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.

Fungal α-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 ß-1,3-glucan. The role of α-1,3-glucan, another fungal cell wall polysaccharide, in modulating the host immune response is not clear. This work investigates the potential of α-1,3-glucan as a fungal PAMP by analyzing the humoral immune response of the greater wax moth Galleria mellonella to Aspergillus niger α-1,3-glucan. We demonstrated that 57-kDa and 61-kDa hemolymph proteins, identified as ß-1,3-glucan recognition proteins, bound to A. niger α-1,3-glucan. Other hemolymph proteins, i.e., apolipophorin I, apolipophorin II, prophenoloxidase, phenoloxidase activating factor, arylphorin, and serine protease, were also identified among α-1,3-glucan-interacting proteins. In response to α-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 α-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 α-1,3-glucan can be postulated.

Morphological and physiological changes in Lentilactobacillus hilgardii cells after cold plasma treatment.

Atmospheric cold plasma (ACP) inactivation of Lentilactobacillus hilgardii was investigated. Bacteria were exposed to ACP dielectric barrier discharge with helium and oxygen as working gases for 5, 10, and 15 min. The innovative approach in our work for evaluation of bacterial survival was the use in addition to the classical plate culture method also flow cytometry which allowed the cells to be sorted and revealed different physiological states after the plasma treatment. Results showed total inhibition of bacterial growth after 10-min of ACP exposure. However, the analysis of flow cytometry demonstrated the presence of 14.4% of active cells 77.5% of cells in the mid-active state and 8.1% of dead cells after 10 min. In addition, some of the cells in the mid-active state showed the ability to grow again on culture medium, thus confirming the hypothesis of induction of VBNC state in L .hilgardii cells by cold plasma. In turn, atomic force microscopy (AFM) which was used to study morphological changes in L. hilgardii after plasma treatment at particular physiological states (active, mid-active, dead), showed that the surface roughness of the mid-active cell (2.70 ± 0.75 nm) was similar to that of the control sample (2.04 ± 0.55 nm). The lack of considerable changes on the cell surface additionally explains the effective cell resuscitation. To the best of our knowledge, AFM was used for the first time in this work to analyze cells which have been sorted into subpopulations after cold plasma treatment and this is the first work indicating the induction of VBNC state in L. hilgardii cells after exposure to cold plasma.

Antifungal Activity of Anionic Defense Peptides: Insight into the Action of Galleria mellonella Anionic Peptide 2.

Anionic antimicrobial peptides constitute an integral component of animal innate immunity, however the mechanisms of their antifungal activity are still poorly understood. The action of a unique Galleria mellonella anionic peptide 2 (AP2) against fungal pathogen Candida albicans was examined using different microscopic techniques and Fourier transform infrared (FTIR) spectroscopy. Although the exposure to AP2 decreased the survival rate of C. albicans cells, the viability of protoplasts was not affected, suggesting an important role of the fungal cell wall in the peptide action. Atomic force microscopy showed that the AP2-treated cells became decorated with numerous small clods and exhibited increased adhesion forces. Intensified lomasome formation, vacuolization, and partial distortion of the cell wall was also observed. FTIR spectroscopy suggested AP2 interactions with the cell surface proteins, leading to destabilization of protein secondary structures. Regardless of the anionic character of the whole AP2 molecule, bioinformatics analyses revealed the presence of amphipathic α-helices with exposed positively charged lysine residues. High content of the α-helical structure was confirmed after deconvolution of the IR absorption spectrum and during circular dichroism measurements. Our results indicated that the antimicrobial properties of G. mellonella AP2 rely on the same general characteristics found in cationic defense peptides.

Studies on the interactions of neutral Galleria mellonella cecropin D with living bacterial cells.

