Autophagy of Candida albicans cells after the action of earthworm Venetin-1 nanoparticle with protease inhibitor activity.

The present studies show the effect of the Venetin-1 protein-polysaccharide complex obtained from the coelomic fluid of the earthworm Dendrobaena veneta on Candida albicans cells. They are a continuation of research on the mechanisms of action, cellular targets, and modes of cell death. After the action of Venetin-1, a reduced survival rate of the yeast cells was noted. The cells were observed to be enlarged compared to the controls and deformed. In addition, an increase in the number of cells with clearly enlarged vacuoles was noted. The detected autophagy process was confirmed using differential interference contrast, fluorescence microscopy, and transmission electron microscopy. Autophagic vesicles were best visible after incubation of fungus cells with the Venetin-1 complex at a concentration of 50 and 100 µg mL-1. The changes in the vacuoles were accompanied by changes in the size of mitochondria, which is probably related to the previously documented oxidative stress. The aggregation properties of Venetin-1 were characterized. Based on the results of the zeta potential at the Venetin-1/KCl interface, the pHiep = 4 point was determined, i.e. the zeta potential becomes positive above pH = 4 and is negative below this value, which may affect the electrostatic interactions with other particles surrounding Venetin-1.

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

In the present research, the effect of a protein-polysaccharide complex Venetin-1 obtained from the coelomic fluid of Dendrobaena veneta earthworm on Candida albicans cells was characterized. The compound destroyed fungal cells without showing cytotoxicity to human skin fibroblasts, which was demonstrated in earlier studies. Since it had an effect on the fungal cell wall and membrane, this complex was compared with the known antifungal antibiotic fluconazole. Both preparations disturbed the division of yeast cells and resulted in the formation of aggregates and chains of unseparated cells, which was illustrated by staining with fluorochromes. Fluorescent staining of the cell wall with Calcofluor white facilitated comparison of the types of aggregates formed after the action of both substances. The analysis performed with the use of Congo red showed that Venetin-1 exposed deeper layers of the cell wall, whereas no such effect was visible after the use of fluconazole. The FTIR analysis confirmed changes in the mannoprotein layer of the cell wall after the application of the Venetin-1 complex. Staining with Rhodamine 123 and the use of flow cytometry allowed comparison of changes in the mitochondria. Significantly elongated mitochondria were observed after the Venetin-1 application, but not after the application of the classic antibiotic. Phase contrast microscopy revealed vacuole enlargement after the Venetin-1 application. The flow cytometry analysis of C. albicans cells treated with Venetin-1 and fluconazole showed that both substances caused a significant decrease in cell viability.

Antimycobacterial Activity of Sida hermaphrodita (L.) Rusby (Malvaceae) Seed Extract.

The current prevalence of such lifestyle diseases as mycobacteriosis and tuberculosis is a result of the growing resistance of microorganisms to the available antibiotics and their significant toxicity. Therefore, plants can successfully become a source of new therapeutic agents. The aim of this study was to investigate the effect of protein extract from Sida hermaphrodita seeds on the morphology, structure, and viability of Mycobacterium smegmatis and to carry out proteomic characterization of the protein extract. The analyses were carried out using fluorescence and transmission microscopy, atomic force microscopy, and spectroscopy. The proteomic studies were performed using liquid chromatography coupled to tandem mass spectrometry. The studies showed that the seed extract applied at concentrations of 50-150 µg/mL exerted a statistically significant effect on M. smegmatis cells, that is, a reduction of the viability of the bacteria and induction of changes in the structure of the mycobacterial cell wall. Additionally, the SEM analysis confirmed that the extract did not have a cytotoxic or cytopathic effect on fibroblast cells. The proteomic analysis revealed the presence of structural, storage, and enzymatic proteins and peptides in the extract, which are typical for seeds. Proteins and peptides with antimicrobial activity identified as vicillins and lipid-transporting proteins were also determined in the protein profile of the extract.

