Mass spectrometry has made significant advances in the analysis of lipid substances, both simple and complex present in extracts of eukaryotic and prokaryotic cells. The development of the ionization techniques of electrospray ionization and matrix assisted laser desorption ionization have both been applied to the analysis of lipids. The example of the types of structural information that can be obtained from MALDI-TOF tandem mass spectrometry is exemplified by the analysis of Kdo2-lipid A, a complex lipopolysaccharide known to activate Toll-like 4-receptors on mammalian cells. Analysis of Kdo2-lipid A obtained from an E. coli WBB06 was found to generate an abundant [M-H]- ion at m/z 2236.4 and a more abundant carbon-13 isotope at m/z 2237.4. Furthermore, collisional activation of the lipid A portion of the molecule at m/z 1796.3 resulted in a series of ions corresponding to the loss of all four fatty acyl groups as neutral carboxylic acids. An altogether different challenge of mass spectrometry applied to the area of lipid analysis is that of quantitative analysis. Two rather different requirements have emerged. One with high precision and accuracy for the measurement of relatively few lipid species that are produced at very low concentrations and typically interact with specific receptor proteins. A rather different challenge is that for the analysis of abundant lipid classes, which are composed of multiple molecular species that can approach several hundred under certain circumstances.
Lipid A of Francisella tularensis subsp. novicida contains a galactosamine (GalN) residue linked to its 1-phosphate group. As shown in the preceding manuscript, this GalN unit is transferred to lipid A from the precursor undecaprenyl phosphate-β-d-GalN. A small portion of the free lipid A of F. novicida is further modified with a glucose residue at position 6′. We now demonstrate that the two F. novicida homologues of E. coli ArnC, designated FlmF1 and FlmF2, are essential for lipid A modification with glucose and GalN, respectively. Recombinant FlmF1 expressed in E. coli selectively condenses undecaprenyl phosphate and UDP-glucose in vitro to form undecaprenyl phosphate-glucose. Recombinant FlmF2 selectively catalyzes the condensation of undecaprenyl phosphate and UDP-N-acetylgalactosamine to generate undecaprenyl phosphate-N-acetylgalactosamine. Based on an analysis of the lipid A composition of flmF1 and flmF2 mutants of F. novicida, we conclude that FlmF1 generates the donor substrate for the modification of F. novicida free lipid A with glucose, whereas FlmF2 generates the immediate precursor of the GalN donor substrate, undecaprenyl phosphate-β-d-GalN. A novel deacetylase, present in membranes of F. novicida, removes the acetyl group from undecaprenyl phosphate-N-acetylgalactosamine to yield undecaprenyl phosphate-β-d-GalN This deacetylase may have an analogous function to the deformylase that generates undecaprenyl phosphate-4-amino-4-deoxy-α-L-arabinose from undecaprenyl phosphate-4-deoxy-4-formylamino-α-L-arabinose in polymyxin-resistant strains of Escherichia coli and Salmonella typhimurium.
Francisella tularensis is a highly infectious pathogen that causes tularemia. Francisella lipid A contains an unusual galactosamine (GalN) unit, attached to its 1-phosphate moiety. Two genes, flmF2 and flmK, are required for the addition of GalN to Francisella lipid A, but the relevant enzymes and the GalN donor substrate have not been characterized. We now report the purification and identification of a novel minor lipid from Francisella novicida that functions as the GalN donor. Based on electrospray ionization mass spectrometry (ESI/MS) and NMR spectroscopy, we propose that this compound is undecaprenyl phosphate-β-D-GalN. Approximately 0.5 mg of pure lipid was obtained from 10 g of F. novicida by chloroform/methanol extraction, followed by DEAE-cellulose chromatography, mild alkaline hydrolysis, and thin layer chromatography. ESI/MS in the negative mode revealed a molecular ion [M-H]- at m/z 1006.699, consistent with undecaprenyl phosphate-GalN. 31P-NMR spectroscopy showed a single phosphorus atom in phosphodiester linkage. Selective inverse decoupling difference spectroscopy demonstrated that the undecaprenyl phosphate group is attached to the anomeric carbon of the sugar. 1H-NMR studies showed the presence of a polyisoprene chain and a sugar consistent with a β-D-GalN unit. Heteronuclear multiple quantum coherence (HMQC) analysis confirmed that nitrogen is attached to C-2 of the sugar. Purified undecaprenyl phosphate-β-D-GalN supports the in vitro modification of lipid IVA by membranes of E. coli cells expressing FlmK, an orthologue of E. coli ArnT, the enzyme that transfers 4- amino-4-deoxy-L-arabinose to lipid A in polymyxin-resistant strains. The discovery of undecaprenyl phosphate-β-D-GalN suggests Francisella modifies lipid A with GalN on the periplasmic surface of the inner membrane.
