Epstein-Barr Virus Large Tegument Protein BPLF1 Contributes to Innate Immune Evasion through Interference with Toll-Like Receptor Signaling

Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV) is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs). The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-κB at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-κB activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts.     

A Cell-penetrating Peptide Derived from Human Lactoferrin with Conformation-dependent Uptake Efficiency

The molecular events that contribute to the cellular uptake of cell-penetrating peptides (CPP) are still a matter of intense research. Here, we report on the identification and characterization of a 22-amino acid CPP derived from the human milk protein, lactoferrin. The peptide exhibits a conformation-dependent uptake efficiency that is correlated with efficient binding to heparan sulfate and lipid-induced conformational changes. The peptide contains a disulfide bridge formed by terminal cysteine residues. At concentrations exceeding 10 μm, this peptide undergoes the same rapid entry into the cytoplasm that was described previously for the arginine-rich CPPs nona-arginine and Tat. Cytoplasmic entry strictly depends on the presence of the disulfide bridge. To better understand this conformation dependence, NMR spectroscopy was performed for the free peptide, and CD measurements were performed for free and lipid-bound peptide. In solution, the peptides showed only slight differences in secondary structure, with a predominantly disordered structure both in the presence and absence of the disulfide bridge. In contrast, in complex with large unilamellar vesicles, the conformation of the oxidized and reduced forms of the peptide clearly differed. Moreover, surface plasmon resonance experiments showed that the oxidized form binds to heparan sulfate with a considerably higher affinity than the reduced form. Consistently, membrane binding and cellular uptake of the peptide were reduced when heparan sulfate chains were removed.     

Inhibition of lipoxygenase affects induction of both direct and indirect plant defences against herbivorous insects

Herbivore-induced plant defences influence the behaviour of insects associated with the plant. For biting–chewing herbivores the octadecanoid signal-transduction pathway has been suggested to play a key role in induced plant defence. To test this hypothesis in our plant—herbivore—parasitoid tritrophic system, we used phenidone, an inhibitor of the enzyme lipoxygenase (LOX), that catalyses the initial step in the octadecanoid pathway. Phenidone treatment of Brussels sprouts plants reduced the accumulation of internal signalling compounds in the octadecanoid pathway downstream of the step catalysed by LOX, i.e. 12-oxo-phytodienoic acid (OPDA) and jasmonic acid. The attraction of Cotesia glomerata parasitoids to host-infested plants was significantly reduced by phenidone treatment. The three herbivores investigated, i.e. the specialists Plutella xylostella, Pieris brassicae and Pieris rapae, showed different oviposition preferences for intact and infested plants, and for two species their preference for either intact or infested plants was shown to be LOX dependent. Our results show that phenidone inhibits the LOX-dependent defence response of the plant and that this inhibition can influence the behaviour of members of the associated insect community.     

Glycosylation and Sialylation of Macrophage-derived Human Apolipoprotein E Analyzed by SDS-PAGE and Mass Spectrometry: EVIDENCE FOR A NOVEL SITE OF GLYCOSYLATION ON SER290*

