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1.  Mechanistic characterization of the tetraacyldisaccharide-1-phosphate 4'-kinase LpxK involved in lipid A biosynthesis 
Biochemistry  2013;52(13):2280-2290.
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.
doi:10.1021/bi400097z
PMCID: PMC3670694  PMID: 23464738
2.  Biosynthesis of Undecaprenyl Phosphate-Galactosamine and Undecaprenyl Phosphate-Glucose in Francisella novicida 
Biochemistry  2009;48(6):1173-1182.
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.
doi:10.1021/bi802212t
PMCID: PMC2708000  PMID: 19166326
3.  Identification of Undecaprenyl Phosphate-β-D-Galactosamine in Francisella novicida and Its Function in Lipid A Modification 
Biochemistry  2009;48(6):1162-1172.
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.
doi:10.1021/bi802211k
PMCID: PMC2701470  PMID: 19166327
4.  NaxD is a deacetylase required for lipid A modification and Francisella pathogenesis 
Molecular microbiology  2012;86(3):10.1111/mmi.12004.
Summary
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.
doi:10.1111/mmi.12004
PMCID: PMC3841722  PMID: 22966934
5.  Pathogenicity of Yersinia pestis Synthesis of 1-Dephosphorylated Lipid A 
Infection and Immunity  2013;81(4):1172-1185.
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.
doi:10.1128/IAI.01403-12
PMCID: PMC3639600  PMID: 23357387
6.  Regulated accumulation of desmosterol integrates macrophage lipid metabolism and inflammatory responses 
Cell  2012;151(1):138-152.
Summary
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.
doi:10.1016/j.cell.2012.06.054
PMCID: PMC3464914  PMID: 23021221
7.  Activity and Crystal Structure of Arabidopsis thaliana UDP-N-acetylglucosamine Acyltransferase 
Biochemistry  2012;51(21):4322-4330.
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.
doi:10.1021/bi3002242
PMCID: PMC3383772  PMID: 22545860
8.  Assignment of 1H, 13C and 15N backbone resonances of Escherichia coli LpxC bound to L-161,240 
Biomolecular NMR assignments  2009;4(1):37-40.
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.
doi:10.1007/s12104-009-9201-5
PMCID: PMC3631426  PMID: 19941092
Escherichia coli; Antibiotic; Lipid A; Inhibitor; Deacetylase
9.  Phosphate Groups of Lipid A Are Essential for Salmonella enterica Serovar Typhimurium Virulence and Affect Innate and Adaptive Immunity 
Infection and Immunity  2012;80(9):3215-3224.
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]).
doi:10.1128/IAI.00123-12
PMCID: PMC3418755  PMID: 22753374
10.  LpxI structures reveal how a lipid A precursor is synthesized 
Nature structural & molecular biology  2012;19(11):1132-1138.
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.
doi:10.1038/nsmb.2393
PMCID: PMC3562136  PMID: 23042606
11.  Non-enzymatically derived minor lipids found in Escherichia coli lipid extracts 
Biochimica et biophysica acta  2011;1811(11):827-837.
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.
doi:10.1016/j.bbalip.2011.08.012
PMCID: PMC3205347  PMID: 21925285
mass spectrometry; E. coli; lipids; chloroform; phosgene; artifacts
12.  Role of Francisella Lipid A Phosphate Modification in Virulence and Long-Term Protective Immune Responses 
Infection and Immunity  2012;80(3):943-951.
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.
doi:10.1128/IAI.06109-11
PMCID: PMC3294632  PMID: 22215738
14.  Salmonella synthesizing 1-monophosphorylated LPS exhibits low endotoxic activity while retaining its immunogenicity 
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.
doi:10.4049/jimmunol.1100339
PMCID: PMC3119770  PMID: 21632711
Attenuated Salmonella; Dephosphorylation of Lipid A; LpxE; TLR4; PspA
15.  Mitochondrial Phosphatase PTPMT1 is essential for cardiolipin biosynthesis 
Cell metabolism  2011;13(6):690-700.
Summary
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.
doi:10.1016/j.cmet.2011.04.007
PMCID: PMC3119201  PMID: 21641550
16.  Palmitoylation State Impacts Induction of Innate and Acquired Immunity by the Salmonella enterica Serovar Typhimurium msbB Mutant ▿ †  
Infection and Immunity  2011;79(12):5027-5038.
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.
doi:10.1128/IAI.05524-11
PMCID: PMC3232669  PMID: 21930761
17.  Structure and function of both domains of ArnA, a dual function decarboxylase and a formyltransferase, involved in 4-amino-4-deoxy-L-arabinose biosynthesis 
The Journal of biological chemistry  2005;280(24):23000-23008.
