The Structure of the Neisserial Lipooligosaccharide Phosphoethanolamine Transferase A (LptA) Required for Resistance to Polymyxin

Gram-negative bacteria possess an outer membrane envelope consisting of an outer leaflet of lipopolysaccharides, also called endotoxins, which protect the pathogen from antimicrobial peptides and have multifaceted roles in virulence. Lipopolysaccharide consists of a glycan moiety attached to lipid A, embedded in the outer membrane. Modification of the lipid A headgroups by phosphoethanolamine (PEA) or 4-amino-arabinose residues increases resistance to the cationic cyclic polypeptide antibiotic, polymyxin. Lipid A PEA transferases are members of the YhjW/YjdB/YijP superfamily and usually consist of a transmembrane domain anchoring the enzyme to the periplasmic face of the cytoplasmic membrane attached to a soluble catalytic domain. The crystal structure of the soluble domain of the protein of the lipid A PEA transferase from Neisseria meningitidis has been determined crystallographically and refined to 1.4 Å resolution. The structure reveals a core hydrolase fold similar to that of alkaline phosphatase. Loop regions in the structure differ, presumably to enable interaction with the membrane-localized substrates and to provide substrate specificity. A phosphorylated form of the putative nucleophile, Thr280, is observed. Metal ions present in the active site are coordinated to Thr280 and to residues conserved among the family of transferases. The structure reveals the protein components needed for the transferase chemistry; however, substrate-binding regions are not evident and are likely to reside in the transmembrane domain of the protein.     

Phosphoethanolamine Transferase LptA in Haemophilus ducreyi Modifies Lipid A and Contributes to Human Defensin Resistance In Vitro

Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, β-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. The triple mutant was significantly more susceptible to both α- and β-defensins; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin LL-37 may be more important than defensin resistance to H. ducreyi pathogenesis.     

Identification of Genes Required for the Function of Non-Race-Specific mlo Resistance to Powdery Mildew in Barley

Recessive alleles (mlo) of the Mlo locus in barley mediate a broad, non-race-specific resistance reaction to the powdery mildew fungus Erysiphe graminis f sp hordei. A mutational approach was used to identify genes that are required for the function of mlo. Six susceptible M2 individuals were isolated after inoculation with the fungal isolate K1 from chemically mutagenized seed carrying the mlo-5 allele. Susceptibility in each of these individuals is due to monogenic, recessively inherited mutations in loci unlinked to mlo. The mutants identify two unlinked complementation groups, designated Ror1 and Ror2 (required for mlo-specified resistance). Both Ror genes are required for the function of different tested mlo alleles and for mlo function after challenge with different isolates of E. g. f sp hordei. A quantitative cytological time course analysis revealed that the host cell penetration efficiency in the mutants is intermediate compared with mlo-resistant and Mlo-susceptible genotypes. Ror1 and Ror2 mutants could be differentiated from each other by the same criterion. The spontaneous formation of cell wall appositions in mlo plants, a subcellular structure believed to represent part of the mlo defense, is suppressed in mlo/ror genotypes. In contrast, accumulation of major structural components in the appositions is seemingly unaltered. We conclude that there is a regulatory function for the Ror genes in mlo-specified resistance and propose a model in which the Mlo wild-type allele functions as a negative regulator and the Ror genes act as positive regulators of a non-race-specific resistance response.     

A Multicopper Oxidase Is Required for Copper Resistance in Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis, is one of the most important bacterial pathogens. Recent work has revealed that the natural bactericidal properties of copper are utilized by the host immune system to combat infections with bacteria, including M. tuberculosis. However, M. tuberculosis employs multiple mechanisms to reduce the internal copper amount by efflux and sequestration, which are required for virulence of M. tuberculosis. Here, we describe an alternative mechanism of copper resistance by M. tuberculosis. Deletion of the rv0846c gene increased the susceptibility of M. tuberculosis to copper at least 10-fold, establishing Rv0846c as a major component of copper resistance in M. tuberculosis. In vitro assays showed that Rv0846c oxidized organic substrates and Fe(II). Importantly, mutation of the predicted copper-coordinating cysteine 486 resulted in inactive Rv0846c protein which did not protect M. tuberculosis against copper stress. Hence, Rv0846c is a multicopper oxidase of M. tuberculosis and was renamed mycobacterial multicopper oxidase (MmcO). MmcO is membrane associated, probably by lipidation after export across the inner membrane by the twin-arginine translocation system. However, mutation of the lipidation site did not affect the oxidase activity or the copper protective function of MmcO. Our study revealed MmcO as an important copper resistance mechanism of M. tuberculosis, which possibly acts by oxidation of toxic Cu(I) in the periplasm.     