The lipopolysaccharide (LPS) isolated from certain important Gram-negative pathogens including a human pathogen Yersinia pestis and opportunistic pathogens Burkholderia mallei and Burkholderia pseudomallei contains D-glycero-D-talo-oct-2-ulosonic acid (Ko), an isosteric analog of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). Kdo 3-hydroxylase (KdoO), a Fe2+/α-KG/O2 dependent dioxygenase from Burkholderia ambifaria and Yersinia pestis is responsible for Ko formation with Kdo2-lipid A as a substrate, but in which stage KdoO functions during the LPS biosynthesis has not been established. Here we purify KdoO from B. ambifaria (BaKdoO) to homogeneity for the first time and characterize its substrates. BaKdoO utilizes Kdo2-lipid IVA or Kdo2-lipid A as a substrate, but not Kdo-lipid IVA
in vivo as well as in vitro and Kdo-(Hep)kdo-lipid A in vitro. These data suggest that KdoO is an inner core assembly enzyme that functions after the Kdo-transferase KdtA but before the heptosyl-transferase WaaC enzyme during the Ko-containing LPS biosynthesis.
Ko formation; Kdo hydroxylase; LPS inner core assembly; Burkholderia LPS Fe2+/O2/α-KG dependent dioxygenase
Acyl-carrier-protein (ACP) represents one of the most highly conserved proteins across all domains of life and is nature's way of transporting hydrocarbon-chains in vivo. Notably, type II ACPs serve as a crucial interaction hub within primary cellular metabolism1 by communicating transiently between partner enzymes of the numerous biosynthetic pathways2,3. However, the highly transient nature of such interactions and the inherent conformational mobility of ACP2 have stymied previous attempts to structurally visualize ACP tied to an overall catalytic cycle. This is essential to understanding a fundamental aspect of cellular metabolism leading to compounds that are not only useful to the cell, but are also of therapeutic value. For example, ACP is central to the biosynthesis of the lipid A (endotoxin) component of lipopolysaccharides (LPS) in Gram-negative microorganisms, which is required for their growth and survival4,5 and is an activator of the mammalian host's immune system6,7, thus emerging as an important therapeutic target8-10. During lipid A synthesis (Raetz Pathway), ACP shuttles acyl-intermediates linked to its prosthetic 4′-phosphopantetheine group (4′-PPT)2 among four acyltransferases, including LpxD11. Here we report the crystal structures of three forms of Escherichia coli ACP engaging LpxD, which represent stalled substrate and liberated products along the reaction coordinate. The structures reveal the intricate interactions at the interface that optimally position ACP for acyl-delivery and that directly involve the pantetheinyl group. Conformational differences among the stalled ACPs provide the molecular basis for the association-dissociation process. An unanticipated conformational shift of 4′-phosphopantetheine groups within the LpxD catalytic chamber reveals an unprecedented role of ACP in product release.