Apolipoprotein E (apoE) is a 34-kDa glycoprotein secreted from various cells including hepatocytes and macrophages and plays an important role in remnant lipoprotein clearance, immune responses, Alzheimer disease, and atherosclerosis. Cellular apoE and plasma apoE exist as multiple glycosylated and sialylated glycoforms with plasma apoE being less glycosylated/sialylated than cell-derived apoE. Some of the glycan structures on plasma apoE are characterized; however, the more complicated structures on plasma and cellular/secreted apoE remain unidentified. We investigated glycosylation and sialylation of cellular and secreted apoE from primary human macrophages by one- and two-dimensional gel electrophoresis and mass spectrometry. Our results identify eight different glycoforms with (HexNAc)₂-Hex₂-(NeuAc)₂ being the most complex glycan detected on Thr¹⁹⁴ in both cellular and secreted apoE. Four additional glycans were identified on apoE(283–299), and using β-elimination/alkylation by methylamine in vitro, we identified Ser²⁹⁰ as a novel site of glycan attachment. Comparison of plasma and cellular/secreted apoE from the same donor confirmed that cell-derived apoE is more extensively sialylated than plasma apoE. Given the importance of the C terminus of apoE in regulating apoE solubility, stability, and lipid binding, these results may have important implications for our understanding of apoE biochemistry.Apolipoprotein E (apoE)¹ is a 34-kDa glycosylated apolipoprotein of 299 amino acids. ApoE is synthesized and secreted by most cells including hepatocytes, smooth muscle cells, neuronal cells, and macrophages (1–3) and demonstrates extraordinary functional diversity. It has important roles in remnant lipoprotein clearance, the immune response, Alzheimer disease, cell proliferation, and lymphocyte activation (4, 5). More recent studies suggest that elevated plasma apoE precedes elevation of C-reactive protein and confers increased risk of cardiovascular death in the elderly (6). Proteomics-based approaches have identified elevated high density lipoprotein (HDL)-apoE as being associated with coronary disease (7). In contrast, macrophage-specific expression of apoE protects against atherosclerosis in mice (8, 9). The mechanisms by which macrophage apoE is antiatherogenic may include stimulating the removal of excess cholesterol from macrophage foam cells as well as anti-inflammatory, antiproliferative, and immunomodulatory properties (4, 5, 10–12). An accurate understanding of the structure of apoE secreted from macrophages is important for our understanding of its properties and its role in the atherosclerotic process.Structural studies on apoE have provided important insights into its biological properties (13). Crystallography has demonstrated that the N-terminal domain is structured in a globular four-helix bundle with the helices orientated in an antiparallel alignment (14). The structure of the C terminus has not been resolved by crystallography, but circular dichroism spectroscopy indicates it to be highly α-helical (14). Recently, NMR studies of monomeric, full-length human apoE indicated that the C-terminal domain in the intact protein adopts a more defined structure than it does as an isolated fragment (15). Lipid binding occurs at the C terminus (residues 244–272), resulting in unfolding of the molecule into a helical hairpin with the binding region for the low density lipoprotein (LDL) receptor contained within the N terminus at its apex (16).Mucin-type O-glycosylation is a particularly common, complex, and important post-translational modification of secreted and cell surface glycoproteins (17, 18) that is difficult to accurately characterize; however, several recent reports have facilitated analysis (19, 20). Cellular apoE and plasma apoE exist as multiple glycoforms, which vary in charge because of variable sialylation. The initial analysis of the carbohydrate content of plasma very low density lipoprotein (VLDL)-apoE by colorimetric methods and gas chromatography demonstrated that the major unmodified hexose in apoE was galactose and that N-acetylglucosamine, N-acetylgalactosamine, and sialic acid were present (21, 22). Two-dimensional gel electrophoresis (2-DE) identified up to six sialylated apoE (Es) glycoforms in cells for any given genotype and fewer sialylated glycoforms in plasma (22). ApoE does not contain the consensus sequence (NX(T/S/C)) required for N-linked glycans, and carbohydrate residues are attached to apoE via an O-linkage to residue Thr¹⁹⁴ (23–25). More recent studies using 2-DE and MALDI-TOF/TOF (23) confirmed previous results and identified five glycosylated glycoforms of apoE in plasma VLDL with the most complex sugar structures containing two sialic acid residues (HexNAc-Hex-NeuAc-NeuAc). There were more negatively charged glycoforms present on 2-DE than were distinguished by MALDI-TOF/TOF, raising the possibility that complex structures containing more than two sialic acid residues may be inherently unstable during MS analysis. Importantly, this recent study did not analyze apoE glycoforms in, or secreted from, cells.The purpose of this study was to undertake the first detailed characterization of the glycan structures of apoE from primary human macrophages by 1-DE, 2-DE, and mass spectrometry. We found that cellular and secreted apoE in human macrophages has at least eight different glycoforms with (HexNAc)₂-Hex₂-(NeuAc)₂ being the most complex glycan identified. We extend previous studies by the identification of a novel site of glycan attachment on Ser²⁹⁰ near the functionally important apoE C terminus in addition to glycosylation of Thr¹⁹⁴ and show that a major glycoform is present in each of the spots separated by 2-DE.     

PHYLOGENETIC POSITION OF ATTHEYA LONGICORNIS AND ATTHEYA SEPTENTRIONALIS (BACILLARIOPHYTA)1

The phylogenetic position of diatoms belonging to the genus Attheya is presently under debate. Species belonging to this genus have been placed in the subclasses Chaetocerotophycidae and Biddulphiophycidae, but published phylogenetic trees based on 18S rDNA, morphology, and sexual reproduction indicate that this group of diatoms may be a sister group of the pennates. To clarify the position of Attheya, we studied the morphology, 18S rDNA, 16S rDNA of the chloroplasts, the rbcL large subunit (LSU) sequences of the chloroplasts, and the sterol composition of three different strains of Attheya septentrionalis (Østrup) R. M. Crawford and one strain of Attheya longicornis R. M. Crawford et C. Gardner. These data were compared with data from more than 100 other diatom species, covering the whole phylogenetic tree, with special emphasis on species belonging to the genera that have been suggested to be related to the genus Attheya. All data suggest that the investigated Attheya species form a separate group of diatoms, and there is no indication that they belong to either the Chaetocerotophycidae or the Biddulphiophycidae. Despite applying these various approaches, we were unable to determine the exact phylogenetic position of the investigated Attheya species within the diatoms.     

Isoniazid Prophylactic Therapy for the Prevention of Tuberculosis in HIV Infected Adults: A Systematic Review and Meta-Analysis of Randomized Trials

Background

Infection with Human Immunodeficiency virus (HIV) is an important risk factor for Tuberculosis (TB). Anti-Retroviral Therapy (ART) has improved the prognosis of HIV and reduced the risk of TB infected patients. Isoniazid Preventive Therapy (IPT) aims to reduce the development of active TB in patients with latent TB.