The Role of Lipid Physical Properties in Lipid Barriers

SYNOPSIS.The hydrophobic nature of lipids means that they providegood bar-riers to the movement of charged and polar molecules.Barrier function appears to depend on the physical state ofthe lipids. Two well-investigated examples in-cludecell membranesand epicuticular lipids of arthropods. Ecologically relevantchanges in temperature significantly affect lipid properties,and both evolutionary and acclimatory differences in lipid compositionappear to preserve the physical properties of lipids under differentenvironmental conditions. These differences are generally believedto be beneficial to the organism, but rigorous examination oftheir adaptive significance is rare. Important issues are howlipid properties are regulated; which properties are physiologicallyrelevant, how are these properties sensed, and what biochemicaland molecular mechanisms regulate lipid properties? Progresshas recently been made in understanding how membrane lipid propertiesare regulated, but regulatory mechanisms for cuticular lipidsand other lipid sys-tems remain unknown.     

ABCA1在动脉粥样硬化发生与发展中的作用

腺苷三磷酸结合盒转运体A1(ATP binding cassette transporter A1 ,ABCA1)是一种整合膜蛋白,它以ATP为能源,促进细胞内游离胆固醇和磷脂的流出,在胆固醇逆转运(RCT)和HDL生成的起始步骤中起重要作用,被称作RCT守门人。核受体PPARs、LXRs和FXR对ABCA1蛋白的表达具有调控作用。人体50种组织中存在有ABCA1 mRNA,在胰、肝、肺、肾上腺和胎儿组织中ABCAl表达水平最高,ABCAl功能障碍将导致巨噬细胞内大量的胆固醇沉积而成为泡沫细胞,继而漫润血管壁,促进As的发生发展。     

A Role for Glutathione Transferase Omega 1 (GSTO1-1) in the Glutathionylation Cycle

The glutathionylation of intracellular protein thiols can protect against irreversible oxidation and can act as a redox switch regulating metabolic pathways. In this study we discovered that the Omega class glutathione transferase GSTO1-1 plays a significant role in the glutathionylation cycle. The catalytic activity of GSTO1-1 was determined in vitro by assaying the deglutathionylation of a synthetic peptide by tryptophan fluorescence quenching and in T47-D epithelial breast cancer cells by both immunoblotting and the direct determination of total glutathionylation. Mutating the active site cysteine residue (Cys-32) ablated the deglutathionylating activity of GSTO1-1. Furthermore, we demonstrate that the expression of GSTO1-1 in T47-D cells that are devoid of endogenous GSTO1-1 resulted in a 50% reduction in total glutathionylation levels. Mass spectrometry and immunoprecipitation identified β-actin as a protein that is specifically deglutathionylated by GSTO1-1 in T47-D cells. In contrast to the deglutathionylation activity, we also found that GSTO1-1 is associated with the rapid glutathionylation of cellular proteins when the cells are exposed to S-nitrosoglutathione. The common A140D genetic polymorphism in GSTO1 was found to have significant effects on the kinetics of both the deglutathionylation and glutathionylation reactions. Genetic variation in GSTO1-1 has been associated with a range of diseases, and the discovery that a frequent GSTO1-1 polymorphism affects glutathionylation cycle reactions reveals a common mechanism where it can act on multiple proteins and pathways.     

