Cathelicidins Show Species-Specific Antiviral Effects.

<p>(A,B,D,E) Groups of 5 mice were infected with 10 MLD₅₀ of A/PR/8/34 influenza virus via intranasal administration on day 0. Mice were nebulized with 200 µl of saline (control), zanamivir (500 µg/ml), the murine cathelicidin mCRAMP (500 µg/ml) (A and B) or the porcine cathelicidin Protegrin-1 (500 µg/ml) (D and E) once daily from day -1 to day 7. Mouse body weight (A, D) and survival (B, E) was monitored daily up to 14 days post infection. Data represent mean values ± SEM, for three independent experiments (A and B) or one experiment (D and E). Statistical analysis was performed using Kaplan Meier with a Mantel-Cox (log rank) test. Survival curves obtained with Zanamivir and mCRAMP treatments were significantly different (P≤0.001) compared to saline control treatment. There was no difference between saline treated and Protegrin treated groups. (C, F) Groups of three mice were infected with 10 MLD₅₀ of A/PR/8/34 virus via intranasal administration on day 0. Mice were nebulized with 200 µl of saline (control), zanamivir (500 µg/ml), the murine cathelicidin mCRAMP (500 µg/ml) or the porcine cathelicidin Protegrin-1 (500 µg/ml) once daily from day -1 to day 2. Mice were euthanized on day 3 and viral titer in the lungs was assessed by plaque assay. Figure shows mean values ± SEM. Statistical analysis was performed using an unpaired t-test to compare virus infected animals with virus/peptide and virus/zanamivir treated animals (*P≤0.05, **P≤0.01, ***P≤0.001).</p

Cathelicidins show antiviral activity against influenza virus <i>in vitro.</i>

<p>Influenza virus was pre-incubated with cathelicidin peptide or control peptide scrambled LL-37 (sLL-37) (A) at a range of concentrations as indicated for 1 hour at room temperature and a plaque formation assay was then performed to assess virus titer in MDCK-L cells in the presence of trypsin. Viruses used were A/PR/8/34 (H1N1) (A, B) or A/Udorn/307/72 (C). The antiviral activity of the cathelicidins LL-37 (A, C), mCRAMP (B) and Protegrin-1 (B) was assessed. Figures are representative of at least three independent experiments. Figures show mean values ± SEM. Statistical analysis was performed using an unpaired t-test to compare virus only titer with virus + peptide (*P≤0.05, ** P≤0.01).</p

D-Isomer cathelicidins inhibit influenza virus.

<p>(A,B) Groups of 5 mice were infected with 10 MLD₅₀ of A/PR/8/34 influenza virus via intranasal administration on day 0. Mice were nebulized with 200 µl of saline (control), zanamivir (500 µg/ml), D-LL-37 peptide (500 µg/ml) or D-mCRAMP peptide (500 µg/ml) once daily from day -1 to day 7. Mouse weight and survival was monitored daily up to 14 days post infection. Data represent mean values ± SEM from n = 1 experiment. Statistical analysis was performed using Kaplan Meier with a Mantel-Cox (log rank) test. Survival curves obtained with Zanamivir, D-LL-37 and D-mCRAMP treatments were significantly different (P≤0.001) compared to saline control treatment. (C) Groups of 5 mice were infected with 10MLD₅₀ of A/PR/8/34 virus via intranasal administration on day 0. Mice were nebulized with 200 µl of saline (control), zanamivir (500 µg/ml), D-LL-37 peptide (500 µg/ml) or D-mCRAMP peptide (500 µg/ml) once daily from day -1 to day 2. Mice were euthanized on day 3 and viral titer in the lungs was assessed by plaque assay. Figure is representative of n = 3 independent experiments. Figure shows mean values ± SEM. Statistical analysis was performed using an unpaired t-test to compare virus infected animals with virus/peptide and virus/zanamivir treated animals (*P≤0.05). (D) The antiviral activity of the cathelicidins D-LL-37 and D-mCRAMP was assessed. Figure is representative of n = 3 independent experiments. Figure shows mean values ± SEM. Statistical analysis was performed using an unpaired t-test to compare PR/8 only titer with PR/8 + Peptide (*P≤0.05, ** P≤0.01).</p

Cathelicidins Mediate Changes in Lung Cytokine Concentrations Following Influenza Virus Infection.

<p>Groups of 5 mice were infected with 10 MLD₅₀ of A/PR/8/34 influenza virus via intranasal administration on day 0. Mice were nebulized with 200 µl of saline (control), or LL-37 peptide (500 µg/ml) once daily from day -1 to day 2. Mice were euthanized on day 3 and concentration of the indicated cytokines in the bronchoalveolar lavage (BAL) fluid were measured by BioPlex assay. Figures show mean values ± SEM. Statistical analysis was performed using an two-way analysis of variance (ANOVA) (*P≤0.05).</p

LL-37 Protects Mice Against Influenza Virus Disease.

