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Fig. 4 in Isolation and functional characterization of an antifungal hydrophilic peptide, Skh-AMP1, derived from Satureja khuzistanica leaves

Fig. 4. (a) Primary structure alignment of Skh-AMP1 against other antifungal peptides from different plant sources is shown. Colored residues correspond to the conserved residues. (b) Three-dimensional structure model of Skh-AMP1. Hydrophilic and hydrophobic areas are shown red and blue regions, respectively. (c) Helical wheel projection of Skh-AMP1. The hydrophilic residues as circles, hydrophobic residues as diamonds, potentially negatively charged as triangles, and potentially positively charged as pentagons.

Fig. 1 in Isolation and functional characterization of an antifungal hydrophilic peptide, Skh-AMP1, derived from Satureja khuzistanica leaves

Fig. 1. (a) Purification of peptides from Satureja khuzistanica leaves. Two hundred fifty μL aliquot of lyophilized extract subjected to reverse phase-HPLC column by using a gradient from 5 to 65% (v/v) solution B (0.098% TFA in acetonitrile) combined with solution A (0.1% TFA in water) during 85 min. The absorbance was monitored at wavelength 220 nm. Totally, 15 peaks were collected. (b) The purity peak S2 was evaluated using the same column and method. (c) Tricine-SDS-PAGE profile of (1) protein standards from 2 kDa to 250 kDa (2) peptide extract passed through an ultra membrane with a 10-kDa cutoff, (3) The peak S2 purified from RP-HPLC column.

Fig. 3 in Isolation and functional characterization of an antifungal hydrophilic peptide, Skh-AMP1, derived from Satureja khuzistanica leaves

Fig. 3. Antifungal effects of peak S2 (identified as Skh-AMP1) against Candida albicans (1), Candida krusei (2), Candida glabrata (3), Aspergillus niger (4), Aspergillus flavus (5) and Aspergillus fumigatus (6) are shown. Amphotericin B and Peak S2 were used at 17.31 μM and 36 μM concentrations, respectively. Results represent means ± SD of three independent experiments.

Fig. 2 in Isolation and functional characterization of an antifungal hydrophilic peptide, Skh-AMP1, derived from Satureja khuzistanica leaves

Fig. 2. (a) Mass spectrum of peptide showing multiply protonated ions of the analyte. Note that not the first (monoisotopic) signals are annotated, but the most abundant one where 13C is incorporated. (b) MS/MS spectrum of peptide QELCTWERGSVRQADK (LysC digest, coverage from doubly protonated spectrum in top of list, from triply protonated ion in bottom of list). (c). MS/MS spectrum profile of a) peptide TSKQELCTWER (tryptic digest), b) peptide GSVRQADKTLAG (tryptic digest), c) peptide GRTSKQELCTWER (tryptic digest).

opencc-by-4.0Nov 2023View details →

ZIF-8 encapsulation improves the antifungal activity of benzaldehyde and methyl anthranilate in films

Relevant Data for publication wit DOI <a title="Link to landing page via DOI" href="https://doi.org/10.1039/D3DT03229A">10.1039/D3DT03229A</a>

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Structure-guided discovery of potent antifungals that prevent Ras signaling by inhibiting protein farnesyltransferase

Infections by fungal pathogens are difficult to treat due to a paucity of antifungals and emerging resistances. Next-generation antifungals therefore are needed urgently. We have developed compounds that prevent farnesylation of Cryptoccoccus neoformans Ras protein by inhibiting protein farnesyltransferase with 3–4 nanomolar affinities. Farnesylation directs Ras to the cell membrane and is required for infectivity of this lethal pathogenic fungus. Our high-affinity compounds inhibit fungal growth with 3–6 micromolar minimum inhibitory concentrations, 4- to 8-fold better than Fluconazole, an antifungal commonly used in the clinic. Compounds bound with distinct inhibition mechanisms at two alternative, partially overlapping binding sites, accessed via different inhibitor conformations. We showed that antifungal potency depends critically on the selected inhibition mechanism, because this determines the efficacy of an inhibitor at low in vivo levels of enzyme and farnesyl substrate. We elucidated how chemical modifications of the antifungals encode the desired inhibitor conformation and concomitant inhibitory mechanism.

opencc-zeroOct 2022View details →

Fig. 6 in Structural and functional features of a class VI chitinase from cashew (Anacardium occidentale L.) with antifungal properties

Fig. 6. Morphological alterations induced in Lasiodiplodia theobromae hyphae by Anacardium occidentale chitinase. SEM images of untreated mycelium(A-B), mycelium treated with 500 μg AoChi (C–I) and mycelium treated with 5 μg Carbendazim (J–M) are shown. The assays to evaluate the morphological alterations in L. theobromae grown in vitro in the presence of AoChi and acquisition of SEM images were performed as described in the Methods section. SEM images from fungus grown in the presence of water (A–B) or Carbendazim (J–M) were included for comparison.

