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.