Exploring Peptaibol's Profile, Antifungal, and Antitumor Activity of Emericellipsin A of Emericellopsis Species from Soda and Saline Soils

Abstract

Features of the biochemical adaptations of alkaliphilic fungi to exist in extreme environments could promote the production of active antibiotic compounds with the potential to control microorganisms, causing infections associated with health care. Thirty-eight alkaliphilic and alkalitolerant Emericellopsis strains (E. alkalina, E. cf. maritima, E. cf. terricola, Emericellopsis sp.) isolated from different saline soda soils and belonging to marine, terrestrial, and soda soil ecological clades were investigated for emericellipsin A (EmiA) biosynthesis, an antifungal peptaibol previously described for Emericellopsis alkalina. The analysis of the Emericellopsis sp. strains belonging to marine and terrestrial clades from chloride soils revealed another novel form with a mass of 1032.7 Da, defined by MALDI-TOF Ms/Ms spectrometers, as the EmiA lacked a hydroxyl (dEmiA). EmiA displayed strong inhibitory effects on cell proliferation and viability of HCT 116 cells in a dose- and time-dependent manners and induced apoptosis.

Keywords: Emericellopsis alkalina; alkaliphilic fungi; antifungal activity; antitumor activity; emericellipsin’s complex; peptide.

Figure 1

Maximum likelihood tree for the Emericellopsis genus based on partial sequences for the ITS rDNA (including 5.8S rDNA) region. Branch lengths are proportional to the estimated number of nucleotide substitutions. The BP values are displayed on the nodes (BP; 1000 replicates). Emericellopsis spp. and related species were clustered into a “Marine”, “Soda soil”, or “Terrestrial” clade. Taxa names of the isolates obtained in this study are highlighted in a light color. “T” beside each strain name indicates the strains as the ex-type strain.

Figure 2

(a) Chromatogram of the analyzed sample with the HPLC method. Only two peaks, 1 and 2, were visualized with retention times of 37.4 min and 39.3 min, respectively (on the chromatogram values are presented in volume units). The names of probable compounds are marked at the top of the corresponding peaks. Both peaks are identified as dotted rectangles colored in blue and green and were related to the corresponding MALDI-TOF MS spectra. (b) The MALDI-TOF MS spectra of the chromatographic fraction related to the peak with a retention time of 37.4 min. (c) The MALDI-TOF MS spectra of chromatographic fraction related to the peak with a retention time of 39.3 min. In the red rectangles, the zoomed spectra of the masses are represented, and the major peaks related to the masses of 1050.7 Da and 1032.7 Da, which correlate to the masses of EmiA and dEmiA, which are marked with a green and with yellow star, respectively.

Figure 3

(a,b) The MALDI-TOF Ms/Ms spectra of the molecular ions with masses of 1032.7 Da and of 1050.7 Da are respectively depicted. Molecular mass differences of fragments is presented as a straight black line with the annotation of masses colored in red. The most unlikely fragment with the molecular formula and monoisotopic mass are highlighted in the black rectangle on both spectra. (a) Fragment relates to the 2-amino-6-hydroxy-4-methyl-8-oxodecanoic acid (AHMOA) residue. (b) Fragment relates to the 2-amino-4-methyl-8-oxodec-6-enoic acid (AHMOEA) residue (c). The structure of EmiA is presented without stereocenter indication. The residue of interest in the structure of EmiA was named as X and highlighted in the red dotted rectangle. The right and the left sets of the residues are highlighted in the black rectangles and named as R1 and R2 radical, respectively. Physico-chemical information about the full name of EmiA, its exact molecular weight with the condensed molecular formula, and information on the atom composition is depicted in the high right corner of (d).

Figure 4

(a) Cell index of the HCT116 cell line at different concentrations of emericellipsin A: 0.25 μg/mL; 1.0 µg/mL; 4.0 µg/mL; 16.0 µg/mL. (b) The level of apoptosis of HCT116 cells under the influence of emericellipsin A at a concentration of 0.25 μg/mL: zone B—cells with fragmented DNA (apoptosis); zone D is the percentage of cells in different phases of the cell cycle.