Fig. 7 in Structural and functional features of a class VI chitinase from cashew (Anacardium occidentale L.) with antifungal properties

Fig. 7. Three-dimensional molecular model of AoChi and its interaction with a chitin oligomer. (A) Cartoon representation of the three-dimensional molecular model of AoChi, which was generated by homology modeling. Side chains of putative catalytic residues are shown as sticks. Disulfide bonds are colored orange. (B) Detailed view of a chito-oligosaccharide (yellow) docked in the substrate-binding cleft of AoChi. Side chains of AoChi residues that probably interact with the docked ligand through hydrogen bonds (represented as yellow dotted lines) are shown as sticks (cyan). N and O atoms are colored blue and red, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5 in Structural and functional features of a class VI chitinase from cashew (Anacardium occidentale L.) with antifungal properties

Fig. 5. Effects of temperature (A), pH (B), metal ions (C) and chemical reagents (D) on the hydrolytic activity (A–D) and stability (A–B) of AoChi. Enzymatic assays were performed using the purified recombinant protein (200 μg/mL) and colloidal chitin as substrate, as described in the Methods section. In panels C and D, means that are significantly different (P <0.05; Bonferroni's multiple comparisons test) when compared to control are indicated by asterisks.

Fig. 4 in Structural and functional features of a class VI chitinase from cashew (Anacardium occidentale L.) with antifungal properties

Fig. 4. Polyacrylamide gel electrophoresis (SDS-PAGE) of the purified recombinant chitinase from Anacardium occidentale, a member of class VI that has chitinolytic activity. (A) The recombinant chitinase (lane 1; 35 μg) was reduced with β-mercaptoethanol and subjected to denaturing gel electrophoresis (15% polyacrylamide), as described in the Methods section. Lane M: protein markers. (B) Representative fragmentation mass spectrum (LC-MS/MS) of a tryptic peptide, obtained from in gel-digestion of the 45 kDa protein band, that matched a segment of 17 residues of the primary structure of the recombinant product. (C) Colorimetric enzymatic assay, showing the ability of the recombinant cashew chitinase to degrade colloidal chitin. (D) Plot of initial reaction velocities vs substrate concentration. The enzymatic assays (panels C and D) were performed at pH 6.0 and 40 ◦C, and using colloidal chitin as substrate, as described in the Methods section.

Fig. 3 in Structural and functional features of a class VI chitinase from cashew (Anacardium occidentale L.) with antifungal properties

Fig. 3. Alignment of the amino acid sequence of the CatD of AoChi with known three-dimensional structures of GH19 chitinases from plants. The CatD sequence of AoChi was aligned to GH19 structures from: Carica papaya (PDB ID: 3CQL), Vigna unguiculata subsp. sesquipedalis (PDB ID: 4TX7), Secale cereale (PDB ID: 4DWX), Hordeum vulgare (PDB ID: 2BAA), Oryza sativa subsp. japonica (PDB ID: 2DKV), Canavalia ensiformis (PDB ID: 1DXJ), Brassica juncea (PDB ID: 2Z37), Cryptomeria japonica (PDB ID: 5H7T) and Picea abies (PDB ID: 3HBD). Sites containing residues involved in catalysis (triangles) and substrate-binding (hash marks) are indicated. Disulfide bonds, as observed in the three-dimensional model of AoChi (Fig. 7), are indicated by orange lines. Loop regions (I–V), as assigned by Taira et al. (Taira et al., 2011), are boxed. Loop VI (this work), which is present only in AoChi and other class VI chitinases, is also boxed. Alignment columns are colored according to the ALSCRIPT Calcons convention, as implemented in ALINE (Bond and Schüttelkopf, 2009), using a predefined color scheme, which reflects the conservation of amino acid properties in each column (dark blue: identical residues; white: dissimilar residues). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2 in Structural and functional features of a class VI chitinase from cashew (Anacardium occidentale L.) with antifungal properties

Fig. 2. Maximum likelihood (ML) tree showing the phylogenetic relationship of Anacardium occidentale chitinase with representative plant chitinases from classes I, II, IV, VI and VII. The evolutionary history of the sequences was inferred by using the ML method and Whelan and Goldman + Freq. model (Whelan and Goldman, 2001). The tree with the highest log likelihood (12837.58) is shown. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 1.5328)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 3.91% sites). The percentage of trees in which the associated sequences clustered together, as determined by the bootstrap test (100 replicates), is shown next to the branches. This analysis involved 62 amino acid sequences, and the final dataset contained a total of 371 positions (Table S2 and Fig. S7). Evolutionary analyses were conducted in MEGA X (Kumar et al., 2018).

