Artificial Lipidation of Antifungal Proteins and Antifungal Behavior: A Case Study with Cholesterylation.

Lipid-based formulation of antifungal small drugs is used to mitigate drug toxicity while retaining effective antifungal activity. Our previous work demonstrated a method to enhance the antifungal properties of a chitin-binding domain (LysM) and catalytic domain (CatD) of antifungal chitinase by microbial transglutaminase (MTG)-mediated palmitoylation. Herein, we studied the effect of artificial cholesterylation of LysM and CatD, both of which were site-specifically modified using an MTG-catalyzed crosslinking.

Topical Delivery of Artificial Lipidated Antifungal Proteins for the Treatment of Subcutaneous Fungal Infections Using a Biocompatible Ionic Liquid-Based Microemulsion.

The rising incidence of fungal infections, compounded by the emergence of severe antifungal resistance, has resulted in an urgent need for innovative antifungal therapies. We developed an antifungal protein-based formulation as a topical antifungal agent by combining an artificial lipidated chitin-binding domain of antifungal chitinase (LysM-lipid) with recently developed ionic liquid-in-oil microemulsion formulations (MEFs). Our findings demonstrated that the lipid moieties attached to LysM and the MEFs effectively disrupted the integrity of the stratum corneum in a mouse skin model, thereby enhancing the skin permeability of the LysM-lipids.

Design of Protease-Responsive Antifungal Liposomal Formulation Decorated with a Lipid-Modified Chitin-Binding Domain.

is a prevalent fungal pathogen that displays antibiotic resistance. The polyene antifungal amphotericin B (AmB) has been the gold standard because of its broad antifungal spectra, and its liposomal formulation, AmBisome, has been used widely and clinically in treating fungal infections. Herein, we explored enhancing the antifungal activity of AmBisome by integrating a small chitin-binding domain (LysM) of chitinase A derived from .

Characterization of an antifungal β-1,3-glucanase from Ficus microcarpa latex and comparison of plant and bacterial β-1,3-glucanases for fungal cell wall β-glucan degradation.

Each β-1,3-glucanase with antifungal activity or yeast lytic activity hydrolyzes different structures of β-1,3-glucans in the fungal cell wall, respectively. Plants express several glycoside hydrolases that target chitin and β-glucan in fungal cell walls and inhibit pathogenic fungal infection. An antifungal β-1,3-glucanase was purified from gazyumaru (Ficus microcarpa) latex, designated as GlxGluA, and the corresponding gene was cloned and expressed in Escherichia coli.

GH-16 Type β-1,3-Glucanase from sp. MK9-1 Enhances Antifungal Activity of GH-19 Type Chitinase, and Its Glucan-binding Domain Binds to Fungal Cell-wall.

The GH-16 type β-1,3-glucanase (BgluC16MK) gene of sp. MK9-1 was cloned to study its antifungal activities. BgluC16MK displays amino acid sequence similarity with GluC from strain N4-7.

Liposomal Amphotericin B Formulation Displaying Lipid-Modified Chitin-Binding Domains with Enhanced Antifungal Activity.

Fungal infections affect more than one billion people worldwide and cause more than one million deaths per year. Amphotericin B (AmB), a polyene antifungal drug, has been used as the gold standard for many years because of its broad antifungal spectrum, high activity, and low tendency of drug resistance. However, the side effects of AmB, such as nephrotoxicity and hepatotoxicity, have hampered its widespread use, leading to the development of a liposome-type AmB formulation, AmBisome.

Preparation of amphotericin B-loaded hybrid liposomes and the integration of chitin-binding proteins for enhanced antifungal activity.

Amphotericin B (AMB) is a gold standard antifungal drug because of its broad-spectrum activity toward pathogenic yeasts and molds. Because of its low solubility in water and toxicity toward humans, several lipid-based formulations that either increase the aqueous solubility or decrease the side effects have been employed in practical use. In our previous research, we found that the combination of AMB with an artificial palmitoylated chitin-binding domain from Pteris ryukyuensis chitinase (LysM-Pal) resulted in synergistic antifungal action against Trichoderma viride.

Structure, mechanism, and phylogeny of LysM-chitinase conjugates specifically found in fern plants.

