Pinewood nematode-associated bacteria contribute to oxidative stress resistance of Bursaphelenchus xylophilus

Background: Pine wilt disease (PWD) caused by the pinewood nematode Bursaphelenchus xylophilus is one of the most serious forest diseases in the world. The role of B. xylophilus-associated bacteria in PWD and their interaction with the nematode, have recently been under substantial investigation. Several studies report a potential contribution of the bacteria for the PWD development, either as a helper to enhance the pathogenicity of the nematode or as a pathogenic agent expressing interesting traits related to lifestyle host-adaptation. Results: We investigated the nematode-bacteria interaction under a severe oxidative stress (OS) condition using a pro-oxidant hydrogen peroxide and explored the adhesion ability of these bacteria to the cuticle surface of the nematodes. Our results clearly demonstrated a beneficial effect of the Serratia spp. (isolates LCN-4, LCN-16 and PWN-146) to B. xylophilus under the OS condition. Serratia spp. was found to be extremely OS-resistant, and promote survival of B. xylophilus and down-regulate two B. xylophilus catalase genes (Bxy-ctl-1 and Bxy-ctl-2). In addition, we show that the virulent isolate (Ka4) of B. xylophilus survives better than the avirulent (C14-5) isolate under the OS condition. The bacterial effect was transverse for both B. xylophilus isolates. We could not observe a strong and specific adhesion of these bacteria on the B. xylophilus cuticle surface. Conclusions: We report, for the first time, that B. xylophilus associated bacteria may assist the nematode opportunistically in the disease, and that a virulent B. xylophilus isolate displayed a higher tolerance towards the OS conditions than an avirulent isolate

he genome and genetics of a high oxidative stress tolerant Serratia sp. LCN16 isolated from the plant parasitic nematode Bursaphelenchus xylophilus

Background: Pine wilt disease (PWD) is a worldwide threat to pine forests, and is caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus. Bacteria are known to be associated with PWN and may have an important role in PWD. Serratia sp. LCN16 is a PWN-associated bacterium, highly resistant to oxidative stress in vitro, and which beneficially contributes to the PWN survival under these conditions. Oxidative stress is generated as a part of the basal defense mechanism used by plants to combat pathogenic invasion. Here, we studied the biology of Serratia sp. LCN16 through genome analyses, and further investigated, using reverse genetics, the role of two genes directly involved in the neutralization of H2O2, namely the H2O2 transcriptional factor oxyR; and the H2O2-targeting enzyme, catalase katA. Results: Serratia sp. LCN16 is phylogenetically most closely related to the phytosphere group of Serratia, which includes S. proteamaculans, S. grimessi and S. liquefaciens. Likewise, Serratia sp. LCN16 shares many features with endophytes (plant-associated bacteria), such as genes coding for plant polymer degrading enzymes, iron uptake/ transport, siderophore and phytohormone synthesis, aromatic compound degradation and detoxification enzymes. OxyR and KatA are directly involved in the high tolerance to H2O2 of Serratia sp. LCN16. Under oxidative stress, Serratia sp. LCN16 expresses katA independently of OxyR in contrast with katG which is under positive regulation of OxyR. Serratia sp. LCN16 mutants for oxyR (oxyR::int(614)) and katA (katA::int(808)) were sensitive to H2O2 in relation with wild-type, and both failed to protect the PWN from H2O2-stress exposure. Moreover, both mutants showed different phenotypes in terms of biofilm production and swimming/swarming behaviors. Conclusions: This study provides new insights into the biology of PWN-associated bacteria Serratia sp. LCN16 and its extreme resistance to oxidative stress conditions, encouraging further research on the potential role of this bacterium in interaction with PWN in planta environment

Le notti bianche

La lettura de Le notti bianche, celebre romanzo breve pubblicato da Dostoevskij in patria nel 1848 e da noi nel 1920, ci trascina in un vortice letterario entro cui dialetticamente operano distinte stratificazioni discorsive, di natura personale e di ordine etico-politico. Diversi soggetti, diversi punti di vista e diversi percorsi narrativi intervengono e si compongono nella definizione di un quadro di assoluta tenuta artistica: quadro classico, pu\uf2 ben dirsi, per gli equilibri di struttura compositivi raggiunti e per una perfezione formale conquistata, non scalfita dal trascorrere del tempo