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.
doi:10.1074/jbc.M501534200
PMCID: PMC3326539  PMID: 15809294
site directed mutagenesis; X-ray crystallography; drug design; LPS biosynthesis; polymyxin resistance
18.  A live attenuated strain of Yersinia pestis KIM as a vaccine against plague 
Vaccine  2011;29(16):2986-2998.
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.
doi:10.1016/j.vaccine.2011.01.099
PMCID: PMC3073832  PMID: 21320544
Yersinia pestis; lipid A; regulated delayed attenuation; plague vaccine
19.  Structural Characterization of the Polar Lipids of Clostridium novyi NT. Further Evidence for a Novel Anaerobic Biosynthetic Pathway to Plasmalogens 
Biochimica et biophysica acta  2010;1811(3):186-193.
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.
doi:10.1016/j.bbalip.2010.12.010
PMCID: PMC3033967  PMID: 21195206
20.  Identification of a chloroform-soluble membrane mini-protein in Escherichia coli and its homolog in Salmonella typhimurium 
Analytical biochemistry  2010;409(2):284-289.
Two homologous 29 amino acid-long highly hydrophobic membrane mini-proteins were identified in the Bligh-Dyer lipid extracts of Escherichia coli and Salmonella typhimurium using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The amino acid sequences of the proteins were determined by collision-induced dissociation tandem mass spectrometry, in conjunction with a translating BLAST (tBLASTn) search, i.e. comparing the MS/MS-determined protein query sequence against the six-frame translations of the nucleotide sequences of the E. coli and S. typhimurium genomes. Further MS characterization revealed that both proteins retain the N-terminal initiating formyl-methionines. The methodologies described here may be amendable for detecting and characterizing small hydrophobic proteins in other organisms that are difficult to annotate or analyze by conventional methods.
doi:10.1016/j.ab.2010.10.035
PMCID: PMC3018292  PMID: 21050835
21.  Species-Specific and Inhibitor-Dependent Conformations of LpxC—Implications for Antibiotic Design 
Chemistry & biology  2010;18(1):38-47.
SUMMARY
LpxC is an essential enzyme in the lipid A biosynthetic pathway in Gram-negative bacteria. Several promising antimicrobial lead compounds targeting LpxC have been reported, though they typically display a large variation in potency against different Gram-negative pathogens. We report that inhibitors with a diacetylene scaffold effectively overcome the resistance caused by sequence variation in the LpxC substrate-binding passage. Compound binding is captured in complex with representative LpxC orthologs, and structural analysis reveals large conformational differences that mostly reflect inherent molecular features of distinct LpxC orthologs, whereas ligand-induced structural adaptations occur at a smaller scale. These observations highlight the need for a molecular understanding of inherent structural features and conformational plasticity of LpxC enzymes for optimizing LpxC inhibitors as broad-spectrum antibiotics against Gram-negative infections.
doi:10.1016/j.chembiol.2010.11.011
PMCID: PMC3149848  PMID: 21167751
LpxC; Lipid A; Antibiotic; Protein-inhibitor complex
22.  Syntheses, Structures and Antibiotic Activities of LpxC Inhibitors Based on the Diacetylene Scaffold 
Bioorganic & medicinal chemistry  2010;19(2):852-860.
Compounds inhibiting LpxC in the lipid A biosynthetic pathway are promising leads for novel antibiotics against multidrug-resistant Gram-negative pathogens. We report the syntheses and structural and biochemical characterizations of LpxC inhibitors based on a diphenyl-diacetylene (1,4-diphenyl-1,3-butadiyne) threonylhydroxamate scaffold. These studies provide a molecular interpretation for the differential antibiotic activities of compounds with a substituted distal phenyl ring as well as the absolute stereochemical requirement at the C2, but not C3, position of the threonyl group.
doi:10.1016/j.bmc.2010.12.017
PMCID: PMC3035996  PMID: 21194954
LpxC; inhibitor; antibiotic; diphenyl-diacetylene; 1,4-diphenyl-1,3-butadiyne; hydroxamate
23.  Expression of functional bacterial undecaprenyl pyrophosphate synthase in the yeast rer2Δ mutant and CHO cells 
Glycobiology  2010;20(12):1585-1593.
