Antifungal and anti-biofilm activity of the first cryptic antimicrobial peptide from an archaeal protein against Candida spp. clinical isolates

Candida species cause cutaneous and systemic infections with a high mortality rate, especially in immunocompromised patients. The emergence of resistance to the most common antifungal drugs, also due to bioflm formation, requires the development of alternative antifungal agents. The antimicrobial peptide VLL-28, isolated from an archaeal transcription factor, shows comparable antifungal activity against 10 clinical isolates of Candida spp. Using a fuoresceinated derivative of this peptide, we found that VLL-28 binds to the surface of planktonic cells. This observation suggested that it could exert its antifungal activity by damaging the cell wall. In addition, analyses performed on bioflms via confocal microscopy revealed that VLL-28 is diferentially active on all the strains tested, with C. albicans and C. parapsilosis being the most sensitive ones. Notably, VLL-28 is the frst example of an archaeal antimicrobial peptide that is active towards Candida spp. Thus, this points to archaeal microorganisms as a possible reservoir of novel antifungal agent

The Role of the Conserved Residues His-246, His-199, and Tyr-255 in the Catalysis of Catechol 2,3-Dioxygenase from Pseudomonas stutzeri OX1

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Production of biofunctionalized MoS2 flakes with rationally modified lysozyme: a biocompatible 2D hybrid material

Bioapplications of 2D materials embrace demanding features in terms of environmental impact, toxicity and biocompatibility. Here we report on the use of a rationally modified lysozyme to assist the exfoliation of MoS2 bulk crystals suspended in water through ultrasonic exfoliation. The design of the proposed lysozyme derivative provides this exfoliated 2D-materail with both, hydrophobic groups that interact with the surface of MoS2 and hydrophilic groups exposed to the aqueous medium, which hinders its re-aggregation. This approach, clarified also by molecular docking studies, leads to a stable material (ζ-potential, 27 ± 1 mV) with a yield of up to 430 μg ml−1. The bio-hybrid material was characterized in terms of number of layers and optical properties according to different slots separated by diverse centrifugal forces. Furthermore the obtained material was proved to be biocompatible using human normal keratinocytes and human cancer epithelial cells, whereas the method was demonstrated to be applicable to produce other 2D materials such as graphene. This approach is appealing for the advantageous production of high quality MoS2 flakes and their application in biomedicine and biosensing. Moreover, this method can be applied to different starting materials, taking the denatured lysozyme a promising bio-tool for surface functionalization of 2D materials

THE IMPORTANCE OF DYNAMIC EFFECTS ON THE ENZYME ACTIVITY: X-RAY STRUCTURE AND MOLECULAR DYNAMICS OF ONCONASE MUTANTS.

Onconase (ONC), a member of the RNase A superfamily extracted from oocytes of Rana pipiens, is an effective cancer killer. It is currently used in treatment of various forms of cancer. ONC antitumor properties depend on its ribonucleolytic activity that is low in comparison with other members of the superfamily. The most damaging side effect from Onconase treatment is renal toxicity, which seems to be caused by the unusual stability of the enzyme. Therefore, mutants with reduced thermal stability and/or increased catalytic activity may have significant implications for human cancer chemotherapy. In this context, we have determined the crystal structures of two Onconase mutants (M23L-ONC and C87S,des103-104-ONC) and performed molecular dynamic simulations of ONC and C87S,des103-104-ONC with the aim of explaining on structural grounds the modifications of the activity and thermal stability of the mutants. The results also provide the molecular bases to explain the lower catalytic activity of Onconase compared with RNase A and the unusually high thermal stability of the amphibian enzyme

Modified denatured lysozyme effectively solubilizes fullerene c60 nanoparticles in water

Fullerenes, allotropic forms of carbon, have very interesting pharmacological effects and engineering applications. However, a very low solubility both in organic solvents and water hinders their use. Fullerene C60, the most studied among fullerenes, can be dissolved in water only in the form of nanoparticles of variable dimensions and limited stability. Here the effect on the production of C60 nanoparticles by a native and denatured hen egg white lysozyme, a highly basic protein, has been systematically studied. In order to obtain a denatured, yet soluble, lysozyme derivative, the four disulfides of the native protein were reduced and exposed cysteines were alkylated by 3-bromopropylamine, thus introducing eight additional positive charges. The C60 solubilizing properties of the modified denatured lysozyme proved to be superior to those of the native protein, allowing the preparation of biocompatible highly homogeneous and stable C60 nanoparticles using lower amounts of protein, as demonstrated by dynamic light scattering, transmission electron microscopy and atomic force microscopy studies. This lysozyme derivative could represent an effective tool for the solubilization of other carbon allotropes

The identification of a novel Sulfolobus islandicus CAMP-like peptide points to archaeal microorganisms as cell factories for the production of antimicrobial molecules

