors (TLRs), critical sensors in innate immunity. YopJ inhibited TLR-mediated NF-kappaB and MAP kinase activation, as suggested by previous studies. In addition, induction of the TLR-mediated interferon response was blocked by YopJ, indicating that YopJ also inhibits IRF3 signalling. Examination of the NF-kappaB signalling pathway in detail suggested that YopJ acts at the level of TAK1 (MAP3K7) activation. Further studies revealed a YopJ-dependent decrease in the ubiquitination of TRAF3 and TRAF6. These data support the hypothesis that YopJ is a deubiquitinating protease that acts on TRAF proteins to prevent or remove the K63-polymerized ubiquitin conjugates required for signal transduction. Our data do not directly address the alternative hypothesis that YopJ is an acetyltransferase that acts on the activation loop of IKK and MKK proteins, but support the conclusion that the critical function of YopJ is to deubiquinate TRAF proteins.

Enzymatic synthesis of lipid A molecules with four amide-linked acyl chains. LpxA acyltransferases selective for an analog of UDP-N-acetylglucosamine in which an amine replaces the 3"-hydroxyl group.

LpxA of Escherichia coli catalyzes the acylation of the glucosamine 3-OH group of UDP-GlcNAc, using R-3-hydroxymyristoyl-acyl carrier protein (ACP) as the donor substrate. We now demonstrate that LpxA in cell extracts of Mesorhizobium loti and Leptospira interrogans, which synthesize lipid A molecules containing 2,3-diamino-2,3-dideoxy-d-glucopyranose (GlcN3N) units in place of glucosamine, do not acylate UDP-GlcNAc. Instead, these LpxA acyltransferases require a UDP-Glc-NAc derivative (designated UDP 2-acetamido-3-amino-2,3-dideoxy-alpha-d-glucopyranose or UDP-GlcNAc3N), characterized in the preceding paper, in which an amine replaces the glucosamine 3-OH group. L. interrogans LpxA furthermore displays absolute selectivity for 3-hydroxylauroyl-ACP as the donor, whereas M. loti LpxA functions almost equally well with 10-, 12-, and 14-carbon 3-hydroxyacyl-ACPs. The substrate selectivity of L. interrogans LpxA is consistent with the structure of L. interrogans lipid A. The mechanism of L. interrogans LpxA appears to be similar to that of E. coli LpxA, given that the essential His(125) residue of E. coli LpxA is conserved and is also required for acyltransferase activity in L. interrogans. Acidithiobacillus ferrooxidans (an organism that makes lipid A molecules containing both GlcN and GlcN3N) has an ortholog of LpxA that is selective for UDP-GlcNAc3N, but the enzyme also catalyzes the acylation of UDP-GlcNAc at a slow rate. E. coli LpxA acylates UDP-GlcNAc and UDP-GlcNAc3N at comparable rates in vitro. However, UDP-GlcNAc3N is not synthesized in vivo, because E. coli lacks gnnA and gnnB. When the latter are supplied together with A. ferrooxidans lpxA, E. coli incorporates a significant amount of GlcN3N into its lipid A.

Oxidation and transamination of the 3"-position of UDP-N-acetylglucosamine by enzymes from Acidithiobacillus ferrooxidans. Role in the formation of lipid a molecules with four amide-linked acyl chains.