The lipid A component of lipopolysaccharide from the nitrogen-fixing plant endosymbiont, Rhizobium etli, is structurally very different from that found in most enteric bacteria. The lipid A from free-living R. etli is structurally heterogeneous and exists as a mixture of species which are either pentaacylated or tetraacylated. In contrast, the lipid A from R. etli bacteroids is reported to consist exclusively of tetraacylated lipid A species. The tetraacylated lipid A species in both cases lack a β-hydroxymyristoyl chain at the 3-position of lipid A. Here, we show that the lipid A modification enzyme responsible for 3-O deacylation in R. etli is a homolog of the PagL protein originally described in Salmonella enterica sv. Typhimurium. In contrast to the PagL proteins described from other species, R. etli PagL displays a calcium dependency. To determine the importance of the lipid A modification catalyzed by PagL, we isolated and characterized a R. etli mutant deficient in the pagL gene. Mass spectrometric analysis confirmed that the mutant strain was exclusively tetraacylated and radiochemical analysis revealed that 3-O deacylase activity was absent in membranes prepared from the mutant. The R. etli mutant was not impaired in its ability to form nitrogen-fixing nodules on Phaseolus vulgaris but it displayed slower nodulation kinetics relative to the wild-type strain. The lipid A modification catalyzed by R. etli PagL, therefore, is not required for nodulation but may play other roles such as protecting bacterial endosymbionts from plant immune responses during infection.
Gram-negative bacteria; lipopolysaccharide; Lipid A; Rhizobium etli; PagL
The PhoQ/PhoP two-component system activates many genes for lipopolysaccharide (LPS) modification when cells are grown at low Mg2+ concentrations. An additional target of PhoQ and PhoP is MgrR, an Hfq-dependent small RNA that negatively regulates expression of eptB, also encoding a protein that carries out LPS modification. Examination of LPS confirmed that MgrR effectively silences EptB; the phosphoethanolamine modification associated with EptB is found in ΔmgrR::kan but not mgrR+ cells. Sigma E has been reported to positively regulate eptB, although the eptB promoter does not have the expected Sigma E recognition motifs. The effects of Sigma E and deletion of mgrR on levels of eptB mRNA were independent, and the same 5′ end was found in both cases. In vitro transcription and the behavior of transcriptional and translational fusions demonstrate that Sigma E acts directly at the level of transcription initiation for eptB, from the same start point as Sigma 70. The results suggest that when Sigma E is active, synthesis of eptB transcript outstrips MgrR-dependent degradation; presumably the modification of LPS is important under these conditions. Adding to the complexity of eptB regulation is a second sRNA, ArcZ, which also directly and negatively regulates eptB.
LPS; Sigma E; PhoPQ; sRNA
The sixth step in the lipid A biosynthetic pathway involves phosphorylation of the tetraacyldisaccharide-1-phosphate (DSMP) intermediate by the cytosol-facing inner membrane kinase LpxK, a member of the P-loop containing nucleoside triphosphate (NTP) hydrolase superfamily. We report the kinetic characterization of LpxK from Aquifex aeolicus and the crystal structures of LpxK in complex with ATP in a pre-catalytic binding state, the ATP analog AMP-PCP in the closed catalytically competent conformation, and a chloride anion revealing an inhibitory conformation of the nucleotide-binding P-loop. We demonstrate that LpxK activity in vitro requires the presence of a detergent micelle and formation of a ternary LpxK-ATP/Mg2+-DSMP complex. Using steady-state kinetics, we have identified crucial active site residues, leading to the proposal that the interaction of D99 with H261 acts to increase the pKa of the imidazole moiety, which in turn serves as the catalytic base to deprotonate the 4'-hydroxyl of the DSMP substrate. The fact that an analogous mechanism has not yet been observed for other P-loop kinases highlights LpxK as a distinct member of the P-loop kinase family, a notion that is also reflected through its localization at the membrane, lipid substrate, and overall structure.
