Anti-Candida albicans effect of the protein-carbohydrate fraction obtained from the coelomic fluid of earthworm Dendrobaena veneta.

Marta J Fiołka,Teresa Urbanik-Sypniewska,Tomasz Buchwald,Katarzyna Macur,Jolanta Kutkowska,Zbigniew Kaczyński,Jerzy Wydrych,Paulina Czaplewska,Roman Paduch,Vijai Gupta

doi:10.1371/journal.pone.0212869

Marta J Fiołka, Teresa Urbanik-Sypniewska + Show 8 more

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https://doi.org/10.1371/journal.pone.0212869

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Abstract

An antifungal active fraction (AAF) from the coelomic fluid (CF) of the earthworm Dendrobaena veneta was isolated. The aim of the study was to analyze the antifungal activity of the AAF and to carry out chemical characterization of the fraction. The active fraction showed antifungal activity against a clinical C. albicans isolate, C. albicans ATCC 10231, and C. krusei ATCC 6258. It effectively reduced the metabolic activity of C. albicans cells and influenced their morphology after 48 hours of incubation. Scanning electron microscopy (SEM) images revealed loss of integrity of the cell wall induced by the active fraction. Calcofluor White staining showed changes in the structure of the C. albicans cell wall induced by the AAF. The fungal cells died via apoptosis and necrosis after the treatment with the studied fraction. Electrophoresis under native conditions revealed the presence of two compounds in the AAF, while SDS/PAGE gel electrophoresis showed several protein and carbohydrate compounds. The active fraction was analyzed using Raman spectroscopy, MALDI TOF/TOF, and ESI LC-MS. The Raman analysis confirmed the presence of proteins and determined their secondary structure. The MALDI TOF/TOF analysis facilitated detection of four main compounds with a mass of 7694.9 m/z, 12292.3 m/z, 21628.3 m/z, and 42923.2 m/z in the analyzed fraction. The presence of carbohydrate compounds in the preparation was confirmed by nuclear magnetic resonance (NMR) and gas chromatography (GC-MS). The ATR-FTIR spectrum of the AAF exhibited high similarity to the spectrum of egg white lysozyme. The AAF showed no endotoxicity and cytotoxicity towards normal skin fibroblasts (HSF); therefore, it can be used for the treatment of skin and mucous membrane candidiasis in the future. Given its efficient and selective action, the fraction seems to be a promising preparation with antifungal activity against C. albicans.

Highlights

Candida albicans is a commensal organism, which can be part of the normal microbiota in healthy humans
The active fraction induced a significant decrease in the metabolic activity of the wild type C. albicans cells, even after the incubation at the low concentrations of protein
The number of living cells with red inclusions decreased, while the number of green fluorescing cells and yellow fluorescing cells increased with the increasing concentration of the antifungal active fraction (AAF)

Summary

Introduction

Candida albicans is a commensal organism, which can be part of the normal microbiota in healthy humans. This Ascomycete yeast is able to cause diseases in individuals with a weakened and compromised immune system [1]. It can cause superficial or invasive infections with a high mortality rate. An increase in resistance to antibiotics in the treatment of fungal infections has been observed. This world-scale problem has created a need to search for new antifungal agents

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