Metabolic, structural, and proteomic changes in Candida albicans cells induced by the protein-carbohydrate fraction of Dendrobaena veneta coelomic fluid

Abstract

The isolated protein-polysaccharide fraction (AAF) from the coelomic fluid of Dendrobaena veneta earthworm shows effective activity against Candida albicans yeast. Fungal cells of the clinical strain after incubation with the active fraction were characterized by disturbed cell division and different morphological forms due to the inability to separate the cells from each other. Staining of the cells with acridine orange revealed a change in the pH of the AAF-treated cells. It was observed that, after the AAF treatment, the mitochondrial DNA migrated towards the nuclear DNA, whereupon both merged into a single nuclear structure, which preceded the apoptotic process. Cells with a large nucleus were imaged with the scanning electron cryomicroscopy (Cryo-SEM) technique, while enlarged mitochondria and the degeneration of cell structures were shown by transmission electron microscopy (TEM). The loss of the correct cell shape and cell wall integrity was visualized by both the TEM and SEM techniques. Mass spectrometry and relative quantitative SWATH MS analysis were used to determine the reaction of the C. albicans proteome to the components of the AAF fraction. AAF was observed to influence the expression of mitochondrial and oxidative stress proteins. The oxidative stress in C. albicans cells caused by the action of AAF was demonstrated by fluorescence microscopy, proteomic methods, and XPS spectroscopy. The secondary structure of AAF proteins was characterized by Raman spectroscopy. Analysis of the elemental composition of AAF confirmed the homogeneity of the preparation. The observed action of AAF, which targets not only the cell wall but also the mitochondria, makes the preparation a potential antifungal drug killing the cells of the C. albicans pathogen through apoptosis.

Novel Venetin-1 nanoparticle from earthworm coelomic fluid as a promising agent for the treatment of non-small cell lung cancer

“…Previous chemical analyses carried out in studies on antifungal activity 27 , 28 , 30 showed that proteins of the lysenin family: lysenin-related protein 2 (LRP2) and lysenin, which were identified with an accuracy of 95% and 90% sequence coverage, respectively, were the main components of Venetin-1. Lysenin is a 33 kDa protein found in the coelomic fluid of earthworms 50 .…”

“…The system was controlled by SageElf software version 1.08. It yielded 12 fractions of the preparation, which were then digested using a standard FASP procedure and trypsin (Trypsin Gold, Promega) 27 , 65 . Fractions obtained from two runs were combined for one FASP procedure.…”

Atypical changes in Candida albicans cells treated with the Venetin-1 complex from earthworm coelomic fluid

“…2 µL of the cell suspension was analyzed under the microscope. The dyes were used for visualization of genetic material of the cells in their current state: regular cells showed blue fluorescence of nuclei, apoptotic cells exhibited bright white florescence of fragmented genetic material, and necrotic cells had red fluorescence 39 , 40 .…”

“…Next, the cells were transferred into a sublimation chamber with a temperature of − 92 °C for 12 min. After that, the frozen cultures were cut in the preparation chamber and analyzed with a ZEISS Ultra Plus Field Emission Microscope (Carl Zeiss, Germany) with electron high tension (EHT) 5 kV and under magnification 30.000 × 39 , 42 .…”

“…The cells prepared in this way were dehydrated in a series of acetone dilutions (15%, 30%, 50%, 70%, 100%), transferred onto SEM stages with carbon discs, and dried in a desiccator for 24 h in the presence of roasted silicone gel (for 2 h at 180 °C). Afterwards, the stages with the probes were coated with a gold layer using a K550X sputtering machine (Quorum Technologies, United Kingdom) and observed with the use of a scanning electron microscope Tescan Vega 3 (Tescan Orsay Holding, Czech Republic) 39 .…”