The Role of the Lipid Bilayer in Tau Aggregation

Tau is a microtubule associated protein whose aggregation is implicated in a number of neurodegenerative diseases. We investigate the mechanism by which anionic lipid vesicles induce aggregation of tau in vitro using K18, a fragment of tau corresponding to the four repeats of the microtubule binding domain. Our results show that aggregation occurs when the amount of K18 bound to the lipid bilayer exceeds a critical surface density. The ratio of protein/lipid at the critical aggregation concentration is pH-dependent, as is the binding affinity. At low pH, where the protein binds with high affinity, the critical surface density is independent both of total lipid concentration as well as the fraction of anionic lipid present in the bilayer. Furthermore, the aggregates consist of both protein and vesicles and bind the β-sheet specific dye, Thioflavin T, in the manner characteristic of pathological aggregates. Our results suggest that the lipid bilayer facilitates protein-protein interactions both by screening charges on the protein and by increasing the local protein concentration, resulting in rapid aggregation. Because anionic lipids are abundant in cellular membranes, these findings contribute to understanding tau-lipid bilayer interactions that may be relevant to disease pathology.     

Protective Role of Spore Structural Components in Determining Bacillus subtilis Spore Resistance to Simulated Mars Surface Conditions

Spores of wild-type and mutant Bacillus subtilis strains lacking various structural components were exposed to simulated Martian atmospheric and UV irradiation conditions. Spore survival and mutagenesis were strongly dependent on the functionality of all of the structural components, with small acid-soluble spore proteins, coat layers, and dipicolinic acid as key protectants.     

A Signaling Protease Required for Melanization in Drosophila Affects Resistance and Tolerance of Infections

Organisms evolve two routes to surviving infections—they can resist pathogen growth (resistance) and they can endure the pathogenesis of infection (tolerance). The sum of these two properties together defines the defensive capabilities of the host. Typically, studies of animal defenses focus on either understanding resistance or, to a lesser extent, tolerance mechanisms, thus providing little understanding of the relationship between these two mechanisms. We suggest there are nine possible pairwise permutations of these traits, assuming they can increase, decrease, or remain unchanged in an independent manner. Here we show that by making a single mutation in the gene encoding a protease, CG3066, active in the melanization cascade in Drosophila melanogaster, we observe the full spectrum of changes; these mutant flies show increases and decreases in their resistance and tolerance properties when challenged with a variety of pathogens. This result implicates melanization in fighting microbial infections and shows that an immune response can affect both resistance and tolerance to infections in microbe-dependent ways. The fly is often described as having an unsophisticated and stereotypical immune response where single mutations cause simple binary changes in immunity. We report a level of complexity in the fly's immune response that has strong ecological implications. We suggest that immune responses are highly tuned by evolution, since selection for defenses that alter resistance against one pathogen may change both resistance and tolerance to other pathogens.     

The dlt Operon of Bacillus cereus Is Required for Resistance to Cationic Antimicrobial Peptides and for Virulence in Insects