Erythritol Can Inhibit the Expression of Senescence Molecules in Mouse Gingival Tissues and Human Gingival Fibroblasts

Oral aging causes conditions including periodontal disease. We investigated how the sugar alcohol erythritol, which has anti-caries effects, impacts aging periodontal tissues and gingival fibroblasts in mice and humans in vivo and in vitro. Mice were classified into three groups: control groups of six-week-old (YC) and eighteen-month-old mice (AC) and a group receiving 5% w/w erythritol water for 6 months (AE). After rearing, RNA was extracted from the gingiva, and the levels of aging-related molecules were measured using PCR. Immunostaining was performed for the aging markers p21, γH2AX, and NF-κB p65. p16, p21, γH2AX, IL-1β, and TNFα mRNA expression levels were higher in the gingiva of the AC group than in the YC group, while this enhanced expression was significantly suppressed in AE gingiva. NF-κB p65 expression was high in the AC group but was strongly suppressed in the AE group. We induced senescence in cultured human gingival fibroblasts using H2O2 and lipopolysaccharide before erythritol treatment, which reduced elevated senescence-related marker (p16, p21, SA-β-gal, IL-1β, and TNFα) expression levels. Knockdown of PFK or PGAM promoted p16 and p21 mRNA expression, but erythritol subsequently rescued pyruvate production. Overall, intraoral erythritol administration may prevent age-related oral mucosal diseases

Expression patterns of <i>Bxy-ctl-1</i>::<i>gfp</i> (A) and <i>Bxy-ctl-2</i>::<i>gfp</i> (B) in, respectively, transgenic <i>C</i>. <i>elegans</i> KHA149 and KHA151.

<p>Differential interference contrast (DIC) microscope images and, DIC and fluorescence-merged images (DIC+FL) of <i>C</i>. <i>elegans</i> head, vulva and tail region. Scale bars, 100 μm.</p

Survival percentage of wild-type (N2) and transgenic (KHA149 and KHA151) <i>C</i>. <i>elegans</i> after 24 hours exposition to H₂O₂ (H₂O₂ concentrations ranging between 0 and 500 μM).

<p>Error bars represent standard deviation. Different letters above the columns indicate significant differences (<i>p</i> < 0.05) between <i>C</i>. <i>elegans N2</i>, <i>KHA149 and KHA151</i> survival percentages in each H₂O₂ treatment, according to <i>post-hoc</i> Duncan’s test.</p

Characterization of <i>Bursaphelenchus xylophilus</i> CTLs, and primers used for CTLs expression analysis.

<p>Sec/whole—Normalized secreted protein/whole proteins; ND—not detected.</p><p>¹ ORF, open reading frame.</p><p>²Petersen et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123839#pone.0123839.ref027" target="_blank">27</a>].</p><p>³ Shinya et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123839#pone.0123839.ref019" target="_blank">19</a>].</p><p>Characterization of <i>Bursaphelenchus xylophilus</i> CTLs, and primers used for CTLs expression analysis.</p

mRNA expression patterns of <i>Bxy-ctl-1</i> (A), <i>Bxy-ctl-2</i> (B), and <i>Bxy-eng-1</i> (D) in <i>B</i>. <i>xylophilus</i> Ka4.

<p>No expression signal was observed with <i>Bxy-ctl-1</i> sense probe (C). Light microscope images of <i>B</i>. <i>xylophilus</i> head region. Scale bars, 100 μm.</p

Relative gene expression of <i>Bxy-ctl-1</i> (A) and <i>Bxy-ctl-2</i> (B) of <i>B</i>. <i>xylophilus</i> (high virulence isolates Ka4 and T4; low virulence isolate C14-5) after 24 hours exposition to H₂O₂.

<p>* and ** indicate, respectively, statistical differences at 95% and 99% confidence levels compared to a normalized value of 1.00 for control treatment without H₂O₂. Error bars represent standard deviation.</p