Modification of specific Gram-negative bacterial cell envelope components, such as capsule, O-antigen and lipid A, are often essential for the successful establishment of infection. Francisella species express lipid A molecules with unique characteristics involved in circumventing host defences, which significantly contribute to their virulence. In this study, we show that NaxD, a member of the highly conserved YdjC superfamily, is a deacetylase required for an important modification of the outer membrane component lipid A in Francisella. Mass spectrometry analysis revealed that NaxD is essential for the modification of a lipid A phosphate with galactosamine in Francisella novicida, a model organism for the study of highly virulent Francisella tularensis. Significantly, enzymatic assays confirmed that this protein is necessary for deacetylation of its substrate. In addition, NaxD was involved in resistance to the antimicrobial peptide polymyxin B and critical for replication in macrophages and in vivo virulence. Importantly, this protein is also required for lipid A modification in F. tularensis as well as Bordetella bronchiseptica. Since NaxD homologues are conserved among many Gram-negative pathogens, this work has broad implications for our understanding of host subversion mechanisms of other virulent bacteria.
Clostridium botulinum has been classified into four groupings (groups I to IV) based on physiological characteristics and 16S rRNA sequencing. We have examined the lipid compositions of 11 representative strains of C. botulinum and a strain of Clostridium sporogenes by 2D-TLC and by MS. All strains contained phosphatidylglycerol (PG), cardiolipin (CL) and phosphatidylethanolamine (PE) in both the all-acyl and the alk-1′-enyl (plasmalogen) forms. Five strains in proteolytic group I, which are related to C. sporogenes, contained varying amounts of an ethanolamine-phosphate derivative of N-acetylglucosaminyl-diradylglycerol, which is also present in C. sporogenes. Three strains in group II, which are related to Clostridium butyricum, Clostridium beijerinckii and Clostridium acetobutylicum, contained lipids characteristic of these saccharolytic species: a glycerol acetal and a PG acetal of the plasmalogen form of PE. Two group III strains, which are related to Clostridium novyi, contained amino-acyl derivatives of PG, which are also found in C. novyi. A strain in group IV had PE, PG and CL, but none of the distinguishing lipids. This work shows that the lipidome of C. botulinum is consistent with its classification by other methods.
Synthesis of Escherichia coli LpxL, which transfers a secondary laurate chain to the 2′ position of lipid A, in Yersinia pestis produced bisphosphoryl hexa-acylated lipid A at 37°C, leading to significant attenuation of virulence. Our previous observations also indicated that strain χ10015(pCD1Ap) (ΔlpxP32::PlpxL
lpxL) stimulated a strong inflammatory reaction but sickened mice before recovery and retained virulence via intranasal (i.n.) infection. The development of live, attenuated Y. pestis vaccines may be facilitated by detoxification of its lipopolysaccharide (LPS). Heterologous expression of the lipid A 1-phosphatase, LpxE, from Francisella tularensis in Y. pestis yields predominantly 1-dephosphorylated lipid A, as confirmed by mass spectrometry. Results indicated that expression of LpxE on top of LpxL provided no significant reduction in virulence of Y. pestis in mice when it was administered i.n. but actually reduced the 50% lethal dose (LD50) by 3 orders of magnitude when the strain was administered subcutaneously (s.c.). Additionally, LpxE synthesis in wild-type Y. pestis KIM6+(pCD1Ap) led to slight attenuation by s.c. inoculation but no virulence change by i.n. inoculation in mice. In contrast to Salmonella enterica, expression of LpxE does not attenuate the virulence of Y. pestis.
Inflammation and macrophage foam cells are characteristic features of atherosclerotic lesions, but the mechanisms linking cholesterol accumulation to inflammation and LXR-dependent response pathways are poorly understood. To investigate this relationship, we utilized lipidomic and transcriptomic methods to evaluate the effect of diet and LDL receptor genotype on macrophage foam cell formation within the peritoneal cavities of mice. Foam cell formation was associated with significant changes in hundreds of lipid species and unexpected suppression, rather than activation, of inflammatory gene expression. We provide evidence that regulated accumulation of desmosterol underlies many of the homeostatic responses observed in macrophage foam cells, including activation of LXR target genes, inhibition of SREBP target genes, selective reprogramming of fatty acid metabolism and suppression of inflammatory response genes. These observations suggest that macrophage activation in atherosclerotic lesions results from extrinsic, pro-inflammatory signals generated within the artery wall that suppress homeostatic and anti-inflammatory functions of desmosterol.
The UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase, encoded by lpxA, catalyzes the first step of lipid A biosynthesis in Gram-negative bacteria, the R-3-hydroxyacyl-ACP dependent acylation of the 3-OH group of UDP-GlcNAc. Recently, we demonstrated that the Arabidopsis thaliana orthologs of six enzymes of the bacterial lipid A pathway produce lipid A precursors with structures similar to Escherichia coli lipid A precursors (Li, C., et al. (2011) Proc Natl Acad Sci U S A 108, 11387–11392). To build upon this finding, we have cloned, purified, and determined the crystal structure of the A. thaliana LpxA ortholog (AtLpxA) to 2.1 Å resolution. The overall structure of AtLpxA is very similar to that of E. coli LpxA (EcLpxA) with an alpha helical rich C-terminus and characteristic N-terminal left-handed parallel β-helix (LβH). All key catalytic and chain-length determining residues of EcLpxA are conserved in AtLpxA, however the AtLpxA has an additional coil and loop added to the LβH not seen in EcLpxA. Consistent with the similarities between the two structures, the purified AtLpxA catalyzes the same reaction as EcLpxA. In addition, A. thaliana lpxA complements an E. coli mutant lacking the chromosomal lpxA and promotes the synthesis of lipid A in vivo similar to the lipid A produced in the presence of E. coli lpxA. This work shows that AtLpxA is a functional UDP-GlcNAc acyltransferase able to catalyze the same reaction as EcLpxA, and supports the hypothesis that lipid A molecules are biosynthesized in Arabidopsis and other plants.
The UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase LpxC catalyzes the committed reaction of lipid A biosynthesis, an essential pathway in Gram-negative bacteria. We report the backbone resonance assignments of the 34 kDa LpxC from Escherichia coli in complex with the antibiotic L-161,240 using multidimensional, multinuclear NMR experiments. The 1H chemical shifts of complexed L-161,240 are also determined.
Escherichia coli; Antibiotic; Lipid A; Inhibitor; Deacetylase
Lipid A is a key component of the outer membrane of Gram-negative bacteria and stimulates proinflammatory responses via the Toll-like receptor 4 (TLR4)-MD2-CD14 pathway. Its endotoxic activity depends on the number and length of acyl chains and its phosphorylation state. In Salmonella enterica serovar Typhimurium, removal of the secondary laurate or myristate chain in lipid A results in bacterial attenuation and growth defects in vitro. However, the roles of the two lipid A phosphate groups in bacterial virulence and immunogenicity remain unknown. Here, we used an S. Typhimurium msbB pagL pagP lpxR mutant, carrying penta-acylated lipid A, as the parent strain to construct a series of mutants synthesizing 1-dephosphorylated, 4′-dephosphorylated, or nonphosphorylated penta-acylated lipid A. Dephosphorylated mutants exhibited increased sensitivity to deoxycholate and showed increased resistance to polymyxin B. Removal of both phosphate groups severely attenuated the mutants when administered orally to BALB/c mice, but the mutants colonized the lymphatic tissues and were sufficiently immunogenic to protect the host from challenge with wild-type S. Typhimurium. Mice receiving S. Typhimurium with 1-dephosphorylated or nonphosphorylated penta-acylated lipid A exhibited reduced levels of cytokines. Attenuated and dephosphorylated Salmonella vaccines were able to induce adaptive immunity against heterologous (PspA of Streptococcus pneumoniae) and homologous antigens (lipopolysaccharide [LPS] and outer membrane proteins [OMPs]).
