No abstract

Considering the worldwide demand for colorants of natural origin, the utilization of ascomycete fungi as a prolific pigment producer unfolds a novel way to obtain these pigments for various applications, including food, cosmetic, and medical use. The presence of very few natural red pigment alternatives in the market also attracts research and industry priorities to unearth novel and sustainable red pigment producers. The present work is an attempt to identify a novel source of red color obtained from endophytic fungi isolated from terrestrial and marine habitats. Based upon the fungal capacity for pigment production, seven isolates of endophytic fungi were recognized as prospective pigment producers. Out of all, fungal isolate CPE04 was selected based upon its capacity to produce profuse extracellular red pigment. The isolate was identified as Talaromyces assiutensis, employing morphological features and phylogenetic characterization by internal transcribed spacer (ITS) sequences. To understand the chemical behavior of pigment molecules, an investigation of the chemical profile of fungal culture filtrate dried powder (CFDP) was performed using ultra-high-performance liquid chromatography-diode array detector-mass spectrometry (UPLC–DAD–MS). In total, eight compounds having pigment and pharmaceutical application were tentatively identified using UPLC–DAD–MS. Considering the commercial aspect of the stated work, an effort was also made for standardizing the upscaling of the pigment molecule. Investigations were performed for optimum medium and culturing conditions for maximum pigment production. CFDP was found to have a significant antibacterial activity against the bacterial pathogens Staphylococcus aureus (MTCC737), Vibrio cholerae (N16961), and methicillin-resistant S. aureus (MRSA) (ATCC BAA811). The CFDP showed a minimum inhibitory concentration at 64, 128, and 256 μg/ml against S. aureus, MRSA, and V. cholerae. A concentration-dependent (50–400 μg/ml) anticancer effect on HeLa cancer line was also observed, having a half-maximal inhibitory concentration (IC50) at 300 μg/ml. The antioxidant potential of CFDP has also been proven with the help of an antioxidant assay against 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) radical (IC50, 32.01 μg/ml); DNA nicking assay and reactive oxygen species were generated in HeLa cancer line cells. The CFDP was also found to have no cytotoxicity toward HEK 293 T cell line using alamar blue (resazurin), a cell metabolic activity reagent.

VopK, a type III effector protein, has been implicated in the pathogenesis of Vibrio cholerae strains belonging to diverse serogroups. Ectopic expression of this protein exhibits strong toxicity in yeast model system. In order to map critical residues in VopK, we scanned the primary sequence guided by available data on various toxins and effector proteins. Our in silico analysis of VopK indicated the presence of predicted MCF1-SHE (SHxxxE) serine peptidase domain at the C-terminus region of the protein. Substitution of each of the predicted catalytic triad residues namely Ser314, His353 and Glu357 with alanine resulted in recombinant VopK proteins varying in lethality as evaluated in yeast model system. We observed that replacement of glutamate357 to alanine causes complete loss in toxicity while substitutions of serine314 and histidine353 with alanine exhibited partial loss in toxicity without affecting the stability of variants. In addition, replacement of another conserved serine residue at position 354 (S354) within predicted S314H353E357 did not affect toxicity of VopK. In essence, combined in silico and site directed mutagenesis, we have identified critical amino acids contributing to the lethal activity of VopK in yeast model system.

VopE, a mitochondrial targeting T3SS effector protein of Vibrio cholerae, perturbs innate immunity by modulating mitochondrial dynamics. In the current study, ectopic expression of VopE was found to be toxic in a yeast model system and toxicity was further aggravated in the presence of various stressors. Interestingly, a VopE variant lacking predicted mitochondrial targeting sequence (MTS) also exhibited partial lethality in the yeast system. With the aid of yeast genetic tools and different stressors, we have demonstrated that VopE and its derivative VopEΔMTS modulate cell wall integrity (CWI-MAPK) signaling pathway and have identified several critical residues contributing to the lethality of VopE. Furthermore, co-expression of two effectors VopEΔMTS and VopX, interfering with the CWI-MAPK cellular pathway can partially suppress the VopX mediated yeast growth inhibition. Taken together, these results suggest that VopE alters signaling through the CWI-MAPK pathway, and demonstrates the usefulness of yeast model system to gain additional insights on the functionality of VopE.

