Halophytes-associated endophytic and rhizospheric bacteria: diversity, antagonism and metabolite production

<p>In Saudi Arabia, halophytes occupy tidal and intertidal forest ecosystems. They and their associated microflora have immense potential to yield novel and important useful natural products. Three halophytes (<i>Avicennia marina</i>, <i>Halocnemum strobilaceum</i>, <i>Zygophyllum qatarense</i>) were targeted for the isolation and identification of populations of endophytic and rhizospheric bacteria having antimicrobial potential. A total 554 bacterial isolates were initially screened against oomycetes fungal pathogens, <i>Phytophthora capsici</i> and <i>Pythium ultimum</i>. Of these, only 57 rhizospheric and endophytic bacteria exhibited inhibition against the targeted bioassay oomycetes<i>.</i> Tentative identification of the bacteria was on the basis of 16S rRNA gene sequences which revealed 92–100% sequence identity to type strains of related species and placed these organisms in six major classes: <i>Actinobacteria, γ-Proteobacteria, Firmicutes</i>, <i>α-Proteobacteria</i>, <i>Flavobacteriia</i> and <i>β-Proteobacteria</i>. When checked for lytic enzyme production, mostly the isolates of <i>Actinobacteria</i> and <i>Firmicutes</i> were potential enzyme producers. Detection of secondary metabolite biosynthetic genes – type I polyketide synthases, type II polyketide synthases and nonribosomal peptide synthetases – confirmed that 21 (35.5%) isolates were positive for at least one type of the biosynthetic gene. In order to identify metabolites, three isolates, <i>Alteromonas australica</i> (EA73), <i>Aidingimonas halophila</i> (EA105) and <i>Halomonas zincidurans</i> (EA127), were selected and subjected to chemical analyses using liquid chromatography–mass spectrometry and gas chromatography–mass spectrometry. Both analyses showed the presence of different bioactive compounds in the culture extracts of isolates some of which are already reported for their diverse biological activities such as 2, 4-Diacetylphloroglucinol. Our results demonstrated that halophytes represent an important source of potentially active bacteria producing antifungal metabolites of medical significance.</p

Co-treatment effect of nicotinamide on thiotepa-induced neuronal cell death.

<p>Light micrographs of cresyl violet-stained neurons in tissue sections of developing rat brain after thiotepa with nicotinamide. The majority of thiotepa-induced degenerating neuronal cells (generally shrunken in appearance, as is commonly observed) are present in the anterior cingulate cortex, CA1 of hippocampus and LDN of thalamus (F, G, and H). The arrows indicate shrunken and damaged neurons. Almost complete protection was observed (I, J and K), when nicotinamide was administered with thiotepa. Images are representative of staining obtained in sections (3–5/group) prepared from at least 5–6 animals/group. (C–K) of Nissl-stained brain tissue at higher magnification with 40× objective field, Scale bar = 20 µm. Statistical difference was determined using one-way analysis of variance (ANOVA) followed by Student's t-test. (^aSignificantly different from CTL, [cortex P<0.001, hippocampus P<0.05 and thalamus P<0.001]; ^bSignificantly different from Thiotepa + Nicotinamide, [cortex P<0.01, hippocampus P<0.05 and thalamus P<0.01]; ^cSignificantly different from Thiotepa, [cortex P<0.01, hippocampus P<0.05 and thalamus P<0.01]).</p

Co-treatment effect of nicotinamide (Nic) on thiotepa-induced expression of Bax and Bcl-2 protein levels.

<p>Representative Western blot analysis shows the expression levels of (<b>A</b>) Bax and (<b>B</b>) Bcl-2 protein in the cortex and thalamus of a 7-day-old rat. Treatment with (30 mg/kg) of thiotepa for 4 h increased Bax protein levels in the cortex and thalamus. Treatment with (1 mg/g) of nicotinamide for 4 h inhibited thiotepa-induced upregulation of Bax protein levels in the cortex and thalamus, as shown. The protein bands of the Western blot were quantified using Sigma gel software; their differences are represented in the graph. Actin was used as a protein loading control. Density values, expressed as mean ± SEM (n = 5–6 animals/group), of Bcl-2 and Bax proteins are presented. The density values on the Y-axis are expressed as arbitrary units (AU). Statistical difference was determined using one-way analysis of variance (ANOVA) followed by Student's t-test. (^aSignificantly different from CTL, [cortex P<0.001 and thalamus P<0.05]; ^bSignificantly different from Nicotinamide, [cortex P<0.01 and thalamus P<0.001]; ^cSignificantly different from Thiotepa + Nicotinamide, [cortex P<0.01 and thalamus P<0.05]; ^dSignificantly different from Thiotepa, [cortex P<0.01 and thalamus P<0.05]).</p

Co-treatment effect of nicotinamide on thiotepa-induced cytosolic cytochrome c levels.

<p>Representative Western blot analysis of cytochrome c levels in the cortex and thalamus of 7-day-old rats. Significant increases in the cytosolic level of cytochrome c in the cortex and thalamus after administration of thiotepa are shown. Treatment with nicotinamide for 4 h significantly reduced thiotepa-induced cytosolic cytochrome c levels in cortex and thalamus, as shown. The protein bands of the Western blot were quantified using Sigma gel software analysis; their differences are represented in the graph. Actin reactivity was used as a protein loading control. Density values, expressed as mean ± SEM (n = 5–6 animals/group), for cytochrome c and activated caspase-9 are presented. The density values on the Y-axis are expressed as arbitrary units (AU). Statistical difference was determined using one-way analysis of variance (ANOVA) followed by Student's t-test. (^aSignificantly different from CTL, [cortex P<0.05 and thalamus P<0.05]); ^bSignificantly different from Nicotinamide, [cortex P<0.05 and thalamus P<0.05]; ^cSignificantly different from Thiotepa + Nicotinamide, [cortex P<0.01 and thalamus P<0.01]; ^dSignificantly different from Thiotepa, [cortex P<0.01 and thalamus P<0.01]).</p

Co-treatment effect of nicotinamide (Nic) on thiotepa-induced Bax and Bcl-2 mRNA levels.