Enzymes in lipid metabolism acquire and deliver hydrophobic substrates and products from within lipid bilayers. The structure at 2.55 Å of one isozyme of a constitutive enzyme in lipid A biosynthesis, LpxI from Caulobacter crescentus, has a novel fold. Two domains close around a completely sequestered substrate, UDP-2,3-diacylglucosamine, and open to release products either to the neighboring enzyme in a putative multi-enzyme complex, or to the bilayer. Mutation identifies Asp225 as key to Mg2+ catalyzed diphosphate hydrolysis. These structures provide snapshots of the enzymatic synthesis of a critical lipid A precursor.
Electrospray ionization mass spectrometry is a powerful technique to analyze lipid extracts especially for the identification of new lipid metabolites. A hurdle to lipid identification is the presence of solvent contaminants that hinder the identification of low abundance species or covalently modify abundant lipid species. We have identified several non-enzymatically derived minor lipid species in lipid extracts of Escherichia coli, phosphatidylmethanol, ethyl and methyl carbamates of PE and N-succinyl PE were identified in lipid extracts of Escherichia coli. Phosphatidylmethanol (PM) was identified by exact mass measurement and collision induced dissociation tandem mass spectrometry (MS/MS). Extraction in the presence of deuterated methanol leads to a 3 atomic mass unit shift in the [M-H]- ions of PM indicating its formation during extraction. Ethyl and methyl carbamates of PE, also identified by exact mass measurement and MS/MS, are likely to be formed by phosgene, a breakdown product of chloroform. Addition of phosgene to extractions containing synthetic PE significantly increases the levels of PE-MC detected in the lipid extracts by ESI-MS. Extraction in the presence of methylene chloride significantly reduced the levels of these lipid species. N-succinyl PE is formed from reaction of succinyl-CoA with PE during extraction. Interestingly N-succinyl PE can be formed in an aqueous reaction mixture in the absence of added E. coli proteins. This work highlights the reactivity of the amine of PE and emphasizes that careful extraction controls are required to ensure that new minor lipid species identified using mass spectrometry are indeed endogenous lipid metabolites.
mass spectrometry; E. coli; lipids; chloroform; phosgene; artifacts
Lipopolysaccharide (LPS) structural modifications have been shown to specifically affect the pathogenesis of many Gram-negative pathogens. In Francisella, modification of the lipid A component of LPS resulted in a molecule with no to low endotoxic activity. The role of the terminal lipid A phosphates in host recognition and pathogenesis was determined using a Francisella novicida mutant that lacked the 4′ phosphatase enzyme (LpxF). The lipid A of this strain retained the phosphate moiety at the 4′ position and the N-linked fatty acid at the 3′ position on the diglucosamine backbone. Studies were undertaken to determine the pathogenesis of this mutant strain via the pulmonary and subcutaneous routes of infection. Mice infected with the lpxF-null F. novicida mutant by either route survived primary infection and subsequently developed protective immunity against a lethal wild-type (WT) F. novicida challenge. To determine the mechanism(s) by which the host controlled primary infection by the lpxF-null mutant, the role of innate immune components, including Toll-like receptor 2 (TLR2), TLR4, caspase-1, MyD88, alpha interferon (IFN-α), and gamma interferon(IFN-γ), was examined using knockout mice. Interestingly, only the IFN-γ knockout mice succumbed to a primary lpxF-null F. novicida mutant infection, highlighting the importance of IFN-γ production. To determine the role of components of the host adaptive immune system that elicit the long-term protective immune response, T- and B-cell deficient RAG1−/− mice were examined. All mice survived primary infection; however, RAG1−/− mice did not survive WT challenge, highlighting a role for T and B cells in the protective immune response.
