Abstract
BackgroundDevelopment of resistant variants to existing antifungal drugs continues to be the serious problem in Candida albicans-induced fungal pathogenesis, which has a considerable impact on animal and human health. Identification and characterization of newer drugs against C. albicans is, therefore, essential. MMGP1 is a direct cell-penetrating peptide recently identified from marine metagenome, which was found to possess potent antifungal activity against C. albicans.Methodology/Principal FindingsIn this study, we investigated the mechanism of antifungal action of MMGP1 against C. albicans. Agarose gel shift assay found the peptide to be having a remarkable DNA-binding ability. The modification of the absorption spectra and fluorescence quenching of the tryptophyl residue correspond to the stacking between indole ring and nucleotide bases. The formation of peptide–DNA complexes was confirmed by fluorescence quenching of SYTO 9 probe. The interaction of peptide with plasmid DNA afforded protection of DNA from enzymatic degradation by DNase I. In vitro transcription of mouse β-actin gene in the presence of peptide led to a decrease in the level of mRNA synthesis. The C. albicans treated with MMGP1 showed strong inhibition of biosynthetic incorporation of uridine analog 5-ethynyluridine (EU) into nascent RNA, suggesting the peptide’s role in the inhibition of macromolecular synthesis. Furthermore, the peptide also induces endogenous accumulation of reactive oxygen species (ROS) in C. albicans. MMGP1 supplemented with glutathione showed an increased viability of C. albicans cells. The hyper-produced ROS by MMGP1 leads to increased levels of protein carbonyls and thiobarbituric acid reactive substances and it also causes dissipation of mitochondrial membrane potential and DNA fragmentation in C. albicans cells.Conclusion And Significance: Therefore, the antifungal activity of MMGP1 could be attributed to its binding with DNA, causing inhibition of transcription followed by endogenous production of ROS, which triggers cascade of events that leads to cell death.
Highlights
Fungal disease, in a majority of the cases, in immunocompromised individuals is caused by the opportunistic fungal pathogen, Candida albicans
The results revealed that in the absence of DNA, MMGP1 exhibited a fluorescence emission maximum at 330 nm, and addition of DNA caused the fluorescence intensity to decrease and the emission maximum to shift from 330 nm to 310 nm, indicating that the conformational changes had occurred in the environment of tryptophan residues on DNA binding
It was reported in our laboratory that the MMGP1 peptide induces cell death in C. albicans cells in a nondisruptive manner through energy-independent direct penetration mechanism [12]
Summary
In a majority of the cases, in immunocompromised individuals is caused by the opportunistic fungal pathogen, Candida albicans. The mechanism of action of antifungal peptides involves membrane permeabilization/translocation, inhibition of cellular processes and formation of reactive oxygen species (ROS) in the fungal species [3]. In our laboratory, we have identified a novel peptide [MLWSASMRIFASAFSTRGLGTRMLMYCSLPSRCWRK (MMGP1)] from marine metagenome, which exhibited chitin binding and direct cell-penetrating property and was found to possess potent antifungal activity against C. albicans and A. niger. The hyper-produced ROS by MMGP1 leads to increased levels of protein carbonyls and thiobarbituric acid reactive substances and it causes dissipation of mitochondrial membrane potential and DNA fragmentation in C. albicans cells. Conclusion: And Significance: the antifungal activity of MMGP1 could be attributed to its binding with DNA, causing inhibition of transcription followed by endogenous production of ROS, which triggers cascade of events that leads to cell death
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