Synthesis and study of antifungal properties of new cationic beta-glucan derivatives

The interaction of positively charged polymers (polycations) with a biological membrane is considered to be the cause of the frequently observed toxicity of these macromolecules. If it is possible to obtain polymers with a predominantly negative effect on bacterial and fungal cells, such systems would have great potential in the treatment of infectious diseases, especially now when reports indicate the growing risk of fungal co-infections in COVID-19 patients. We describe in this article cationic derivatives of natural beta-glucan polymers obtained by reacting the polysaccharide isolated from Saccharomyces boulardii (SB) and Cetraria islandica (CI) with glycidyl trimethyl ammonium chloride (GTMAC). Two synthesis strategies were applied to optimize the product yield. Fungal diseases particularly affect low-income countries, hence the emphasis on the simplicity of the synthesis of such drugs so they can be produced without outside help. The three structures obtained showed selective anti-mycotic properties (against, i.e., Scopulariopsis brevicaulis, Aspergillus brasiliensis, and Fusarium solani), and their toxicity established using fibroblast 3T3-L1 cell line was negligible in a wide range of concentrations. For one of the polymers (SB derivative), using in vivo model of Aspergillus brasiliensis infection in Galleria mellonella insect model, we confirmed the promising results obtained in the preliminary study

Fungal α\alpha-1,3-glucan as a new pathogen-associated molecular pattern in the insect model host Galleria mellonella

Recognition of pathogen-associated molecular patterns (PAMPs) by appropriate pattern recognition receptors (PRRs) is a key step in activating the host immune response. The role of a fungal PAMP is attributed to beta-1,3-glucan. The role of $\alpha$-1,3-glucan, another fungal cell wall polysaccharide, in modulating the host immune response is not clear. This work investigates the potential of $\alpha$-1,3-glucan as a fungal PAMP by analyzing the humoral immune response of the greater wax moth Galleria mellonella to Aspergillus niger $\alpha$-1,3-glucan. We demonstrated that 57-kDa and 61-kDa hemolymph proteins, identified as $\beta$-1,3-glucan recognition proteins, bound to A. niger $\alpha$-1,3-glucan. Other hemolymph proteins, i.e., apolipophorin I, apolipophorin II, prophenoloxidase, phenoloxidase activating factor, arylphorin, and serine protease, were also identified among $\alpha$-1,3-glucan-interacting proteins. In response to $\alpha$-1,3-glucan, a 4.5-fold and 3-fold increase in the gene expression of antifungal peptides galiomicin and gallerimycin was demonstrated, respectively. The significant increase in the level of five defense peptides, including galiomicin, corresponded well with the highest antifungal activity in hemolymph. Our results indicate that A. niger $\alpha$-1,3-glucan is recognized by the insect immune system, and immune response is triggered by this cell wall component. Thus, the role of a fungal PAMP for $\alpha$-1,3-glucan can be postulated

Synergistic action of Galleria mellonella apolipophorin III and lysozyme against Gram-negative bacteria

AbstractInsect immune response relies on the humoral and cellular mechanisms of innate immunity. The key factors are the antimicrobial polypeptides that act in concert against invading pathogens. Several such components, e.g. apolipophorin III (apoLp-III), lysozyme, and anionic peptide 2, are present constitutively in the hemolymph of non-challenged Galleria mellonella larvae. In the present study, we demonstrate an evidence for a synergistic action of G. mellonella lysozyme and apoLp-III against Gram-negative bacteria, providing novel insights into the mode of action of these proteins in insect antimicrobial defense. It was found that the muramidase activity of G. mellonella lysozyme considerably increased in the presence of apoLp-III. Moreover, apoLp-III enhanced the permeabilizing activity of lysozyme toward Escherichia coli cells. As shown using non-denaturing PAGE, the proteins did not form intermolecular complexes in vivo and in vitro, indicating that the effect observed was not connected with the intermolecular interactions between the proteins. Analysis of AFM images of E. coli cells exposed to G. mellonella lysozyme and/or apoLp-III revealed evident alterations in the bacterial surface structure accompanied by the changes in their biophysical properties. The bacterial cells demonstrated significant differences in elasticity, reflected by Young's modulus, as well as in adhesive forces and roughness values in comparison to the control ones. The constitutive presence of these two defense molecules in G. mellonella hemolymph and the fact that apoLp-III enhances lysozyme muramidase and perforating activities indicate that they can be regarded as important antibacterial factors acting at the early stage of infection against Gram-negative as well as Gram-positive bacteria

Amphotericin B-Silver Hybrid Nanoparticles Help to Unveil the Mechanism of Biological Activity of the Antibiotic: Disintegration of Cell Membranes

Amphotericin B is a popular antifungal antibiotic, and despite decades of pharmacological application, the exact mode of its biological activity is still a matter of debate. Amphotericin B-silver hybrid nanoparticles (AmB-Ag) have been reported to be an extremely effective form of this antibiotic to combat fungi. Here, we analyze the interaction of AmB-Ag with C. albicans cells with the application of molecular spectroscopy and imaging techniques, including Raman scattering and Fluorescence Lifetime Imaging Microscopy. The results lead to the conclusion that among the main molecular mechanisms responsible for the antifungal activity of AmB is the disintegration of the cell membrane, which occurs on a timescale of minutes