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

The present research shows the antitumor activity of a protein-polysaccharide complex Venetin-1 obtained from the coelomic fluid of Dendrobaena veneta earthworms against A549 cancer cells. The investigations are a continuation of experiments on the antitumor activity of coelomic fluid obtained from this species. The Venetin-1 nanoparticle was obtained after thermal treatment of the coelomic fluid, separation from coelomocytes, filtration, and lyophilization. The preparation showed a selective effect on cancer cells, whereas normal cells were unaffected. Venetin-1 was effective against the lung cancer cells at doses of 31.3 and 62.5 µg/ml, and the results were imaged using light microscopy and scanning electron microscopy (SEM). The cells died mainly via the apoptosis pathway. Necrotic cells appeared sporadically in the microscopic view. SEM imaging revealed complete destruction of the A549 cells after the incubation with Venetin-1. The atomic force microscopy (AFM) analyses showed changes in the topography, peak force error images, and Young's modulus (elasticity) of the A549 cells after the incubation with Venetin-1. The transmission electron cryomicroscopy (Cryo-TEM) analysis indicated a polymeric nature of the analyzed preparation. The samples of Venetin-1 showed a very homogeneous size profile with the microparticle size of approximately 58.23 nm. A significant decrease in Venetin-1 binding to sphingomyelin was observed. Venetin-1 lost its pore-forming activity or deactivation of the pore-forming activity occurred. This confirms the absence of hemolytic capacity of Venetin-1 towards red blood cells. The conducted analyses show the suitability of the obtained complex for biomedical research. The next step will consist in analyses of the effect of Venetin-1 on the immune system in mice.

Resistance of Dickeya solani strain IPO 2222 to lytic bacteriophage ΦD5 results in fitness tradeoffs for the bacterium during infection.

Resistance to bacteriophage infections protects bacteria in phage-replete environments, enabling them to survive and multiply in the presence of their viral predators. However, such resistance may confer costs for strains, reducing their ecological fitness as expressed as competitiveness for resources or virulence or both. There is limited knowledge about such costs paid by phage-resistant plant pathogenic bacteria in their natural habitats. This study analyzed the costs of phage resistance paid by the phytopathogenic pectinolytic bacterium Dickeya solani both in vitro and in potato (Solanum tuberosum L.) plants. Thirteen Tn5 mutants of D. solani IPO 2222 were identified that exhibited resistance to infection by lytic bacteriophage vB_Dsol_D5 (ΦD5). The genes disrupted in these mutants encoded proteins involved in the synthesis of bacterial envelope components (viz. LPS, EPS and capsule). Although phage resistance did not affect most of the phenotypes of ΦD5-resistant D. solani such as growth rate, production of effectors, swimming and swarming motility, use of various carbon and nitrogen sources and biofilm formation evaluated in vitro, all phage resistant mutants were significantly compromised in their ability to survive on leaf surfaces as well as to grow within and cause disease symptoms in potato plants.

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

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.

Effect of Low Temperature on Changes in AGP Distribution during Development of Bellis perennis Ovules and Anthers.

Arabinogalactan proteins (AGPs) are a class of heavily glycosylated proteins occurring as a structural element of the cell wall-plasma membrane continuum. The features of AGPs described earlier suggest that the proteins may be implicated in plant adaptation to stress conditions in important developmental phases during the plant reproduction process. In this paper, the microscopic and immunocytochemical studies conducted using specific antibodies (JIM13, JIM15, MAC207) recognizing the carbohydrate chains of AGPs showed significant changes in the AGP distribution in female and male reproductive structures during the first stages of Bellis perennis development. In typical conditions, AGPs are characterized by a specific persistent spatio-temporal pattern of distribution. AGP epitopes are visible in the cell walls of somatic cells and in the megasporocyte walls, megaspores, and embryo sac at every stage of formation. During development in stress conditions, the AGP localization is altered, and AGPs entirely disappear in the embryo sac wall. In the case of male development, AGPs are present in the tapetum, microsporocytes, and microspores in normal conditions. In response to development at lower temperature, AGPs are localized in the common wall of microspores and in mature pollen grains. Additionally, they are accumulated in remnants of tapetum cells.

Candida albicans cell wall as a target of action for the protein-carbohydrate fraction from coelomic fluid of Dendrobaena veneta.

The protein-polysaccharide fraction (AAF) isolated from the coelomic fluid of the earthworm Dendrobaena veneta destroys C. albicans cells by changing their morphology, disrupting cell division, and leading to cell death. Morphological changes in C. albicans cells induced by treatment with AAF were documented using DIC, SEM, and AFM. Congo Red staining showed that the fungal wall structure was changed after incubation with AAF. The effect on C. albicans cell walls was shown by AFM analysis of the surface roughness of fungal cell walls and changes in the wall thickness were visualized using Cryo-SEM. The FTIR analysis of C. albicans cells incubated with AAF indicated attachment of protein or peptide compounds to the fungal walls. The intact LC-ESI-MS analysis allowed accurate determination of the masses of molecules present in AAF. As shown by the chromatographic study, the fraction does not cross biological membranes. The Cryo-TEM analysis of AAF demonstrated the ability of smaller subunits to combine into larger agglomerates. AAF is thermally stable, which was confirmed by Raman spectroscopy. AAF can be considered as a potential antifungal antibiotic with activity against clinical C. albicans strains.

Sida hermaphrodita seeds as the source of anti - Candida albicans activity.