<p>(A,B) Groups of 5 mice were inoculated with 10 MLD₅₀ of A/Puerto Rico/8/1934 influenza virus by the intranasal route on day 0. Mice were nebulized with 200 µl of saline (control), zanamivir (500 µg/ml), LL-37 peptide (500 µg/ml) or scrambled LL-37 control peptide (500 µg/ml) once daily from day -1 to day 7. Mouse body weight (A) and survival (B) was monitored daily up to 14 days post infection. Data represent mean values ± SEM, for three independent experiments. Statistical analysis was performed using Kaplan Meier with a Mantel-Cox (log rank) test. Survival curves obtained with Zanamivir and LL-37 treatments were significantly different (P≤0.001) compared to saline control treatment. There was no difference between saline treated and sLL-37 treated groups. (C) Groups of three mice (Female, 6–8 week old Balb/c) were inoculated with 10 MLD₅₀ of A/PR/8/34 virus intranasally on day 0. Mice were nebulized with 200 µl of saline (control) zanamivir (500 µg/ml), LL-37 peptide (500 µg/ml) or scrambled LL-37 control peptide (500 µg/ml) once daily from day -1 to day 2. Mice were euthanized on day 3 and viral titer in the lungs was assessed by plaque assay. Figure is representative of three independent experiments. Figure shows mean values ± SEM. Statistical analysis was performed using a Student t-test to compare virus infected animals with virus/peptide and virus/zanamivir treated animals (*P≤0.05).</p

Structural and Functional Characterization of an Ancient Bacterial Transglutaminase Sheds Light on the Minimal Requirements for Protein Cross-Linking

Transglutaminases are best known for their ability to catalyze protein cross-linking reactions that impart chemical and physical resilience to cellular structures. Here, we report the crystal structure and characterization of Tgl, a transglutaminase from the bacterium <i>Bacillus subtilis.</i> Tgl is produced during sporulation and cross-links the surface of the highly resilient spore. Tgl-like proteins are found only in spore-forming bacteria of the <i>Bacillus</i> and <i>Clostridia</i> classes, indicating an ancient origin. Tgl is a single-domain protein, produced in active form, and the smallest transglutaminase characterized to date. We show that Tgl is structurally similar to bacterial cell wall endopeptidases and has an NlpC/P60 catalytic core, thought to represent the ancestral unit of the cysteine protease fold. We show that Tgl functions through a unique partially redundant catalytic dyad formed by Cys116 and Glu187 or Glu115. Strikingly, the catalytic Cys is insulated within a hydrophobic tunnel that traverses the molecule from side to side. The lack of similarity of Tgl to other transglutaminases together with its small size suggests that an NlpC/P60 catalytic core and insulation of the active site during catalysis may be essential requirements for protein cross-linking

Acetylation by Eis and Deacetylation by Rv1151c of <i>Mycobacterium tuberculosis</i> HupB: Biochemical and Structural Insight

Bacterial nucleoid-associated proteins (NAPs) are critical to genome integrity and chromosome maintenance. Post-translational modifications of bacterial NAPs appear to function similarly to their better studied mammalian counterparts. The histone-like NAP HupB from <i>Mycobacterium tuberculosis</i> (<i>Mtb</i>) was previously observed to be acetylated by the acetyltransferase Eis, leading to genome reorganization. We report biochemical and structural aspects of acetylation of HupB by Eis. We also found that the SirT-family NAD⁺-dependent deacetylase Rv1151c from <i>Mtb</i> deacetylated HupB <i>in vitro</i> and characterized the deacetylation kinetics. We propose that activities of Eis and Rv1151c could regulate the acetylation status of HupB to remodel the mycobacterial chromosome in response to environmental changes

Induction of IFN and IFN-stimulated genes is inhibited by reduction of RIG-I but not PKR.

<p>A549cells were first transfected with the siRNAs against RIG-I or PKR(3 µg)as shown or mock transfected. 24 hr later, the cells were transfected again with the indicated IVT RNAs (3 µg) and processed 24 hr later for RNA as well as protein. Protein lysates are used to determine the levels of PKR and RIG-I proteins by western blot analyses and RNA is used to determine the levels of mRNA for IFN-β. (A) Western blots using the indicated antibodies of protein extracts from treated cells are shown. (B) and (C) IFN-β mRNA levels were measured using qRT-PCR. Error bars represent the standard deviation of triplicate qRT-PCR runs using RNAs from one of three representative experiments.</p

K_d (nM) of RIG-I to RNA from NS1.

<p>Affinities were measured using a fluorescence polarization assay. Values represent the mean ± standard deviation of at least two independent experiments. The RNA molecules with the lowest affinities (in bold) neither bound RIG-I <i>in vivo</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032661#pone-0032661-g006" target="_blank">Fig. 6</a>) nor induced IFN expression (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032661#pone-0032661-g002" target="_blank">Fig. 2</a>).</p

Specificity of the Toxocariasis Luminex assay.

<p>Specificity of the Toxocariasis Luminex assay.</p