During evolution the average chain length of polyisoprenoid glycosyl carrier lipids increased from C55 (prokaryotes) to C75 (yeast) to C95 (mammalian cells). In this study, the ability of the E. coli enzyme, undecaprenyl pyrophosphate synthase (UPPS), to complement the loss of the yeast cis-isoprenyltransferase in the rer2Δ mutant was tested to determine if (55)dolichyl phosphate (Dol-P) could functionally substitute in the protein N-glycosylation pathway for (75)Dol-P, the normal isoprenologue synthesized in S. cerevisiae. First, expression of UPPS in the yeast mutant was found to complement the growth and the hypoglycosylation of carboxypeptidase Y defects suggesting that the (55)polyprenyl-P-P intermediate was converted to (55)Dol-P and that (55)Dol-P could effectively substitute for (75)Dol-P in the biosynthesis and function of Man-P-Dol, Glc-P-Dol and Glc3Man9GlcNAc2-P-P-Dol (mature DLO) in the protein N-glycosylation pathway and glycosylphosphatidylinositol anchor assembly. In support of this conclusion, mutant cells expressing UPPS (1) synthesized (55)Dol-P based on MS analysis, (2) utilized (55)Dol-P to form Man-P-(55)Dol in vitro and in vivo, and (3) synthesized N-linked glycoproteins at virtually normal rates as assessed by metabolic labeling with [3H]mannose. In addition, an N-terminal GFP-tagged construct of UPPS was shown to localize to the endoplasmic reticulum of Chinese hamster ovary cells. Consistent with the synthesis of (55)Dol-P by the transfected cells, microsomes from the transfected cells synthesized the [14C](55)polyprenyl-P-P intermediate when incubated with [14C]isopentenyl pyrophosphate and [3H]Man-P-(55)Dol when incubated with GDP-[3H]Man. These results indicate that (C55)polyisoprenoid chains, significantly shorter than the natural glycosyl carrier lipid, can function in the transbilayer movement of DLOs in the endoplasmic reticulum of yeast and mammalian cells, and that conserved sequences in the cis-isoprenyltransferases are recognized by, yet to be identified, binding partners in the endoplasmic reticulum of mammalian cells.
doi:10.1093/glycob/cwq107
PMCID: PMC3003547  PMID: 20685834
CHO cells; cis-isoprenyltransferase; dolichyl phosphate; undecaprenyl pyrophosphate synthase; yeast mutant
24.  Removal of the outer Kdo from Helicobacter pylori lipopolysaccharide and its impact on the bacterial surface 
Molecular microbiology  2010;78(4):837-852.
Helicobacter pylori produces a unique surface lipopolysaccharide (LPS) characterized by strikingly low endotoxicity that is thought to aid the organism in evading the host immune response. This reduction in endotoxicity is predicted to arise from the modification of the Kdo-lipid A domain of Helicobacter LPS by a series of membrane bound enzymes including a Kdo (3-deoxy-D-manno-octulosonic acid) hydrolase responsible for the modification of the core-oligosaccharide. Here we report that Kdo hydrolase activity is dependent upon a putative two-protein complex composed of proteins Hp0579 and Hp0580. Inactivation of Kdo hydrolase activity produced two phenotypes associated with cationic antimicrobial peptide (CAMP) resistance and O-antigen expression. Kdo hydrolase mutants were highly sensitive to polymyxin B, which could be attributed to a defect in downstream modifications to the lipid A 4′-phosphate group. Production of a fully extended O-antigen was also diminished in a Kdo hydrolase mutant, with a consequent increase in core-lipid A. Finally, expression of O-antigen Lewis X and Y epitopes, known to mimic glycoconjugates found on human tissues, was also affected. Taken together, we have demonstrated that loss of Kdo hydrolase activity affects all three domains of H. pylori LPS, thus highlighting it’s role in the maintenance of the bacterial surface.
doi:10.1111/j.1365-2958.2010.07304.x
PMCID: PMC2978256  PMID: 20659292
25.  A Two-component Kdo Hydrolase in the Inner Membrane of Francisella novicida 
Molecular microbiology  2010;78(4):820-836.
Lipid A coats the outer surface of the outer membrane of Gram-negative bacteria. In Francisella tularensis subspecies novicida lipid A is present either as the covalently attached anchor of lipopolysaccharide (LPS) or as free lipid A. The lipid A moiety of Francisella LPS is linked to the core domain by a single 2-keto-3-deoxy-D-manno-octulosonic acid (Kdo) residue. F. novicida KdtA is bifunctional, but F. novicida contains a membrane-bound Kdo hydrolase that removes the outer Kdo unit. The hydrolase consists of two proteins (KdoH1 and KdoH2), which are expressed from adjacent, co-transcribed genes. KdoH1 (related to sialidases) has a single predicted N-terminal transmembrane segment. KdoH2 contains 7 putative transmembrane sequences. Neither protein alone catalyzes Kdo cleavage when expressed in E. coli. Activity requires simultaneous expression of both proteins or mixing of membranes from strains expressing the individual proteins under in vitro assay conditions in the presence of non-ionic detergent. In E. coli expressing KdoH1 and KdoH2, hydrolase activity is localized in the inner membrane. WBB06, a heptose-deficient E. coli mutant that makes Kdo2-lipid A as its sole LPS, accumulates Kdo-lipid A when expressing the both hydrolase components, and 1-dephospho-Kdo-lipid A when expressing both the hydrolase and the Francisella lipid A 1-phosphatase (LpxE).
doi:10.1111/j.1365-2958.2010.07305.x
PMCID: PMC2978271  PMID: 20662782

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