A unique GH18 chitinase containing two N-terminal lysin motifs (PrLysM1 and PrLysM2) was first found in fern, Pteris ryukyuensis (Onaga and Taira, Glycobiology, 18, 414-423, 2008). This type of LysM-chitinase conjugates is not usually found in plants but in fungi. Here, we produced a similar GH18 chitinase with one N-terminal LysM module (EaLysM) from the fern, Equisetum arvense (EaChiA, Inamine et al.

Structural Analysis and Construction of a Thermostable Antifungal Chitinase.

Chitin is a biopolymer of -acetyl-d-glucosamine with β-1,4-bond and is the main component of arthropod exoskeletons and the cell walls of many fungi. Chitinase (EC 3.2.

Enhancement of the Antifungal Activity of Chitinase by Palmitoylation and the Synergy of Palmitoylated Chitinase with Amphotericin B.

Combinations of antifungal drugs can have synergistic antifungal activity, achieving high therapeutic efficacy while minimizing the side effects. Amphotericin B (AMB) has been used as a standard antifungal drug for fungal infections; however, because of its high toxicity, new strategies to minimize the required dose are desirable. Chitinases have recently received attention as alternative safe antifungal agents.

Identification of Genes Involved in the Synthesis of the Fungal Cell Wall Component Nigeran and Regulation of Its Polymerization in Aspergillus .

Certain Aspergillus and spp. produce the fungal cell wall component nigeran, an unbranched d-glucan with alternating α-1,3- and α-1,4-glucoside linkages, under nitrogen starvation. The mechanism underlying nigeran biosynthesis and the physiological role of nigeran in fungal survival are not clear.

Orthogonal Enzymatic Conjugation Reactions Create Chitin Binding Domain Grafted Chitinase Polymers with Enhanced Antifungal Activity.

Enzymatic reaction offers site-specific conjugation of protein units to form protein conjugates or protein polymers with intrinsic functions. Herein, we report horseradish peroxidase (HRP)- and microbial transglutaminase (MTG)-catalyzed orthogonal conjugation reactions to create antifungal protein polymers composed of chitinase-A (ChiA) and its two domains, catalytic domain, CatD, and chitin-binding domain, LysM. We engineered the ChiA and CatD by introducing a peptide tag containing tyrosine (Y-tag) at N-termini and a peptide tag containing lysine and tyrosine (KY-tag) at C-termini to construct Y-ChiA-KY and Y-CatD-KY.

cDNA cloning, expression, and antifungal activity of chitinase from Ficus microcarpa latex: difference in antifungal action of chitinase with and without chitin-binding domain.

A chitin-binding domain could contribute to the antifungal ability of chitinase through its affinity to the fungal lateral wall by hydrophobic interactions. Complementary DNA encoding the antifungal chitinase of gazyumaru (Ficus microcarpa), designated GlxChiB, was cloned and expressed in Escherichia coli cells. The results of cDNA cloning showed that the precursor of GlxChiB has an N-terminal endoplasmic reticulum targeting signal and C-terminal vacuolar targeting signal, whereas mature GlxChiB is composed of an N-terminal carbohydrate-binding module family-18 domain (CBM18) and a C-terminal glycoside hydrolase family-19 domain (GH19) with a short linker.

Cloning, expression, and characterization of a GH 19-type chitinase with antifungal activity from Lysobacter sp. MK9-1.

The chitin-assimilating gram-negative bacterium, Lysobacter sp. MK9-1, was isolated from soil and was the source of a glycoside hydrolase family 19-type chitinase (Chi19MK) gene that is 933-bp long and encodes a 311-residue protein. The deduced amino acid sequence of Chi19MK includes a signal peptide, an uncharacterized sequence, a carbohydrate-binding module family 12-type chitin binding domain, and a catalytic domain.

Hyperglycemic Crisis in Patients With Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like Episodes (MELAS).

Background: Diabetes mellitus is the most commonly encountered endocrinopathy in patients with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), which manifests as multisystemic organ failure. Whether the management of diabetes mellitus in MELAS requires special consideration is not fully clarified.

Methods: In this single-center study, we retrospectively reviewed the medical records of patients with MELAS to elucidate the clinical characteristics of MELAS-associated diabetes mellitus.