The dlt operon encodes proteins that alanylate teichoic acids, the major components of cell walls of gram-positive bacteria. This generates a net positive charge on bacterial cell walls, repulsing positively charged molecules and conferring resistance to animal and human cationic antimicrobial peptides (AMPs) in gram-positive pathogenic bacteria. AMPs damage the bacterial membrane and are the most effective components of the humoral immune response against bacteria. We investigated the role of the dlt operon in insect virulence by inactivating this operon in Bacillus cereus, which is both an opportunistic human pathogen and an insect pathogen. The Δdlt_Bc mutant displayed several morphological alterations but grew at a rate similar to that for the wild-type strain. This mutant was less resistant to protamine and several bacterial cationic AMPs, such as nisin, polymyxin B, and colistin, in vitro. It was also less resistant to molecules from the insect humoral immune system, lysozyme, and cationic AMP cecropin B from Spodoptera frugiperda. Δdlt_Bc was as pathogenic as the wild-type strain in oral infections of Galleria mellonella but much less virulent when injected into the hemocoels of G. mellonella and Spodoptera littoralis. We detected the dlt operon in three gram-negative genera: Erwinia (Erwinia carotovora), Bordetella (Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), and Photorhabdus (the entomopathogenic bacterium Photorhabdus luminescens TT01, the dlt operon of which did not restore cationic AMP resistance in Δdlt_Bc). We suggest that the dlt operon protects B. cereus against insect humoral immune mediators, including hemolymph cationic AMPs, and may be critical for the establishment of lethal septicemia in insects and in nosocomial infections in humans.Gram-positive bacteria are generally enclosed by cell walls consisting of macromolecular assemblies of cross-linked peptidoglycan (murein), polyanionic teichoic acids (TAs), and surface proteins (69). TAs are polymers of repeating glycerophosphate residues. They may be covalently anchored to either peptidoglycan (wall-associated TAs) or the cytoplasmic membrane via glycolipids (lipoteichoic acids [LTAs]). TAs may be involved in controlling cell shape, autolytic enzyme activity, and cation homeostasis (69). They make a significant contribution to the overall negative charge of the bacterial cell wall, attracting negatively charged compounds, including the cationic antimicrobial peptides (AMPs) of the innate humoral immune systems of higher organisms (69).Many of the gram-positive bacterial species pathogenic to humans display resistance to cationic AMPs because of a decrease in the net negative charge of bacterial cell envelopes (75). Modifications to the TAs at the bacterial surface involving the incorporation of positively charged residues, such as d-alanine, prevent cationic AMPs from reaching their target, thereby protecting the organism against these compounds. This process involves the Dlt proteins encoded by the dltABCD operon present in most of the genome sequences established to date for gram-positive bacteria (44, 58, 74). d-Alanine is incorporated into LTAs through a two-step reaction involving a d-alanine-d-alanyl carrier protein ligase (Dcl) and a d-alanyl carrier protein (Dcp), encoded by the dltA and dltC genes, respectively (18, 44, 45, 70). The dltB and dltD genes encode other proteins required for the d-alanylation of LTAs. DltD is involved in selection of the Dcp carrier protein for ligation with d-alanine (19), whereas DltB is thought to be involved in d-alanyl-Dcp secretion (69). d-Alanine may be transferred from d-alanylated LTAs to wall-associated TAs by transacylation. For many human gram-positive bacterial pathogens, dlt operon inactivation has been shown to affect bacterial resistance to cationic AMPs and virulence. Indeed, Listeria monocytogenes, Bacillus anthracis, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Lactobacillus reuteri, and group B streptococci harboring mutations in dlt genes all have a higher negative charge on the cell surface and are more susceptible to cationic AMPs of various origins (1, 34, 56, 58, 59, 77, 78, 89). The inactivation of dlt genes in these pathogenic bacterial species also decreases interactions with phagocytic and nonphagocytic cells (1, 13, 34, 78).The impact of Dlt proteins on cationic AMP resistance and virulence in insect bacterial pathogens has never before been studied, despite the major role of cationic AMPs in insect humoral immunity (9, 61). Insect bacterial pathogens also termed entomopathogenic bacteria are able to multiply in the insect hemocoel from small inocula (<10,000 viable cells) and produce fatal septicemia (8, 57). Entomopathogenic bacteria entering the hemolymph are targeted by an array of immune system mediators of both cellular and humoral reactions. The cellular response results in bacterial phagocytosis or encapsulation by circulating hemocytes, whereas the humoral response generates cationic AMPs (61). These peptides are small, inducible molecules produced in large amounts in hemolymph by the fat body (9, 26). They participate to the insect antimicrobial defense in a systemic response. Many AMP have been reported to cause damage in microbial membranes, which may ultimately lead to bacterial cell lysis (94).We investigated the role of the dlt operon in cationic AMP resistance and virulence in Bacillus cereus, a human opportunistic and insect facultative bacterial pathogen. B. cereus sensu stricto is a spore-forming gram-positive bacterium. The B. cereus sensu lato group of bacteria also includes the closely related insect pathogen Bacillus thuringiensis and the human pathogen B. anthracis. Genomic data have shown that B. thuringiensis and B. cereus have almost identical chromosomal genetic backgrounds (54, 55) but that B. thuringiensis carries a plasmid encoding entomopathogenic cytoplasmic crystalline δ-endotoxins (Cry proteins) specifically active against insect larvae upon ingestion (22, 23, 83). B. cereus can cause opportunistic food-borne gastroenteritis and local/systemic infections in immunocompromised humans (85). Both B. thuringiensis (with and without Cry toxins) and B. cereus strains are highly pathogenic when injected directly into the hemocoels of insect larvae, in which they cause lethal septicemia (46, 82, 86, 96). The occurrence, structure, and glycosylation of LTAs were studied for different Bacillus species, including B. cereus strains containing LTAs (built up of polyglycerol phosphate chains and hydrophobic anchors) and d-alanine (11, 50, 51, 62). Therefore, the presence of a dlt operon in the B. cereus 14579 genome suggests that the LTAs may be alanylated.We report here that the dlt operon of B. cereus is required for resistance to cationic AMPs of bacterial or insect origin. The dlt operon is required for full B. cereus virulence following bacterial injection into two lepidopteran insects, the caterpillar Spodoptera littoralis and the wax moth Galleria mellonella. We also detected the dlt operon in three gram-negative bacterial genera: Erwinia (Erwinia carotovora), Bordetella (Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), and Photorhabdus (the entomopathogenic bacterium Photorhabdus luminescens TT01).     