Objective

Systematically review and synthesize effect estimates of IPT for TB prevention in adult HIV patients. Secondary objectives were to assess the effect of IPT on HIV disease progression, all-cause mortality and adverse drug reaction (ADR).

Search Strategy

Electronic databases were searched to identify relevant articles in English available by September 11^th 2015.

Selection Criteria

Research articles comparing IPT to placebo or no treatment in HIV infected adults using randomized clinical trials.

Data Analysis

A qualitative review included study-level information on randomization and treatment allocation. Effect estimates were pooled using random-effects models to account for between-study heterogeneity.

Main Results

This review assessed ten randomized clinical trials that assigned 7619 HIV patients to IPT or placebo. An overall 35% of TB risk reduction (RR = 0.65, 95% CI (0.51, 0.84)) was found in all participants, however, larger benefit of IPT was observed in Tuberculin Skin Test (TST) positive participants, with pooled relative risk reduction of 52% [RR = 0.48; 95% CI (0.29, 0.82)] and with a prediction interval ranging from 0.13 to 1.81. There was no statistically significant effect of IPT on TB occurrence in TST negative or unknown participants. IPT also reduced the risk of HIV disease progression in all participants (RR = 0.69; 95% CI (0.48, 0.99)) despite no benefits observed in TST strata. All-cause mortality was not affected by IPT although participants who had 12 months of IPT tend to have a reduced risk (RR = 0.65; 95% CI(0.47, 0.90)). IPT had an elevated, yet statistically non-significant, risk of adverse drug reaction [RR = 1.20; 95% CI (1.20, 1.71)]. Only a single study assessed the effect of IPT in combination with ART in preventing TB and occurrence of multi-drug resistant tuberculosis.

Conclusions

IPT use substantially contributes in preventing TB in persons with HIV in general and in TST positive individuals in particular. More evidence is needed to explain discrepancies in the protective effect of IPT in these individuals.     

The Impact of Lactobacillus plantarum WCFS1 Teichoic Acid D-Alanylation on the Generation of Effector and Regulatory T-cells in Healthy Mice

To date it remains unclear how probiotics affect the immune system. Bacterial envelope components may play an essential role, as these are the first to establish bacterial-host cell interactions. Teichoic acids (TAs), and especially lipoteichoic acids, are the most pro-inflammatory components of the gram-positive bacterial envelope. This effect is dependent on D-alanyl substitution of the TA backbone and interactions with TLR2 on host cells. Although the pro-inflammatory properties of TAs have been established in vitro, it remains unclear how TAs affect immunomodulation in vivo. In this study, we investigated the role of TA D-alanylation on L. plantarum–induced intestinal and systemic immunomodulation in vivo. For this, we compared the effect of L. plantarum WCFS1 and its TA D-Alanylation negative derivative (dltX-D) on the distribution of dendritic cell and T cell populations and responses in healthy mice. We demonstrated that the majority of the L. plantarum-induced in vivo immunomodulatory effects were dependent on D-alanylation (D-Ala), as some L. plantarum WCFS1-induced immune changes were not observed in the dltX-D-treated group and some were only observed after treatment with dltX-D. Strikingly, not only pro-inflammatory immune responses were abolished in the absence of D-Ala substitution, but also anti-inflammatory responses, such as the L. plantarum-induced generation of regulatory T cells in the spleen. With this study we provide insight in host-microbe interactions, by demonstrating the involvement of D-alanylation of TAs on the bacterial membrane in intestinal and systemic immunomodulation in healthy mice.     

A Highlights from MBoC Selection: Nesprin-3 connects plectin and vimentin to the nuclear envelope of Sertoli cells but is not required for Sertoli cell function in spermatogenesis

Nesprin-3 is a nuclear envelope protein that connects the nucleus to intermediate filaments by interacting with plectin. To investigate the role of nesprin-3 in the perinuclear localization of plectin, we generated nesprin-3–knockout mice and examined the effects of nesprin-3 deficiency in different cell types and tissues. Nesprin-3 and plectin are coexpressed in a variety of tissues, including peripheral nerve and muscle. The expression level of nesprin-3 in skeletal muscle is very low and decreases during myoblast differentiation in vitro. Of interest, plectin was concentrated at the nuclear envelope in only a few cell types. This was most prominent in Sertoli cells of the testis, in which nesprin-3 is required for the localization of both plectin and vimentin at the nuclear perimeter. Testicular morphology and the position of the nucleus in Sertoli cells were normal, however, in the nesprin-3–knockout mice and the mice were fertile. Furthermore, nesprin-3 was not required for the polarization and migration of mouse embryonic fibroblasts. Thus, although nesprin-3 is critical for the localization of plectin to the nuclear perimeter of Sertoli cells, the resulting link between the nuclear envelope and the intermediate filament system seems to be dispensable for normal testicular morphology and spermatogenesis.