Lipid A, a major component of the outer membranes of Escherichia coli and other Gram-negative bacteria, is usually constructed around a beta-1',6-linked glucosamine disaccharide backbone. However, in organisms like Acidithiobacillus ferrooxidans, Leptospira interrogans, Mesorhizobium loti, and Legionella pneumophila, one or both glucosamine residues are replaced with the sugar 2,3-diamino-2,3-dideoxy-d-glucopyranose. We now report the identification of two proteins, designated GnnA and GnnB, involved in the formation of the 2,3-diamino-2,3-dideoxy-d-glucopyranose moiety. The genes encoding these proteins were recognized because of their location between lpxA and lpxB in A. ferrooxidans. Based upon their sequences, the 313-residue GnnA protein was proposed to catalyze the NAD(+)-dependent oxidation of the glucosamine 3-OH of UDP-GlcNAc, and the 369-residue GnnB protein was proposed to catalyze the subsequent transamination to form UDP 2-acetamido-3-amino-2,3-dideoxy-alpha-d-glucopyranose (UDP-GlcNAc3N). Both gnnA and gnnB were cloned and expressed in E. coli using pET23c+. In the presence of l-glutamate and NAD(+), both proteins were required for the conversion of [alpha-(32)P]UDP-GlcNAc to a novel, less negatively charged sugar nucleotide shown to be [alpha-(32)P]UDP-GlcNAc3N. The latter contained a free amine, as judged by modification with acetic anhydride. Using recombinant GnnA and GnnB, approximately 0.4 mg of the presumptive UDP-GlcNAc3N was synthesized. The product was purified and subjected to NMR analysis to confirm the replacement of the GlcNAc 3-OH group with an equatorial NH(2). As shown in the accompanying papers, UDP-GlcNAc3N is selectively acylated by LpxAs of A. ferrooxidans, L. interrogans, and M. loti. UDP-GlcNAc3N may be useful as a substrate analog for diverse enzymes that utilize UDP-GlcNAc.

Activation of the plant mitochondrial alternative oxidase: insights from site-directed mutagenesis.

The homodimeric cyanide-resistant alternative oxidase of plant mitochondria reduces oxygen to water without forming ATP. Arabidopsis thaliana alternative oxidase AOX1a is stimulated by pyruvate or other alpha-keto acids associating with a regulatory cysteine at position 78, by succinate in a serine-78 mutant, and by site-directed mutation of position 78 to glutamate. The mechanism of activation was explored with additional amino acid substitutions made at Cys-78 in AOX1a, which was functionally expressed in Escherichia coli. Oxidases with positively charged substitutions (Lys and Arg) were insensitive to pyruvate or succinate but were more active than the wild type without pyruvate. Uncharged substitutions (Gln, Leu) produced an inactive enzyme. These results indicate that activation may be due to conformational changes caused by charge repulsion between the dimer subunits and not through a direct role of alpha-keto acids in catalysis. Oxygen isotope fractionation experiments suggest that the charge of the amino acid at position 78 also affects the entry of oxygen into the active site. Therefore, the N-terminal portion of the protein containing residue 78 can indirectly affect both catalysis at the diiron active site and the path of oxygen to that site. In addition, both positively and negatively substituted alternative oxidases were stimulated by glyoxylate, suggesting the presence of a second activation site, possibly Cys-128.

Relaxed acyl chain specificity of Bordetella UDP-N-acetylglucosamine acyltransferases.

Lipid A (endotoxin) is a major structural component of Gram-negative outer membranes. It also serves as the hydrophobic anchor of lipopolysaccharide and is a potent activator of the innate immune response. Lipid A molecules from the genus Bordetella are reported to exhibit unusual structural asymmetry with respect to the acyl chains at the 3- and 3'-positions. These acyl chains are attached by UDP-N-acetylglucosamine acyltransferase (LpxA). To determine the origin of the acyl variability, the single lpxA ortholog present in each of the genomes of Bordetella bronchiseptica (lpxA(Br)), Bordetella parapertussis (lpxA(Pa)), and Bordetella pertussis (lpxA(Pe)) was cloned and expressed in Escherichia coli. In contrast to all LpxA proteins studied to date, LpxA(Br) and LpxA(Pe) display relaxed acyl chain length specificity in vitro, utilizing C(10)OH-ACP, C(12)OH-ACP, and C(14)OH-ACP at similar rates. Furthermore, hybrid lipid A molecules synthesized at 42 degrees C by an E. coli lpxA mutant complemented with lpxA(Pe) contain C(10)OH, C(12)OH, and C(14)OH at both the 3- and 3'-positions, as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In contrast, LpxA from B. parapertussis did not display relaxed specificity but was selective for C(10)OH-ACP. This study provides an enzymatic explanation for some of the unusual acyl chain variations found in Bordetella lipid A.