The development of safe live, attenuated Salmonella vaccines may be facilitated by detoxification of its lipopolysaccharide. Recent characterization of the lipid A 1-phosphatase, LpxE, from Francisella tularensis allowed us to construct recombinant, plasmid-free strains of Salmonella that produce predominantly 1-dephosphorylated lipid A, similar to the adjuvant approved for human use. Complete lipid A 1-dephosphorylation was also confirmed under low pH, low Mg2+ culture conditions, which induce lipid A modifications. lpxE expression in Salmonella reduced its virulence in mice by five orders of magnitude. Moreover, mice inoculated with these detoxified strains were protected against wild-type challenge. Candidate Salmonella vaccine strains synthesizing pneumococcal surface protein A (PspA) were also confirmed to possess nearly complete lipid A 1-dephosphorylation. After inoculation by the LpxE/PspA strains, mice produced robust levels of anti-PspA antibodies and showed significantly improved survival against challenge with wild-type Streptococcus pneumoniae WU2 as compared to vector-only immunized mice, validating Salmonella synthesizing 1-dephosphorylated lipid A as an antigen delivery system.
Attenuated Salmonella; Dephosphorylation of Lipid A; LpxE; TLR4; PspA
PTPMT1 was the first protein tyrosine phosphatase found localized to the mitochondria, but its biological function was unknown. Herein, we demonstrate that whole body deletion of Ptpmt1 in mice leads to embryonic lethality, suggesting an indispensable role for PTPMT1 during development. Ptpmt1-deficiency in mouse embryonic fibroblasts compromises mitochondrial respiration and results in abnormal mitochondrial morphology. Lipid analysis of Ptpmt1-deficient fibroblasts reveals an accumulation of phosphatidylglycerophosphate (PGP) along with a concomitant decrease in phosphatidylglycerol. PGP is an essential intermediate in the biosynthetic pathway of cardiolipin, a mitochondrial-specific phospholipid regulating the membrane integrity and activities of the organelle. We further demonstrate that PTPMT1 specifically dephosphorylates PGP in vitro. Loss of PTPMT1 leads to dramatic diminution of cardiolipin, which can be partially reversed by the expression of catalytic active PTPMT1. Our study identifies PTPMT1 as the mammalian PGP phosphatase and points to its role as a regulator of cardiolipin biosynthesis.
Lipopolysaccharide (LPS), composed of lipid A, core, and O-antigen, is a major virulence factor of Salmonella enterica serovar Typhimurium, with lipid A being a major stimulator to induce the proinflammatory response via the Toll-like receptor 4 (TLR4)-MD2-CD14 pathway. While Salmonella msbB mutants lacking the myristate chain in lipid A were investigated widely as an anticancer vaccine, inclusion of the msbB mutation in a Salmonella vaccine to deliver heterologous antigens has not yet been investigated. We introduced the msbB mutation alone or in combination with mutations in other lipid A acyl chain modification genes encoding PagL, PagP, and LpxR into wild-type S. enterica serovar Typhimurium. The msbB mutation reduced virulence, while the pagL, pagP, and lpxR mutations did not affect virulence in the msbB mutant background when administered orally to BALB/c mice. Also, all mutants exhibited sensitivity to polymyxin B but did not display sensitivity to deoxycholate. LPS derived from msbB mutants induced less inflammatory responses in human Mono Mac 6 and murine macrophage RAW264.7 cells in vitro. However, an msbB mutant did not decrease the induction of inflammatory responses in mice compared to the levels induced by the wild-type strain, whereas an msbB pagP mutant induced less inflammatory responses in vivo. The mutations were moved to an attenuated Salmonella vaccine strain to evaluate their effects on immunogenicity. Lipid A modification caused by the msbB mutation alone and in combination with pagL, pagP, and lpxR mutations led to higher IgA production in the vaginal tract but still retained the same IgG titer level in serum to PspA, a test antigen from Streptococcus pneumoniae, and to outer membrane proteins (OMPs) from Salmonella.