Background: Pathogenic bacteria easily develop resistance to conventional antibiotics so that even relatively new molecules are quickly losing efficacy. This strongly encourages the quest of new antimicrobials especially for the treatment of chronic infections. Cationic antimicrobial peptides (CAMPs) are small positively charged peptides with an amphipathic structure, active against Gram-positive and Gram-negative bacteria, fungi, as well as protozoa.Results: A novel (CAMP)-like peptide (VLL-28) was identified in the primary structure of a transcription factor, Stf76, encoded by pSSVx, a hybrid plasmid-virus from the archaeon Sulfolobus islandicus. VLL-28 displays chemical, physical and functional properties typical of CAMPs. Indeed, it has a broad-spectrum antibacterial activity and acquires a defined structure in the presence of membrane mimetics. Furthermore, it exhibits selective leakage and fusogenic capability on vesicles with a lipid composition similar to that of bacterial membranes. VLL-28 localizes not only on the cell membrane but also in the cytoplasm of Escherichia coli and retains the ability to bind nucleic acids. These findings suggest that this CAMP-like peptide could exert its antimicrobial activity both on membrane and intra cellular targets.Conclusions: VLL-28 is the first CAMP-like peptide identified in the archaeal kingdom, thus pointing to archaeal microorganisms as cell factories to produce antimicrobial molecules of biotechnological interest. Furthermore, results from this work show that DNA/RNA-binding proteins could be used as sources of CAMPs

Denatured lysozyme-coated carbon nanotubes: a versatile biohybrid material

Carbon nanotubes (CNTs) are among the most versatile nanomaterials, but their exploitation is hindered by limited dispersibility, especially in aqueous solvents. Here, we show that AP-LYS, a highly cationic soluble derivative of denatured hen egg lysozyme, is a very effective tool for the unbundling and solubilisation of CNTs. AP-LYS proved to mediate the complete and stable dispersion of CNTs at protein: CNT ratios ≥1: 3 (w:w) in very mild conditions (10-20 minutes sonication in ammonium acetate buffer, pH 5.0). Electrophoretic mobility and ζ-potential measurements confirmed that dispersed CNTs were coated by the protein, whereas molecular docking was used to study the interactions between AP-LYS and CNTs. AP-LYS-coated CNTs proved to be a very effective microbial cell-flocculating agent with an efficiency similar to that of chitosan, one of the best available flocculating agents, thus suggesting that this hybrid could find industrial applications in the treatment of wastewaters contaminated by microbial cells, or to remove microbial cells after fermentation processes. Moreover, we exploited the low stability of AP-LYS-coated CNT dispersions in eukaryotic cell culture media to prepare scaffolds with an extracellular matrix-like rough surface for the cultivation of eukaryotic cells

Host defence peptides identified in human apolipoprotein B as promising antifungal agents

Therapeutic options to treat invasive fungal infections are still limited. This makes the development of novel antifungal agents highly desirable. Naturally occurring antifungal peptides represent valid candidates, since they are not harmful for human cells and are endowed with a wide range of activities and their mechanism of action is different from that of conventional antifungal drugs. Here, we characterized for the first time the antifungal properties of novel peptides identified in human apolipoprotein B. ApoB-derived peptides, here named r(P)ApoBLPro, r(P)ApoBLAla and r(P)ApoBSPro, were found to have significant fungicidal activity towards Candida albicans (C. albicans) cells. Peptides were also found to be able to slow down metabolic activity of Aspergillus niger (A. niger) spores. In addition, experiments were carried out to clarify the mechanism of fungicidal activity of ApoB-derived peptides. Peptides immediately interacted with C. albicans cell surfaces, as indicated by fluorescence live cell imaging analyses, and induced severe membrane damage, as indicated by propidium iodide uptake induced upon treatment of C. albicans cells with ApoB-derived peptides. ApoB-derived peptides were also tested on A. niger swollen spores, initial hyphae and branched mycelium. The effects of peptides were found to be more severe on swollen spores and initial hyphae compared to mycelium. Fluorescence live cell imaging analyses confirmed peptide internalization into swollen spores with a consequent accumulation into hyphae. Altogether, these findings open interesting perspectives to the application of ApoB-derived peptides as effective antifungal agents

Protective Effects of Recombinant Human Angiogenin in Keratinocytes: New Insights on Oxidative Stress Response Mediated by RNases

Human angiogenin (ANG) is a 14-kDa ribonuclease involved in different pathophysiological processes including tumorigenesis, neuroprotection, inflammation, innate immunity, reproduction, the regeneration of damaged tissues and stress cell response, depending on its intracellular localization. Under physiological conditions, ANG moves to the cell nucleus where it enhances rRNA transcription; conversely, recent reports indicate that under stress conditions, ANG accumulates in the cytoplasmic compartment and modulates the production of tiRNAs, a novel class of small RNAs that contribute to the translational inhibition and recruitment of stress granules (SGs). To date, there is still limited and controversial experimental evidence relating to a hypothetical role of ANG in the epidermis, the outermost layer of human skin, which is continually exposed to external stressors. The present study collects compelling evidence that endogenous ANG is able to modify its subcellular localization on HaCaT cells, depending on different cellular stresses. Furthermore, the use of recombinant ANG allowed to determine as this special enzyme is effectively able to counter at various levels the alterations of cellular homeostasis in HaCaT cells, actually opening a new vision on the possible functions that this special enzyme can support also in the stress response of human skin