Phenolic acid decarboxylase of Aspergillus luchuensis plays a crucial role in 4-vinylguaiacol production during awamori brewing.

Aspergillus luchuensis has been used to produce awamori, a distilled liquor, in Okinawa, Japan. Vanillin, derived from ferulic acid (FA) in rice grains, is one of the characteristic flavors in aged and matured awamori, known as kusu. Decarboxylation of FA leads to the production of 4-vinylguaiacol (4-VG), which is converted to vanillin by natural oxidization.

Antifungal activities of LysM-domain multimers and their fusion chitinases.

PrChiA is an antifungal chitinase obtained from Pteris ryukyuensis, a fern plant. It consists of two N-terminal lysin motif (LysM) domains and a C-terminal catalytic domain of glycoside hydrolase family 18. Previous studies have shown that the deletion of LysM domains or loss of hydrolytic activity causes the loss of the antifungal activity of chitinases.

Mode of action and specificity of a chitinase from unicellular microalgae, Euglena gracilis.

Euglena gracilis is a unicellular microalga showing characteristics of both plants and animals, and extensively used as a model organism in the research works of biochemistry and molecular biology. Biotechnological applications of E. gracilis have been conducted for production of numerous important compounds.

Purification, cDNA cloning, and characterization of plant chitinase with a novel domain combination from lycophyte Selaginella doederleinii.

Unlabelled: Chitinase-A from a lycophyte Selaginella doederleinii (SdChiA), having molecular mass of 53 kDa, was purified to homogeneity by column chromatography. The cDNA encoding SdChiA was cloned by rapid amplification of cDNA ends and polymerase chain reaction. It consisted of 1477 nucleotides and its open reading frame encoded a polypeptide of 467 amino acid residues.

Structure and Enzymatic Properties of a Two-Domain Family GH19 Chitinase from Japanese Cedar ( Cryptomeria japonica) Pollen.

CJP-4 is an allergen found in pollen of the Japanese cedar Cryptomeria japonica. The protein is a two-domain family GH19 (class IV) Chitinase consisting of an N-terminal CBM18 domain and a GH19 catalytic domain. Here, we produced recombinant CJP-4 and CBM18-truncated CJP-4 (CJP-4-Cat) proteins.

Evoked potential studies for predicting functional recovery in a case of acute necrotizing encephalopathy.

Normal-appearing evoked potentials during the acute stage of the disease despite persistent coma may predict subsequent functional recovery of the brain in a pediatric case of acute necrotizing encephalopathy, indicating that evoked potential studies are useful for predicting functional outcome of the brain.

Unique GH18 chitinase from Euglena gracilis: full-length cDNA cloning and characterization of its catalytic domain.

A cDNA of putative chitinase from Euglena gracilis, designated EgChiA, encoded 960 amino acid residues, which is arranged from N-terminus in the order of signal peptide, glycoside hydrolase family 18 (GH18) domain, carbohydrate binding module family 18 (CBM18) domain, GH18 domain, CBM18 domain, and transmembrane helix. It is likely that EgChiA is anchored on the cell surface. The recombinant second GH18 domain of EgChiA, designated as CatD2, displayed optimal catalytic activity at pH 3.

Comparative multi-omics analysis reveals diverse latex-based defense strategies against pests among latex-producing organs of the fig tree (Ficus carica).

Latexes in immature fruit, young petioles and lignified trunks of fig trees protect the plant using toxic proteins and metabolites in various organ-dependent ways. Latexes from plants contain high amounts of toxic proteins and metabolites, which attack microbes and herbivores after exudation at pest-induced wound sites. The protein and metabolite constituents of latexes are highly variable, depending on the plant species and organ.

Crystal structure and thermodynamic dissection of chitin oligosaccharide binding to the LysM module of chitinase-A from Pteris ryukyuensis.

We determined the crystal structure of a LysM module from Pteris ryukyuensis chitinase-A (PrLysM2) at a resolution of 1.8 Å. Structural and binding analysis of PrLysM2 indicated that this module recognizes chitin oligosaccharides in a shallow groove comprised of five sugar-binding subsites on one side of the molecule.