The Antifungal Plant Defensin HsAFP1 from Heuchera sanguinea Induces Apoptosis in Candida albicans

Plant defensins are active against plant and human pathogenic fungi (such as Candida albicans) and baker's yeast. However, they are non-toxic to human cells, providing a possible source for treatment of fungal infections. In this study, we characterized the mode of action of the antifungal plant defensin HsAFP1 from coral bells by screening the Saccharomyces cerevisiae deletion mutant library for mutants with altered HsAFP1 sensitivity and verified the obtained genetic data by biochemical assays in S. cerevisiae and C. albicans. We identified 84 genes, which when deleted conferred at least fourfold hypersensitivity or resistance to HsAFP1. A considerable part of these genes were found to be implicated in mitochondrial functionality. In line, sodium azide, which blocks the respiratory electron transport chain, antagonized HsAFP1 antifungal activity, suggesting that a functional respiratory chain is indispensable for HsAFP1 antifungal action. Since mitochondria are the main source of cellular reactive oxygen species (ROS), we investigated the ROS-inducing nature of HsAFP1. We showed that HsAFP1 treatment of C. albicans resulted in ROS accumulation. As ROS accumulation is one of the phenotypic markers of apoptosis in yeast, we could further demonstrate that HsAFP1 induced apoptosis in C. albicans. These data provide novel mechanistic insights in the mode of action of a plant defensin

Structure-activity relationship study of the plant-derived decapeptide OSIP108 inhibiting Candida albicans biofilm formation

We performed a structure-activity relationship study of the antibiofilm plant-derived decapeptide OSIP108. Introduction of positively charged amino acids R, H, and K resulted in an up-to-5-fold-increased antibiofilm activity against Candida albicans compared to native OSIP108, whereas replacement of R9 resulted in complete abolishment of its antibiofilm activity. By combining the most promising amino acid substitutions, we found that the double-substituted OSIP108 analogue Q6R/G7K had an 8-fold-increased antibiofilm activity

Potentiation of Antibiofilm Activity of Amphotericin B by Superoxide Dismutase Inhibition

This study demonstrates a role for superoxide dismutases (Sods) in governing tolerance of Candida albicans biofilms to amphotericin B (AmB). Coincubation of C. albicans biofilms with AmB and the Sod inhibitors N,N′-diethyldithiocarbamate (DDC) or ammonium tetrathiomolybdate (ATM) resulted in reduced viable biofilm cells and increased intracellular reactive oxygen species levels as compared to incubation of biofilm cells with AmB, DDC, or ATM alone. Hence, Sod inhibitors can be used to potentiate the activity of AmB against C. albicans biofilms

Increasing the fungicidal action of Amphotericin B by inhibiting the Nitric Oxide-Dependent tolerance pathway

Amphotericin B (AmB) induces oxidative and nitrosative stresses, characterized by production of reactive oxygen and nitrogen species, in fungi. Yet, how these toxic species contribute to AmB-induced fungal cell death is unclear. We investigated the role of superoxide and nitric oxide radicals in AmB's fungicidal activity in Saccharomyces cerevisiae, using a digital microfluidic platform, which enabled monitoring individual cells at a spatiotemporal resolution, and plating assays. The nitric oxide synthase inhibitor L-NAME was used to interfere with nitric oxide radical production. L-NAME increased and accelerated AmB-induced accumulation of superoxide radicals, membrane permeabilization, and loss of proliferative capacity in S. cerevisiae. In contrast, the nitric oxide donor S-nitrosoglutathione inhibited AmB's action. Hence, superoxide radicals were important for AmB's fungicidal action, whereas nitric oxide radicals mediated tolerance towards AmB. Finally, also the human pathogens Candida albicans and Candida glabrata were more susceptible to AmB in the presence of L-NAME, pointing to the potential of AmB-L-NAME combination therapy to treat fungal infections.Kim Vriens acknowledges the receipt of a predoctoral grant from the Flanders Innovation & Entrepeneurship Agency (IWT-SB 111016); Karin Thevissen acknowledges the receipt of a mandate of Industrial Research Fund (KU Leuven). In addition, the research leading to these results has received funding from the Research Foundation - Flanders (FWO G086114N and G080016N) and the KU Leuven (OT 13/ 058 and IDO 10/012, IOF KP/12/009 Atheromix, IOF KP/ 12/002 Nanodiag). This work was partially developed under the scope of the project NORTE-01-0145-FEDER-000013, supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). Belém Sampaio-Marques is supported by the fellowship SFRH/BPD/90533/2012 funded by Fundação para a Ciência e Tecnologia (FCT, Portugal).info:eu-repo/semantics/publishedVersio

Skn1 and Ipt1 negatively regulate autophagy in Saccharomyces cerevisiae

We demonstrated that a yeast deletion mutant in IPT1 and SKN1 , encoding proteins involved in the biosynthesis of mannosyldiinositolphosphoryl ceramides, is characterized by increased autophagy and DNA fragmentation upon nitrogen (N) starvation as compared with the single deletion mutants or wild type (WT). Apoptotic features were not significantly different between single and double deletion mutants upon N starvation, pointing to increased autophagy in the double Δ ipt1 Δ skn1 deletion mutant independent of apoptosis. We observed increased basal levels of phytosphingosine in membranes of the double Δ ipt1 Δ skn1 deletion mutant as compared with the single deletion mutants or WT. These data point to a negative regulation of autophagy by both Ipt1 and Skn1 in yeast, with a putative involvement of phytosphingosine in this process.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/78623/1/j.1574-6968.2009.01869.x.pd

The plant decapeptide OSIP108 can alleviate mitochondrial dysfunction induced by cisplatin in human cells

We investigated the effect of the Arabidopsis thaliana-derived decapeptide OSIP108 on human cell tolerance to the chemotherapeutic agent cisplatin (Cp), which induces apoptosis and mitochondrial dysfunction. We found that OSIP108 increases the tolerance of HepG2 cells to Cp and prevents Cp-induced changes in basic cellular metabolism. More specifically, we demonstrate that OSIP108 reduces Cp-induced inhibition of respiration, decreases glycolysis and prevents Cp-uptake in HepG2 cells. Apart from its protective action against Cp in human cells, OSIP108 also increases the yeast Saccharomyces cerevisiae tolerance to Cp. A limited yeast-based study of OSIP108 analogs showed that cyclization does not severely affect its activity, which was further confirmed in HepG2 cells. Furthermore, the similarity in the activity of the D-stereoisomer (mirror image) form of OSIP108 with the L stereoisomer suggests that its mode of action does not involve binding to a stereospecific receptor. In addition, as OSIP108 decreases Cp uptake in HepG2 cells and the anti-Cp activity of OSIP108 analogs without free cysteine is reduced, OSIP108 seems to protect against Cp-induced toxicity only partly via complexation. Taken together, our data indicate that OSIP108 and its cyclic derivatives can protect against Cp-induced toxicity and, thus, show potential as treatment options for mitochondrial dysfunction- and apoptosis-related conditions

Embry-Riddle Fly Paper 1943-01-15

https://commons.erau.edu/fly-paper/1159/thumbnail.jp