Mechanisms of the Antifungal Action of Marine Metagenome-Derived Peptide, MMGP1, against Candida albicans

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.

Inhibitory effect ofBacillus subtilisWL-2 and its IturinA lipopeptides againstPhytophthora infestans

“…Next, the JC-1 solution was removed(62), and the mycelium was resuspended in PBS. The fluorescence of JC-1 (red fluorescence and green fluorescence) was monitored at Ex/Em = 490/525 nm and 490/590 nm using a CFM system(64).…”

Microbial flow cytometry: An ideal tool for prospective antimicrobial drug development

“…bacteria or yeast) can be studied based on the biosynthetic incorporation of EU into nascent RNA using flow cytometry [25,26]. RNA incorporated with EU can then be detected by tetramethyl rhodamine-azide (TMR-A) (10 µM) staining in the presence of CuSO 4 .…”

“…Dissipation of mitochondrial membrane potential in AMP-treated microbial cells can be analyzed by rhodamine 123 staining using flow cytometry [26]. Rhodamine 123 is a cellpermeant cationic fluorescent dye sequestered by active mitochondria.…”

Chasing New Drugs against Infectious Diseases: A Herculean Task

“…In recent past, pharmaceutical industries have slashed investment in antibiotics due to poor sales of existing drugs, whose overuse has spurred the spread of resistance. Industries claim that "bug-killing drugs" are severely undervalued, if it fails to fight against infections [5][6][7]. Thus, the global health impact of antimicrobial resistance together with rapid depletion of antibiotic arsenal has revived interest in the discovery and development of new therapeutic drugs.…”

“…In recent years, metagenomic approaches are being developed to access untapped reservoir of chemical diversity and for discovery of novel resistance determinants in clinical and natural environments [6]. As an alternative, metagenomics can be used as reliable approach to explore prospective reservoir of natural AMPs from unexplored environments, which might be the potential source of therapeutic peptides in near future.…”