Sida hermaphrodita is a perennial herbaceous plant with potential economic importance; however, there is no information about its antimicrobial properties. The aim of our study was to analyze the morphology and metabolic activity of Candida albicans cells after exposure to the extract from S. hermaphrodita seeds, determine its cytotoxicity against human skin fibroblasts and carry out chemical analysis of the extract. Microscopic analysis showed that the crude seed extract (CSE) caused a significant decrease in the metabolic activity of fungal cells, clear cell deformation, and budding disturbances. The analysis of cytotoxicity showed no influence of the extract on the fibroblasts. The CSE and seed extract after dialysis (DSE) were analyzed using electrophoretic, chromatographic, and spectroscopic methods. SDS-PAGE electrophoresis showed the presence of proteins and carbohydrate compounds in the extract. The Raman spectroscopy analysis of the DSE confirmed the presence of proteins, while FTIR analyses revealed the occurrence of albumin-type proteins. The NMR and GC-MS analyses showed the presence of carbohydrates in the seed extract. The MALDI and ESI LC-MS/MS analysis of the CSE and the DSE fractions revealed the occurrence of vicilin-type and plant lipid transfer proteins. The seed extract is a promising formulation to use in C. albicans infections.

Antitumor activity and apoptotic action of coelomic fluid from the earthworm Dendrobaena veneta against A549 human lung cancer cells.

It is known that earthworm coelomic fluid (CF) can affect not only cancer but also normal cells. The study demonstrated that the CF of the earthworm Dendrobaena veneta exhibited cytotoxicity against A549 lung cancer cells but did not toward the bronchial epithelial cell line BEAS-2B. The selective effect on the tumor cells was achieved after a short-term CF heat pre-treatment at 70 °C. The cytotoxic effect of the CF was time- and concentration-dependent. The CF noticeably decreased the viability and affected the morphology of the A549 cells. Scanning electron microscopy revealed a different degree of destruction of the nucleus and cytoplasm of A549 cells. As determined by atomic force microscopy, the cell surface roughness increased while the cell stiffness was reduced upon the CF treatment. A twofold increase in the caspase 3, 4, 5, and 10 levels was observed in the A549 cells after the incubation with the CF. The results obtained by flow cytometry using Annexin V confirmed the proapoptotic effect of the earthworm CF on A549 lung cancer cells. The D. veneta CF and active fraction obtained with cytotoxicity toward A549 lung cancer is an interesting and promising preparation for further biological, chemical, and biomedical research.

Analysis of antifungal and anticancer effects of the extract from Pelargonium zonale.

The extract from Pelargonium zonale stalks exhibits activity against Candida albicans and exerts an effect on the HeLa cell line. The action against C. albicans cells was analysed using light, CLSM, SEM, and TEM microscopes. The observations indicate that the extract influenced fungal cell morphology and cell metabolic activity. The morphological changes include cell wall damage, deformations of cell surfaces, and abnormalities in fungal cell shape and size. Cells of C. albicans treated with the extract exhibited disturbances in the budding pattern and a tendency to form agglomerates and multicellular chains. The P. zonale extract caused a significant decrease in the metabolic activity of C. albicans cells. Cells died via both apoptosis and necrosis. The antitumor activity of the extract was analysed using the MTT assay. The P. zonale extract exhibited minor cytotoxicity against the HeLa cell line but a dose-dependent cytopathic effect was noticed. The P. zonale extract is a promising source for the isolation of antifungal and anticancer compounds.

Antifungal and anticancer effects of a polysaccharide-protein complex from the gut bacterium Raoultella ornithinolytica isolated from the earthworm Dendrobaena veneta.

The polysaccharide-protein complex (PPC) isolated from metabolites of gut bacteria Raoultella ornithinolytica from Dendrobaena veneta earthworms exhibits activity against Candida albicans, in breast ductal carcinoma (line T47D) and in the endometrioid ovarian cancer line (TOV-112D) in vitro. The action against C. albicans was analyzed using light, SEM, TEM, and AFM microscopes. The changes observed indicated two directions of the action of the complex, that is, disturbance of metabolic activity and cell wall damage. The PPC is an adhesion-promoting complex inducing death of C. albicans cells by necrosis. Owing to its significant effect on C. albicans, the complex is a promising source of antifungal compounds. The PPC showed a minimal cytotoxic effect against human skin fibroblasts; however, the cytotoxicity against the T47D line was determined at 20% and 15% against the TOV-112D line. The action of the PPC against the T47D line exerted a cytopathic effect, whereas in the TOV-112D line, it caused a reduction in the cell number. The PPC induced death of tumor cells by apoptosis and necrosis. In view of the negligible cytotoxicity on fibroblasts, the PPC will be subjected to chemical modifications to increase its antitumor activity for prospective medical applications.