Defence of Agaricus bisporus against toxic secondary metabolites from Trichoderma aggressivum.

Trichoderma spp are effective competitors against other fungi because they are mycoparasitic and produce hydrolytic enzymes and secondary metabolites that inhibit the growth of their competitors. Inhibitory compounds produced by Trichoderma aggressivum, the causative agent of green mold disease, are more toxic to the hybrid off-white strains of Agaricus bisporus than the commercial brown strains, consistent with the commercial brown strain’s increased resistance to the disease. This project looked at the response of hybrid off-white and commercial brown strains of A. bisporus to the presence of T. aggressivum metabolites with regard to three A. bisporus genes: laccase 1, laccase 2, and manganese peroxidase. The addition of T. aggressivum toxic metabolites had no significant effect on MnP or lcc1 transcript abundance. Alternatively, laccase 2 appears to be involved in resistance to T. aggressivum because the presence of T. aggressivum metabolites results in higher lcc2 transcript abundance and laccase activity, especially in the commercial brown strain. The difference in laccase expression and activity between A. bisporus strains was not a result of regulatory or coding sequence differences. Alteration of laccase transcription by RNAi resulted in transformants with variable levels of laccase transcript abundance. Transformants with a low number of lcc transcripts were very sensitive to T. aggressivum toxins, while those with a high number of lcc transcripts had increased resistance. These results indicated that laccase activity, in particular that encoded by lcc2, serves as a defense response of A. bisporus to T. aggressivum toxins and contributes to green mold disease resistance in commercial brown strains

A Key Role for Neurotensin in Chronic-Stress-Induced Anxiety-Like Behavior in Rats

Accepted ManuscriptChronic stress is a major cause of anxiety disorders that can be reliably modeled preclinically, providing insight into alternative therapeutic targets for this mental health illness. Neuropeptides have been targeted in the past to no avail possibly due to our lack of understanding of their role in pathological models. In this study we use a rat model of chronic stress-induced anxiety-like behaviors and hypothesized that neuropeptidergic modulation of synaptic transmission would be altered in the bed nucleus of the stria terminalis (BNST), a brain region suspected to contribute to anxiety disorders. We use brain slice neurophysiology and behavioral pharmacology to compare the role of locally released endogenous neuropeptides on synaptic transmission in the oval (ov) BNST of non-stressed (NS) or chronic unpredictably stressed (CUS) rats. We found that in NS rats, post-synaptic depolarization induced the release of vesicular neurotensin (NT) and corticotropin-releasing factor (CRF) that co-acted to increase ovBNST inhibitory synaptic transmission in 59% of recorded neurons. CUS bolstered this potentiation (100% of recorded neurons) through an enhanced contribution of NT over CRF. In contrast, locally released opioid neuropeptides decreased ovBNST excitatory synaptic transmission in all recorded neurons, regardless of stress. Consistent with CUS-induced enhanced modulatory effects of NT, blockade of ovBNST NT receptors completely abolished stress-induced anxiety-like behaviors in the elevated plus maze paradigm. The role of NT has been largely unexplored in stress and our findings highlight its potential contribution to an important behavioral consequence of chronic stress, that is, exaggerated avoidance of open space in rats.CPN was funded by CIHR Vanier Graduate Scholarship (338319); APVS was funded by Fundação para a Ciência e Tecnologia (SFRH/BPD/52078/2013); ERH was funded by CIHR Postdoctoral Fellowship (MFE-123712); SA was funded by a Queen Elizabeth II Graduate Scholarship in Science and Technology; ÉCD was funded by the Canadian Institute of Health Research (MOP-25953)info:eu-repo/semantics/publishedVersio

Honey glycoproteins containing antimicrobial peptides, Jelleins of the Major Royal Jelly Protein 1, are responsible for the cell wall lytic and bactericidal activities of honey.