A Conserved Role for Atlastin GTPases in Regulating Lipid Droplet Size

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The Bacillus anthracis Protein MprF Is Required for Synthesis of Lysylphosphatidylglycerols and for Resistance to Cationic Antimicrobial Peptides

During inhalational anthrax, Bacillus anthracis survives and replicates in alveolar macrophages, followed by rapid invasion into the host's bloodstream, where it multiplies to cause heavy bacteremia. B. anthracis must therefore defend itself from host immune functions encountered during both the intracellular and the extracellular stages of anthrax infection. In both of these niches, cationic antimicrobial peptides are an essential component of the host's innate immune response that targets B. anthracis. However, the genetic determinants of B. anthracis contributing to resistance to these peptides are largely unknown. Here we generated Tn917 transposon mutants in the ΔANR strain (pXO1⁻ pXO2⁻) of B. anthracis and screened them for altered protamine susceptibility. A protamine-sensitive mutant identified carried the transposon inserted in the BA1486 gene encoding a putative membrane protein homologous to MprF known in several gram-positive pathogens. A mutant strain with the BAS1375 gene (the orthologue of BA1486) deleted in the Sterne 34F2 strain (pXO1⁺ pXO2⁻) of B. anthracis exhibited hypersusceptibility not only to protamine but also to α-helical cathelicidin LL-37 and β-sheet defensin human neutrophil peptide 1 compared to the wild-type Sterne strain. Analysis of membrane lipids using isotopic labeling demonstrated that the BAS1375 deletion mutant is unable to synthesize lysinylated phosphatidylglycerols, and this defect is rescued by genetic complementation. Further, we determined the structures of these lysylphosphatidylglycerols by using various mass spectrometric analyses. These results demonstrate that in B. anthracis a functional MprF is required for the biosynthesis of lysylphosphatidylglycerols, which is critical for resistance to cationic antimicrobial peptides.     

A Global In Vivo Drosophila RNAi Screen Identifies a Key Role of Ceramide Phosphoethanolamine for Glial Ensheathment of Axons

Glia are of vital importance for all complex nervous system. One of the many functions of glia is to insulate and provide trophic and metabolic support to axons. Here, using glial-specific RNAi knockdown in Drosophila, we silenced 6930 conserved genes in adult flies to identify essential genes and pathways. Among our screening hits, metabolic processes were highly represented, and genes involved in carbohydrate and lipid metabolic pathways appeared to be essential in glia. One critical pathway identified was de novo ceramide synthesis. Glial knockdown of lace, a subunit of the serine palmitoyltransferase associated with hereditary sensory and autonomic neuropathies in humans, resulted in ensheathment defects of peripheral nerves in Drosophila. A genetic dissection study combined with shotgun high-resolution mass spectrometry of lipids showed that levels of ceramide phosphoethanolamine are crucial for axonal ensheathment by glia. A detailed morphological and functional analysis demonstrated that the depletion of ceramide phosphoethanolamine resulted in axonal defasciculation, slowed spike propagation, and failure of wrapping glia to enwrap peripheral axons. Supplementing sphingosine into the diet rescued the neuropathy in flies. Thus, our RNAi study in Drosophila identifies a key role of ceramide phosphoethanolamine in wrapping of axons by glia.