Cecropins constitute an important family of insect antimicrobial peptides involved in humoral innate immune response. In comparison with the highly basic cecropins A and B, cecropins D are less cationic and more hydrophobic. Interestingly, cecropins D were described only in lepidopteran insects, e.g., the greater wax moth Galleria mellonella. In the present study, interactions of neutral cecropin D (pI 6.47) purified from hemolymph of G. mellonella with living Escherichia coli cells were investigated. Fluorescence lifetime imaging microscopy using fluorescein isothiocyanate-labeled cecropin D revealed very fast binding of the peptide to E. coli cells. Fourier transform infrared spectroscopy analyses showed that G. mellonella cecropin D interacted especially with E. coli LPS and probably other lipid components of the bacterial cell envelope and exhibited an ordering effect with regard to lipid chains. This effect is consistent with the peptide binding mechanism based upon its incorporation into the lipid phase of the cell membrane. The interaction resulted in permeabilization of the bacterial cell membrane. Upon cecropin D binding, the cells lost characteristic surface topography, which was accompanied by altered nanomechanical properties, as revealed by atomic force microscopy. The interaction of the peptide with the bacterial cells also led to intracellular damage, i.e., loss of the cell envelope multilayer structure, formation of membrane vesicles, and enlargement of periplasmic space, which eventually caused death of the bacteria. In summary, it can be concluded that amphipathic character of α-helices, exposure of small positively charged patches on their polar surfaces and hydrophobic interactions are important physicochemical characteristics related to effective binding to E. coli cells and antibacterial activity of neutral G. mellonella cecropin D.

Galleria mellonella lysozyme induces apoptotic changes in Candida albicans cells.

The greater wax moth Galleria mellonella has been increasingly used as a model host to determine Candida albicans virulence and efficacy of antifungal treatment. The G. mellonella lysozyme, similarly to its human counterpart, is a member of the c-type family of lysozymes that exhibits antibacterial and antifungal activity. However, in contrast to the relatively well explained bactericidal action, the mechanism of fungistatic and/or fungicidal activity of lysozymes is still not clear. In the present study we provide the direct evidences that the G. mellonella lysozyme binds to the protoplasts as well as to the intact C. albicans cells and decreases the survival rate of both these forms in a time-dependent manner. No enzymatic activity of the lysozyme towards typical chitinase and ß-glucanase substrates was detected, indicating that hydrolysis of main fungal cell wall components is not responsible for anti-Candida activity of the lysozyme. On the other hand, pre-treatment of cells with tetraethylammonium, a potassium channel blocker, prevented them from the lysozyme action, suggesting that lysozyme acts by induction of programmed cell death. In fact, the C. albicans cells treated with the lysozyme exhibited typical apoptotic features, i.e. loss of mitochondrial membrane potential, phosphatidylserine exposure in the outer leaflet of the cell membrane, as well as chromatin condensation and DNA fragmentation.

Analysis of nanomechanical properties of Borrelia burgdorferi spirochetes under the influence of lytic factors in an in vitro model using atomic force microscopy.

BACKGROUND: Atomic force microscopy (AFM) is an experimental technique which recently has been used in biology, microbiology, and medicine to investigate the topography of surfaces and in the evaluation of mechanical properties of cells. The aim of this study was to evaluate the influence of the complement system and specific anti-Borrelia antibodies in in vitro conditions on the modification of nanomechanical features of B. burgdorferi B31 cells. MATERIAL AND METHODS: In order to assess the influence of the complement system and anti-Borrelia antibodies on B. burgdorferi s.s. B31 spirochetes, the bacteria were incubated together with plasma of identified status. The samples were applied on the surface of mica disks. Young's modulus and adhesive forces were analyzed with a NanoScope V, MultiMode 8 AFM microscope (Bruker) by the PeakForce QNM technique in air using NanoScope Analysis 1.40 software (Bruker). RESULTS/CONCLUSION: The average value of flexibility of spirochetes' surface expressed by Young's modulus was 10185.32 MPa, whereas the adhesion force was 3.68 nN. AFM is a modern tool with a broad spectrum of observational and measurement abilities. Young's modulus and the adhesion force can be treated as parameters in the evaluation of intensity and changes which take place in pathogenic microorganisms under the influence of various lytic factors. The visualization of the changes in association with nanomechanical features provides a realistic portrayal of the lytic abilities of the elements of the innate and adaptive human immune system.

Single-Layer Graphene as an Effective Mediator of the Metal-Support Interaction.

Single-layer chemical vapor deposition (CVD)-grown graphene was transferred onto a ZnO (0001) substrate forming a large-area, low-defect density, protective layer. The quality of the graphene layer and its effect on the interaction between the ZnO support and vapor-deposited cobalt particles was investigated by spectroscopic and microscopic techniques. We demonstrate that the in-between graphene layer influences both the oxidation state and the morphology of cobalt upon annealing in vacuum. In particular, cobalt strongly interacts with the bare ZnO substrate forming flat particles, which are readily oxidized and redispersed upon annealing in ultrahigh vacuum conditions. In contrast, in the presence of the graphene interlayer, cobalt forms highly dispersed nanoparticles, which are resistant to oxidation, but prone to surface diffusion and agglomeration. The graphene layer exhibits remarkable stability upon cobalt deposition and vacuum annealing, while interaction with reactive gases can facilitate the formation of defects.

