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.     

Phosphoethanolamine Modification of Neisseria gonorrhoeae Lipid A Reduces Autophagy Flux in Macrophages

Autophagy, an ancient homeostasis mechanism for macromolecule degradation, performs an important role in host defense by facilitating pathogen elimination. To counteract this host defense strategy, bacterial pathogens have evolved a variety of mechanisms to avoid or otherwise dysregulate autophagy by phagocytic cells so as to enhance their survival during infection. Neisseria gonorrhoeae is a strictly human pathogen that causes the sexually transmitted infection, gonorrhea. Phosphoethanolamine (PEA) addition to the 4'' position of the lipid A (PEA-lipid A) moiety of the lipooligosaccharide (LOS) produced by gonococci performs a critical role in this pathogen’s ability to evade innate defenses by conferring decreased susceptibility to cationic antimicrobial (or host-defense) peptides, complement-mediated killing by human serum and intraleukocytic killing by human neutrophils compared to strains lacking this PEA decoration. Heretofore, however, it was not known if gonococci can evade autophagy and if so, whether PEA-lipid A contributes to this ability. Accordingly, by using murine macrophages and human macrophage-like phagocytic cell lines we investigated if PEA decoration of gonococcal lipid A modulates autophagy formation. We report that infection with PEA-lipid A-producing gonococci significantly reduced autophagy flux in murine and human macrophages and enhanced gonococcal survival during their association with macrophages compared to a PEA-deficient lipid A mutant. Our results provide further evidence that PEA-lipid A produced by gonococci is a critical component in the ability of this human pathogen to evade host defenses.     

The Meningococcal Vaccine Candidate Neisserial Surface Protein A (NspA) Binds to Factor H and Enhances Meningococcal Resistance to Complement

Complement forms an important arm of innate immunity against invasive meningococcal infections. Binding of the alternative complement pathway inhibitor factor H (fH) to fH-binding protein (fHbp) is one mechanism meningococci employ to limit complement activation on the bacterial surface. fHbp is a leading vaccine candidate against group B Neisseria meningitidis. Novel mechanisms that meningococci employ to bind fH could undermine the efficacy of fHbp-based vaccines. We observed that fHbp deletion mutants of some meningococcal strains showed residual fH binding suggesting the presence of a second receptor for fH. Ligand overlay immunoblotting using membrane fractions from one such strain showed that fH bound to a ∼17 kD protein, identified by MALDI-TOF analysis as Neisserial surface protein A (NspA), a meningococcal vaccine candidate whose function has not been defined. Deleting nspA, in the background of fHbp deletion mutants, abrogated fH binding and mAbs against NspA blocked fH binding, confirming NspA as a fH binding molecule on intact bacteria. NspA expression levels vary among strains and expression correlated with the level of fH binding; over-expressing NspA enhanced fH binding to bacteria. Progressive truncation of the heptose (Hep) I chain of lipooligosaccharide (LOS), or sialylation of lacto-N-neotetraose LOS both increased fH binding to NspA-expressing meningococci, while expression of capsule reduced fH binding to the strains tested. Similar to fHbp, binding of NspA to fH was human-specific and occurred through fH domains 6–7. Consistent with its ability to bind fH, deleting NspA increased C3 deposition and resulted in increased complement-dependent killing. Collectively, these data identify a key complement evasion mechanism with important implications for ongoing efforts to develop meningococcal vaccines that employ fHbp as one of its components.     

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.     

Hyperleptinemia Is Required for the Development of Leptin Resistance

Leptin regulates body weight by signaling to the brain the availability of energy stored as fat. This negative feedback loop becomes disrupted in most obese individuals, resulting in a state known as leptin resistance. The physiological causes of leptin resistance remain poorly understood. Here we test the hypothesis that hyperleptinemia is required for the development of leptin resistance in diet-induced obese mice. We show that mice whose plasma leptin has been clamped to lean levels develop obesity in response to a high-fat diet, and the magnitude of this obesity is indistinguishable from wild-type controls. Yet these obese animals with constant low levels of plasma leptin remain highly sensitive to exogenous leptin even after long-term exposure to a high fat diet. This shows that dietary fats alone are insufficient to block the response to leptin. The data also suggest that hyperleptinemia itself can contribute to leptin resistance by downregulating cellular response to leptin as has been shown for other hormones.     

Role of Endothelial Cell Ion Channels in the Resistance Artery Function

Endothelium-dependent hyperpolarizing factor (EDHF) underlies nitric oxide and prostacyclin-independent arterial relaxation. As the influence of EDHF increases with decreasing artery size, it plays an important role in vascular regulation. Initially suggested to represent a diffusible factor, EDHF is now thought to represent a variable input in different arteries from a factor(s) and the spread of hyperpolarizing current from the endothelium to the smooth muscle. Key to unravelling this pathway has been the demonstration that hyperpolarization within the endothelium can be blocked using a combination of the K_Ca channel blockers, apamin and charibdotoxin. As a consequence, the relaxation of vascular smooth muscle, which represents the end point of the EDHF pathway, is blocked. This review discusses the evidence that a differential distribution of ion channels between the smooth muscle and endothelial cells underlies the EDHF pathway. Also, that a diffusible factor, which may well be K ions released by the endothelium, acts alongside the spread of hyperpolarization through myoendothelial gap junctions to explain EDHF-evoked smooth muscle relaxation. While the relative importance of each of these two components can vary between arteries, together they can explain the EDHF phenomenon.     

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.     

A Simple Colorimetric Assay for Specific Detection of Glutathione-S Transferase Activity Associated with DDT Resistance in Mosquitoes

Background

Insecticide-based methods represent the most effective means of blocking the transmission of vector borne diseases. However, insecticide resistance poses a serious threat and there is a need for tools, such as diagnostic tests for resistance detection, that will improve the sustainability of control interventions. The development of such tools for metabolism-based resistance in mosquito vectors lags behind those for target site resistance mutations.

Methodology/Principal Findings

We have developed and validated a simple colorimetric assay for the detection of Epsilon class Glutathione transferases (GST)-based DDT resistance in mosquito species, such as Aedes aegypti, the major vector of dengue and yellow fever worldwide. The colorimetric assay is based on the specific alkyl transferase activity of Epsilon GSTs for the haloalkene substrate iodoethane, which produces a dark blue colour highly correlated with AaGSTE2-2-overexpression in individual mosquitoes. The colour can be measured visually and spectrophotometrically.

Conclusions/Significance

The novel assay is substantially more sensitive compared to the gold standard CDNB assay and allows the discrimination of moderate resistance phenotypes. We anticipate that it will have direct application in routine vector monitoring as a resistance indicator and possibly an important impact on disease vector control.     

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.