Modification of the lipid A moiety of lipopolysaccharide by the addition of the sugar, 4-amino-4-deoxy-L-arabinose (L-Ara4N), is a strategy adopted by pathogenic Gram-negative bacteria to evade cationic antimicrobial peptides produced by the innate immune system. L-Ara4N biosynthesis is therefore a potential anti-infective target, as inhibiting its synthesis would render certain pathogens more sensitive to the immune system. The bifunctional enzyme ArnA, which is required for L-Ara4N biosynthesis, catalyzes the NAD+-dependent oxidative-decarboxylation of UDP-glucuronic acid to generate a UDP-4′-keto-pentose sugar, and also catalyzes transfer of a formyl group from N-10-formyltetrahydrofolate to the 4′-amine of UDP-L-Ara4N. We now report the crystal structure of the N-terminal formyltransferase domain in a complex with uridine monophosphate and N-5-formyltetrahydrofolate. Using this structure we identify the active site of formyltransfer in ArnA including the key catalytic residues N102, H104, and D140. Additionally, we have shown that residues S433 and E434 of the decarboxylase domain are required for the oxidative-decarboxylation of UDP-GlcUA. An E434Q mutant is inactive suggesting that chemical rather than steric properties of this residue are crucial in the decarboxylation reaction. Our data suggests that the decarboxylase domain catalyzes both hydride abstraction (oxidation) from the C-4′ position and the subsequent decarboxylation.
site directed mutagenesis; X-ray crystallography; drug design; LPS biosynthesis; polymyxin resistance
Yersinia pestis, the causative agent of plague, is a potential weapon of bioterrorism. Y. pestis evades the innate immune system by synthesizing tetra-acylated lipid A with poor Toll-like receptor 4 (TLR4)-stimulating activity at 37°C, whereas hexa-acylated lipid A, a potent TLR4 agonist, is made at lower temperatures. Synthesis of Escherichia coli LpxL, which transfers the secondary laurate chain to the 2′-position of lipid A, in Y. pestis results in production of hexa-acylated lipid A at 37°C, leading to significant attenuation of virulence. Previously, we described a Y. pestis vaccine strain in which crp expression is under the control of the arabinose-regulated araC PBAD promoter, resulting in a 4-5 log reduction in virulence. To reduce the virulence of the crp promoter mutant further, we introduced E. coli lpxL into the Y. pestis chromosome. The χ10030(pCD1Ap) (ΔlpxP32::PlpxL lpxL ΔPcrp21::TT araC PBAD crp) construct likewise produced hexa-acylated lipid A at 37°C and was significantly more attenuated than strains harboring each individual mutation. The LD50 of the mutant in mice, when administered subcutaneously or intranasally was >107-times and >104-times greater than wild type, respectively. Mice immunized subcutaneously with a single dose of the mutant were completely protected against a subcutaneous challenge of 3.6 × 107 wild-type Y. pestis and significantly protected (80% survival) against a pulmonary challenge of 1.2 × 104 live cells. Intranasal immunization also provided significant protection against challenges by both routes. This mutant is an immunogenic, highly attenuated live Y. pestis construct that merits further development as a vaccine candidate.
Yersinia pestis; lipid A; regulated delayed attenuation; plague vaccine
A study of the polar lipids of Clostridium novyi NT has revealed the presence of phosphatidylethanolamine (PE) and cardiolipin as major phospholipids with smaller amounts of phosphatidylglycerol (PG), lysyl-PG and alanyl-PG. Other minor phospholipids included phosphatidic acid, CDP-diacylglycerol, phosphatidylserine (PS) and phosphatidylthreonine (PT). PE, PG and amino acyl PG were present in both the diacyl and alk-1’-enyl acyl (plasmalogen) forms and cardiolipin plasmalogens were found to contain one or two alk-1’-enyl chains. In contrast, the precursor lipids phosphatidic acid, CDP-diacylglycerol and PS were present almost exclusively as diacyl phospholipids. These findings are consistent with the hypothesis that plasmalogens are formed from diacylated phospholipids at a late stage of phospholipid formation in Clostridium species. This novel pathway contrasts with the route in animals in which a saturated ether bond is formed at an early stage of plasmalogen biosynthesis and the alk-1-enyl bond is formed by an aerobic mechanism.