Macelignan inhibits bee pathogenic fungi Ascophaera apis growth through HOG1 pathway

Abstract

Ascosphaera apis is a bee pathogen that causes bee larvae infection disease, to which treatment is not yet well investigated. The aim of this study was to investigate antifungal susceptibility in vitro against A. apis and to identify a new antifungal agent for this pathogen through minimal inhibitory concentration (MIC) assay and western blot analysis. Macelignan had 1.56 and 3.125 μg/mL MIC against A. apis after 24 and 48 h, respectively, exhibiting the strongest growth inhibition against A. apis among the tested compounds (corosolic acid, dehydrocostus lactone, loganic acid, tracheloside, fangchinoline and emodin-8-O-β-D-glucopyranoside). Furthermore, macelignan showed a narrow-ranged spectrum against various fungal strains without any mammalian cell cytotoxicity. In spite of miconazole having powerful broad-ranged anti-fungal activity including A. apis, it demonstrated strong cytotoxicity. Therefore, even if macelignan alone was effective as an antifungal agent to treat A. apis, combined treatment with miconazole was more useful to overcome toxicity, drug resistance occurrence and cost effectiveness. Finally, HOG1 was revealed as a target molecule of macelignan in the anti-A. apis activity by inhibiting phosphorylation using S. cerevisiae as a model system. Based on our results, macelignan, a food-grade antimicrobial compound, would be an effective antifungal agent against A. apis infection in bees.

Ampicillin activates Mpk1 phosphorylation in Saccharomyces cerevisiaeand ERK1/2 phosphorylation in HepG2 cells

“…Escherichia coli DH5α harboring YEp351-MPK1-FLAG (p2313; Shin and Kim, 2016) was cultivated in Luria broth medium (1% Bacto tryptone, 0.5% Bacto yeast extract, and 1% NaCl) supplemented with ampicillin at 37 °C. Ampicillin-resistant clones were used to propagate the plasmid by standard methods.…”

“…Immunoblotting was performed as described by Shin and Kim (2016) with slight modification. Y595 and Y698 strains harboring YEp351-MPK1-FLAG were grown overnight in SD medium.…”

“…Total Mpk1 was detected with anti-FLAG antibodies (dilution 1:4000; Cell Signaling Technology, Danvers, MA, USA) in the presence of 5% nonfat milk for 1 h followed by horseradish peroxide-conjugated antimouse secondary antibodies (dilution 1:4000; antimouse IgG, HRP-linked antibody; Cell Signaling Technology) for 1 h. After stringently washing the membrane, an enhanced chemiluminescence (ECL) detection system (Santa Cruz Biotechnology, Houston, TX, USA) was used to detect signals. Phosphorylation of Mpk1 was detected by incubation with rabbit polyclonal anti-phospho-p44/ p42 MAPK (Thr 202 /Tyr 204 ) antibodies (New England Biolabs, Ipswich, MA, USA) at a dilution of 1:3000 as described previously (Kim et al, 2008;Shin and Kim, 2016) overnight, followed by incubation with horseradish peroxide-conjugated secondary antibodies (donkey antirabbit antibody; GE Healthcare) at a dilution of 1:4000 for 1 h. Signals were detected using an ECL kit (Bio-Rad). Experiments were performed at least three times, and a representative image was selected.…”

“…Antifungal activity against S. cerevisiae or C. albicans was tested using the broth microdilution assay based on the standard NCCLS document M27-A (NCCLS, 1997) as described in Kim et al (2004) and Shin and Kim (2016). A stock of miconazole (10 g/L) was first serially 2-fold diluted with or without 3 mM ampicillin and dispensed in duplicate into wells of a 96-well microtiter plate with a total volume of 100 µL of RPMI 1640 medium (RPMI tissue culture medium supplemented with glutamine; Sigma) with 2% glucose, buffered to pH 7.0 with 0.156 M 3-N-morpholinopropanesulfonic acid (MOPS; Sigma).…”

“…Two pathways-the high-osmolarity glycerol (HOG1) pathway and the cell wall integrity (CWI) pathway-are important for maintenance of cellular integrity. Of these, the CWI pathway is considered the most essential for recognizing cell wall perturbations in many cell stress conditions (Levin, 2005;Kim et al, 2010;Kim and Levin, 2011;Shin and Kim, 2016). The CWI pathway is composed of cell surface receptors (Wsc1, Wsc2 Wsc3, Mid2, and Mtl1), transducer proteins (Rom2 and Rho1), calcium-dependent protein kinase (Pkc1), mitogen-activated protein (MAP) kinase kinase kinase (Bck1), MAP kinase kinases (Mkk1/2), and MAP kinase (Mpk1).…”