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.

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

Insect 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.

Synergistic action of Galleria mellonella anionic peptide 2 and lysozyme against Gram-negative bacteria.

Lysozyme and antimicrobial peptides are key factors of the humoral immune response in insects. In the present work lysozyme and anionic defense peptide (GMAP2) were isolated from the hemolymph of the greater wax moth Galleria mellonella and their antibacterial activity was investigated. Adsorption of G. mellonella lysozyme on the cell surface of Gram-positive and Gram-negative bacteria was demonstrated using immunoblotting with anti-G. mellonella lysozyme antibodies. Lysozyme effectively inhibited the growth of selected Gram-positive bacteria, which was accompanied by serious alterations of the cell surface, as revealed by atomic force microscopy (AFM) imaging. G. mellonella lysozyme used in concentrations found in the hemolymph of naive and immunized larvae, perforated also the Escherichia coli cell membrane and the level of such perforation was considerably increased by GMAP2. GMAP2 used alone did not perforate E. coli cells nor influence lysozyme muramidase activity. However, the peptide induced a decrease in the turgor pressure of the bacterial cell. Moreover, in the samples of bacteria treated with a mixture of lysozyme and GMAP2 the sodium chloride crystals were found, suggesting disturbance of ion transport across the membrane leading to cell disruption. These results clearly indicated the synergistic action of G. mellonella lysozyme and anionic peptide 2 against Gram-negative bacteria. The reported results suggested that, thanks to immune factors constitutively present in hemolymph, G. mellonella larvae are to some extent protected against infection caused by Gram-negative bacteria.

The effect of Galleria mellonella apolipophorin III on yeasts and filamentous fungi.

Galleria mellonella apolipophorin III (apoLp-III) has been implicated in the innate immune response against bacterial infections. The protein binds components of bacterial cell wall and inhibits growth of selected Gram-positive and Gram-negative bacteria. Interaction of apoLp-III with fungal ß-1,3-glucan suggests antifungal properties of the protein. In the present study, the effect of apoLp-III on the growth, metabolic activity and cell surface characteristics of selected yeasts and filamentous fungi was investigated using light, confocal and atomic force microscopy. ApoLp-III bound to the cell surface of different yeasts and filamentous fungi as confirmed by immunoblotting with anti-apoLp-III antibodies. Incubation of the fungi in the presence of apoLp-III induced alterations in growth morphology. Candida albicans underwent transition from yeast-like to hyphal growth with formation of true hyphae, whereas Fusarium oxysporum hyphae exhibited decreased metabolic activity, increased vacuolization and appearance of numerous monophialids with microconidia. Atomic force microscopy imaging demonstrated evident alterations in the fungal cell surface after incubation with apoLp-III, suggesting that the protein affected the cell wall components.

Synthesis and characterisation of coating polyurethane cationomers containing fluorine built-in hard urethane segments.

Polyurethane cationomers were synthesised in the reaction of 4,4'-methylenebis(phenyl isocyanate) with polyoxyethylene glycol (M = 2,000) or poly(tetrafluoroethyleneoxide-co-difluoromethylene oxide) α,ω-diisocyanate and N-methyl diethanolamine. Amine segments were built-in to the urethane-isocyanate prepolymer in the reaction with 1-bromobutane or formic acid, and then they were converted to alkylammonium cations. The obtained isocyanate prepolymers were then extended in the aqueous medium that yielded stable aqueous dispersions which were applied on the surfaces of test poly(tetrafluoroethylene) plates. After evaporation of water, the dispersions formed thin polymer coatings. (1)H, (13)C NMR and IR spectral methods were employed to confirm chemical structures of synthesised cationomers. Based on (1)H NMR and IR spectra, the factors κ and α were calculated, which represented the polarity level of the obtained cationomers. The DSC, wide angle X-ray scattering and atom force microscopy methods were employed for the microstructural assessment of the obtained materials. Changes were discussed in the surface free energy and its components, as calculated independently according to the method suggested by van Oss-Good, in relation to chemical and physical structures of cationomers as well as morphology of coating surfaces obtained from those cationomers. Fluorine incorporated into cationomers (about 30%) contributed to lower surface free energy values, down to about 15 mJ/m(2). That was caused by gradual weakening of long-range interactions within which the highest share is taken by dispersion interactions.