Fig. 1 in Structural and functional features of a class VI chitinase from cashew (Anacardium occidentale L.) with antifungal properties

Fig. 1. Domain architecture of Anacardium occidentale chitinase (AoChi). (A) Graphical representation of AoChi domains (SP: signal peptide; GH19 CatD: GH19 catalytic domain). The catalytic residues of AoChi, Lys(K)128, Glu(E)150 and Tyr(Y)183, are highlighted. (B) Alignment of a stretch of 61 residues from the primary structure of AoChi (from His124 to Trp184), which contains the 3 catalytic residues, with corresponding segments from homologous proteins belonging to plant species from different families and orders (Table S5). Alignment columns are colored according to the ALSCRIPT Calcons convention, as implemented in ALINE (Bond and Schüttelkopf, 2009), using a predefined color scheme, which reflects the conservation of amino acid properties in each column (dark red: identical residues; white: dissimilar residues). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

opennotspecifiedJun 2020View details →

Fig. 2 in Terpenoids from the Chinese liverwort Odontoschisma grosseverrucosum and their antifungal virulence activity

Fig. 2. Selected HMBC (H→C), 1H–1H COSY (H▬H) and NOESY correlations (H↔H) of 1 (A/B), 4 (D/E), 8 (F\G) and the X-ray crystallographic structure of 3 (C).

opennotspecifiedJun 2020View details →

Fig. 3 in Terpenoids from the Chinese liverwort Odontoschisma grosseverrucosum and their antifungal virulence activity

Fig. 3. (A) The inhibition effect of compound 1 on the morphologic transition. (B) The inhibitory effect of compound 1 on the adhesion of C. albicans to A549 cells. (C) The number of adherent cells was determined by measuring the total size of the picture. The bars represent means ± SDs. * denotes P <0.05, **P <0.01, and ***P <0.001. (D) The inhibition effect of compound 1 on C. albicans biofilm formation. (E) The biofilm was detected with a CCK-8 reduction assay. The bars represent means ± SDs. * denotes P <0.05, and ** denotes P <0.01. (F) The downregulation of genes that are involved in virulence of C. albicans. The bars represent means ± SDs. * denotes P <0.05, **P <0.01, and ***P <0.001.

Fig. 2 in Identification, characterization, and antifungal activity of cysteine peptidases from Calotropis procera latex

Fig. 2. Three-dimensional models of cysteine peptidases from Calotropis procera (CpCP A, CpCP C and CpCP D). The models were generated with the SWISSMODEL platform and show their active sites for proteolytic activity, which are constituted by the triad Cys, Asn and His. The cysteine peptidase (PDB: 1BY8) was used as a reference.

Fig. 7 in Identification, characterization, and antifungal activity of cysteine peptidases from Calotropis procera latex

Fig. 7. Detection of ROS in spores of F. oxysporum after treatment with latex peptidases (CpCP1, CpCP2 and CpCP3) or inhibited with iodoacetamide (CpCP 1-IAA, CpCP 2-IAA and CpCP 3-IAA). Uptake of DAB was identified by the presence of a reddish-brown precipite. Samples (50 μg/mL) were incubated with spores for 30 min at 27 ̊C, pH 7.0. Control: 50 mM sodium phosphate buffer (pH 7.0). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 1 in Identification, characterization, and antifungal activity of cysteine peptidases from Calotropis procera latex

Fig. 1. Multiple amino acid sequence alignment of C. procera cysteine peptidases. Sequence alignment was performed with C. procera cysteine peptidases (CpCP A-E), (SnuCalCp 01–20), procerain B, procerain and papain. The highly conserved cysteine residues that participate in the disulfide bond formation are highlighted in gray and the conserved residues involved in the active site are indicated by vertical black arrows.

Fig. 4 in Identification, characterization, and antifungal activity of cysteine peptidases from Calotropis procera latex

Fig. 4. Antifungal activity of three cysteine peptidases (CpCP1, CpCP2 and CpCP3) from Calotropis procera latex on Fusarium oxysporum and Colletotrichum acutatum. The fungal growth was measured by absorbance at 620 nm after 72 h at 27 ̊C. Control: 50 mM sodium phosphate buffer (pH 7.0, containing 1 mM L-Cysteine). The samples were dissolved in 50 mM sodium phosphate buffer, containing 1 mM L-cysteine, at 50 μg/mL. Different letters indicate statistical difference compared with the control (p <0.05).

Fig. 6 in Identification, characterization, and antifungal activity of cysteine peptidases from Calotropis procera latex

Fig. 6. Membrane permeabilization induced by cysteine peptidases from Calotropis procera latex in spores of Fusarium oxysporum. Peptidases (50 μg/mL) were incubated with spore suspensions (2 × 106 spores/mL) for 30 min at 27 ̊C and then the fluorescence of propidium iodide was detected using fluorescence microscopy. Bars: 5 μm. Control: 50 mM sodium phosphate buffer (pH 7.0).