HapR is a quorum-sensing master regulatory protein in Vibrio cholerae. Though many facts are known regarding its structural and functional aspects, much still can be learnt from natural variants of this wild-type protein. While unraveling the underlying cause of functional inertness of a natural variant (HapRV2), the significance of a conserved glycine residue at position 39 in a glycine-rich linker in DNA-binding domain comes into light. This work aims at investigating how the length of glycine-rich linker (R(33)GIGRGG(39)) bridging helices α1 and α2 modulates the functionality of HapR. In pursuit of our interest, glycine residues were inserted after terminal glycine (G39) of the linker in a sequential manner. To evaluate functionality, all the glycine linker variants were subjected to a battery of performance tests under various conditions. Combined in vitro and in vivo results clearly demonstrated a gradual functional impairment of HapR linker variants coupled with increasing length of glycine-rich linker and finally, linker variant harboring four glycine residues resulted in a functionally compromised protein with significant loss of communication with cognate DNAs. Molecular dynamics studies of modeled HapR linker variants in complex with cognate promoter region show that residues namely Ser50, Thr53 and Asn56 are involved in varying degree of interactions with different nucleotides of HapR-DNA complex. The diminished functionality between variants and DNA appears to result from reduced or no interactions between Phe55 and nucleotides of cognate DNA as observed during simulations.

: Antioxidants are the substances that interact inside and outside of a biological system, against the damaging effects of highly reactive molecules called free radicals produced during metabolism. Among various natural alternative sources of bioactive metabolites, endophytic fungi have emerged as a significant reservoir of potent antioxidant compounds. These scantly explored micro-organisms are prolific producers of novel metabolites and have the capability to produce compounds that are exclusively isolated from Plantae. A wide array of compounds like nucleobases, polyketides, terpenoids, flavonoids, coumarins, xanthones, semiquinones, peptides, and phenolic acids have been identified as natural antioxidants. Some of these free radical scavengers have shown a wide application in the food and pharmaceutical industry as potential food preservatives, nutraceuticals, antibacterial, anticancer and antifungal agents. This review aims to highlight some of the novel compounds from endophytic fungi, discovered between the years 2013 – 2018 and their application as potential antioxidant candidates discussed through various biological assays.

No abstract

Food acceptability and appeal are significantly influenced by colour. Harmful effects associated with synthetic colorants are well established, and research is currently focused on developing natural, synthetic chemical-free substitutes from fungal sources, with broad applications in food, medicine, textiles and agriculture. Additionally, the market’s dearth of natural red colour substitutes requires the creation of novel red pigment alternatives from secure and scalable sources. The goal of the current research was to establish new endophytic marine fungi that are naturally occurring bio-sources of the red pigment. Based on its profuse extracellular red pigment-producing capacity, the fungus CPEF02 was selected and identified as Monascus purpureus CPEF02 via internal transcribed spacer (ITS) sequences and phylogenetic analysis. The chemical moieties of the pigmented extracts were identified by liquid chromatography-high resolution mass spectrometry (LC-HRMS). The optimal culture conditions for maximum pigment production were investigated by surveying various media compositions. The methanolic fungal colourant extract was shown to have substantial antibacterial and antifungal activities against anthropogenic pathogens, Staphylococcus aureus (MTCC 1430), methicillin-resistant Staphylococcus aureus (ATCCBAA811), Salmonella typhimurium (MTCC 3241) and Vibrio cholerae (N16961) at a 100 µg/mL concentration and at a 1 mg/mL concentration for Alternaria solani (ITCC 4632) and Rhizoctonia solani (AG1-IA). This extract also exhibited antioxidant activity against the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical with an IC50 of 14.42 µg/mL and a Trolox equivalent antioxidant capacity of 0.571 µM Trolox/µg of the methanolic colourant extract. The findings suggested that M. purpureus’s pigment could be a source of an industrially useful natural red colourant.