We have recently identified the bacterial cell wall as the cellular target for honey antibacterial compounds; however, the chemical nature of these compounds remained to be elucidated. Using Concavalin A-affinity chromatography, we found that isolated glycoprotein fractions (glps), but not flow-through fractions, exhibited strong growth inhibitory and bactericidal properties. The glps possessed two distinct functionalities: (a) specific binding and agglutination of bacterial cells, but not rat erythrocytes and (b) non-specific membrane permeabilization of both bacterial cells and erythrocytes. The isolated glps induced concentration- and time-dependent changes in the cell shape of both E. coli and B. subtilis as visualized by light and SEM microscopy. The appearance of filaments and spheroplasts correlated with growth inhibition and bactericidal effects, respectively. The time-kill kinetics showed a rapid, >5-log10 reduction of viable cells within 15 min incubation at 1xMBC, indicating that the glps-induced damage of the cell wall was lethal. Unexpectedly, MALDI-TOF and electrospray quadrupole time of flight mass spectrometry, (ESI-Q-TOF-MS/MS) analysis of glps showed sequence identity with the Major Royal Jelly Protein 1 (MRJP1) precursor that harbors three antimicrobial peptides: Jelleins 1, 2, and 4. The presence of high-mannose structures explained the lectin-like activity of MRJP1, while the presence of Jelleins in MRJP1 may explain cell wall disruptions. Thus, the observed damages induced by the MRJP1 to the bacterial cell wall constitute the mechanism by which the antibacterial effects were produced. Antibacterial activity of MRJP1 glps directly correlated with the overall antibacterial activity of honey, suggesting that it is honey's active principle responsible for this activity

Time-dependent phenotypic changes observed after application of honey to log phase <i>E. coli</i> cultures.

<p>The changes paralleled the growth kinetics shown in Fig. 1D. (A) lag phase <i>E. coli</i> culture, (B) log phase <i>E. coli</i> culture at the time of honey application, (C) <i>E. coli</i> phenotypes after 30 min incubation with honey H208 (12.5% v/v), (D). <i>E. coli</i> phenotypes observed after 1 h incubation with honey H210 (12.5% v/v), (F) and (G) <i>E. coli</i> spheroplasts formed after 2 h incubation with honey H208 and H210 at 50% v/v, (H) spheroplasts observed after 18 h incubation, lag phase. The size-scale is the same for all micrographs in the figure, obtained under the same 400x magnification.</p

Summary of fully annotated matched peptides between the MRJP1 and 61 kDa glycoproteins G208 and G210.

<p>Blue bars indicate sequence coverage and confirmed identifications.</p

Glycoprotein- induced cell wall damage and cell shape changes in log-phase <i>E. coli</i> and <i>B. subtilis</i> cultures.

<p>A. <i>E. coli</i>. a. untreated control, b. <i>E. coli</i> incubated with G177, c. G208 and d. G210. B. <i>B. subtilis</i>. a. untreated control, b. <i>B. subtilis</i> incubated with G177, c. G208 and d. G210.</p

Morphological changes in ampicillin-resistant <i>E. coli</i> after exposure to honey or ampicillin.

<p>Ampicillin-resistant <i>E. coli</i> before (A) and after treatment with honey, H210, 25%w/v, (B) and ampicillin-resistant <i>E. coli</i> before (C) and after treatment with 2.5 ug/ml of ampicillin (D). The size-scale is the same for all micrographs in the figure, obtained under the same 400x magnification.</p

MIC and MBC values of glycoproteins.

<p>MIC and MBC values of glycoproteins.</p

Summary of ESI-Q-TOF- MS/MS statistical data for 61 kDa glycoproteins G208 and G210.

<p>Summary of ESI-Q-TOF- MS/MS statistical data for 61 kDa glycoproteins G208 and G210.</p

Susceptibility of ampicillin-resistant <i>E. coli</i> to honey and ampicillin.

<p>The bars represent the mean ± standard deviation (n = 9).</p