<p>Representative RT-PCR analysis shows expression levels of (<b>A</b>) Bax and (<b>B</b>) Bcl-2 mRNA in the cortex and thalamus of 7-day-old rat pups. Treatment with thiotepa (30 mg/kg for 4 h) increased Bax mRNA levels in the cortex and thalamus. Treatment with (1 mg/g) of nicotinamide for 4 h significantly reduced thiotepa-induced up-regulation of Bax mRNA levels in the cortex and thalamus, as shown. Detailed procedures are described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027093#s4" target="_blank">Materials and Methods</a> section. The mRNA bands of RT-PCR were quantified using Sigma gel software; these differences are represented in the graph. GAPDH was used as mRNA loading control. Density values are expressed as mean ± SEM (n = 5 animals/group). The density values on the Y-axis are expressed as arbitrary units (AU). Statistical difference was determined using one-way analysis of variance (ANOVA) followed by Student's t-test. (^aSignificantly different from CTL, [cortex P<0.01 and thalamus P<0.01]; ^bSignificantly different from Nicotinamide, [cortex P<0.001 and thalamus P<0.05]; ^cSignificantly different from Thiotepa + Nicotinamide, [cortex P<0.01 and thalamus P<0.05]; ^dSignificantly different from Thiotepa, [cortex P<0.01 and thalamus P<0.05]).</p

Co-treatment effect of nicotinamide on thiotepa-induced apoptotic neurodegeneration.

<p>Representative photomicrographs of TUNEL staining show apoptotic dead neuronal cells after thiotepa administration with nicotinamide. The arrows indicate thiotepa-induced TUNEL stained apoptotic dead neurons (D and F) counterstained with DAPI (G and I) in the cortex and thalamus. Nicotinamide treatment effectively blocked thiotepa-induced apoptosis, as evident from the lack of TUNEL-positive cells (J and L). Images are representative of staining obtained in sections (3–5/group) prepared from at least 5–6 animals/group. (A–L) of TUNEL-stained brain tissue at higher magnification with 40× objective field, Scale bar = 20 µm. Statistical difference was determined using one-way analysis of variance (ANOVA) followed by Student's t-test. (^aSignificantly different from CTL, [cortex P<0.001 and thalamus P<0.001]; ^bSignificantly different from Thiotepa + Nicotinamide, [cortex P<0.01 and thalamus P<0.01]; ^cSignificantly different from Thiotepa, [cortex P<0.01 and thalamus P<0.01]).</p

A schematic diagram representing the hypothetical mechanism by which nicotinamide protects against thiotepa-induced neurodegeneration in the brain of the developing rat.

<p>Thiotepa-induced neurodegeneration is caused by the overactivation of mitochondria-dependent apoptosis, beginning with the down-regulation of Bax, an increase in cytochrome c release from mitochondria to cytosol, expression of activated capase-3, cleavage of PARP-1 and necrosis by overactivation and cleavage of PARP-1 and depletion of NAD⁺ (blue arrow). PARP-1 activation and formation of Poly ADP-ribose PAR in the nucleus, which translocates to the cytosol to induce caspase-independent cell death (black arrow). Nicotinamide, as indicated by the X sign, inhibits several key elements in the apoptotic cascade beginning with the down-regulation of Bax, a decrease in cytochrome c release from mitochondria to cytosol, inhibition of activated caspase-3 and cleavage of PARP-1 and necrosis by the inhibition of PARP-1 activation and prevention of ATP and NAD⁺ depletion (red arrow), resulting in protection against thiotepa-induced apoptotic and possible necrotic cell death.</p

BAD, a Proapoptotic Protein, Escapes ERK/RSK Phosphorylation in Deguelin and siRNA-Treated HeLa Cells

<div><p>This study has been undertaken to explore the therapeutic effects of deguelin and specific siRNAs in HeLa cells. The data provided clearly show the silencing of ERK 1/2 with siRNAs and inhibition of ERK1/2 with deguelin treatment in HeLa cells. Additionally, we are providing information that deguelin binds directly to anti-apoptotic Bcl-2, Bcl-xl and Mcl-1 in the hydrophobic grooves, thereby releasing BAD and BAX from dimerization with these proteins. This results in increased apoptotic activity through the intrinsic pathway involved in rupture of mitochondrial membrane and release of cytochrome C. Evidence for inhibition of ERK1/2 by deguelin and escape of BAD phosphorylation at serine 112 through ERK/RSK pathway has been further fortified by obtaining similar results by silencing ERK 1/2 each with specific siRNAs. Increase in BAD after treatment with deguelin or siRNAs has been interpreted to mean that deguelin acts through several alternative pathways and therefore can be used as effective therapeutic agent.</p></div

Docking of deguelin on anti-apoptotic proteins.

<p>(A) Structure of deguelin arrow indicates the binding of deguelin on Bcl-2 (B) The docking of deguelin on BH3 domain of hydrophobic group of anti-apoptotic protein Bcl-xl (C) Binding of deguelin on MCL-1 (D) Binding of deguelin molecule on ERK-1 near the ATP binding site. Docking was carried out using bio-informative tools as described in materials and methods.</p

The binding of deguelin molecule on ERK-1 and ERK-2 near the ATP binding site.

<p>Docking was carried out using bio-informative tools as described in materials and methods. It may be observed that the molecule binds to ERK1/2 in the ATP pocket, thereby, preventing the phosphorylation of ERK.</p